WO2013026021A2 - Trka as a target for inhibiting app cleavage and/or progression of alzheimer's disease - Google Patents

Trka as a target for inhibiting app cleavage and/or progression of alzheimer's disease Download PDF

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Publication number
WO2013026021A2
WO2013026021A2 PCT/US2012/051426 US2012051426W WO2013026021A2 WO 2013026021 A2 WO2013026021 A2 WO 2013026021A2 US 2012051426 W US2012051426 W US 2012051426W WO 2013026021 A2 WO2013026021 A2 WO 2013026021A2
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compound
inhibitor
group
trka
addn
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PCT/US2012/051426
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French (fr)
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WO2013026021A3 (en
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Varghese John
Qiang Zhang
Dale E. Bredesen
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Buck Institute For Research On Aging
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2999/00—Further aspects of viruses or vectors not covered by groups C12N2710/00 - C12N2796/00 or C12N2800/00
    • C12N2999/005—Biological teaching, e.g. a link between protein and disease, new virus causing pandemic

Definitions

  • AD Alzheimer's disease
  • MCI mild cognitive impairment
  • the disease broadly falls into two categories: a) late onset AD, which occurs generally at 65 years or older and is correlated to numerous risk factors including presence of an APOE ⁇ 4 allele; and b) early onset AD, which develops early on between 30 and 60 years of age and is generally associated with familial Alzheimer's disease (FAD) mutations in the amyloid precursor protein (APP) gene or in the presenilin gene.
  • FAD familial Alzheimer's disease
  • APP amyloid precursor protein
  • the disease is characterized by at least two types of lesions in the brain, senile plaques composed of the ⁇ peptide (and other components, typically at lower concentrations than the ⁇ peptide) and neurofibrillary tangles composed primarily of intracellular deposits of microtubule associated tau protein (especially hyperphosphorylated tau).
  • senile plaques composed of the ⁇ peptide (and other components, typically at lower concentrations than the ⁇ peptide)
  • neurofibrillary tangles composed primarily of intracellular deposits of microtubule associated tau protein (especially hyperphosphorylated tau).
  • CSF cerebrospinal fluid
  • AD Alzheimer's disease
  • ⁇ peptide a small peptide that damages brain cells by physical and chemical properties, such as the binding of damaging metals, reactive oxygen species production, and direct damage to cell membranes. While such effects of ⁇ have been clearly demonstrated, they do not offer a physiological role for the peptide.
  • AD physiological signaling properties
  • APP has all of the characteristics of a dependence-receptor, i.e., a receptor that mediates cell-death in the presence of an anti-trophin (in this case, AB) but supports cell survival in the presence of a trophic-factor (such as laminin).
  • a dependence-receptor i.e., a receptor that mediates cell-death in the presence of an anti-trophin (in this case, AB) but supports cell survival in the presence of a trophic-factor (such as laminin).
  • AD Alzheimer's disease
  • Alzheimer's disease results from an imbalance between two normal processes: memory formation and normal forgetting.
  • the data show that the ⁇ peptide can play a role in modulating, processing and signaling through binding to the amyloid precursor protein (APP), and thus play a central role in the pathogenesis of Alzheimer's disease through signaling rather than chemical and physical effects.
  • APP amyloid precursor protein
  • APP695 can be cleaved by caspases at an intracellular site (Asp664), leading to the release of a small C31 peptide and an APPneo (APP664) fragment, and both products are potentially proapoptotic (Galavan et al. supra).
  • Immunohistochemical analysis of AD brain demonstrates that this cytoplasmic cleavage occurs 4-fold more in AD brain than normal, and the products are found around plaques and tangles in key brain areas affected by the disease (Bredesen et al. (2006) Nature 443: 796-802).
  • methods for inhibiting (partially or fully) the C-terminal cleavage of APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal.
  • the methods typically involve administering, or causing to be administered, to the mammal a TrkA kinase inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a APP-C31 peptide and/ APPneo.
  • the inhibitor comprises ADDN-1351 or a derivative thereof.
  • APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal are provided where the methods comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a APP-C31 peptide and/or APPneo (APP664).
  • methods of mitigating in a mammal one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms in a mammal comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to mitigate said one or more symptoms.
  • methods of reducing the risk, lessening the severity, or delaying the progression or onset of a disease e.g.,
  • Alzheimer's disease age-related macular degeneration (AMD), Cerebrovascular dementia, Parkinson's disease, Huntington's disease, Cerebral amyloid angiopathy, etc.
  • AMD age-related macular degeneration
  • Cerebrovascular dementia Parkinson's disease
  • Huntington's disease Cerebral amyloid angiopathy, etc.
  • beta-amyloid deposits in the brain of a mammal where the methods comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to reduce the risk, lessen the severity, and/or delay the progression or onset of the disease.
  • methods of preventing or delaying the onset of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or preventing or delaying the progression of a pre -Alzheimer's condition or cognitive dysfunction to Alzheimer's disease in a mammal comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway.
  • APP amyloid precursor protein
  • methods of promoting the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway as characterized by increasing sAPPa and/or the sAPPa/Ap42 ratio in a mammal comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway.
  • APP amyloid precursor protein
  • a method comprising administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a APP-C31 peptide and/or APPneo (APP664); and/or administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to mitigate said one or more symptoms associated with a disease (e.g., Alzheimer's disease, age-related macular degeneration (AMD), Cerebrovascular dementia, Parkinson's disease, Huntington's disease, Cerebral amyloid angiopathy, etc.) characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms; and/or administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to reduce the risk, lessen the severity, and/or delay the progression or onset of a disease characterized by beta-amyloid deposits in the brain of the
  • a disease
  • the TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siRNA.
  • the TrkA inhibitor comprises a small organic molecule inhibitor of TrkA.
  • the TrkA kinase inhibitor has the formula:
  • R 1 is selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy, thioalkyl, and aminoalkyl; and R 2 and R 3 are independently selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl.
  • R 1 is a Ci_ 6 alkyl. In certain embodiments R 1 is substituted alkyl. In certain embodiments R 1 is unsubstituted alkyl. In certain embodiments R 1 is substituted alkenyl. In certain embodiments R 1 is unsubstituted alkenyl. In certain embodiments R 1 is substituted alkynyl. In certain embodiments R 1 is
  • R 1 is substituted alkoxy. In certain embodiments R 1 is unsubstituted alkoxy. In certain embodiments R 1 is substituted thioalkyl. In certain embodiments R 1 is unsubstituted thioalkyl. In certain embodiments R 1 is substituted aminoalkyl. In certain embodiments R 1 is unsubstituted aminoalkyl. In certain embodiments R 2 is aryl or substituted aryl, or heteroaryl, or substituted heteroaryl. In certain of any of these embodiments R 3 is aryl, or is substituted aryl, or heteroaryl, or substituted heteroaryl.
  • the TrkA kinase inhibitor is ADDN- 1351 or an analogue thereof. In certain embodiments the TrkA kinase inhibitor is ADDN- 1351. In certain embodiments the TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN-1351d, ADDN-1351e, ADDN- 135 If, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN-1351j, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN- 135 lp as shown in Table 4. In certain embodiments the TrkA inhibitor comprises a compound according to the formula:
  • R 1 is selected from the group consisting of cyclopropyl, O'Pr, SMe, Me, OPr, H, and 3 ⁇ 4u
  • R 2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and halogen
  • R 3 is selected from the group consisting of H, (S)-MQ, (R)-MQ, (S)-CH 2 OH, (i?)-CH 2 OH, (5)-Me, (i?)-CH 2 OH, (5)-CH 2 CONMe 2 , and (5)-CH 2 CONHMe
  • Y 1 and Y 2 are independent selected from the group consisting of CH, and N
  • R 7 i selected from the group consisting of H, OH, CH 3
  • R 2 is F.
  • Y 1 is CH and Y 2 is N, or Y 1 and Y 2 are both N, or Y 1 and Y 2 are both CH.
  • the TrkA inhibitor comprises a compound according to the formula:
  • X is selected from the group consisting of Me, H, and halogen
  • R 2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and F
  • R 3 is selected from the group consisting of H, (S)-MQ, (R)-MQ, (5)-CH 2 OH, ( ?)-CH 2 OH, (S)-MQ, ( ?)-CH 2 OH, (5)-CH 2 CONMe 2 , and (S)-CH 2 CONHMe.
  • X is CI or Br.
  • the compound is a compound selected from the group consisting of compound 10a, compound 10b, compound 10c, compound lOd, compound lOe, compound lOf, compound lOg, compound lOh, compound lOi, compound lOj, compound 10k, compound 101, compound 10m, and compound 10 ⁇ , as shown in Table 5.
  • the TrkA inhibitor comprises a compound according to the formula:
  • TrkA inhibitor comprises a compound according to the formula:
  • X is selected from the group consisting of Me, H, and halogen
  • R 10 is selected from the group consisting of H, and OH
  • R 1 is selected from the group consisting of cyclopropyl, O'Pr, Me, OPr, H, and fuse
  • R 7 is selected from the group consisting of H, OH, CH 3 ,
  • X is CI or H.
  • R 1 is selected from the group consisting of Cp, Me, and O'Pr.
  • R 7 is selected from the group consisting of
  • the compound is a compound selected from the group consisting of compound 15a, compound 15b, compound 15c, compound 15d, compound 15e, compound 15f, and compound 15g, as shown in Table 7.
  • the TrkA inhibitor comprises a compound according to the formula:
  • X is selected from the group consisting of Me, H, and halogen
  • R 1 is selected from the group consisting of Cp, and O'Pr
  • Y 1 and Y 2 are independent selected from the group consisting of CH, and CH
  • R 7 is selected from the group consisting of H, OH, CH 3 ,
  • X is F or CI.
  • Y 1 is CH and Y 2 is N, or Y 1 and Y 2 are both N.
  • R 7 is selected from the group consisting of H,
  • the compound is a compound selected from the group consisting of compound lOx, compound lOy, compound lOz, compound 15h, compound 15i, and compound 1 la, as shown in Table 8.
  • the TrkA inhibitor comprises a compound according to the formula:
  • TrkA inhibitor comprises a compound selected from the group consisting of compound 1 , compound 5a, compound 5b, compound 5c, compound 5d, compound 5e, compound 5f, compound 5g, compound 5h, and compound 6, as shown in Table 9.
  • TrkA inhibitor comprises a compound selected from the group consisting of compoundl3a, compound 13b, compound 13c, compound 14a, compound 14b, compound 13d, compound 14c, compound 13e, compound 13f, and compound 13g, as shown in Table 10.
  • TrkA inhibitor comprises a compound according to the formula:
  • the TrkA inhibitor comprises a compound selected from the group consisting of compound 19i, compound 19j, compound 191, compound 191, compound 19m, compound 19n, and compound 19o, as shown in Table XI. In certain embodiments, the TrkA inhibitor comprises a compound according to the formula:
  • the TrkA inhibitor comprises a compound according to the formula:
  • the TrkA inhibitor comprises a compound according to the formula:
  • the TrkA inhibitor comprises a compound according to the formula:
  • the TrkA inhibitor comprises a compound according to the formula
  • R 1 is selected from the group consisting of Cp, CH 3 , O'Pr, O'Pr, O'Pr, O'Pr, OEt, and OCH 3 ; and R 2 , when present, is selected from the group consisting of CH 3 , and CH 2 OH.
  • the inhibitor comprises a compound selected from the group consisting of compound 17a, compound 17b, compound 17c, compound 17d, compound 17e, compound 18a, compound 18b, and compound 18c, as shown in Table 12.
  • the inhibitor is a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11.
  • the mammal is a human.
  • the mammal is diagnosed as a pre -Alzheimer's and/or a pre- MCI cognitive impairment. In certain embodiments of these methods, administration of the TrkA kinase inhibitor delays or prevents the progression of cognitive dysfunction to MCI. In certain embodiments of these methods, the mammal is diagnosed as having mild cognitive impairment (MCI). In certain embodiments of these methods, TrkA kinase inhibitor delays or prevents the progression of MCI to Alzheimer's disease. In certain embodiments of these methods, the mammal is diagnosed as having moderately severe cognitive decline (Moderate or mid- stage Alzheimer's disease).
  • MCI mild cognitive impairment
  • TrkA kinase inhibitor delays or prevents the progression of MCI to Alzheimer's disease. In certain embodiments of these methods, the mammal is diagnosed as having moderately severe cognitive decline (Moderate or mid- stage Alzheimer's disease).
  • the mammal is diagnosed as having severe cognitive decline (moderately severe or mid-stage Alzheimer's disease). In certain embodiments of these methods, the mammal is diagnosed as having Alzheimer's disease. In certain embodiments of these methods, the mammal is at risk of developing Alzheimer's disease. In certain embodiments of these methods, the mammal has a familial risk for having Alzheimer's disease. In certain embodiments of these methods, mammal has a familial Alzheimer's disease (FAD) mutation. In certain embodiments of these methods, the mammal has the APOE ⁇ 4 allele. In certain embodiments of these methods, the mammal is free of and does not have genetic risk factors of Parkinson's disease or schizophrenia.
  • FAD familial Alzheimer's disease
  • the mammal is not diagnosed as having or at risk for Parkinson's disease or schizophrenia. In certain embodiments of these methods, the mammal does not have a neurological disease or disorder other than Alzheimer's disease. In certain embodiments of these methods, the mammal is not diagnosed as having or at risk for a neurological disease or disorder other than Alzheimer's disease. In certain embodiments of these methods, the mammal is not diagnosed as having, and/or at risk for, and/or under treatment for one or more indications selected from the group consisting of cancer, pain, inflammation, rheumatoid arthritis, and an immunological disorder. In certain embodiments of these methods, mammal is a mammal not diagnosed as having cancer and/or not being treated for cancer.
  • method produces a reduction in the CSF of levels of one or more additional components selected from the group consisting of Tau, phospho-Tau (pTau), soluble ⁇ 40 and soluble ⁇ 42.
  • the method results in a reduction of the plaque load in the brain of the mammal.
  • the method results in a reduction in the rate of plaque formation or deposition in the brain of the mammal.
  • the method results in an improvement in the cognitive abilities of the mammal.
  • the mammal is a human and said method produces a perceived improvement in quality of life by said human.
  • the TrkA kinase inhibitor is administered orally.
  • the administering is over a period of at least three weeks. In certain embodiments of these methods the administering is over a period of at least 6 months. In certain embodiments of these methods the TrkA kinase inhibitor is formulated for administration via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration. In certain embodiments the TrkA kinase inhibitor is administered via a route selected from the group consisting of isophoretic delivery, cannula, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration.
  • the TrkA inhibitor is administered before the onset of mild Alzheimer's disease. In certain of these embodiments the TrkA inhibitor is administered to a subject diagnosed with MCI. In certain of these embodiments the TrkA inhibitor is administered before the onset of MCI. In certain of these embodiments the TrkA inhibitor is
  • the TrkA inhibitor is administered in conjunction with another neuropharmaceutical (e.g., in conjunction with an agent selected from the group consisting of NGF, an NGF mimetic, a tropinol ester, tropisetron, a tropisetron analog, disulfiram, a disulfiram analog, honokiol, a honokiol analog, nimetazepam, a nimetazepam analog, donepezil, rivastigmine,
  • another neuropharmaceutical e.g., in conjunction with an agent selected from the group consisting of NGF, an NGF mimetic, a tropinol ester, tropisetron, a tropisetron analog, disulfiram, a disulfiram analog, honokiol, a honokiol analog, nimetazepam, a nimetazepam analog, donepezil, rivastigmine,
  • galantamine tacrine, memantine, solanezumab, bapineuzmab, alzemed, flurizan, ELND005, valproate, semagacestat, rosiglitazone, phenserine, cernezumab, dimebon, egcg,
  • PF3084014 V950, E2609, MK0752, CTS21 166, AZD-3839, LY2886721 , CHF5074, an anti-inflammatory, dapsone, an anti-TNF antibody, and a statin).
  • the subject is in severe stage Alzheimer's disease and the TrkA inhibitor is administered in conjunction with NGF and/or an NGF mimetic.
  • pharmaceutical formulations are provided.
  • the formulations comprise a TrkA kinase inhibitor according to Formula I as described and/or claimed herein, or a polymorph thereof, enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a
  • this pharmaceutical formulation excludes ADDN-1351, or a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • this pharmaceutical formulation comprises ADDN-1351, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • the TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN-1351d, ADDN-1351e, ADDN-1351f, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN-1351j, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN- 135 lp as shown in Table 4, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • the formulation is a unit dosage formulation. In certain embodiments the formulation is a sterile formulation. In certain embodiments the TrkA kinase inhibitor is formulated for administration via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration.
  • a TrkA kinase inhibitor for use in inhibiting the C-terminal cleavage of APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal; and/or mitigating in a mammal one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms; and/or reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal; and/or preventing or delaying the onset of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or preventing or delaying the progression of a pre- Alzheimer's condition or cognitive dysfunction to
  • the TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siRNA as described and/or claimed herein.
  • the TrkA inhibitor comprises a small organic molecule inhibitor of TrkA according to any of Formulas I-XVIII, and/or Tables 4- 12, as described and/or claimed herein and/or a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11, and/or a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • the TrkA inhibitor is an inhibitor according to Formula I as described and/or claimed herein, or a polymorph thereof, enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • this Formula excludes excludes ADDN-1351.
  • the TrkA inhibitor is ADDN-1351, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • the TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN- 1351c, ADDN-1351d, ADDN-1351e, ADDN-1351f, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN-1351j, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN-1351p as shown in Table 4, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a
  • TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siRNA as described and/or claimed herein.
  • the TrkA inhibitor comprises a small organic molecule inhibitor of TrkA according to any of Formulas I-XVIII, and/or Tables 4-12, as described and/or claimed herein and/or a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11, and/or a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • TrkA inhibitor is an inhibitor according to Formula I as described and/or claimed herein, or a polymorph thereof, enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a
  • this Formula excludes ADDN-1351.
  • the TrkA inhibitor is ADDN-1351, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • the TrkA kinase inhibitor is selected from the group consisting of ADDN- 1351a, ADDN- 1351b, ADDN- 1351c, ADDN-135 Id, ADDN-135 le, ADDN-135 If, ADDN-135 lg, ADDN-135 lh, ADDN-135 li, ADDN-135 lj, ADDN-135 lk, ADDN-13511, ADDN-135 lm, ADDN-135 In, ADDN-135 lo, AND ADDN-135 lp as shown in Table 4, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a
  • Kits are also provided.
  • the kits comprise a container containing a TrkA inhibitor and instructions for use of that inhibitor in the treatment and/or prophylaxis of an amyloidogenic pathology.
  • the TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siRNA as described and/or claimed herein.
  • the TrkA inhibitor comprises a small organic molecule inhibitor of TrkA according to any of Formulas I-XVIII, and/or Tables 4- 12, as described and/or claimed herein and/or a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11, and/or a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • the TrkA inhibitor is an inhibitor according to Formula I as described and/or claimed herein, or a polymorph thereof, enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • this Formula excludes excludes ADDN-1351.
  • the TrkA inhibitor is ADDN-1351, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
  • TrkA kinase inhibitor is selected from the group consisting of ADDN-135 la, ADDN-135 lb, ADDN- 1351c, ADDN-135 Id, ADDN-135 le, ADDN-135 If, ADDN-135 lg, ADDN-135 lh,
  • the methods described and/or claimed herein expressly exclude administration of the active agents (TrkA kinase inhibitors) for the treatment of one or more indications selected from the group consisting of Parkinson's disease, psychosis, Schizophrenia, a neurological disease or disorder other than Alzheimer's disease, cancer, pain, inflammation, rheumatoid arthritis, and/or an immunological disorder.
  • active agents TrkA kinase inhibitors
  • the methods described and/or claimed herein expressly exclude the use of TrkA kinase inhibitors to promote myelination, neuronal survival, and oligodendrocyte differentiation and/or to treat demyelination and
  • dysmyelination disease(s) including, but not limited to multiple sclerosis (MS), progressive multi focal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), Wallerian Degeneration and some inherited diseases such as
  • Tyrosine kinase receptor A is a plasma member receptor composed of an extracellular domain (responsible for high affinity binding to nerve growth factor, NGF), a transmembrane segment and an intracellular protein tyrosine kinase domain (responsible to transmit the NGF signal to initiate and coordinate neuronal responses). NGF binding induces TrkA clustering on the membrane and activates the kinase. The kinase initiates a cascade of protein phosphorylation events through multiple pathways including SHC/Ras/MAPK, PI3K and PLCgl .
  • TrkA kinase inhibitor generally refers to a molecule that decreases, reduces, or inhibits TrkA autophosphorylation and/or TrkA kinase activity.
  • Illustrative TrkA kinase inhibitors include, but are not limited to small organic molecule inhibitors, TrkA inhibitory peptides, anti-TrkA antibodies.
  • the term "TrkA inhibitor” refers to a TrkA kinase inhibitor.
  • a "TrkA inhibitor” is not a "TrkA kinase inhibitor” and inhibits TrkA receptor signaling activity and/or TrkA mRNA or protein expression.
  • TrkA inhibitors include, but are not limited to small organic molecule inhibitors, TrkA inhibitory peptides, anti-TrkA antibodies, and TrkA siRNA.
  • reference to a certain element such as hydrogen or H is meant to include all isotopes of that element.
  • an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Accordingly, isotopically labeled compounds are within the scope of this invention.
  • substituted refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms.
  • Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom.
  • a substituted group will be substituted with one or more substituents, unless otherwise specified.
  • a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents.
  • substituent groups include: halogens (e.g., F, CI, Br, and I); hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxy amines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N- oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (i
  • Ci_ 6 alkyl group refers to a straight chain or branched chain alkyl group having 1 to 6 carbon atoms, and may be exemplified by a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert- butyl group, a sec-butyl group, an n-pentyl group, a tert-amyl group, a 3-methylbutyl group, a neopentyl group, and an n-hexyl group.
  • Aryl groups are cyclic aromatic hydrocarbons that do not contain
  • Aryl groups include monocyclic, bicyclic and polycyclic ring systems.
  • aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups.
  • aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups.
  • aryl groups includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like), it does not include aryl groups that have other groups, such as alkyl or halo groups, bonded to one of the ring members. Rather, groups such as tolyl are referred to as substituted aryl groups.
  • Representative substituted aryl groups may be mono-substituted or substituted more than once.
  • monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.
  • heteroaryl group refers to a monocyclic or condensed-ring aromatic heterocyclic group containing one or more hetero-atoms selected from O, S and N. If the aromatic heterocyclic group has a condensed ring, it can include a partially
  • a heteroaryl group examples include a pyrazolyl group, a thiazolyl group, an isothiazolyl group, a thiadiazolyl group, an imidazolyl group, a furyl group, a thienyl group, an oxazolyl group, an isoxazolyl group, a pyrrolyl group, an imidazolyl group, a (1,2,3)- and (l,2,4)-triazolyl group, a tetrazolyl group, a pyranyl group, a pyridyl group, a pyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a quinolyl group, an isoquinolyl group, a benzofuranyl group, an isobenzofuranyl group, an indolyl group, an isoindolyl group, an indazolyl group, a benzofuranyl group,
  • a “derivative" of a compound means a chemically modified compound wherein the chemical modification takes place at one or more functional groups of the compound. The derivative however, is expected to retain, or enhance, the pharmacological activity of the compound from which it is derived.
  • a subject in need thereof refers to a subject, as described infra, that suffers or is at a risk of suffering ⁇ e.g., pre-disposed such as genetically pre-disposed) from the diseases or conditions listed herein.
  • the terms "subject”, “individual”, and “patient” may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals ⁇ e.g., canine or feline), laboratory mammals ⁇ e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals ⁇ e.g., equine, bovine, porcine, ovine).
  • the subject can be a human ⁇ e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker.
  • An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a “therapeutically effective amount” of a TrkA inhibitor, e.g., ADDN-1351 or an analogue thereof, optionally in combination with one or more other pharmaceuticals, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the treatment to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of a treatment are substantially absent or are outweighed by the therapeutically beneficial effects.
  • the term "therapeutically effective amount” refers to an amount of an active agent or composition comprising the same that is effective to "treat" a disease or disorder in a mammal ⁇ e.g., a patient).
  • a therapeutically effective amount is an amount sufficient to improve at least one symptom associated with a neurological disorder, improve neurological function, improve cognition, or one or more markers of a neurological disease, or to enhance the efficacy of one or more pharmaceuticals administered for the treatment or prophylaxis of a neurodegenerative pathology.
  • an effective amount is an amount sufficient alone, or in combination with a pharmaceutical agent to prevent advancement or the disease, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by the disease,
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
  • the prophylactically effective amount is less than the therapeutically effective amount.
  • treatment refers to actions that produce a desirable effect on the symptoms or pathology of a disease or condition, particularly those that can be effected utilizing the TrkA kinase inhibitors described herein, and may include, but are not limited to, even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. Treatments also refers to delaying the onset of, retarding or reversing the progress of, reducing the severity of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition.
  • Treatment does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
  • treatment comprises improvement of at least one symptom of a disease being treated.
  • the improvement may be partial or complete.
  • the subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.
  • mitigating refers to reduction or elimination of one or more symptoms of that pathology or disease, and/or a reduction in the rate or delay of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease.
  • the phrases "improve at least one symptom” or “improve one or more symptoms” or equivalents thereof, refer to the reduction, elimination, or prevention of one or more symptoms of pathology or disease.
  • Illustrative symptoms of pathologies treated, ameliorated, or prevented by the compositions described herein e.g., small molecule TrkA kinase inhibitors, including, but not limited to, ADDN-1351, ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN-1351d, ADDN-1351e, ADDN-1351f, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN-1351j, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, and ADDN-1351p) include, but are not limited to, reduction, elimination, or prevention of one or more markers that are characteristic of the pathology or disease (e.g., of total-Tau (tTau), phospho-Tau (tTau), phospho
  • ⁇ 42/ ⁇ 40 ratio ⁇ 42/ ⁇ 38 ratio
  • sAPPa ⁇ / ⁇ ratio
  • ⁇ / ⁇ 40 ratio ⁇ / ⁇ 42 ratio
  • CDR clinical dementia rating
  • Illustrative measures for improved neurological function include, but are not limited to the use of the mini-mental state examination (MMSE) or Folstein test (a questionnaire test that is used to screen for cognitive impairment), the General Practitioner Assessment of Cognition (GPCOG), a brief screening test for cognitive impairment described by Brodaty et ah, (2002) Geriatrics Society 50(3): 530-534, and the like.
  • MMSE mini-mental state examination
  • GCOG General Practitioner Assessment of Cognition
  • the phrase "cause to be administered” refers to the actions taken by a medical professional (e.g., a physician), or a person prescribing and/or controlling medical care of a subject, that control and/or determine, and/or permit the administration of the agent(s)/compound(s) at issue to the subject.
  • Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing particular agent(s)/compounds for a subject.
  • Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like.
  • administering in conjunction with when used, for example with respect to the TrkA kinase inhibitors and another active agent (e.g., NGF, NGF mimetics, etc.), refers to administration of the TrkA inhibitor(s) and the other active agent(s) such that both can simultaneously achieve a physiological effect.
  • the TrkA inhibitor and the active agent composition need not be administered together, either temporally or at the same site; moreover, the TrkA inhibitor and the other active agent(s) need not be administered by the same method, e.g., the TrkA inhibitor may be administered orally and the other active agent(s) may be administered intravenously or orally.
  • the TrkA inhibitor(s) and other the active agent(s) are administered at different times and by different methods of administration. In certain embodiments, administration of one can precede administration of the other. Simultaneous physiological effect need not necessarily require presence of the TrkA inhibitors and the other active agent in the circulation at the same time. However, in certain embodiments, co-administering typically results in both the TrkA inhibitor(s) and the other active agent(s) being simultaneously present in the body (e.g., in the plasma) at a significant fraction (e.g., 20% or greater, preferably 30% or 40% or greater, more preferably 50%> or 60%> or greater, most preferably 70%> or 80%> or 90%> or greater) of their maximum serum concentration for any given dose.
  • a significant fraction e.g. 20% or greater, preferably 30% or 40% or greater, more preferably 50%> or 60%> or greater, most preferably 70%> or 80%> or 90%> or greater
  • Figure 1 illustrates induction of APPneo fragment produced by C-terminal cleavage of APP, upon co-transfection with TrkA.
  • Figure 2 shows that APPneo fragment is not induced by a kinase-dead TrkA.
  • Figure 3 shows inhibition of TrkA induction by a TrkA inhibitor.
  • FIG. 4 shows that ADDN- 1351 is a TrkA inhibitor.
  • Figure 5 illustrates pyrazole derivative TrkA inhibitors from AstraZeneca.
  • Figure 6 illustrates various TrkA inhibitors from Bayer and BMS.
  • Figure 7 illustrates various TrkA inhibitors from Cephalon.
  • FIG. 8 illustrates various TrkA inhibitors from GSK, Japan Tobacco, and J
  • Figure 9 illustrates TrkA inhibitors from Nerviano, Novartis/IRM, Pfizer, and Plexxikon.
  • Figure 10 illustrates various TrkA inhibitors (see, e.g., Raeppel et al. (2012)
  • FIG 11 illustrates CEP-701 and analogues (see, e.g., Wang et al. (2008) 51 : 4672-4684).
  • FIGS 12A-12D illustrate how APP-C31 cleavage inhibition screening identified a TrkA inhibitor.
  • Figure 12A Scattergraph of results from APP-C31 cleavage inhibition screen of a CNS-focused small molecule library. 5000 compounds were screened and 52 inhibited the APPneo signal (the neo epitope exposed after APP-C31 cleavage, APP 1-664) by over 70%. One of them, ADDN-1351, inhibited APPneo signal by over 90%.
  • Figure 12B ADDN-1351 is a TrkA inhibitor. At a low concentration of O.luM, ADDN-1351 was tested against a panel of 24 kinases.
  • TrkA was inhibited by ADDN- 1351 in this kinase inhibition assay.
  • Figure 12C Chemical structures of TrkA inhibitors ADDN-1351, PHA-739358, and GW441756.
  • Figure 12D IC50-Data for TrkA inhibition: ADDN-1351, PHA-739358, and GW441756.
  • FIGS 13A-13D show that TrkA induced APP-C31 cleavage is mediated through TrkA kinase activity.
  • Figure 13 A TrkA induced APP-C31 cleavage was blocked by the kinase dead TrkA(K538A) mutation.
  • CHO cells stably over-expressing human APP was transiently transfected with empty vector, TrkA or TrkA(K538A) mutant constructs, and the level of APPneo was detected by Western blot. TrkA over-expression induced
  • TrkA induced APP-C31 cleavage was blocked by the TrkA inhibitor GW441756.
  • CHO cells stably over-expressing human APP was transiently transfected with empty vector or TrkA, and treated with vehicle (DMSO) or GW441756 ( ⁇ ). The level of APPneo was detected by Western blot. TrkA over-expression induced APP-C31 cleavage, and this induction was abolished by GW441756 treatment.
  • NGF treatment enhanced TrkA induced APP-C31 cleavage.
  • TrkA induced APP-C31 cleavage was increased by NGF treatment, in the absence or presence of TrkA co-receptor p75 NTR .
  • Figure 13D TrkA over-expression induced cell death was blocked by the kinase dead TrkA(K538A) mutation.
  • TrkA TrkA over-expression induced significant cell death
  • Figures 14A-14E show that TrkA interacts with APP.
  • TrkA co-immunoprecipitated with wild type APP Fig. 14A
  • APP carrying the Swedish and Indiana FAD mutations APPsi, Fig. 14A
  • APP carrying the D664A mutation Fig. 14B
  • APP with the C-terminal 31 amino acids deleted Fig. 14B
  • 293T cells transiently expressing TrkA and wild type APP Fig. 14A
  • APPsi Fig. 14A
  • APP(D664A) Fig. 14B
  • APPAC31 Fig. 14B
  • TrkA Over-expression of TrkA inhibited the production of ⁇ 40 and ⁇ 42.
  • NGF treatment (5 nM) increased ⁇ 40 and ⁇ 42 production in the presence of TrkA.
  • Figure 16C TrkA over-expression inhibited the production of sAPPa, while increasing the level of full-length APP.
  • TrkA over-expression decreased sAPPa production and increased the levels of full length APP.
  • Figure 16D TrkA(K538 A) over-expression also inhibited the production of sAPPa.
  • NGF treatment (5 nM) increased sAPPa production in the presence of TrkA but not TrkA(K538A).
  • Figure 16E TrkA over-expression increased the level of ⁇ - CTF (APP-C99).
  • FIGS 17A-17E show that TrkA inhibitor GW441756 treatment decreases
  • NGF Alzheimer's disease
  • BFCN Alzheimer's disease
  • synapses of BFCN take up NGF and retrogradely transport it back to the cell bodies in the form of NGF - TrkA complexes.
  • NGF trophic support maintain the normal function of BFCN.
  • the retrograde transport system efficiency is reduced, which leads to lower levels of active NGF-TrkA complexes in the BFCN, and higher levels of NGF-TrkA complexes in the target regions of BFCN, including the entorhinal cortex, hippocampus and neo-cortex.
  • NGF levels are not, and it is likely that local hyper-activation of TrkA in the target regions of BFCN still exists. Therefore anti-TrkA treatment will still have anti-AD effects in the target regions of BFCN. However, because of the prominent BFCN degeneration problems at this stage, a combination of anti-TrkA treatment and targeted BFCN NGF delivery may be ideal.
  • Figures 18 illustrates a synthesis scheme for ADDN-1351 and analogs thereof.
  • AD Alzheimer's disease
  • Competing with ⁇ is a novel APP ligand, netrin-1, which mediates neurite extension.
  • a CNS-focused library was used to identify compounds that block APP-C31.
  • ADDN1351 was identified as a TrkA kinase inhibitor. Complementing these results, TrkA co-expressed with APP increased APP-C31 production and cell death. APP-C31 cleavage did not occur with the kinase-dead TrkA (mutation of lysine 538 to alanine) mutant or in the presence of TrkA inhibitor GW441756, whereas induction was enhanced by NGF.
  • TrkA is a dependence receptor, that induces cell death in response to reduced
  • NGF concentration (Nikoletopoulou et al. (2010) Nature 467: 59-63). Previous work showed that p75NTR interacts with APP and modulates its signaling (Fombonne et al. (2009) Ann. Neurol., 65: 294-303). The present inventors discovered that TrkA interacts with APP.
  • the intracellular domain of APP (AICD) interacts with several adaptor proteins. AICD/Fe65/Tip60 and AICD/Mint3 /Y AP complexes are transcriptionally active (Cao and Sudhof (2001) Scie/ice 293: 115-120; Swistowski et al. (2009) J. Neurosci., 29: 15703- 15712).
  • TrkA co-immunoprecipitated with APP, and this did not require the C31 region of APP. TrkA also co-immunoprecipitated with APP-C99 but not C83. Thus, the first 16 amino acids of the ⁇ region are important in TrkA- APP interaction. In APPGal4 transactivation assays, co-expression of TrkA inhibited the transactivation directed by Fe65 or Mint3/YAP by over 90%.
  • Basal forebrain cholinergic neurons (BFCN), hippocampus and entorhinal cortex are the earliest targets of AD.
  • TrkA inhibition can alleviate AD associated damage in the cortex and hippocampus, where active NGF-TrkA complexes accumulate.
  • methods of inhibiting the C-terminal cleavage of APP resulting in the formation of APP-C31 peptide and APPneo are provided.
  • C-terminal cleavage of full-length ⁇ 65 9 results in the proteolytic fragments APP neo (APPneo) and the APP-C31 terminal peptide.
  • induction of the APPneo fragment correlates with increases in cell death in APP transfected cells, and cotransfection of TrkA in APP transfected cells results in increased APPneo production.
  • TrkA kinase dead mutant did not inhibit APPneo production (see, e.g., Figure 1). and cotransfection of a kinase dead TrkA kinase did induce APP cleavage to form APPneo (see, e.g., Figure 2).
  • TrkA kinase by a potent small molecule inhibitor GW441756 inhibited APPneo production in cells transfected with TrkA kinase (see, e.g., Figure 3).
  • methods provided herein typically involve administering to a mammal, a tropomyosin-related kinase A (TrkA) inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a C31 peptide and/or APPneo.
  • TrkA tropomyosin-related kinase A
  • inhibition of expression and/or activity of a TrkA kinase particularly in neurological tissue, will inhibit the processing of APP to APPneo and APP-C31 and thereby reduce the rate of onset, severity, or possibly reverse one or more symptoms of Alzheimer's disease, and potentially other disorders characterized by amyloid plaque formation.
  • one or more TrkA kinase inhibitors are prophylactically administered to a mammal (e.g., a human) at risk for Alzheimer's disease, or are therapeutically administered to a mammal (e.g. , a human diagnosed as having pre-, early stage, mid stage, or late stage Alzheimer's disease.
  • TrkA inhibitors for mitigating in a mammal one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms, said method comprising and/or for reducing the risk, lessening the severity, or delaying the progression or onset of a disease (e.g., Alzheimer's disease, age-related macular
  • TrkA kinase inhibitors are also contemplated for use in preventing or delaying the onset of a pre- Alzheimer's condition and/or cognitive
  • TrkA kinase inhibitors can be used to promote the processing of amyloid precursor protein (APP) by the non- amyloidogenic pathway as characterized, for example, by increasing sAPPa and/or the sAPPa/A 42 ratio in a mammal.
  • APP amyloid precursor protein
  • the methods comprise administering one or more
  • TrkA inhibitors e.g. , a TrkA inhibitor as described herein, in an amount sufficient to produce the desired activity (e.g., mitigating one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms, and/or reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal, and/or promoting the processing of amyloid precursor protein (APP) by the non- amyloidogenic pathway).
  • the desired activity e.g., mitigating one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms, and/or reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal, and/or promoting the processing of amyloid precursor protein (APP) by
  • the timing of administration of the TrkA kinase inhibitors can offer significant benefits. For example, during the stage of mild AD (or a pre- AD condition (e.g., MCI), there is no (or little) cholinergic deficit, suggesting that at least the function of neurotransmission is preserved in the basal forebrain cholinergic neurons (BFCN) at this stage. However, at this same stage, in the BFCN target regions, progressive ⁇ accumulation and Tau pathology are developing and are associated with clinical symptoms. In certain embodiments, it may be preferred to use TrkA inhibitors at this stage of the disease, in order to slow or reverse AD progression at the target regions.
  • MCI basal forebrain cholinergic neurons
  • the present invention contemplates, in part, that administration of TrkA inhibitors at mild or pre-AD stages protects BFCN target regions and prevents deficits in memory and other cognitive functions; potentially blocks the positive feedback loops at BFCN target regions; reduces the levels of ⁇ and p-Tau;
  • TrkA inhibition can be a viable therapeutic strategy in a stage- dependent fashion and in combination therapy with NGF or NGF mimetics, or as part of a therapeutic cocktail that targets multiple mechanisms of AD pathogenesis (see, Example 1 herein for further discussion).
  • ADDN-1351 (see, Figure 12C) is a potent TrkA inhibitor, easily crosses the blood barrier, and is effective to inhibit cleavage of APP to form APPneo and APP-C31. Accordingly, in various embodiments, pharmaceutical formulations comprising ADDN-1351 and various derivatives thereof are contemplated.
  • Subjects/patients amenable to treatment using the methods described herein include individuals at risk of disease (e.g., a pathology characterized by amyloid plaque formation) but not showing symptoms, as well as subjects presently showing symptoms. Accordingly, certain subjects include subjects at increased risk for the onset of a pre-
  • Alzheimer's condition and/or cognitive dysfunction e.g., MCI
  • subjects diagnosed as having a pre- Alzheimer's condition and/or cognitive dysfunction e.g., MCI
  • therapeutic and/or prophylactic methods are provided that utilize the active agent(s) (e.g., TrkA kinase inhibitors) are provided.
  • the methods involve administering one or more active agent(s) (e.g., TrkA kinase inhibitors) to a subject (e.g., to a human in need thereof) in an amount sufficient to realize the desired therapeutic or prophylactic result.
  • active agent(s) e.g., TrkA kinase inhibitors
  • TrkA inhibitors can be used to prevent or delay the onset of a pre -Alzheimer's cognitive dysfunction, and/or to ameliorate one more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or to prevent or delay the progression of a pre- Alzheimer's condition and/or cognitive dysfunction to Alzheimer's disease.
  • the prophylactic methods described herein are contemplated for subjects identified as “at risk” and/or as having evidence of early Alzheimer's Disease (AD) pathological changes, but who do not meet clinical criteria for MCI or dementia.
  • AD Alzheimer's Disease
  • AD-P AD-pathophysiological process(es)
  • AD-P see, e.g., Sperling et al, (201 1) Alzheimer 's & Dementia, 1-13) at risk for progression to AD dementia to biomarker-positive individuals who are already demonstrating very subtle decline but not yet meeting standardized criteria for MCI (see, e.g., Albert et al., (2011) Alzheimer's and Dementia, 1-10
  • this continuum of pre-symptomatic AD can also encompass (1) individuals who carry one or more apolipoprotein E (APOE) ⁇ 4 alleles who are known or believed to have an increased risk of developing AD dementia, at the point they are AD-P biomarker-positive, and (2) carriers of autosomal dominant mutations, who are in the presymptomatic biomarker-positive stage of their illness, and who will almost certainly manifest clinical symptoms and progress to dementia.
  • APOE apolipoprotein E
  • a biomarker model has been proposed in which the most widely validated biomarkers of AD-P become abnormal and likewise reach a ceiling in an ordered manner (see, e.g., Jack et al, (2010) Lancet Neurol, 9: 119-128.).
  • This biomarker model parallels proposed pathophysiological sequence of (pre- AD/ AD), and is relevant to tracking the preclinical (asymptomatic) stages of AD (see, e.g., Figure 3 in Sperling et al, (2011) Alzheimer's & Dementia, 1-13).
  • Biomarkers of brain amyloidosis include, but are not limited to reductions in CSF ⁇ 42 and increased amyloid tracer retention on positron emission tomography (PET) imaging.
  • Elevated CSF tau is not specific to AD and is thought to be a biomarker of neuronal injury.
  • Decreased fluorodeoxyglucose 18F (FDG) uptake on PET with a temporoparietal pattern of hypometabolism is a biomarker of AD- related synaptic dysfunction.
  • Brain atrophy on structural magnetic resonance imaging (MRI) in a characteristic pattern involving the medial temporal lobes, paralimbic and temporoparietal cortices is a biomarker of AD-related neurodegeneration.
  • markers include, but are not limited to volumetric MRI, FDG-PET, or plasma biomarkers (see, e.g., Vemuri et al, (2009) Neurology, 73: 294-301; Yaffe et al, (2011) JAMA 305: 261-266).
  • the subjects suitable for the prophylactic methods contemplated herein include, but are not limited to subject characterized as having asymptomatic cerebral amyloidosis.
  • these individuals have biomarker evidence of ⁇ accumulation with elevated tracer retention on PET amyloid imaging and/or low ⁇ 42 in CSF assay, but typically no detectable evidence of additional brain alterations suggestive of neurodegeneration or subtle cognitive and/or behavioral symptomatology.
  • CSF and PET imaging biomarkers of ⁇ primarily provide evidence of amyloid accumulation and deposition of fibrillar forms of amyloid.
  • Data suggest that soluble or oligomeric forms of ⁇ are likely in equilibrium with plaques, which may serve as reservoirs.
  • oligomeric forms of amyloid may be critical in the pathological cascade, and provide useful markers.
  • early synaptic changes may be present before evidence of amyloid accumulation.
  • the subjects suitable for the prophylactic methods contemplated herein include, but are not limited to, subjects characterized as amyloid positive with evidence of synaptic dysfunction and/or early neurodegeneration. In various embodiments, these subjects have evidence of amyloid positivity and presence of one or more markers of "downstream" AD-P-related neuronal injury.
  • Illustrative, but non-limiting markers of neuronal injury include, but are not limited to (1) elevated CSF tau or phospho- tau, (2) hypometabolism in an AD-like pattern ⁇ e.g., posterior cingulate, precuneus, and/or temporoparietal cortices) on FDG-PET, and (3) cortical thinning/gray matter loss in a specific anatomic distribution ⁇ e.g., lateral and medial parietal, posterior cingulate, and lateral temporal cortices) and/or hippocampal atrophy on volumetric MRI.
  • Other markers include, but are not limited to fMRI measures of default network connectivity.
  • early synaptic dysfunction as assessed by functional imaging techniques such as FDG-PET and fMRI, can be detectable before volumetric loss.
  • functional imaging techniques such as FDG-PET and fMRI
  • amyloid-positive individuals with evidence of early neurodegeneration may be farther down the trajectory ⁇ e.g. , in later stages of preclinical (asymptomatic) AD).
  • the subjects suitable for the prophylactic methods contemplated herein include, but are not limited to, subjects characterized as amyloid positive with evidence of neurodegeneration and subtle cognitive decline. Without being bound to a particular theory, it is contemplated that those individuals with biomarker evidence of amyloid accumulation, early neurodegeneration, and evidence of subtle cognitive decline are in the last stage of preclinical (asymptomatic) AD, and are
  • MCI mild cognitive impairment
  • criteria include, but are not limited to, self-complaint of memory decline or other subtle neurobehavioral changes.
  • subjects/patients amenable to prophylactic methods described herein include individuals at risk of disease (e.g. , a pathology characterized by amyloid plaque formation such as MCI) but not showing symptoms, as well as subjects presently showing certain symptoms or markers. It is known that the risk of MCI and later Alzheimer's disease generally increases with age.
  • MCI amyloid plaque formation
  • prophylactic application is contemplated for subjects over 50 years of age, or subjects over 55 years of age, or subjects over 60 years of age, or subjects over 65 years of age, or subjects over 70 years of age, or subjects over 75 years of age, or subjects over 80 years of age, in particular to prevent or slow the onset or ultimate severity of mild cognitive impairment (MCI), and/or to slow or prevent the progression from MCI to early stage Alzheimer's disease (AD).
  • MCI mild cognitive impairment
  • AD Alzheimer's disease
  • the methods described herein present methods are especially useful for individuals who do have a known genetic risk of Alzheimer's disease (or other amyloidogenic pathologies), whether they are asymptomatic or showing symptoms of disease.
  • individuals include those having relatives who have experienced MCI or AD (e.g., a parent, a grandparent, a sibling), and those whose risk is determined by analysis of genetic or biochemical markers.
  • Genetic markers of risk toward Alzheimer's disease include, for example, mutations in the APP gene, particularly mutations at position 717 and positions 670 and 671 referred to as the Hardy and Swedish mutations respectively (see, e.g., Hardy (1997) Trends. Neurosci., 20: 154-159).
  • markers of risk include mutations in the presenilin genes (PS1 and PS2), family history of AD, having the familial Alzheimer's disease (FAD) mutation, the APOE ⁇ 4 allele, hypercholesterolemia or atherosclerosis. Further susceptibility genes for the development of Alzheimer's disease are reviewed, e.g., in Sleegers, et al, (2010) Trends Genet. 26(2): 84-93.
  • the subject is asymptomatic but has familial and/or genetic risk factors for developing MCI or Alzheimer's disease.
  • treatment can begin at any age (e.g., 20, 30, 40, 50 years of age). Usually, however, it is not necessary to begin treatment until a patient reaches at least about 40, 50, 60 or 70 years of age.
  • the subject is exhibiting symptoms, for example, of mild cognitive impairment (MCI) or Alzheimer's disease (AD).
  • MCI mild cognitive impairment
  • AD Alzheimer's disease
  • Individuals presently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors described above.
  • a number of diagnostic tests are available for identifying individuals who have AD. These include measurement of CSF Tau, phospho-tau (pTau), ⁇ 42 levels and C-terminally cleaved APP fragment (APPneo).
  • the subject or patient is diagnosed as having MCI.
  • NTP neural thread protein
  • a2M macroglobulin
  • CHC complement factor H
  • subjects amenable to treatment may have age- associated memory impairment (AAMI), or mild cognitive impairment (MCI).
  • AAMI age-associated memory impairment
  • MCI mild cognitive impairment
  • the methods described herein are particularly well-suited to the prophylaxis and/or treatment of MCI. In such instances, the methods can delay or prevent the onset of MCI, and or reduce one or more symptoms characteristic of MCI and/or delay or prevent the progression from MCI to early-, mid- or late- stage Alzheimer's disease or reduce the ultimate severity of the disease.
  • MCI Mild Cognitive Impairment
  • TrkA inhibitors alone or in conjunction with other active agents, are contemplated in the treatment and/or prophylaxis of age-related cognitive decline and/or in the treatment and/or prophylaxis of mild cognitive impairment (MCI).
  • Mild cognitive impairment also known as incipient dementia, or isolated memory impairment
  • MCI mild cognitive impairment
  • MCI can present with a variety of symptoms, when memory loss is the predominant symptom it is termed "amnestic MCI” and is frequently seen as a risk factor for Alzheimer's disease (see, e.g., Grundman et al., (2004) Arch. Neurol. 61(1): 59- 66; and on the internet at en.wikipedia.org/wiki/Mild_cognitive_impairment - cite note- Grundman-1).
  • individuals have impairments in domains other than memory it is often classified as non-amnestic single- or multiple-domain MCI and these individuals are believed to be more likely to convert to other dementias (e.g. dementia with Lewy bodies).
  • dementias e.g. dementia with Lewy bodies
  • patients may be in a transitional stage of evolving Alzheimer's disease; patients in this hypothesized transitional stage demonstrated diffuse amyloid in the neocortex and frequent neurofibrillary tangles in the medial temporal lobe (see, e.g., Petersen et al, (2006) Arch. Neurol, 63(5): 665-72).
  • MCI MCI-associated neurological disorders
  • diagnosis of MCI typically involves a comprehensive clinical assessment including clinical observation, neuroimaging, blood tests and
  • diagnostic criteria for MCI include, but are not limited to those described by Albert et al, (201 1) Alzheimer 's & Dementia. 1- 10. As described therein, diagnostic criteria include (1) core clinical criteria that could be used by healthcare providers without access to advanced imaging techniques or
  • cerebrospinal fluid analysis and (2) research criteria that could be used in clinical research settings, including clinical trials.
  • the second set of criteria incorporate the use of biomarkers based on imaging and cerebrospinal fluid measures.
  • the final set of criteria for mild cognitive impairment due to AD has four levels of certainty, depending on the presence and nature of the biomarker findings.
  • clinical evaluation/diagnosis of MCI involves: (1) concern reflecting a change in cognition reported by patient or informant or clinician (e.g. , historical or observed evidence of decline over time); (2) objective evidence of impairment in one or more cognitive domains, typically including memory (e.g., formal or bedside testing to establish level of cognitive function in multiple domains); (3) preservation of independence in functional abilities; (4) not demented; and in certain embodiments, (5) an etiology of mci consistent with ad pathophysiological processes, typically vascular, traumatic, medical causes of cognitive decline are ruled out where possible, in certain embodiments, evidence of longitudinal decline in cognition is identified, when feasible. Diagnosis is reinforced by a history consistent with AD genetic factors, where relevant.
  • the cognitive changes should be sufficiently mild that there is no evidence of a significant impairment in social or occupational functioning. If an individual has only been evaluated once, change will be inferred from the history and/or evidence that cognitive performance is impaired beyond what would have been expected for that individual.
  • Cognitive testing is optimal for objectively assessing the degree of cognitive impairment for an individual. Scores on cognitive tests for individuals with MCI are typically 1 to 1.5 standard deviations below the mean for their age and education matched peers on culturally appropriate normative data (e.g., for the impaired domain(s), when available).
  • Episodic memory i.e., the ability to learn and retain new information
  • MCI patients who subsequently progress to a diagnosis of AD dementia.
  • episodic memory tests that are useful for identifying those MCI patients who have a high likelihood of progressing to AD dementia within a few years. These tests typically assess both immediate and delayed recall, so that it is possible to determine retention over a delay.
  • wordlist learning tests with multiple trials. Such tests reveal the rate of learning over time, as well as the maximum amount acquired over the course of the learning trials.
  • MCI it is desirable to examine domains in addition to memory.
  • domains include, but are not limited to executive functions (e.g., set-shifting, reasoning, problem-solving, planning), language (e.g., naming, fluency, expressive speech, and comprehension), visuospatial skills, and attentional control (e.g., simple and divided attention).
  • executive functions e.g., set-shifting, reasoning, problem-solving, planning
  • language e.g., naming, fluency, expressive speech, and comprehension
  • visuospatial skills e.g., simple and divided attention.
  • neuropsychological measures are available to assess these cognitive domains, including (but not limited to the Trail Making Test (executive function), the Boston Naming Test, letter and category fluency (language), figure copying (spatial skills), and digit span forward (attention). [0089] As indicated above, genetic factors can be incorporated into the diagnosis of
  • MCI MCI. If an autosomal dominant form of AD is known to be present (e.g., mutation in APP, PS1, PS2), then the development of MCI is most likely the precursor to AD dementia. The large majority of these cases develop early onset AD (e.g., onset below 65 years of age).
  • AD dementia there are genetic influences on the development of late onset AD dementia.
  • APOE apolipoprotein E
  • the presence of one or two ⁇ 4 alleles in the apolipoprotein E (APOE) gene is a genetic variant broadly accepted as increasing risk for late -onset AD dementia.
  • Evidence suggests that an individual who meets the clinical, cognitive, and etiologic criteria for MCI, and is also APOE ⁇ 4 positive, is more likely to progress to AD dementia within a few years than an individual without this genetic characteristic.
  • additional genes play an important, but smaller role than APOE and also confer changes in risk for progression to AD dementia (see, e.g., Bertram et al., (2010) Neuron, 21 : 270-281).
  • subjects suitable for the prophylactic methods described herein include, but need not be limited to subjects identified having one or more of the core clinical criteria described above and/or subjects identified with one or more "research criteria" for MCI, e.g., as described below.
  • "Research criteria" for the identification/prognosis of MCI include, but are not limited to biomarkers that increase the likelihood that MCI syndrome is due to the pathophysiological processes of AD. Without being bound to a particular theory, it is contemplated that the conjoint application of clinical criteria and biomarkers can result in various levels of certainty that the MCI syndrome is due to AD pathophysiological processes.
  • biomarkers have been the most studied and applied to clinical outcomes are contemplated. These include “ ⁇ ” (which includes CSF ⁇ 42 and/or PET amyloid imaging) and “biomarkers of neuronal injury” (which include, but are not limited to CSF tau/p-tau, hippocampal, or medial temporal lobe atrophy on MRI, and temporoparietal/ precuneus hypometabolism or hypoperfusion on PET or SPECT).
  • ⁇ which includes CSF ⁇ 42 and/or PET amyloid imaging
  • biomarkers of neuronal injury which include, but are not limited to CSF tau/p-tau, hippocampal, or medial temporal lobe atrophy on MRI, and temporoparietal/ precuneus hypometabolism or hypoperfusion on PET or SPECT.
  • biomarker findings may be contradictory. Accordingly, particular biomarker combinations may be indicative of a differential diagnosis, but not itself dispositive. It is recognized that varying severities of an abnormality may confer different likelihoods or prognoses that are difficult to quantify accurately for broad application.
  • a positive ⁇ biomarker and a positive biomarker of neuronal injury provide an indication that the MCI syndrome is due to AD processes and the subject is well suited for the methods described herein.
  • a positive ⁇ biomarker in a situation in which neuronal injury biomarkers have not been or cannot be tested or a positive biomarker of neuronal injury in a situation in which ⁇ biomarkers have not been or cannot be tested indicate an intermediate likelihood that the MCI syndrome is due to AD.
  • Such subjects are believed to be is well suited for the methods described herein
  • Negative biomarkers for both ⁇ and neuronal injury suggest that the MCI syndrome is not due to AD. In such instances the subjects may not be well suited for the methods described herein.
  • PiB PET imaging is used to clearly show the sites and shapes of beta amyloid deposits in living subjects using a CI 1 tracer that binds selectively to such deposits (see, e.g., Jack et ah, (2008) Brain 131 (Pt 3): 665-680).
  • mci is typically diagnosed when there is 1) evidence of memory impairment; 2) preservation of general cognitive and functional abilities; and 3) absence of diagnosed dementia.
  • MCI and stages of Alzheimer's disease can be identified/categorized, in part by Clinical Dementia Rating (CDR) scores.
  • CDR Clinical Dementia Rating
  • the CDR is a five point scale used to characterize six domains of cognitive and functional performance applicable to Alzheimer disease and related dementias: 1) memory, orientation, 2) judgment & problem solving, 3) community affairs, 4) home & hobbies, and 5) personal care.
  • the information to make each rating is obtained through a semi-structured interview of the patient and a reliable informant or collateral source (e.g., family member).
  • the CDR table provides descriptive anchors that guide the clinician in making appropriate ratings based on interview data and clinical judgment.
  • An illustrative CDR table is shown in Table 2.
  • Table 2 Illustrative clinical dementia rating (CDR) table.
  • Solving problems & in solving handling handling judgments handles problems, problems, problems, or solve business & similarities, similarities similarities problems financial and and and
  • a CDR rating of -0.5 or -0.5 to 1.0 is often considered clinically relevant
  • administering is deemed effective when there is a reduction in the CSF of levels of one or more components selected from the group consisting of Tau, phospho-Tau (pTau), APPneo, soluble ⁇ 40, soluble ⁇ 42, and/or ⁇ 42/ ⁇ 40 ratio, and/or when there is a reduction of the plaque load in the brain of the subject, and/or when there is a reduction in the rate of plaque formation in the brain of the subject, and/or when there is an improvement in the cognitive abilities of the subject, and/or when there is a perceived improvement in quality of life by the subject, and/or when there is a significant reduction in clinical dementia rating (CDR), and/or when the rate of increase in clinical dementia rating is slowed or stopped and/or when the progression from MCI to early stage AD is slowed or stopped.
  • CDR clinical dementia rating
  • a diagnosis of MCI can be determined by considering the results of several clinical tests. For example, Grundman, et al., (2004) Arch Neurol 61 : 59-66, report that a diagnosis of MCI can be established with clinical efficiency using a simple memory test (paragraph recall) to establish an objective memory deficit, a measure of general cognition (Mini-Mental State Exam (MMSE), discussed in greater detail below) to exclude a broader cognitive decline beyond memory, and a structured clinical interview (CDR) with patients and caregivers to verify the patient's memory complaint and memory loss and to ensure that the patient was not demented.
  • MMSE Mini-Mental State Exam
  • CDR structured clinical interview
  • Patients with MCI perform, on average, less than 1 standard deviation (SD) below normal on nonmemory cognitive measures included in the battery. Tests of learning, attention, perceptual speed, category fluency, and executive function may be impaired in patients with MCI, but these are far less prominent than the memory deficit.
  • AD Alzheimer's Disease
  • the TrkA kinase inhibitor(s) are contemplated for the treatment of Alzheimer's disease.
  • the methods described herein are useful in preventing or slowing the onset of Alzheimer's disease (AD), in reducing the severity of AD when the subject has transitioned to clinical AD diagnosis, and/or in mitigating one or more symptoms of Alzheimer's disease.
  • the methods can reduce or eliminate one or more symptoms characteristic of AD and/or delay or prevent the progression from MCI to early or later stage Alzheimer's disease.
  • Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors described above.
  • diagnostic tests are available for identifying individuals who have AD.
  • Individuals presently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors described above.
  • diagnostic tests are available for identifying individuals who have AD. These include measurement of CSF Tau, phospho-tau (pTau), sAPPa, sAPPp, ⁇ 40, ⁇ 42 levels and/or C terminally cleaved APP fragment (APPneo).
  • Elevated Tau, pTau, sAPPp and/or APPneo, and/or decreased sAPPa, soluble ⁇ 40 and/or soluble ⁇ 42 levels, particularly in the context of a differential diagnosis, can signify the presence of AD.
  • subjects amenable to treatment may have
  • Alzheimer's disease Individuals suffering from Alzheimer's disease can also be diagnosed by Alzheimer's disease and Related Disorders Association (ADRDA) criteria.
  • ADRDA Alzheimer's disease and Related Disorders Association
  • NINCDS-ADRDA Alzheimer's criteria were proposed in 1984 by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (now known as the Alzheimer's Association) and are among the most used in the diagnosis of Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • NINCDS-ADRDA Alzheimer's Criteria specify eight cognitive domains that may be impaired in AD: 1) memory, 2) language, 3) perceptual skills, 4) attention, 5) constructive abilities, 6) orientation, 7) problem solving, and 8) functional abilities. These criteria have shown good reliability and validity.
  • MMSE Mini-Mental State Exam
  • Alzheimer's Disease Assessment Scale ADAS
  • MMSE Mini-Mental State Exam
  • Any score greater than or equal to 25 points (out of 30) is effectively normal (intact).
  • scores can indicate severe ( ⁇ 9 points), moderate (10-20 points) or mild (21-24 points) Alzheimer's disease.
  • Moderate cognitive decline Mild or early-stage Alzheimer's disease
  • Moderately severe cognitive decline Moderate or mid-stage Alzheimer's disease
  • Severe cognitive decline Modevere cognitive decline
  • Very severe cognitive decline Severe or late-stage Alzheimer's disease as shown in Table 3.
  • administration of one or more agents described herein to subjects diagnosed with Alzheimer's disease is deemed effective when the there is a reduction in the CSF of levels of one or more components selected from the group consisting of Tau, phospho-Tau (pTau), APPneo, soluble ⁇ 40, soluble ⁇ 42, and/or and ⁇ 42/ ⁇ 40 ratio, and/or when there is a reduction of the plaque load in the brain of the subject, and/or when there is a reduction in the rate of plaque formation in the brain of the subject, and/or when there is an improvement in the cognitive abilities of the subject, and/or when there is a perceived improvement in quality of life by the subject, and/or when there is a significant reduction in clinical dementia rating (CDR) of the subject, and/or when the rate of increase in clinical dementia rating is slowed or stopped and/or when the progression of AD is slowed or stopped ⁇ e.g. , when the transition from one stage to another as listed in Table 3 is slowed or stopped).
  • CDR clinical dementia rating
  • subjects amenable to the present methods generally are free of a neurological disease or disorder other than Alzheimer's disease.
  • the subject does not have and is not at risk of developing a neurological disease or disorder such as Huntington's Disease, and/or Parkinson's disease, and/or schizophrenia, and/or psychosis.
  • the effectiveness of treatment can be determined by comparing a baseline measure of a parameter of disease before administration of the TrkA kinase inhibitor(s) is commenced to the same parameter one or more time points after the formulation has been administered.
  • a biomarker ⁇ e.g., a peptide oligomer
  • Such biomarkers include, but are not limited to increased levels of sAPPa, p3 ( ⁇ 17-42 or ⁇ 17-40), ⁇ , soluble ⁇ 40, and/or soluble ⁇ 42 in the blood, plasma, serum, urine, mucous or cerebrospinal fluid (CSF).
  • Detection of increased levels of sAPPa and/or p3, and decreased levels of ⁇ and/or APPneo is an indicator that the treatment is effective. Conversely, detection of decreased levels of sAPPa and/or p3, and/or increased levels of ⁇ , APPneo, Tau or phospho-Tau (pTau) is an indicator that the treatment is not effective.
  • Amyloid plaques can be determined using any method known in the art, e.g., as determined by CT, PET, PIB-PET and/or MRI.
  • administration of the active agent(s) described herein can result in a reduction in the rate of plaque formation, and even a retraction or reduction of plaque deposits in the brain. Effectiveness of treatment can also be determined by observing a stabilization and/or improvement of cognitive abilities of the subject.
  • Cognitive abilities can be evaluated using any art-accepted method, including for example, Clinical Dementia Rating (CDR), the mini-mental state examination (MMSE) or Folstein test, evaluative criteria listed in the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) or DSM-V, and the like.
  • CDR Clinical Dementia Rating
  • MMSE mini-mental state examination
  • Folstein test evaluative criteria listed in the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) or DSM-V, and the like.
  • the monitoring methods can entail determining a baseline value of a measurable biomarker or parameter (e.g., amyloid plaque load or cognitive abilities) in a subject before administering a dosage of the TrkA inhibitor described herein alone or in combination with one or more pharmaceuticals, and comparing this biomarker or parameter with a value for the same measurable biomarker or parameter after treatment.
  • a control value e.g., a mean and standard deviation
  • the individuals in the control population have not received prior treatment and do not have AD, MCI, nor are at risk of developing AD or MCI.
  • treatment is considered efficacious.
  • the individuals in the control population have not received prior treatment and have been diagnosed with AD or MCI. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered inefficacious.
  • a subject who is not presently receiving treatment but has undergone a previous course of treatment is monitored for one or more of the biomarkers or clinical parameters to determine whether a resumption of treatment is required.
  • the measured value of one or more of the biomarkers or clinical parameters in the subject can be compared with a value previously achieved in the subject after a previous course of treatment.
  • the value measured in the subject can be compared with a control value (mean plus standard deviation/ ANOVA) determined in population of subjects after undergoing a course of treatment.
  • the measured value in the subject can be compared with a control value in populations of prophylactically treated subjects who remain free of symptoms of disease, or populations of therapeutically treated subjects who show amelioration of disease characteristics.
  • a control value in populations of prophylactically treated subjects who remain free of symptoms of disease, or populations of therapeutically treated subjects who show amelioration of disease characteristics.
  • a significant difference relative to the control level e.g. , more than a standard deviation is an indicator that treatment should be resumed in the subject.
  • the tissue sample for analysis is typically blood, plasma, serum, urine, mucous or cerebrospinal fluid from the subject.
  • TrkA provides a good target to inhibit APP processing to APP-C31 and APPneo. Moreover, it is contemplated that inhibition of TrkA kinase activity and/or expression can mitigate the severity or delay the onset or reverse certain symptoms of Alzheimer's disease.
  • TrkA inhibitors include, but are not limited to peptide inhibitors, anti-TrkA antibodies, TrkA siRNA, TrkA ribozymes, and small organic molecule inhibitors of TrkA kinase activity.
  • ADDN-1351 and derivatives are ADDN-1351 and derivatives.
  • TrkA kinase inhibitors ADPN-1351
  • IC50-700 nM TrkA kinase inhibitors
  • ADDN-1351 type inhibitors include, but are not limited to molecules according to Formula I:
  • R 1 is alkyl (e.g., a Ci_ 6 alkyl, or substituted alkyl group); alkoxy or substituted alkoxy; alkenyl or substituted alkenyl; or alkynyl or substituted alkynyl; and R 2 and R 3 are independently aryl, substituted aryl, or heteroaryl and substituted heteroaryl.
  • the compounds encompassed by Formula I exclude ADDN-1351. [0123] ADDN 1351 and illustrative analogues (compounds ADDN- 1351 a-ADDN-
  • TrkA kinase inhibitors are well known to those of skill in the art. A number of such inhibitors are illustrated herein in Figures 7-11.
  • TrkA inhibitors include for example 4- aminopyrazolylpyrimidines.
  • Wang et al. (2008) J. Med. Chem., 51 : 4672-4684 describe the synthesis and screening of a number of TrkA inhibitors based on the lead compound of Formula II:
  • R 1 is selected from the group consisting of cyclopropyl, O'Pr, SMe, Me, OPr, H, and 3 ⁇ 4u
  • R 2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and halogen
  • R 3 is selected from the group consisting of H, (S)-Me, (R)-Me, (S)-CH 2 OH, (R)-CH 2 OH, (S)-Me, (i?)-CH 2 OH, (5)-CH 2 CONMe 2 , and (5)-CH 2 CONHMe
  • Y 1 and Y 2 are independent selected from the group consisting of CH, and N
  • R 7 is selected from the group consisting of H, OH, CH 3 ,
  • R 2 is F.
  • Y 1 and Y 2 are both N or Y 1 and Y 2 are both CH.
  • particular compounds comprise substitutions at the pyrimidine 2-position (see, e.g., of Table 5.
  • TrkA solvent channel A wide variety of functional groups were tolerated in the TrkA solvent channel including terminal basic rings (such as 15a) and neutral linear (15c) and branched hydroxyl side chains (15b, 15d-g) (see Table 7).
  • Table 7 Functional group substitutions in the TrkA solvent channel (Formula VI) (see, e.g. Wang et al. (2008) supra.). [0130] Table 8 shows introduction of nitrogens at the 2 and/or 6 position of the phenyl ring. These substitutions are tolerated in terms of cellular potency.
  • TrkA kinase inhibitors include, but are not limited to isothiazoles such as the compound of Formula VIII (see e.g., Lippa et al. (2006) Bioorg. Med. Chem. Lett. 16: 3444-3448): and related isothiazole compounds as shown below in Table 9 and aminoheterocycle isothiazole analogs shown in Table 10.
  • TrkA kinase activity inhibition for isothiazoles 1, 5a-h, and 6 (Formula IX) (Lippa et al. (2006), supra).
  • TrkA kinase activity inhibition for aminoheterocycle isothiazole analogs lOa-g (Formula X) (shown here as 13a-g) and 14a-c (Lippa et al. (2006), supra).
  • TrkA kinase inhibitors with sulfur linked C3 bicycles are shown in Table 11. Table 11. TrkA kinase and cell inhibition for isothiazoles with sulfur linked C3 bicycles (Formula XI) (corresponding to compounds 5i-o in Lippa et al. (2006), supra).
  • bValues are means of at least two experiments, assay error is ⁇ 2x.
  • TrkA kinase inhibitors include, but are not limited to (2E)-
  • TrkA kinase inhibitor Another illustrative, but non-limiting example of a TrkA kinase inhibitor is
  • GNF-5837 (see, e.g., Albaugh et al. (2012) ACS Med. Chem. Lett., 3(2): 140-145) shown below as Formula XV.
  • TrkA kinase inhibitors also include, but are not limited to disubstituted imidazo[4,5-£]pyridines and purines. The synthesis and characterization of these compounds is described in Wang et al. (2012) ACS Med. Chem. Lett., OI:
  • TrkA inhibitors are illustrative and not intended to be limiting.
  • Other small molecule TrkA inhibitors will be known to those of skill in the art.
  • TrkA antagonist polypeptides are contemplated.
  • Such peptides include any polypeptide which blocks, inhibits, interferes, or reduces the kinase activity of TrkA.
  • such peptides include TrkA peptide fragments that bind a TrkA receptor but lack kinase activity. Such peptides would act as competitive inhibitors of TrkA kinase.
  • TrkA expression can be downregulated or entirely inhibited by the use of antisense molecules.
  • An "antisense sequence or antisense nucleic acid” is a nucleic acid that is complementary to the coding TrkA mRNA nucleic acid sequence or a subsequence thereof. Binding of the antisense molecule to the TrkA mRNA interferes with normal translation of the TrkA transcription factor.
  • antisense molecules include oligonucleotides and oligonucleotide analogs that are hybridizable with TrkA messenger RNA. This relationship is commonly denominated as "antisense.”
  • the oligonucleotides and oligonucleotide analogs are able to inhibit the function of the RNA, either its translation into protein, its translocation into the cytoplasm, or any other activity necessary to its overall biological function. The failure of the messenger RNA to perform all or part of its function results in a reduction or complete inhibition of expression of TrkA polypeptides.
  • oligonucleotide refers to a polynucleotide formed from naturally-occurring bases and/or cyclofuranosyl groups joined by native phosphodiester bonds. This term effectively refers to naturally-occurring species or synthetic species formed from naturally-occurring subunits or their close homo logs.
  • oligonucleotide may also refer to moieties which function similarly to
  • oligonucleotides but which have non naturally-occurring portions.
  • oligonucleotides may have altered sugar moieties or inter-sugar linkages.
  • Exemplary among these are the phosphorothioate and other sulfur containing species that are known for use in the art.
  • at least one of the phosphodiester bonds of the oligonucleotide has been substituted with a structure which functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA whose activity is to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures.
  • the phosphodiester bonds are substituted with structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in the practice of the invention.
  • the internucleotide phosphodiester linkage is replaced with a peptide linkage.
  • peptide nucleic acids tend to show improved stability, penetrate the cell more easily, and show enhances affinity for their target.
  • Methods of making peptide nucleic acids are known to those of skill in the art (see, e.g., U.S. Patent Nos: 6,015,887, 6,015,710, 5,986,053, 5,977,296, 5,902,786, 5,864,010, 5,786,461, 5,773,571, 5,766,855, 5,736,336, 5,719,262, and 5,714,331).
  • Oligonucleotides may also include species that include at least some modified base forms.
  • oligonucleotides in accordance with certain embodiments of this invention comprise from about 3 to about 50 subunits. It is more preferred that such oligonucleotides and analogs comprise from about 8 to about 25 subunits and still more preferred to have from about 12 to about 20 subunits.
  • a subunit is a base and sugar combination suitably bound to adjacent subunits through phosphodiester or other bonds.
  • the oligonucleotides used in accordance with this invention can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such syntheses is sold by several vendors (e.g. Applied
  • TrkA expression can be inhibited by the use of ribozymes.
  • ribozymes include RNA molecules that contain antisense sequences for specific recognition, and an RNA-cleaving enzymatic activity. The catalytic strand cleaves a specific site in a target (TrkA) RNA, preferably at greater than
  • ribozymes Two “types” of ribozymes are particularly useful in this invention, the hammerhead ribozyme (Rossi et al. (1991) Pharmac. Ther. 50: 245-254) and the hairpin ribozyme (Hampel et al. (1990) Nucl. Acids Res. 18: 299-304, and U.S. Pat. No. 5,254,678).
  • ribozyme technology has emerged as a potentially powerful extension of the antisense approach to gene inactivation.
  • the ribozymes of the invention typically consist of RNA, but such ribozymes may also be composed of nucleic acid molecules comprising chimeric nucleic acid sequences (such as DNA/RNA sequences) and/or nucleic acid analogs ⁇ e.g., phosphorothioates).
  • ribozymes have the ability to inhibit TrkA expression.
  • Such ribozymes may be in the form of a "hammerhead" (for example, as described by Forster and Symons (1987) Cell 48: 211-220; Haseloff and Gerlach (1988) Nature 328: 596-600; Walbot and Bruening (1988) Nature 334: 196;
  • Ribozymes as well as DNA encoding such ribozymes and other suitable nucleic acid molecules can be chemically synthesized using methods well known in the art for the synthesis of nucleic acid molecules. Alternatively, Promega, Madison, Wis., USA, provides a series of protocols suitable for the production of RNA molecules such as ribozymes.
  • the ribozymes also can be prepared from a DNA molecule or other nucleic acid molecule (which, upon transcription, yields an RNA molecule) operably linked to an RNA polymerase promoter, e.g., the promoter for T7 RNA polymerase or SP6 RNA polymerase. Such a construct may be referred to as a vector.
  • nucleic acid molecules e.g., DNA or cDNA, coding for the ribozymes of this invention.
  • the vector also contains an RNA polymerase promoter operably linked to the DNA molecule, the ribozyme can be produced in vitro upon incubation with the RNA polymerase and appropriate nucleotides.
  • the DNA may be inserted into an expression cassette (see, e.g., Cotten and Birnstiel (1989) EMBO J 8(12): 3861-3866; Hempel et al. (1989) Biochem. 28: 4929-4933, etc.).
  • the ribozyme can be modified by ligation to a DNA molecule having the ability to stabilize the ribozyme and make it resistant to RNase.
  • the ribozyme can be modified to the phosphothio analog for use in liposome delivery systems. This modification also renders the ribozyme resistant to endonuclease activity.
  • the ribozyme molecule also can be in a host prokaryotic or eukaryotic cell in culture or in the cells of an organism/patient. Appropriate prokaryotic and eukaryotic cells can be transfected with an appropriate transfer vector containing the DNA molecule encoding a ribozyme of this invention.
  • the ribozyme molecule, including nucleic acid molecules encoding the ribozyme may be introduced into the host cell using traditional methods such as transformation using calcium phosphate precipitation
  • microprojectile bombardment (Williams et al. (1991) Proc. Natl. Acad. Sci., USA, 88: 2726- 2730), polycation compounds such as polylysine, receptor specific ligands, liposomes entrapping the nucleic acid molecules, spheroplast fusion whereby E coli containing the nucleic acid molecules are stripped of their outer cell walls and fused to animal cells using polyethylene glycol, viral transduction, (Cline et al, (1985) Pharmac. Ther. 29: 69; and Friedmann et al. (1989) Science 244: 1275), and DNA ligand (Wu et al (1989) J. Biol. Chem. 264: 16985-16987), as well as psoralen inactivated viruses such as Sendai or
  • the ribozyme is introduced into the host cell utilizing a lipid, a liposome or a retroviral vector.
  • the RNA can be produced in the host cell when the host cell is grown under suitable conditions favoring transcription of the DNA molecule.
  • the vector can be, but is not limited to, a plasmid, a virus, a retrotransposon or a cosmid. Examples of such vectors are disclosed in U.S. Pat. No. 5,166,320.
  • Other representative vectors include, but are not limited to adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Kolls et al. (1994) PNAS 91(l):215-219; Kass-Eisler et a/., (1993) Proc.
  • nucleotide sequences coding for ribozymes are preferably placed under the control of a strong promoter such as the lac, SV40 late, SV40 early, or lambda promoters. Ribozymes are then produced directly from the transfer vector in vivo
  • RNA interference refers to a mechanism by which double-stranded (sense strand) RNA (dsRNA) specifically blocks expression of its homologous gene when injected, or otherwise introduced into cells.
  • dsRNA double-stranded RNA
  • the discovery of this incidence came with the observation that injection of antisense or sense RNA strands into Caenorhabditis elegans cells resulted in gene-specific inactivation (Guo and Kempheus (1995) Cell 81 : 611-620). While gene inactivation by the antisense strand was expected, gene silencing by the sense strand came as a surprise. Adding to the surprise was the finding that this gene-specific inactivation actually came from trace amounts of contaminating dsRNA (Fire et al. (1998) Nature 391 : 806-811).
  • RNAi activity has been associated with functions as disparate as transposon-silencing, anti-viral defense mechanisms, and gene regulation (Grant (1999) Cell 96: 303-306).
  • dsRNA By injecting dsRNA into tissues, one can inactivate specific genes not only in those tissues, but also during various stages of development. This is in contrast to tissue- specific knockouts or tissue-specific dominant-negative gene expressions, which do not allow for gene silencing during various stages of the developmental process (see, e.g., Gura (2000) Nature 404: 804-808).
  • the double-stranded RNA is cut by a nuclease activity into 21-23 nucleotide fragments. These fragments, in turn, target the homologous region of their corresponding mRNA, hybridize, and result in a double-stranded substrate for a nuclease that degrades it into fragments of the same size (Hammond et al. (2000) N ' ature, 404: 293- 298; Zamore et al. (2000). Cell 101 : 25-33).
  • siRNAs small inhibitory RNAs
  • TrkA siRNAs can readily be produced.
  • siRNA that inhibit TrkA can comprise partially purified RNA, substantially pure RNA, synthetic RNA,
  • RNA recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include, for example, addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.
  • one or both strands of the siRNA can comprise a 3' overhang.
  • a "3' overhang” refers to at least one unpaired nucleotide extending from the 3 '-end of an RNA strand.
  • the siRNA comprises at least one 3' overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, from 1 to about 5 nucleotides in length, from 1 to about 4 nucleotides in length, or about 2 to about 4 nucleotides in length.
  • the length of the overhangs can be the same or different for each strand.
  • the 3' overhang is present on both strands of the siRNA, and is one, two, or three nucleotides in length.
  • each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
  • TT dithymidylic acid
  • uu diuridylic acid
  • the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine nucleotides in the 3' overhangs with 2'-deoxythymidine, is tolerated and does not affect the efficiency of R Ai degradation.
  • the absence of a 2' hydroxyl in the 2'-deoxythymidine can significantly enhance the nuclease resistance of the 3' overhang [0161]
  • the siRNA comprises the sequence AA(N19)TT
  • siRNA comprise approximately 30%-70% GC, and preferably comprise approximately 50% G/C.
  • the sequence of the sense siRNA strand corresponds to (N19)TT or N21 (i.e., positions 3 to 23), respectively. In the latter case, the 3' end of the sense siRNA is converted to TT.
  • the rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense strand 3' overhangs.
  • the antisense RNA strand is then synthesized as the complement to positions 1 to 21 of the sense strand.
  • the 3 '-most nucleotide residue of the antisense strand can be chosen deliberately.
  • the penultimate nucleotide of the antisense strand (complementary to position 2 of the 23 -nt sense strand in either embodiment) is generally complementary to the targeted sequence.
  • the siRNA comprises the sequence NAR(N 17)YNN (SEQ ID NO :4), where R is a purine (e.g. , A or G) and Y is a pyrimidine (e.g., C or U/T).
  • R is a purine
  • Y is a pyrimidine
  • the respective 21-nt sense and antisense RNA strands of this embodiment therefore generally begin with a purine nucleotide.
  • Such siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine.
  • the siRNA of the invention can be targeted to any stretch of approximately 10-30, or 15-25, or 19-25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence”).
  • target sequence any of the target mRNA sequences.
  • Techniques for selecting target sequences for siRNA are given, for example, in Tuschl et al, "The siRNA User Guide,” revised May 6, 2004.
  • the "siRNA User Guide” is available on the world wide web at a website maintained by Dr. Thomas Tuschl, and can be found by accessing the website of
  • siRNA User Guide can be located by performing a google search for "siRNA User Guide” and can also be found at "www.rockefeller.edu/labheads/tuschl/sirna.html. Techniques for selecting target sequences for siRNA and miRNA can also be found in Sioud (2008) siRNA and miRNA Gene Silencing: From Bench to Bedside (Methods in Molecular Biology) , Humana Press.
  • the sense strand of the present siRNA comprises a nucleotide sequence identical to any contiguous stretch of about 19 to about 25 nucleotides in the target tyrosine kinase receptor A (TrkA) mRNA.
  • a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nucleotides downstream ⁇ i.e., in the 3' direction) from the start codon.
  • the target sequence can, however, be located in the 5 ' or 3' untranslated regions, or in the region nearby the start.
  • TrkA silencing siRNAs can be obtained using a number of techniques known to those of skill in the art.
  • the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. published application US 2002/0086356.
  • the siRNAs are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • the siRNAs can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • Commercial suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, III, USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).
  • Custom siRNA can be obtained from commercial suppliers ⁇ see, e.g., Thermo Fisher Scientific, Lafayette CO; Qiagen, Valencia, CA; Applied Biosystems, Foster City, CA; and the like).
  • siRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • suitable promoters for expressing siRNA from a plasmid include, for example, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant plasmids can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
  • siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly at or near the target area(s) in vivo.
  • the use of recombinant plasmids to deliver siRNA to cells in vivo is discussed in more detail below.
  • siRNA can be expressed from a recombinant plasmid either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Selection of plasmids suitable for expressing siRNAs, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art ⁇ see, e.g., Tuschl (2002) Nat. Biotechnol, 20: 446-448; Brummelkamp et al. (2002) Science 296: 550 553; Miyagishi et al. (2002) Nat. Biotechnol.
  • a plasmid comprising nucleic acid sequences for expressing an siRNA for inhibiting TrkA comprises a sense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter, and an antisense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter.
  • the plasmid is ultimately intended for use in producing an recombinant adeno- associated viral vector comprising the same nucleic acid sequences for expressing the siRNA [0172]
  • "in operable connection with a polyT termination sequence” means that the nucleic acid sequences encoding the sense or antisense strands are adjacent to the polyT termination signal in the 5' direction or sufficiently close so that during transcription of the sense or antisense sequences from the plasmid, the polyT termination signals act to terminate transcription after the desired product is transcribed.
  • promoter under the control of a promoter means that the nucleic acid sequences encoding the sense or antisense strands are located 3' of the promoter, so that the promoter can initiate transcription of the sense or antisense coding sequences.
  • the siRNA can be expressed from recombinant viral vectors intracellularly at or near the target site(s) in vivo.
  • the recombinant viral vectors comprise sequences encoding the siRNA of the invention and any suitable promoter for expressing the siRNA sequences. Suitable promoters include, but are not limited to, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant viral vectors can also comprise inducible or regulatable promoters for expression of the siR A in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver siRNA of the invention to cells in vivo is discussed in more detail below.
  • the siRNA can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • Any viral vector capable of accepting the coding sequences for the siRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses ⁇ e.g. lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.
  • the tropism of the viral vectors can also be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses.
  • an AAV vector can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
  • suitable viral vectors include those derived from AV and AAV.
  • the siRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter.
  • a suitable AV vector for expressing the siRNA, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells are described in Xia et al. (2002) Nat. Biotech. 20: 1006 1010.
  • Suitable AAV vectors for expressing the siRNA, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are also described in Samulski et al. (1987) J. Virol. 61 : 3096-3101; Fisher et al. (1996) J. Virol, 70: 520-532; Samulski et al. (1989) J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479 and 5,139,941; International Patent Application Nos. WO 1994/013788; and WO 1993/024641, and the like.
  • siRNA containing a given target sequence can be evaluated using standard techniques for measuring the levels of R A or protein in cells.
  • siRNA can be delivered to cultured cells, and the levels of target mRNA can be measured by Northern blot or dot blotting techniques, or by quantitative RT-PCR.
  • the levels of TrkA in cells can be measured by ELISA or Western blot.
  • RNAi-mediated degradation of target TrkA mRNA by an siRNA containing a given target sequence can also be evaluated with suitable animal models of aging.
  • the siRNA can be delivered as a small hairpin RNA or short hairpin RNA (shRNA).
  • shRNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA that is bound to it.
  • RISC RNA-induced silencing complex
  • the shRNA/siRNA described herein target and cause the RNAi-mediated degradation of TrkA, or alternative splice forms, mutants or cognates thereof. Degradation of the target mRNA by the present siRNA reduces the production of a functional gene product from the TrkA gene.
  • methods for inhibiting expression of TrkA in a subject comprising administering an effective amount of an TrkA siRNA to the subject, such that the target mRNA is degraded.
  • siRNA of described herein can degrade the target mRNA in substoichiometric amounts. Without wishing to be bound by any theory, contemplated that the siRNA described herein cause degradation of the target mRNA in a catalytic manner.
  • siRNA of the invention can readily determine an effective amount of the siRNA of the invention to be administered to a given subject, by taking into account factors such as the size and weight of the subject; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic. Pharmaceutical formulations.
  • one or more active agents are administered to a mammal in need thereof, e.g., to a mammal at risk for or suffering from a pathology characterized by abnormal processing of amyloid precursor proteins, a mammal at risk for progression of MCI to Alzheimer's disease, and so forth.
  • the active agent(s) can be administered in the "native" form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically, e.g., effective in the present method(s).
  • Salts, esters, amides, prodrugs and other derivatives of the active agents can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience.
  • a pharmaceutically acceptable salt can be prepared for any compound described herein having a functionality capable of forming a salt, such as the carboxylic acid or tetrazole functionality of the compounds described herein.
  • a pharmaceutically acceptable salt is any salt which retains the activity of the parent compound and does not impart any deleterious or untoward effect on the subject to which it is administered and in the context in which it is administered.
  • pharmaceutically acceptable salts may be derived from organic or inorganic bases.
  • the salt may be a mono or polyvalent ion.
  • the inorganic ions lithium, sodium, potassium, calcium, and magnesium.
  • Organic salts may be made with amines, particularly ammonium salts such as mono-, di- and trialkyl amines or ethanol amines. Salts may also be formed with caffeine,
  • salts can be prepared from the free base using conventional methodology that typically involves reaction with a suitable acid.
  • a suitable acid such as methanol or ethanol
  • the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto.
  • the resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent.
  • Suitable acids for preparing acid addition salts include, but are not limited to both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • organic acids e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, cit
  • An acid addition salt can be reconverted to the free base by treatment with a suitable base.
  • Certain particularly preferred acid addition salts of the active agents herein include halide salts, such as may be prepared using hydrochloric or hydrobromic acids.
  • preparation of basic salts of the active agents described herein are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like.
  • Particularly preferred basic salts include alkali metal salts, e.g., the sodium salt, and copper salts.
  • the pKa of the counterion is preferably at least about 2 pH units lower than the pKa of the drug.
  • the pKa of the counterion is preferably at least about 2 pH units higher than the pKa of the drug. This permits the counterion to bring the solution's pH to a level lower than the pH max to reach the salt plateau, at which the solubility of salt prevails over the solubility of free acid or base.
  • the generalized rule of difference in pKa units of the ionizable group in the active pharmaceutical ingredient (API) and in the acid or base is meant to make the proton transfer energetically favorable.
  • the counterion is a pharmaceutically acceptable counterion.
  • Suitable anionic salt forms include, but are not limited to acetate, benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate (embonate), phosphate and diphosphate, salicylate and disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like, while suitable cationic salt forms include, but are not limited to aluminum, benzathine, calcium, ethylene diamine
  • esters typically involves functionalization of hydroxyl and/or carboxyl groups that are present within the molecular structure of the active agent.
  • the esters are typically acyl-substituted derivatives of free alcohol groups, e.g., moieties that are derived from carboxylic acids of the formula RCOOH where R is alky, and preferably is lower alkyl.
  • Esters can be reconverted to the free acids, if desired, by using conventional hydrogeno lysis or hydrolysis procedures.
  • Amides can also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine.
  • the active agents identified herein are useful for parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment of one or more of the pathologies/indications described herein (e.g., amyloidogenic pathologies).
  • the active agents described herein can also be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition.
  • Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s).
  • Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protection and uptake enhancers such as lipids, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
  • physiologically acceptable compounds particularly of use in the preparation of tablets, capsules, gel caps, and the like include, but are not limited to binders, diluent/fillers, disentegrants, lubricants, suspending agents, and the like.
  • an oral dosage form e.g., a tablet
  • an excipient e.g., lactose, sucrose, starch, mannitol, etc.
  • an optional disintegrator e.g., calcium carbonate, carboxymethylcellulose calcium, sodium starch glycollate, crospovidone etc.
  • a binder e.g., alpha-starch, gum arabic, microcrystalline cellulose
  • active component or components e.g., TrkA inhibitors described herein
  • Suitable coating materials include, but are not limited to ethyl-cellulose, hydroxymethylcellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, and Eudragit (Rohm & Haas, Germany;
  • physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid.
  • the choice of pharmaceutically acceptable carrier(s), including a physiologically acceptable compound depends, for example, on the route of administration of the active agent(s) and on the particular physio-chemical characteristics of the active agent(s).
  • the excipients are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. For various oral dosage form excipients such as tablets and capsules sterility is not required. The USP/NF standard is usually sufficient.
  • compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • suitable unit dosage forms include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectable, implantable sustained-release formulations, mucoadherent films, topical varnishes, lipid complexes, etc.
  • compositions comprising the TrkA inhibitors can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate processing of the active agents into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active agents described herein can be formulated as solutions, gels, ointments, creams, suspensions, and the like as are well- known in the art.
  • Systemic formulations include, but are not limited to, those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.
  • the active agents described herein can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer and/or in certain emulsion formulations.
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active agent(s) can be provided in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be readily formulated by combining the active agent(s) with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds described herein ⁇ e.g., TrkA inhibitors) to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
  • PVP polyvinylpyrrolidone
  • disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
  • suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like can be added.
  • the compositions may take the form of tablets, lozenges, etc.
  • the active agent(s) are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane,
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the active agent(s) can be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing
  • the compounds may also be formulated as a depot preparation. Such long acting formulations can be
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well known examples of delivery vehicles that may be used to protect and deliver pharmaceutically active compounds.
  • Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
  • the active agents described herein are administered orally. This is readily accomplished by the use of tablets, caplets, lozenges, liquids, and the like.
  • the active agents described herein are administered systemically (e.g., orally, or as an injectable) in accordance with standard methods well known to those of skill in the art.
  • the agents can also be delivered through the skin using conventional transdermal drug delivery systems, e.g., transdermal "patches" wherein the active agent(s) are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin.
  • the drug composition is typically contained in a layer, or "reservoir,” underlying an upper backing layer. It will be appreciated that the term “reservoir” in this context refers to a quantity of "active ingredient(s)" that is ultimately available for delivery to the surface of the skin.
  • the "reservoir” may include the active ingredient(s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art.
  • the patch may contain a single reservoir, or it may contain multiple reservoirs.
  • the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery.
  • suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
  • the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.
  • the backing layer in these laminates which serves as the upper surface of the device, preferably functions as a primary structural element of the "patch" and provides the device with much of its flexibility.
  • the material selected for the backing layer is preferably substantially impermeable to the active agent(s) and any other materials that are present.
  • one or more active agents described herein can be provided as a "concentrate”, e.g., in a storage container ⁇ e.g., in a premeasured volume) ready for dilution, or in a soluble capsule ready for addition to a volume of water, alcohol, hydrogen peroxide, or other diluent.
  • the active agents described herein are preferably suitable for oral administration.
  • the active agent(s) in the oral compositions can be either coated or non- coated.
  • the preparation of enteric-coated particles is disclosed for example in U.S. Pat. Nos. 4,786,505 and 4,853,230.
  • compositions contemplated herein typically comprise one or more of the various TrkA inhibitors described herein in an effective amount to achieve a pharmacological effect or therapeutic improvement without undue adverse side effects.
  • Various effects deemed therapeutic are described above.
  • Illustrative pharmacological effects or therapeutic improvements include, but are not limited to a reduction in the CSF of levels of one or more components selected from the group consisting of Tau, phospho-Tau (pTau), APPneo, soluble ⁇ 40 and soluble ⁇ 42, and/or when a reduction of the plaque load in the brain of the subject, and/or a reduction in the rate of plaque formation in the brain of the subject, and/or an improvement in the cognitive abilities of the subject, and/or a perceived improvement in quality of life by the subject, and/or a significant reduction in clinical dementia rating (CDR) of the subject, and/or a slowing in the rate of increase in clinical dementia rating, and/or when a slowing or stopping in the progression of AD (e.g., when the transition from one stage to another as listed in Table 3 is slowed or stopped).
  • CDR clinical dementia rating
  • the typical daily dose of compound(s) varies and will depend on various factors such as the individual requirements of the patients and the disease to be treated.
  • the daily dose of compounds can be in the range of 0.1 mg to about 5,000 mg or to about 2,500 mg, or to about 2,000 mg, or to about 1 ,500 mg, or about 1 mg, or about 5 mg, or about 10 mg, to about 1 ,000 mg, or about 1 mg, or about 5 mg, or about 10 mg, to about 800 mg, or about 1 mg, or about 5 mg, or about 10 mg to about 600 mg , or about 1 mg, or about 5 mg, or about 10 mg, to about 500 mg, or about 1-500 mg, or 1-400 mg, or 1-300 mg, or 1-200 mg, or 1-100 mg.
  • the various TrkA inhibitor(s) described above present in the composition can be typically about 0.1 or about 1 to about lOOmg, or to about 400, or to about 1 ,000, or to about 2,000 mg, more preferably about 5 to 500 mg, and most preferably about 10 to 100 mg administered once a day, in certain embodiments, administered twice a day, in certain embodiments, administered 3 times/day, and in certain embodiments, administered 4, or 6, or 6 or 7, or 8 times/day.
  • the dosage ranges from about 0.01 or about 0.1 , or about 1 mg/kg to about 100 mg/kg, or to about 50 mg/kg, or to about 40 mg/kg, or to about 30 mg/kg, or to about 20 mg/kg, or to about 10 mg/kg, or to about 30 mg/kg, administered once a day, in certain embodiments, administered twice a day, in certain embodiments, administered 3 times/day, and in certain embodiments, administered 4, or 6, or 6 or 7, or 8 times/day.
  • the active ingredients of the are preferably formulated in a single oral dosage form containing all active ingredients.
  • Such oral formulations include solid and liquid forms. It is noted that solid formulations typically provide improved stability as compared to liquid formulations and can often afford better patient compliance.
  • the one or more of the various TrkA inhibitors described above are formulated in a single solid dosage form such as single- or multi-layered tablets, suspension tablets, effervescent tablets, powder, pellets, granules or capsules comprising multiple beads as well as a capsule within a capsule or a double chambered capsule.
  • the active agents may be formulated in a single liquid dosage form such as suspension containing all active ingredients or dry suspension to be reconstituted prior to use.
  • the compound(s) are formulated as enteric-coated delayed-release granules or as granules coated with non-enteric time-dependent release polymers in order to avoid contact with the gastric juice.
  • enteric-coated delayed-release granules or as granules coated with non-enteric time-dependent release polymers in order to avoid contact with the gastric juice.
  • suitable pH-dependent enteric-coated polymers are: cellulose acetate phthalate,
  • hydroxypropylmethylcellulose phthalate polyvinylacetate phthalate, methacrylic acid copolymer, shellac, hydroxypropylmethylcellulose succinate, cellulose acetate trimellitate, and mixtures of any of the foregoing.
  • a suitable commercially available enteric material for example, is sold under the trademark EUDRAGIT L 100-55®. This coating can be spray coated onto a substrate.
  • Illustrative non-enteric-coated time-dependent release polymers include, for example, one or more polymers that swell in the stomach via the absorption of water from the gastric fluid, thereby increasing the size of the particles to create thick coating layer.
  • the time-dependent release coating generally possesses erosion and/or diffusion properties that are independent of the pH of the external aqueous medium.
  • the active ingredient is slowly released from the particles by diffusion or following slow erosion of the particles in the stomach.
  • Illustrative non-enteric time-dependent release coatings are for example: film-forming compounds such as cellulosic derivatives, such as methylcellulose, hydroxypropyl methylcellulose (HPMC), hydroxyethylcellulose, and/or acrylic polymers including the non-enteric forms of the EUDRAGIT® brand polymers.
  • film-forming materials can be used alone or in combination with each other or with the ones listed above.
  • These other film forming materials generally include, for example, poly(vinylpyrrolidone), Zein, poly(ethylene glycol), poly(ethylene oxide), poly( vinyl alcohol), poly( vinyl acetate), and ethyl cellulose, as well as other pharmaceutically acceptable hydrophilic and hydrophobic film-forming materials.
  • These film-forming materials may be applied to the substrate cores using water as the vehicle or, alternatively, a solvent system. Hydro- alcoholic systems may also be employed to serve as a vehicle for film formation.
  • Other materials suitable for making the time-dependent release coating of the compounds described herein include, by way of example and without limitation, water soluble polysaccharide gums such as carrageenan, fucoidan, gum ghatti, tragacanth, arabinogalactan, pectin, and xanthan; water-soluble salts of polysaccharide gums such as sodium alginate, sodium tragacanthin, and sodium gum ghattate; water-soluble polysaccharide gums such as carrageenan, fucoidan, gum ghatti, tragacanth, arabinogalactan, pectin, and xanthan; water-soluble salts of polysaccharide gums such as sodium alginate, sodium tragacanthin, and sodium gum ghattate; water-soluble polysaccharide gums such as carrageenan, fucoidan, gum ghatti, tragacanth, arabinogalactan, pectin, and
  • hydroxyalkylcellulose wherein the alkyl member is straight or branched of 1 to 7 carbons such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose;
  • synthetic water-soluble cellulose-based lamina formers such as methyl cellulose and its hydroxyalkyl methylcellulose cellulose derivatives such as a member selected from the group consisting of hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, and hydroxybutyl methylcellulose; other cellulose polymers such as sodium
  • lamina forming materials that can be used for this purpose include, but are not limited to poly(vinylpyrrolidone), polyvinylalcohol, polyethylene oxide, a blend of gelatin and polyvinyl-pyrrolidone, gelatin, glucose, saccharides, povidone, copovidone,
  • compositions and methods are described herein with respect to use in humans, they are also suitable for animal, e.g., veterinary use.
  • animal e.g., veterinary use.
  • certain illustrative organisms include, but are not limited to humans, non-human primates, canines, equines, felines, porcines, ungulates, largomorphs, and the like.
  • one or more of the TrkA inhibitors described herein are administered in conjunction with one or more additional active agent that are known, or believed, to have utility in the treatment of neurodegenerative diseases including, but not limited to Alzheimer's disease, age-related cognitive impairment, MCI, and the like.
  • the two agents ⁇ e.g., TrkA inhibitors and additional agent
  • TrkA inhibitors and additional agent can be administered simultaneously or sequentially.
  • the two agents are typically administered so that both achieve a physiologically relevant concentration and/or effect over a similar time period ⁇ e.g., so that both agents are active at some common time).
  • one or more of the TrkA inhibitors described herein are administered before the one or more additional active agents or they are administered after the one or more additional active agents. In certain embodiments one or more of the TrkA inhibitors described herein are administered simultaneously with one or more additional active agents and in such instances may be formulated as a compound formulation.
  • Suitable additional active agents include, but are not limited to, Donepezil ⁇ e.g. , Aricept), Rivastigmine ⁇ e.g. , EXELON®), Galantamine ⁇ e.g. , RAZADINE®), Tacrine ⁇ e.g., COGNEX®), Memantine ⁇ e.g., NAMENDA®), Solanezumab, Bapineuzmab, Alzemed, Flurizan, ELND005, Valproate, Semagacestat, Rosiglitazone, Phenserine, Cernezumab, Dimebon, EGCg, Gammagard, PBT2, PF04360365, NIC5-15, Bryostatin-1, AL-108, Nicotinamide, EHT-0202, BMS708163, NP12, Lithium, ACCOOl, AN1792, ABT089, NGF, CAD106, AZD3480, SB742457, AD02, Huperzine-
  • MABT5102A Talsaclidine, UB311, Begacestat, R1450, PF3084014, V950, E2609, MK0752, CTS21166, AZD-3839, LY2886721, CHF5074, anti-inflammatories (e.g., Flurizan (Myriad Genetics), Dapsone, anti-TNF antibodies (e.g., etanercept
  • statins e.g., atorvastatin (LIPITOR®), simvastatin
  • treatment methods comprising administration of one or more TrkA inhibitors in conjunction with any one of the foregoing additional active agents is contemplated. In certain embodiments, treatment methods comprising administration of one or more TrkA inhibitors in conjunction with any one or more of the foregoing additional active agents is contemplated.
  • combination formulations comprising one or more
  • TrkA inihibitors described herein, derivatives thereof, analogs thereof, polymorphs thereof, in combination with any one of the foregoing additional active agents is contemplated.
  • combination formulations comprising one or more TrkA inhibitors with any one or more of the foregoing additional active agents is contemplated.
  • the additional agent comprises NGF or an NGF mimetic.
  • treatment methods comprising administration of one or more TrkA inhibitors derivatives thereof, analogs thereof, polymorphs thereof, and the like described herein in conjunction with additional therapeutic agents such as tropinol esters (e.g., as described in PCT Publication PCT/US2012/049223, which is incorporated herein by reference for the compounds described therein disulfiram and/or analogues thereof, honokiol and/or analogues thereof, tropisetron and/or analogues thereof, nimetazepam and/or analogues thereof (e.g., as described in USSN 13/213,960 (U.S.
  • the treatment method comprises administration of tropisetron in conjunction with of one or more TrkA inhibitors.
  • combination formulations comprising
  • TrkA inhibitors described herein derivatives thereof, analogs thereof, polymorphs thereof, and the like in combination with additional therapeutic agents such as tropinol esters (e.g., as described in PCT Publication PCT/US2012/049223), disulfiram and/or analogues thereof, honokiol and/or analogues thereof, tropisetron and/or analogues thereof, nimetazepam and/or analogues thereof (e.g., as described in USSN 13/213,960 (U.S.
  • the combination formulation comprises tropisetron in combination with of one or more TrkA inhibitors.
  • TrkA inhibitors promote processing of APP by the nonamyloidogenic pathway and/or reduce or inhibit processing of APP by the amyloidogenic pathway.
  • APP is first cleaved by a- secretase within the ⁇ sequence, releasing the APPsa ectodomain ("sAPPa").
  • sAPPa APPsa ectodomain
  • amyloidogenic pathway is initiated when ⁇ -secretase cleaves APP at the amino terminus of the ⁇ , thereby releasing the APPsP ectodomain ("sAPPP").
  • APP processing by the nonamyloidogenic and amyloidogenic pathways is known in the art and reviewed, e.g., by Xu (2009) J. Alzheimer's Dis., 16(2):211 -224 and De Strooper et al, (2010) Nat Rev Neurol, 6(2): 99-107.
  • One method to evaluate the efficacy of TrkA inhibitors described herein is to determine whether or not the compound(s) in question produce a reduction or elimination in the level of APP processing by the amyloidogenic pathway, e.g. , a reduction or elimination in the level of APP processing by ⁇ -secretase cleavage.
  • Assays for determining the extent of APP cleavage at the ⁇ -secretase cleavage site are well known in the art. Illustrative assays, are described, for example, in U.S. Pat. Nos. 5,744,346 and 5,942,400. Kits for determining the presence and levels in a biological sample of sAPPa and sAPPp, as well as APPneo and ⁇ commercially available, e.g., from PerkinElmer. Cell Free Assays
  • TrkA inhibitors are described, for example, in PCT Publication Nos: WO 2000/017369, and WO 2000/003819, and in U.S. Patent Nos: 5,942,400 and 5,744,346.
  • assays can be performed in cell-free incubations or in cellular incubations using cells expressing an alpha-secretase and/or beta-secretase and an APP substrate having an alpha- secretase and beta-secretase cleavage sites.
  • One illustrative assay test the compound(s) of interest utilizing an APP substrate containing alpha-secretase and beta-secretase cleavage sites of APP, for example, a complete APP or variant, an APP fragment, or a recombinant or synthetic APP substrate containing the amino acid sequence: KM-DA or NL-DA, which is incubated in the presence of an a-secretase and/or ⁇ -secretase enzyme, a fragment thereof, or a synthetic or recombinant polypeptide variant having alpha-secretase or beta-secretase activity and effective to cleave the alpha-secretase or beta-secretase cleavage sites of APP, under incubation conditions suitable for the cleavage activity of the enzyme.
  • an APP substrate containing alpha-secretase and beta-secretase cleavage sites of APP for example, a complete APP or variant, an APP fragment, or a
  • Suitable substrates optionally include derivatives that may be fusion proteins or peptides that contain the substrate peptide and a modification useful to facilitate the purification or detection of the peptide or its a-secretase and/or ⁇ -secretase cleavage products.
  • Useful modifications include the insertion of a known antigenic epitope for antibody binding; the linking of a label or detectable moiety, the linking of a binding substrate, and the like.
  • Suitable incubation conditions for a cell-free in vitro assay include, for example, approximately 200 nanomolar to 10 micromolar substrate, approximately 10 to 200 picomolar enzyme, and approximately 0.1 nanomolar to 10 micromolar TrkA inhibitor, in aqueous solution, at an approximate pH of 4-7, at approximately 37°C, for a time period of approximately 10 minutes to 3 hours.
  • These incubation conditions are illustrative only, and can be varied as required for the particular assay components and/or desired
  • optimization of the incubation conditions for the particular assay components can account for the specific alpha-secretase and/or beta-secretase enzyme used and its pH optimum, any additional enzymes and/or markers that might be used in the assay, and the like. Such optimization is routine and does not require undue experimentation.
  • One useful assay utilizes a fusion peptide having maltose binding protein (MBP) fused to the C-terminal 125 amino acids of APP-SW.
  • MBP maltose binding protein
  • the MBP portion is captured on an assay substrate by anti-MBP capture antibody.
  • Incubation of the captured fusion protein in the presence of alpha-secretase and/or beta-secretase results in cleavage of the substrate at the alpha-secretase and/or beta-secretase cleavage sites, respectively.
  • Analysis of the cleavage activity can be, for example, by immunoassay of cleavage products.
  • One such immunoassay detects a unique epitope exposed at the carboxy terminus of the cleaved fusion protein, for example, using the antibody SW192. This assay is described, for example, in U.S. Pat. No. 5,942,400.
  • Numerous cell-based assays can be used to evaluate the effect of the compounds described herein on the ratio of relative alpha-secretase activity to beta- secretase activity and/or on the processing of APP to release amyloidogenic versus non- amyloidogenic ⁇ oligomers.
  • Contact of an APP substrate with an alpha-secretase and/or beta-secretase enzyme within the cell and in the presence or absence of compound(s) in question can be used to demonstrate a-secretase and/or ⁇ -secretase inhibitory activity of the compound(s).
  • the assay in the presence of compound(s) provides at least about 30%, most preferably at least about 50% inhibition of the enzymatic activity, as compared with a non-inhibited control.
  • cells that naturally express alpha-secretase and/or beta-secretase are used.
  • cells can be modified to express a
  • the APP substrate can be added to the culture medium and in certain embodiments, the substrate is preferably expressed in the cells.
  • Cells that naturally express APP, variant or mutant forms of APP, or cells transformed to express an isoform of APP, mutant or variant APP, recombinant or synthetic APP, APP fragment, or synthetic APP peptide or fusion protein containing the ⁇ -secretase and/or ⁇ -secretase APP cleavage sites can be used, provided that the expressed APP is permitted to contact the enzyme and enzymatic cleavage activity can be analyzed.
  • Human cell lines that normally process ⁇ from APP provide a useful means to assay inhibitory activities of the compound(s) described herein. Production and release of ⁇ and/or other cleavage products into the culture medium can be measured, for example by immunoassay, such as Western blot or enzyme-linked immunoassay (EIA) such as by ELISA.
  • immunoassay such as Western blot or enzyme-linked immunoassay (EIA) such as by ELISA.
  • cells expressing an APP substrate and an active a- secretase and/or ⁇ -secretase can be incubated in the presence of the compound(s) being tested to demonstrate the effect of the compound(s) on relative enzymatic activity of the a- secretase and/or ⁇ -secretase as compared with a control.
  • Relative activity of the alpha- secretase to the beta-secretase can be measured by analysis of one or more cleavage products of the APP substrate.
  • inhibition of ⁇ -secretase activity against the substrate APP would be expected to decrease release of specific ⁇ -secretase induced APP cleavage products such as ⁇ , ⁇ and APPneo.
  • Promotion or enhancement of a- secretase activity against the substrate APP would be expected to increase release of specific a-secretase induced APP cleavage products such as sAPPa and p3 peptide.
  • APP-SW Swedish Mutant form of APP
  • APP-KK APP containing an ER retention signal (-K QN-, (SEQ ID NO:5)
  • APP-SW-K provides cells having enhanced beta-secretase activity and producing amounts of ⁇ that can be readily measured.
  • the cells expressing APP, alpha-secretase and/or beta-secretase are incubated in a culture medium under conditions suitable for a-secretase and/or ⁇ -secretase enzymatic activity at its cleavage site on the APP substrate.
  • TrkA inhibitor On exposure of the cells to TrkA inhibitor, the amount of ⁇ released into the medium and/or the amount of CTF99 fragments of APP in the cell lysates is reduced as compared with the control.
  • the cleavage products of APP can be analyzed, for example, by immune reactions with specific antibodies, as discussed above.
  • preferred cells for analysis of ⁇ -secretase and/or ⁇ - secretase activity include primary human neuronal cells, primary transgenic animal neuronal cells where the transgene is APP, and other cells such as those of a stable 293 cell line expressing APP, for example, APP-SW.
  • TrkA inhibitor alone or in combination with other therapeutic agents described herein on the relative alpha- secretase and/or beta-secretase activity and/or processing of APP to release ⁇ .
  • transgenic animals expressing APP substrate, alpha-secretase and/or beta- secretase enzyme can be used to demonstrate inhibitory activity of the TrkA inhibitor.
  • Certain transgenic animal models have been described, for example, in U.S. Pat. Nos. 5,877,399; 5,612,486; 5,387,742; 5,720,936; 5,850,003; 5,877,015, and 5,811,633, and in Games et al., (1995) Nature 373: 523-527.
  • animals that exhibit characteristics associated with the pathophysiology of AD.
  • Administration of the TrkA inhibitor to the transgenic mice described herein provides an alternative method for demonstrating the inhibitory activity of the compound(s) in question.
  • administration of the TrkA inhibitor(s) in a pharmaceutically effective carrier and via an administrative route that reaches the target tissue in an appropriate therapeutic amount is preferred.
  • Inhibition of beta-secretase mediated cleavage of APP at the beta-secretase cleavage site and of ⁇ release can be analyzed in these animals by measure of cleavage fragments in the animal's body fiuids such as cerebral fluid or tissues.
  • promotion or enhancement of alpha-secretase mediated cleavage of APP at the alpha-secretase cleavage site and of release of sAPPa can be analyzed in these animals by measure of cleavage fragments in the animal's body fluids such as cerebral fluid or tissues.
  • analysis of brain tissues for ⁇ deposits or plaques is preferred.
  • an APP substrate is contacted with an alpha- secretase and/or beta-secretase enzyme in the presence of the TrkA inhibitor(s) under conditions sufficient to permit enzymatic mediated cleavage of APP and/or release of ⁇ from the substrate.
  • the TrkA inhibitor is deemed effective when it reduces beta-secretase- mediated cleavage of APP at the ⁇ -secretase cleavage site and/or reduces released amounts of ⁇ .
  • TrkA inhibitor(s) are also deemed effective if they enhance a-secretase- mediated cleavage of APP at the a-secretase cleavage site and to increase released amounts of sAPPa and/or to reduce ⁇ deposition in brain tissues of the animal, and to reduce the number and/or size of beta amyloid plaques.
  • clinical efficacy can be monitored using any method known in the art.
  • Measurable biomarkers to monitor efficacy include, but are not limited to, monitoring blood, plasma, serum, mucous or cerebrospinal fluid (CSF) levels of sAPPa, ⁇ , ⁇ 42, ⁇ 40, APPneo and p3 (e.g., ⁇ 17-42 or ⁇ 17-40). Detection of increased levels of sAPPa and/or p3 and decreased levels of ⁇ and APPneo are indicators that the treatment or prevention regime is efficacious.
  • CSF cerebrospinal fluid
  • Efficacy can also be determined by measuring amyloid plaque load in the brain. The treatment or prevention regime is considered efficacious when the amyloid plaque load in the brain does not increase or is reduced. Conversely, the treatment or prevention regime is considered inefficacious when the amyloid plaque load in the brain increases. Amyloid plaque load can be determined using any method known in the art, e.g., including magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • Efficacy can also be determined by measuring the cognitive abilities of the subject. Cognitive abilities can be measured using any method known in the art.
  • One test is the clinical dementia rating (CDR) described above, while another is the mini mental state examination (MMSE) (Folstein, et al., Journal of Psychiatric Research 12 (3): 189-98).
  • CDR clinical dementia rating
  • MMSE mini mental state examination
  • subjects who maintain the same score or who achieve a higher score on a CDR and/or on an MMSE indicate that the treatment or prevention regime is efficacious.
  • subjects who score lower on a CDR and/or on an MMSE indicate that the treatment or prevention regime has not been efficacious.
  • the monitoring methods can entail determining a baseline value of a measurable biomarker or parameter (e.g., amyloid plaque load or cognitive ability) in a subject before administering a dosage of TrkA inhibitor, and comparing this with a value for the same measurable biomarker or parameter after treatment.
  • a measurable biomarker or parameter e.g., amyloid plaque load or cognitive ability
  • a control value (e.g., a mean and standard deviation) of the measurable biomarker or parameter is determined for a control population.
  • the individuals in the control population have not received prior treatment and do not have AD, MCI, nor are at risk of developing AD or MCI. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered efficacious.
  • the individuals in the control population have not received prior treatment and have been diagnosed with AD or MCI. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered inefficacious.
  • a subject who is not presently receiving treatment, but has undergone a previous course of treatment is monitored for one or more of the biomarkers or clinical parameters to determine whether a resumption of treatment is required.
  • the measured value of one or more of the biomarkers or clinical parameters in the subject can be compared with a value previously achieved in the subject after a previous course of treatment.
  • the value measured in the subject can be compared with a control value (mean plus standard deviation) determined in population of subjects after undergoing a course of treatment.
  • the measured value in the subject can be compared with a control value in populations of prophylactically treated subjects who remain free of symptoms of disease, or populations of therapeutically treated subjects who show amelioration of disease characteristics.
  • the tissue sample for analysis is typically blood, plasma, serum, mucous or cerebrospinal fluid from the subject.
  • TrkA-APP interaction as a therapeutic target in Alzheimer's disease
  • ADDN1351 An unbiased screen for compounds that block amyloid- ⁇ precursor protein (APP) caspase cleavage identified ADDN1351 , which reduced APP-C31 by 90%.
  • Target identification studies showed that ADDN1351 is a TrkA inhibitor, and, in complementary studies, TrkA over-expression increased APP-C31 and cell death. TrkA was shown to interact with APP and suppress APP -mediated transcriptional activation.
  • TrkA inhibitor GW441756 Treatment of PDAPP transgenic mice with the known TrkA inhibitor GW441756 not only decreased ⁇ , but also increased sAPPa.
  • TrkA inhibition rather than NGF activation, can be used as a novel therapeutic approach, and may counteract the hyperactive signaling resulting from the accumulation of active NGF -TrkA complexes due to reduced retrograde transport.
  • the optimal therapy for AD may involve a combination of the delivery of NGF or NGF mimetics to basal forebrain cholinergic neuron somata and a TrkA inhibitor that is active more distally.
  • AD Alzheimer's disease
  • senile plaques characterized by senile plaques, neurofibrillary tangles, and loss of synapses and neurons.
  • the predominant components of senile plaques are the ⁇ -amyloid peptides ( ⁇ 40 & ⁇ 42), peptides generated from the ⁇ -precursor protein (APP).
  • APP ⁇ -precursor protein
  • a major neuroanatomical feature of AD is the selective degeneration of basal forebrain cholinergic neurons (BFCN). These neurons provide cholinergic innervation to the neocortex, hippocampus, and entorhinal cortex. BFCN express nerve growth factor (NGF) receptors p75 NTR and TrkA, and NGF trophic support is required for the normal function and survival of BFCN. NGF is produced by target regions of the BFCN and retrogradely transported to the cell body in the form of NGF-TrkA complexes. It has been proposed that this retrograde transport deficit is one of the major causes of BFCN
  • NGF nerve growth factor
  • AD Alzheimer's disease
  • APP generates another cytotoxic peptide, APP-C31 , by intracytoplasmic cleavage at D664 (Lu et al. (2000) Nat. Med., 6: 397-404).
  • TrkA is reported to be reduced in BFCN in AD, and failure of NGF-TrkA signaling has been considered the major cause of BFCN degeneration. Therefore, NGF and TrkA agonists have been proposed as potential treatments for AD (Williams et al. (2006) Prog. Neurobiol, 80: 114-128; Mufson et a/. (2008) Expert Rev. Neurother., 8: 1703-1718).
  • TrkA hyper-activation in neuronal processes
  • TrkA hypo- activation in neuronal somata
  • TrkA may promote pro-AD signaling through its kinase activity.
  • TrkA over-expression induces APP-C31 production, which could be prevented by a kinase-dead TrkA mutant or by TrkA inhibitor GW441756 (Wood et al. (2004) Bioorg. Med. Chem. Lett., 14: 953-957).
  • TrkA also interacts with APP and modulates APP processing.
  • TrkA by NGF induces increased ⁇ production in vitro. Further confirmation of the role of TrkA in APP processing was provided by in vivo testing of GW441756. Treatment with this TrkA inhibitor resulted in decreased ⁇ and increased sAPPa levels in the PDAPP AD transgenic mouse model. NGF and NGF mimetics may have detrimental as well as beneficial effects on AD pathophysiology, and thus, an optimal therapy for AD may comprise the combined delivery of NGF or NGF mimetics to BFCN somata, along with a TrkA inhibitor that is active more distally.
  • TrkA kinase may prove to be part of an optimal therapeutic cocktail for AD, by negating the hyperactivation and resulting toxicity produced from accumulation of active NGF-TrkA complexes, as a result of the retrograde transport deficits that occur early in AD.
  • Constructs pCMV5-TrkA and pCMV5-TrkA(K538A) were kindly provided by Dr. Moses Chao.
  • Construct pcDNA3-flag-rat-TrkA was a gift from Dr. Francis Lee.
  • Constructs pCMV5-Mint3, pMst-APP, pG5ElB-luc, pCMV-LacZ, and pCMV-Fe65 were generously provided by Dr. Thomas Sudhof, Dr. Patrick Mehlen, and Dr. Veronique Corset.
  • Construct pcDNA4-His-MaxB-hYAPl was a kind gift from Dr. Marius Sudol.
  • 6E10 anti-APP antibody was purchased from Covance.
  • CT15 anti-APP C- terminus antibody was a kind gift from Dr. Edward Koo.
  • Anti-APPneo antibody was described previously (Galvan et al. (2002) J. Neurochem., 82: 283-294).
  • Anti-TrkA antibody was purchased from Santa Cruz Biotech.
  • Nerve Growth Factor (NGF) was purchased from Sigma.
  • GW441756 was purchased from Tocris Bioscience.
  • PHA739358 was purchased from EMD.
  • MTT was purchased from Sigma. Cell culture and co-immunoprecipitations.
  • H4 neuroglioma cell line over- expressing human APP was a kind gift from Dr. Todd Golde.
  • HEK293T and B103 cell lines were described previously (Lu et al. (2000) Nat. Med., 6: 397-404; Lu et al. (2003) Ann. Neurol, 54: 781-789). Plasmid constructs were transiently transfected into
  • HEK293T or 7W cells with Lipofectamine 2000 (Invitrogen). Coimmunoprecipitation and Western analysis were performed as previously described (Swistowski et al. (2009) J.
  • a high-throughput DELFIA screen was employed to identify small molecule modulators of the APPneo generation.
  • the assay utilizes the DELFIA microtiter plates coated with the capture 6E10 antibody. After stimulation of the H4APPwt cells with staurosporine (STS), the assay measures the formation of the APPneo fragment using anti- APPneo polyclonal antibody and anti-rabbit-Europium (Eu) labeled reporter antibody.
  • STS staurosporine
  • Eu anti-rabbit-Europium
  • the screening assay protocol involves: a) Use of H4APPwt cells to seed 96-well dishes with 40,000 cells/well; b) Preincubate with small molecules and treat with STS, lyse and assay cell lysates; c) Coat DELFIA microtiter plates with 6E10 mAb; d) Develop with lOOng/well anti-APPneo Ab and lOng/well anti-rabbit-Eu reporter antibody; e) Perform single data point screen of ADDN library; f) Analysis of the data is done as a function of percent activity of positive control (+ STS); Set positive control to 100%; g) Cherry pick 'hits' from daughter plates and then assay compounds at 10 ⁇ in triplicate. Kinase inhibition assays were performed by the Reaction Biology Corporation, Malvern, PA.
  • 293T cells were transfected with pcDNA3, pCMV5-TrkA or pCMV5- TrkA(K538A) constructs. 48 hours after transfection, cell viability was measured by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, as previously described(Descamps et al). Briefly, 100 ⁇ g/ml of MTT was added to the transfected cells and the cells were incubated in dark for 2 hours. Then media were replaced with DMSO and the cells were incubated for 5 minutes with vigorous shaking. Optical densities were read at 570nm. Transactivation assay.
  • MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • HEK293T cells were co-transfected with five or six plasmids: (1) pG5ElB- luc, O ⁇ g; (2) pCMV-LacZ, O. ⁇ g; (3) pMst-APP (APP-Gal4), 0.3 ⁇ ; (4) pCMV5-TrkA or pCMV5-TrkA(K538A), l g; (5) pcDNA4-His-MaxB-hYAPl or pCMV-Fe65, l .( ⁇ g. Where indicated, a sixth plasmid was co-transfected: pCMV5-Mint3, 1.0 ⁇ .
  • the expression vector pcDNA3 was used without insert.
  • Cells were harvested 48 hr after transfection in 0.2ml per well Cell Culture Lysis Buffer (Promega), and their luciferase and ⁇ -galactosidase activities were determined with the Promega luciferase assay kit and the Promega ⁇ -galactosidase assay kit, respectively.
  • the luciferase activity was standardized by the ⁇ -galactosidase activity to control for transfection efficiency and general effects on transcription. Values shown are averages of transactivation assays performed in duplicate or triplicate and repeated at least three times each constructs.
  • mice 6-6.5 months old J20 mice were treated with DMSO control or GW441756 at lOmg/kg/day (i.p.) for 5 days. 2 hours after the last injection, mice were sacrificed and hippocampi were dissected and homogenized. The levels of sAPPa were detected by AlphaLISA assay (PerkinElmer, Waltham, MA). ⁇ 40 and ⁇ 42 levels were quantified by ELISA (BioSource, Camarillo, CA).
  • TrkA induces APP cleavage at Asp664 and cell death
  • a screen for small molecules that inhibit the production of APP-C31 and identified 52 compounds from a 5000-compound CNS-focused library.
  • One compound, ADDN-1351 reduced APP-C31 by over 90% (Fig. 12A). Since ADDN-1351 displays structural similarities to some kinase inhibitors, it was evaluated against a panel of 24 kinases at a low dose of 0.1 ⁇ , to determine whether it interacts with any specific kinase from this panel. Using this approach TrkA was identified as a specific target for ADDN- 1351 (Fig. 12B).
  • TrkA transfection induced a marked increase (more than 10-fold) in APP-C31 production, as detected by an antibody specific for the APPneo epitope (the epitope exposed after APP-C31 cleavage) (Galvan et al. (2002) J. Neurochem., 82: 283-294). This induction did not occur when a kinase dead TrkA (K538A) mutant construct was transfected (Fig. 13 A).
  • TrkA in a kinase activity dependent manner, activates APP cleavage to produce APP-C31.
  • the APP-C31 peptide is toxic, and blocking this cleavage by mutating the cleavage site in transgenic mice ameliorated the AD phenotype, without altering plaque load or ⁇ concentration.
  • TrkA expression and TrkA induced APP-C31 cleavage significantly decreased cell survival, and this effect required the kinase activity of TrkA (Fig. 13D).
  • TrkA has been found to act as a dependence receptor and induce neuronal death in the absence of NGF, while transducing trophic signals in the presence of NGF (Nikoletopoulou et al. (2010) Nature 467: 59-63). Surprisingly, NGF treatment further enhanced TrkA induced APP-C31 cleavage, indicating that NGF-TrkA signaling positively regulates the production of APP-C31 (Fig. 13C).
  • TrkA interacts with APP
  • TrkA interacts with APP. Indeed, TrkA not only co- immunoprecipitated with wild type APP (Fig. 14A), but also interacted with different mutant forms of APP, including the APP carrying the Swedish and Indiana familial Alzheimer's disease (FAD) mutations (Fig. 14A), APP carrying the D664A mutation that is resistant to caspase cleavage (Fig. 14B), and a fragment of APP with the C-terminal 31 amino acids deleted (Fig. 14B).
  • FAD familial Alzheimer's disease
  • TrkA inhibits APP-Gal4 transactivation in a kinase independent manner
  • TrkA inhibited APP-Gal4 transactivation by over 95% (Fig. 15 A).
  • This effect was confirmed using the APP-Gal4/Fe65 transactivation assay developed in the laboratory of Dr. Thomas Siidhof; potent inhibition of APP-Gal4 transactivation by TrkA (Fig. 15B) was observed.
  • Inhibition of APP-Gal4 transactivation with the kinase dead TrkA(K538A) mutant (Fig. 15C), as well as in the presence of NGF (Fig. 15D) was also observed.
  • TrkA affects APP processing
  • APP is processed through two major pathways: the non-amyloidogenic pathway is initiated by a-secretase cleavage, generating sAPPa and a-CTF (C83), while the amyloidogenic pathway is initiated by ⁇ -secretase cleavage, producing sAPPp and ⁇ -CTF (C99).
  • the ⁇ -CTF is then cleaved by the ⁇ -secretase, which produces ⁇ and AICD. Since TrkA interacts with APP, the effects of TrkA over-expression on APP processing were evaluated.
  • TrkA and APP were co-expressed in 293T cells, and the levels of ⁇ , full- length APP, sAPPa, and ⁇ -CTF were assayed.
  • the level of full-length APP increased, while the levels of ⁇ 40, ⁇ 42 and sAPPa decreased (Figs. 16A-16C).
  • Mint 1 a protein that interacts with and stabilizes APP
  • the level of ⁇ -CTF was increased (Fig. 16E).
  • NGF signaling can enhance sAPPa
  • TrkA(K538A) (Fig. 16B), indicating that the increase in ⁇ production is mediated through TrkA kinase activity and the NGF -TrkA signaling pathways.
  • APP is processed through two major pathways: the non-amyloidogenic pathway is initiated by a-secretase cleavage, generating sAPPa and a-CTF (C83), while the amyloidogenic pathway is initiated by ⁇ -secretase cleavage, producing ⁇ and ⁇ -CTF (C99).
  • the ⁇ -CTF is then cleaved by the ⁇ -secretase, which produces ⁇ and AICD. Since TrkA interacts with APP, the effects of TrkA over-expression on APP processing were evaluated.
  • TrkA and APP were co-expressed in 293T cells, and the levels of ⁇ , full- length APP, sAPPa, and ⁇ -CTF were assayed.
  • the level of full-length APP increased, while the levels of ⁇ 40, ⁇ 42 and sAPPa decreased (Figs. 16A-16C).
  • Mint 1 a protein that interacts with and stabilizes APP
  • the level of ⁇ -CTF was increased (Fig. 16E).
  • NGF signaling can enhance sAPPa
  • TrkA(K538A) (Fig. 16B), indicating that the increase in ⁇ production is mediated through TrkA kinase activity and the NGF -TrkA signaling pathways.
  • TrkA kinase inhibitor the potent APP-C31 cleavage inhibitor identified through library screening, was found to be a TrkA kinase inhibitor.
  • This discovery is somewhat counter-intuitive because an NGF -TrkA signaling deficit has been considered as a potential cause of BFCN degeneration and cholinergic deficit, one of the major features of AD.
  • TrkA hyper-activation not only induced APP-C31 cleavage, but also increased ⁇ production
  • anti-TrkA treatment in an AD transgenic mouse model decreased the levels of ⁇ 40 and ⁇ 42, while increasing sAPPa. Therefore, TrkA inhibition appears to represent a novel therapeutic approach to AD, and indicates that potent brain-permeable TrkA kinase inhibitors can be beneficial
  • BFCN at target regions are contributors to the pathogenesis of AD.
  • ⁇ and Tau pathology begin to appear, and the disease progresses(Frost and Diamond (2010) Nat. Rev. Neurosci., 11 : 155-159), a positive feedback loop arises that further impairs the retrograde transport of NGF-TrkA complexes. This would aggravate the hyperactivation of TrkA and cause more neuronal toxicity and cell death.
  • TrkA is decreased in target regions of BFCN, but the cholinergic markers, including acetylcholine (ACh) and choline acetyltransferase (ChAT), are not reduced(/ ⁇ i).
  • ACh acetylcholine
  • ChAT choline acetyltransferase
  • TrkA levels are decreased in the BFCN target regions of these patients, at a single synapse, given that markers of TrkA downstream signaling like activated ERK1/2 and PLCy are increased in AD, it is likely that TrkA is locally hyper-activated, and in such a case, TrkA inhibitors may potentially retard the progression of AD at the target regions of the BFCN.
  • one optimal treatment for AD may include both TrkA inhibition in the BFCN processes and TrkA activation in the BFCN somata.
  • TrkA activation may be supportive for the BFCN, yet toxic for the target regions of BFCN, and since retrograde transport problems may develop with aging, this might contribute to the exponential increase in incidence of AD from age 65 to 85.
  • One therapeutic consideration is whether TrkA inhibitors may be effective to treat AD without inducing significant side effects, and without augmenting BFCN degeneration. It is important to note that several TrkA inhibitors have entered advanced clinical testing for cancer, and these appear to be well tolerated (Wang et al. (2009) Expert Opin. Ther. Pat., 19: 305-319).
  • timing is important for the treatment of AD with
  • TrkA inhibitors during the stage of mild AD, there is no cholinergic deficit, indicating that at least the function of neurotransmission is preserved in the BFCN at this stage. In addition, at this same stage, in the BFCN target regions, progressive ⁇ accumulation and Tau pathology are developing and are associated with clinical symptoms. Thus, in particular embodiments, use of TrkA inhibitors at this stage of the disease may be preferred, in order to slow or reverse AD progression at the target regions.
  • TrkA inhibition may block the positive feedback loops at BFCN target regions, reduce the levels of ⁇ and p-Tau, and prevent further deterioration of the disrupted retrograde transport, and therefore might bring NGF support back to pre- AD levels.
  • use of TrkA inhibitors to treat mild stage AD is expected to result in protection of the BFCN target regions and prevention of deficits in memory and other cognitive functions.
  • TrkA inhibition is a viable therapeutic strategy in a stage-dependent fashion, optionally in combination therapy with NGF or NGF mimetics, or as part of a therapeutic cocktail that targets multiple mechanisms of AD pathogenesis (Mangialasche et al. (2010) Lancet Neurol., 9: 702-716).
  • TrkA chronic hyper-activation of TrkA, due to the accumulation of active NGF -TrkA complexes in the target regions of BFCN, is an early mechanism in the pathogenesis of AD (Fig. 17E). Accordingly, potent and brain-permeable TrkA kinase inhibitors represent one component of an optimal treatment for AD.
  • Example 2
  • Step 1 of a synthesis scheme for ADDN-1351 4-t-butyl-phenyl hydrazide (10 mmol) was mixed with ethanol (10 ml) and treated with the ethyl isothiocyanate (11 mmol). The mixture was stirred at reflux for approximately 7 hours, cooled to ambient temperature, concentrated in vacuo to one third of the original volume and diluted with two equivalents of deionised water to give an off white precipitate. The precipitate was collected by filtration through a sintered glass funnel and the structure and purity of the product were assessed using 1H-NMR spectroscopy and HPLC.
  • Step 2 the 4-t-butyl-phenyl hydrazinecarbothioamide was isolated in quantitative yield in greater than 90% purity (by HPLC) and no further purification was required.
  • Step 2 the 4-t-butyl-phenyl hydrazinecarbothioamide (7 mmol) was mixed with sulfuric acid (10 mL) and stirred at 80 °C for 6 hours. The reaction was then cooled to ambient temperature and left overnight. The reaction mixture was poured onto ice. Aqueous ammonia (25%) was carefully added until the pH of the solutions was 8-9. The products precipitated and the solids were collected by filtration through sintered glass funnels. The 5-(4-(tert-butyl)phenyl)-N-ethyl-l,3,4-thiadiazol-2-amine was isolated in quantitative yield. The compound was characterized using H-NMR
  • Step 2 the nicotinic chloride (2.1 mmol) was prepared in situ by heating at 60°C in excess thionyl chloride for 60 minutes. The thionyl chloride was removed in vacuo and the relevant thiadiazole (0.7 mmol) in pyridine (2 ml) was carefully added. The solution was heated to 70°C and left stirring for 12 hours. The pyridine was removed in vacuo and the residue was rinsed with saturated sodium bicarbonate solution and extracted into DCM. The solvent was removed in vacuo and a portion of the residue was loaded onto a preparative TLC plate (50% EtOAc/hexane). The product band was scraped from the TLC plate, taken up in ethyl acetate and filtered to remove the silica
  • N-(5-(4-(tert-butyl)phenyl)-l,3,4-thiadiazol-2-yl)-N-ethylpyrimidine-5- carboxamide was isolated in 80% purity and was subsequently recrystallized from ethyl acetate and hexane to give 14 mg of the desired product in 95% purity (by HPLC and 1 H-NMR) with a molecular ion ofm/z 368 [M +l].
  • the analogs were prepared using the same three step procedures with the appropriate hydrazides, and acid chlorides. The yields generally for the three steps were similar to ADDN-1351. The final compounds were isolated and recrystalized and analysed by 1 HNMR and MS.

Abstract

In various embodiments method are provided for inhibiting (partially or fully) the C-terminal cleavage of APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal. The methods typically involve administering or causing to be administered to the mammal a TrkA kinase inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a C31 peptide and/or APPneo. In certain embodiments the inhibitor comprises ADDN-1351 or a derivative thereof.

Description

TRKA AS A TARGET FOR INHIBITING APP CLEAVAGE AND/OR PROGRESSION OF ALZHEIMER'S DISEASE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of and priority to USSN 61/525,076, filed on August 18, 2011 , which is incorporated herein by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This work was supported in part by Grant No R01 AG034427 from the
National Institutes of Health, National Institute on Aging. The Government has certain rights in this invention. BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by rapid cognitive and functional decline in patients diagnosed with the disease. In the early stages of the disease the patients generally suffer from mild cognitive impairment (MCI) that converts over time to full blown AD. The disease broadly falls into two categories: a) late onset AD, which occurs generally at 65 years or older and is correlated to numerous risk factors including presence of an APOE ε4 allele; and b) early onset AD, which develops early on between 30 and 60 years of age and is generally associated with familial Alzheimer's disease (FAD) mutations in the amyloid precursor protein (APP) gene or in the presenilin gene. In both types of disease, the pathology is the same but the abnormalities tend to be more severe and widespread in cases beginning at an earlier age.
[0004] The disease is characterized by at least two types of lesions in the brain, senile plaques composed of the Αβ peptide (and other components, typically at lower concentrations than the Αβ peptide) and neurofibrillary tangles composed primarily of intracellular deposits of microtubule associated tau protein (especially hyperphosphorylated tau). Measurement of the levels of Αβ peptide and Tau/phosphorylated Tau in
cerebrospinal fluid (CSF) along with imaging analysis and cognitive/functional tests are used clinically to determine progression of the disease and conversion to full-blown AD.
[0005] Alzheimer's disease (AD) has been viewed largely as a disease of toxicity, mediated by the collection of a small peptide (the Αβ peptide) that damages brain cells by physical and chemical properties, such as the binding of damaging metals, reactive oxygen species production, and direct damage to cell membranes. While such effects of Αβ have been clearly demonstrated, they do not offer a physiological role for the peptide.
[0006] Recent results from several different laboratories suggest AB has
physiological signaling properties (e.g., via interaction with APP itself, the insulin-receptor, and other receptors), and our results suggest that AD may result from an imbalance between two normal processes: memory formation and normal forgetting. Our studies show that APP has all of the characteristics of a dependence-receptor, i.e., a receptor that mediates cell-death in the presence of an anti-trophin (in this case, AB) but supports cell survival in the presence of a trophic-factor (such as laminin).
[0007] Several significant changes have occurred in the AD landscape recently. Two therapies that showed marked reduction of β- amyloid levels in AD patients with limited to no cognitive improvement (Mangialasche, et. al, (2010) Lancet Neurol. 9, 702-716,). This was unexpected by much of the research community, as AD has been largely viewed as a disease of chemical and physical toxicity of β-amyloid (e.g., generation of reactive oxygen species, metal binding, etc.). However, recent results from multiple laboratories suggest a completely different view of AD as an imbalance in physiological signal transduction.
[0008] For example Lu et al. (2000) Nat. Med., 6: 397-404 and Galvan et al. (2006)
Proc. Natl. Acad. Sci. USA, 103:7130-7135 have shown a role of APP in mechanisms of signal transduction leading to neuronal cell death. These results, coupled with others, argue that Alzheimer's disease results from an imbalance between two normal processes: memory formation and normal forgetting. The data show that the Αβ peptide can play a role in modulating, processing and signaling through binding to the amyloid precursor protein (APP), and thus play a central role in the pathogenesis of Alzheimer's disease through signaling rather than chemical and physical effects.
[0009] APP695 can be cleaved by caspases at an intracellular site (Asp664), leading to the release of a small C31 peptide and an APPneo (APP664) fragment, and both products are potentially proapoptotic (Galavan et al. supra). Immunohistochemical analysis of AD brain demonstrates that this cytoplasmic cleavage occurs 4-fold more in AD brain than normal, and the products are found around plaques and tangles in key brain areas affected by the disease (Bredesen et al. (2006) Nature 443: 796-802). Through a single genetic mutation in the amyloid precursor protein (APP the AD phenotype was reversed in a transgenic mouse model by producing a mutation of aspartic acid residue 664 to alanine of APP695 leading to the complete blockage of the C-terminal cleavage in vivo. In addition, these transgenic mice demonstrate normal synaptic function and normal memory.
Furthermore, it has been shown in cell culture that this C-terminal cleavage requires Αβ- facilitated APP multimerization. The striking result of this research— that blockage of the D664 cleavage of APP leads to abrogation of the characteristic pathophysiological abnormalities and behavioral symptoms associated with Alzheimer's disease— argues recognition of this mechanism can be of fundamental importance in developing novel therapeutic agents for treatment for AD.
SUMMARY
[0010] In various embodiments methods are provided for inhibiting (partially or fully) the C-terminal cleavage of APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal. The methods typically involve administering, or causing to be administered, to the mammal a TrkA kinase inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a APP-C31 peptide and/ APPneo. In certain embodiments the inhibitor comprises ADDN-1351 or a derivative thereof. [0011] In certain embodiments, methods of inhibiting the C-terminal cleavage of
APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal are provided where the methods comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a APP-C31 peptide and/or APPneo (APP664). In certain embodiments, methods of mitigating in a mammal one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms in a mammal are provided where the methods comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to mitigate said one or more symptoms. In certain embodiments, methods of reducing the risk, lessening the severity, or delaying the progression or onset of a disease (e.g.,
Alzheimer's disease, age-related macular degeneration (AMD), Cerebrovascular dementia, Parkinson's disease, Huntington's disease, Cerebral amyloid angiopathy, etc.) characterized by beta-amyloid deposits in the brain of a mammal are provided where the methods comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to reduce the risk, lessen the severity, and/or delay the progression or onset of the disease. In certain embodiments, methods of preventing or delaying the onset of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or preventing or delaying the progression of a pre -Alzheimer's condition or cognitive dysfunction to Alzheimer's disease in a mammal are provided, where the methods comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway. In certain embodiments, methods of promoting the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway as characterized by increasing sAPPa and/or the sAPPa/Ap42 ratio in a mammal are provided, where the methods comprise administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway. In certain
embodiments a method is provided where the method comprises administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a APP-C31 peptide and/or APPneo (APP664); and/or administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to mitigate said one or more symptoms associated with a disease (e.g., Alzheimer's disease, age-related macular degeneration (AMD), Cerebrovascular dementia, Parkinson's disease, Huntington's disease, Cerebral amyloid angiopathy, etc.) characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms; and/or administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to reduce the risk, lessen the severity, and/or delay the progression or onset of a disease characterized by beta-amyloid deposits in the brain of the mammal; and/or administering to a mammal, a TrkA kinase inhibitor in an amount sufficient for preventing or delaying the onset of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or preventing or delaying the progression of a pre- Alzheimer's condition or cognitive dysfunction to Alzheimer's disease in the mammal; and/or administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to increase the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway, wherein the increase is characterized by increasing sAPPa and/or the
sAPPa/A 42 ratio in the mammal. In various embodiments in any of these methods the TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siRNA. In certain embodiments the TrkA inhibitor comprises a small organic molecule inhibitor of TrkA. In certain embodiments the TrkA kinase inhibitor has the formula:
Figure imgf000007_0001
or is an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof, or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof; where R1 is selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy, thioalkyl, and aminoalkyl; and R2 and R3 are independently selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl. In certain embodiments R1 is a Ci_6 alkyl. In certain embodiments R1 is substituted alkyl. In certain embodiments R1 is unsubstituted alkyl. In certain embodiments R1 is substituted alkenyl. In certain embodiments R1 is unsubstituted alkenyl. In certain embodiments R1 is substituted alkynyl. In certain embodiments R1 is
unsubstituted alkynyl. In certain embodiments R1 is substituted alkoxy. In certain embodiments R1 is unsubstituted alkoxy. In certain embodiments R1 is substituted thioalkyl. In certain embodiments R1 is unsubstituted thioalkyl. In certain embodiments R1 is substituted aminoalkyl. In certain embodiments R1 is unsubstituted aminoalkyl. In certain of any of these embodiments, R2 is aryl or substituted aryl, or heteroaryl, or substituted heteroaryl. In certain of any of these embodiments R3 is aryl, or is substituted aryl, or heteroaryl, or substituted heteroaryl. In certain embodiments the TrkA kinase inhibitor is ADDN- 1351 or an analogue thereof. In certain embodiments the TrkA kinase inhibitor is ADDN- 1351. In certain embodiments the TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN-1351d, ADDN-1351e, ADDN- 135 If, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN-1351j, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN- 135 lp as shown in Table 4. In certain embodiments the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000008_0001
where X is selected from the group consisting of Me, H, and halogen; R1 is selected from the group consisting of cyclopropyl, O'Pr, SMe, Me, OPr, H, and ¾u; R2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and halogen; R3 is selected from the group consisting of H, (S)-MQ, (R)-MQ, (S)-CH2OH, (i?)-CH2OH, (5)-Me, (i?)-CH2OH, (5)-CH2CONMe2, and (5)-CH2CONHMe; Y1 and Y2 are independent selected from the group consisting of CH, and N; and R7 i selected from the group consisting of H, OH, CH3
Figure imgf000008_0002
certain embodiments R2 is F. In certain embodiments Y1 is CH and Y2 is N, or Y1 and Y2 are both N, or Y1 and Y2 are both CH. In certain embodiments the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000008_0003
where X is selected from the group consisting of Me, H, and halogen; R2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and F; and R3 is selected from the group consisting of H, (S)-MQ, (R)-MQ, (5)-CH2OH, ( ?)-CH2OH, (S)-MQ, ( ?)-CH2OH, (5)-CH2CONMe2, and (S)-CH2CONHMe. In certain of these embodiments X is CI or Br. In certain embodiments the compound is a compound selected from the group consisting of compound 10a, compound 10b, compound 10c, compound lOd, compound lOe, compound lOf, compound lOg, compound lOh, compound lOi, compound lOj, compound 10k, compound 101, compound 10m, and compound 10η, as shown in Table 5. In certain embodiments the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000009_0001
where X is selected from the group consisting of Me, H, and halogen; R1U is selected from the group consisting of H, and OH; and R1 is selected from the group consisting of cyclopropyl, O'Pr, SMe, Me, OPr, H, and feu. In certain of these embodiments X is selected from the group consisting of Br, Me, H, F, and CI. In certain embodiments the compound is a compound selected from the group consisting of compound 10ο, compound lOp, compound lOq, compound lOr, compound 10s, compound lOt, compound lOu, compound lOv, and compound lOw, as shown in Table 6. In certain embodiments the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000009_0002
where X is selected from the group consisting of Me, H, and halogen; R10 is selected from the group consisting of H, and OH; R1 is selected from the group consisting of cyclopropyl, O'Pr, Me, OPr, H, and feu; and R7 is selected from the group consisting of H, OH, CH3,
Figure imgf000010_0001
IN certain of these embodiments, X is CI or H. In certain of these embodiments R1 is selected from the group consisting of Cp, Me, and O'Pr. In certain of these embodiments R7 is selected from the group consisting of
Figure imgf000010_0002
certain embodiments the compound is a compound selected from the group consisting of compound 15a, compound 15b, compound 15c, compound 15d, compound 15e, compound 15f, and compound 15g, as shown in Table 7. In certain embodiments the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000010_0003
where X is selected from the group consisting of Me, H, and halogen; R1 is selected from the group consisting of Cp, and O'Pr; Y1 and Y2 are independent selected from the group consisting of CH, and CH; and R7 is selected from the group consisting of H, OH, CH3,
Figure imgf000010_0004
. In certain of these embodiments, X is F or CI. In certain of these embodiments Y1 is CH and Y2 is N, or Y1 and Y2 are both N. In certain of these embodiments, R7 is selected from the group consisting of H,
Figure imgf000011_0001
jn certain embodiments the compound is a compound selected from the group consisting of compound lOx, compound lOy, compound lOz, compound 15h, compound 15i, and compound 1 la, as shown in Table 8. In certain embodiments the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000011_0002
where X is selected from the group consisting of CN, and CONH2; and R is selected from the group consisting of H, Me, Et, z'-Pr, n-Vr, Ph, and H. In certain embodiments the TrkA inhibitor comprises a compound selected from the group consisting of compound 1 , compound 5a, compound 5b, compound 5c, compound 5d, compound 5e, compound 5f, compound 5g, compound 5h, and compound 6, as shown in Table 9. In certain
embodiments the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000011_0003
where R1 is selected from the group consisting of /?-chlorobenzyl, CH(Me)/?-chlorobenzyl, CH2(cyclohexyl), and o-chlorobenzyl; and R2 is selected from the group consisting of H, 2- pyr, 3-pyr, 3-pyr, 3-p -pyr, 4-pyr, 4-pyramidine,
Figure imgf000011_0004
, and . In certain embodiments the TrkA inhibitor comprises a compound selected from the group consisting of compoundl3a, compound 13b, compound 13c, compound 14a, compound 14b, compound 13d, compound 14c, compound 13e, compound 13f, and compound 13g, as shown in Table 10. In certain embodiments the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000012_0001
where X is selected from the group consisting of halogen, and H; n is 1, 2, or 3; and R is H or Me. In certain embodiments X is H. In certain embodiments X is CI. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain of these embodiments, R is H or Me. In certain embodiments, the TrkA inhibitor comprises a compound selected from the group consisting of compound 19i, compound 19j, compound 191, compound 191, compound 19m, compound 19n, and compound 19o, as shown in Table XI. In certain embodiments, the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000012_0002
In certain embodiments, the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000012_0003
In certain embodiments, the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000013_0001
In certain embodiments, the TrkA inhibitor comprises a compound according to the formula:
Figure imgf000013_0002
In certain embodiments, the TrkA inhibitor comprises a compound according to the formula
Figure imgf000013_0003
where R1 is selected from the group consisting of Cp, CH3, O'Pr, O'Pr, O'Pr, O'Pr, OEt, and OCH3; and R2, when present, is selected from the group consisting of CH3, and CH2OH. In certain embodiments the inhibitor comprises a compound selected from the group consisting of compound 17a, compound 17b, compound 17c, compound 17d, compound 17e, compound 18a, compound 18b, and compound 18c, as shown in Table 12. In certain embodiments the inhibitor is a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11. In certain embodiments of these methods, the mammal is a human. In certain embodiments of these methods, the mammal is diagnosed as a pre -Alzheimer's and/or a pre- MCI cognitive impairment. In certain embodiments of these methods, administration of the TrkA kinase inhibitor delays or prevents the progression of cognitive dysfunction to MCI. In certain embodiments of these methods, the mammal is diagnosed as having mild cognitive impairment (MCI). In certain embodiments of these methods, TrkA kinase inhibitor delays or prevents the progression of MCI to Alzheimer's disease. In certain embodiments of these methods, the mammal is diagnosed as having moderately severe cognitive decline (Moderate or mid- stage Alzheimer's disease). In certain embodiments of these methods, the mammal is diagnosed as having severe cognitive decline (moderately severe or mid-stage Alzheimer's disease). In certain embodiments of these methods, the mammal is diagnosed as having Alzheimer's disease. In certain embodiments of these methods, the mammal is at risk of developing Alzheimer's disease. In certain embodiments of these methods, the mammal has a familial risk for having Alzheimer's disease. In certain embodiments of these methods, mammal has a familial Alzheimer's disease (FAD) mutation. In certain embodiments of these methods, the mammal has the APOE ε4 allele. In certain embodiments of these methods, the mammal is free of and does not have genetic risk factors of Parkinson's disease or schizophrenia. In certain embodiments of these methods, the mammal is not diagnosed as having or at risk for Parkinson's disease or schizophrenia. In certain embodiments of these methods, the mammal does not have a neurological disease or disorder other than Alzheimer's disease. In certain embodiments of these methods, the mammal is not diagnosed as having or at risk for a neurological disease or disorder other than Alzheimer's disease. In certain embodiments of these methods, the mammal is not diagnosed as having, and/or at risk for, and/or under treatment for one or more indications selected from the group consisting of cancer, pain, inflammation, rheumatoid arthritis, and an immunological disorder. In certain embodiments of these methods, mammal is a mammal not diagnosed as having cancer and/or not being treated for cancer. In certain embodiments of these methods, method produces a reduction in the CSF of levels of one or more additional components selected from the group consisting of Tau, phospho-Tau (pTau), soluble Αβ40 and soluble Αβ 42. In certain embodiments of these methods, the method results in a reduction of the plaque load in the brain of the mammal. In certain embodiments of these methods the method results in a reduction in the rate of plaque formation or deposition in the brain of the mammal. In certain embodiments of these methods the method results in an improvement in the cognitive abilities of the mammal. In certain embodiments of these methods the mammal is a human and said method produces a perceived improvement in quality of life by said human. In certain embodiments of these methods the TrkA kinase inhibitor is administered orally. In certain embodiments of these methods the administering is over a period of at least three weeks. In certain embodiments of these methods the administering is over a period of at least 6 months. In certain embodiments of these methods the TrkA kinase inhibitor is formulated for administration via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration. In certain embodiments the TrkA kinase inhibitor is administered via a route selected from the group consisting of isophoretic delivery, cannula, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration. In certain of these embodiments the the TrkA inhibitor is administered before the onset of mild Alzheimer's disease. In certain of these embodiments the TrkA inhibitor is administered to a subject diagnosed with MCI. In certain of these embodiments the TrkA inhibitor is administered before the onset of MCI. In certain of these embodiments the TrkA inhibitor is
administered prophylactically to a healthy subject. In certain of these embodiments the TrkA inhibitor is administered in conjunction with another neuropharmaceutical (e.g., in conjunction with an agent selected from the group consisting of NGF, an NGF mimetic, a tropinol ester, tropisetron, a tropisetron analog, disulfiram, a disulfiram analog, honokiol, a honokiol analog, nimetazepam, a nimetazepam analog, donepezil, rivastigmine,
galantamine, tacrine, memantine, solanezumab, bapineuzmab, alzemed, flurizan, ELND005, valproate, semagacestat, rosiglitazone, phenserine, cernezumab, dimebon, egcg,
gammagard, PBT2, PF04360365, NIC5-15, bryostatin-1 , AL-108, nicotinamide, EHT-0202, BMS708163, NP12, lithium, ACCOOl , AN 1792, ABT089, NGF, CAD 106, AZD3480, SB742457, AD02, huperzine-A, EVP6124, PRX03140, PUFA, HF02, MEM3454, TTP448, PF-04447943, GSK933776, MABT5102A, talsaclidine, UB31 1 , begacestat, R1450,
PF3084014, V950, E2609, MK0752, CTS21 166, AZD-3839, LY2886721 , CHF5074, an anti-inflammatory, dapsone, an anti-TNF antibody, and a statin). In certain of these embodiments the subject is in severe stage Alzheimer's disease and the TrkA inhibitor is administered in conjunction with NGF and/or an NGF mimetic. [0012] In various embodiments pharmaceutical formulations are provided. In certain embodiments the formulations comprise a TrkA kinase inhibitor according to Formula I as described and/or claimed herein, or a polymorph thereof, enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a
pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments this pharmaceutical formulation excludes ADDN-1351, or a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments this pharmaceutical formulation comprises ADDN-1351, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments the TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN-1351d, ADDN-1351e, ADDN-1351f, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN-1351j, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN- 135 lp as shown in Table 4, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments the formulation is a unit dosage formulation. In certain embodiments the formulation is a sterile formulation. In certain embodiments the TrkA kinase inhibitor is formulated for administration via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration.
[0013] In various embodiments a TrkA kinase inhibitor is provided for use in inhibiting the C-terminal cleavage of APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal; and/or mitigating in a mammal one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms; and/or reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal; and/or preventing or delaying the onset of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or preventing or delaying the progression of a pre- Alzheimer's condition or cognitive dysfunction to
Alzheimer's disease in a mammal; and/or for promoting the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway as characterized by increasing sAPPa and/or the sAPPa/A 42 ratio in a mammal. In certain embodiments the TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siRNA as described and/or claimed herein. In certain embodiments the TrkA inhibitor comprises a small organic molecule inhibitor of TrkA according to any of Formulas I-XVIII, and/or Tables 4- 12, as described and/or claimed herein and/or a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11, and/or a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments the TrkA inhibitor is an inhibitor according to Formula I as described and/or claimed herein, or a polymorph thereof, enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments this Formula excludes excludes ADDN-1351. In certain embodiments the TrkA inhibitor is ADDN-1351, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments the TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN- 1351c, ADDN-1351d, ADDN-1351e, ADDN-1351f, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN-1351j, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN-1351p as shown in Table 4, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a
pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
[0014] Also provided are methods for the treatment or prophylaxis of age-related macular degeneration (AMD), where the methods comprise administering, or causing to be administered, to a mammal thereof a TrkA kinase inhibitor in an amount sufficient to ameliorate one or more symptoms of AMD and/or to slow or prevent the progression of AMD. In certain embodiments the TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siRNA as described and/or claimed herein. In certain embodiments the TrkA inhibitor comprises a small organic molecule inhibitor of TrkA according to any of Formulas I-XVIII, and/or Tables 4-12, as described and/or claimed herein and/or a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11, and/or a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments the TrkA inhibitor is an inhibitor according to Formula I as described and/or claimed herein, or a polymorph thereof, enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a
pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments this Formula excludes ADDN-1351. In certain embodiments the TrkA inhibitor is ADDN-1351, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments the TrkA kinase inhibitor is selected from the group consisting of ADDN- 1351a, ADDN- 1351b, ADDN- 1351c, ADDN-135 Id, ADDN-135 le, ADDN-135 If, ADDN-135 lg, ADDN-135 lh, ADDN-135 li, ADDN-135 lj, ADDN-135 lk, ADDN-13511, ADDN-135 lm, ADDN-135 In, ADDN-135 lo, AND ADDN-135 lp as shown in Table 4, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a
pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof.
[0015] Kits are also provided. In certain embodiments the kits comprise a container containing a TrkA inhibitor and instructions for use of that inhibitor in the treatment and/or prophylaxis of an amyloidogenic pathology. In certain embodiments the TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siRNA as described and/or claimed herein. In certain embodiments the TrkA inhibitor comprises a small organic molecule inhibitor of TrkA according to any of Formulas I-XVIII, and/or Tables 4- 12, as described and/or claimed herein and/or a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11, and/or a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments the TrkA inhibitor is an inhibitor according to Formula I as described and/or claimed herein, or a polymorph thereof, enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments this Formula excludes excludes ADDN-1351. In certain embodiments the TrkA inhibitor is ADDN-1351, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. In certain embodiments the TrkA kinase inhibitor is selected from the group consisting of ADDN-135 la, ADDN-135 lb, ADDN- 1351c, ADDN-135 Id, ADDN-135 le, ADDN-135 If, ADDN-135 lg, ADDN-135 lh,
ADDN-135 li, ADDN-135 lj, ADDN-135 lk, ADDN-13511, ADDN-135 lm, ADDN-135 In, ADDN-135 lo, AND ADDN-135 lp as shown in Table 4, a polymorph, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a
pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof. [0016] In certain embodiments the methods described and/or claimed herein expressly exclude administration of the active agents (TrkA kinase inhibitors) for the treatment of one or more indications selected from the group consisting of Parkinson's disease, psychosis, Schizophrenia, a neurological disease or disorder other than Alzheimer's disease, cancer, pain, inflammation, rheumatoid arthritis, and/or an immunological disorder.
[0017] In certain embodiments ,the methods described and/or claimed herein expressly exclude the use of TrkA kinase inhibitors to promote myelination, neuronal survival, and oligodendrocyte differentiation and/or to treat demyelination and
dysmyelination disease(s) including, but not limited to multiple sclerosis (MS), progressive multi focal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), Wallerian Degeneration and some inherited diseases such as
adrenoleukodystrophy, Alexander's disease, and Pelizaeus Merzbacher disease (PMZ).
DEFINITIONS
[0018] Tyrosine kinase receptor A (TrkA) is a plasma member receptor composed of an extracellular domain (responsible for high affinity binding to nerve growth factor, NGF), a transmembrane segment and an intracellular protein tyrosine kinase domain (responsible to transmit the NGF signal to initiate and coordinate neuronal responses). NGF binding induces TrkA clustering on the membrane and activates the kinase. The kinase initiates a cascade of protein phosphorylation events through multiple pathways including SHC/Ras/MAPK, PI3K and PLCgl .
[0019] A "TrkA kinase inhibitor" generally refers to a molecule that decreases, reduces, or inhibits TrkA autophosphorylation and/or TrkA kinase activity. Illustrative TrkA kinase inhibitors include, but are not limited to small organic molecule inhibitors, TrkA inhibitory peptides, anti-TrkA antibodies. In particular embodiments, the term "TrkA inhibitor" refers to a TrkA kinase inhibitor. In certain embodiments, a "TrkA inhibitor" is not a "TrkA kinase inhibitor" and inhibits TrkA receptor signaling activity and/or TrkA mRNA or protein expression. Thus, illustrative TrkA inhibitors include, but are not limited to small organic molecule inhibitors, TrkA inhibitory peptides, anti-TrkA antibodies, and TrkA siRNA. [0020] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Accordingly, isotopically labeled compounds are within the scope of this invention. [0021] In general, "substituted" refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group will be substituted with one or more substituents, unless otherwise specified. In some
embodiments, a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents.
Examples of substituent groups include: halogens (e.g., F, CI, Br, and I); hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxy amines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N- oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (i.e., CN), and the like. [0022] The term "alkyl" refers to and covers any and all groups that are known as normal alkyl, branched-chain alkyl, cycloalkyl and also cycloalkyl-alkyl.
[0023] The term "Ci_6 alkyl group" refers to a straight chain or branched chain alkyl group having 1 to 6 carbon atoms, and may be exemplified by a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert- butyl group, a sec-butyl group, an n-pentyl group, a tert-amyl group, a 3-methylbutyl group, a neopentyl group, and an n-hexyl group.
[0024] Aryl groups are cyclic aromatic hydrocarbons that do not contain
heteroatoms. Aryl groups include monocyclic, bicyclic and polycyclic ring systems. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. Although the phrase "aryl groups" includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like), it does not include aryl groups that have other groups, such as alkyl or halo groups, bonded to one of the ring members. Rather, groups such as tolyl are referred to as substituted aryl groups. Representative substituted aryl groups may be mono-substituted or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.
[0025] The term "heteroaryl group" refers to a monocyclic or condensed-ring aromatic heterocyclic group containing one or more hetero-atoms selected from O, S and N. If the aromatic heterocyclic group has a condensed ring, it can include a partially
hydrogenated monocyclic group. Examples of such a heteroaryl group include a pyrazolyl group, a thiazolyl group, an isothiazolyl group, a thiadiazolyl group, an imidazolyl group, a furyl group, a thienyl group, an oxazolyl group, an isoxazolyl group, a pyrrolyl group, an imidazolyl group, a (1,2,3)- and (l,2,4)-triazolyl group, a tetrazolyl group, a pyranyl group, a pyridyl group, a pyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a quinolyl group, an isoquinolyl group, a benzofuranyl group, an isobenzofuranyl group, an indolyl group, an isoindolyl group, an indazolyl group, a benzoimidazolyl group, a benzotriazolyl group, a benzoxazolyl group, a benzothiazolyl group, a benzo[b]thiophenyl group, a thieno[2,3-b]thiophenyl group, a (1,2)- and (l,3)-benzoxathiol group, a chromenyl group, a 2-oxochromenyl group, a benzothiadiazolyl group, a quinolizinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, and a carbazolyl group.
[0026] A "derivative" of a compound means a chemically modified compound wherein the chemical modification takes place at one or more functional groups of the compound. The derivative however, is expected to retain, or enhance, the pharmacological activity of the compound from which it is derived.
[0027] As used herein, the phrase "a subject in need thereof refers to a subject, as described infra, that suffers or is at a risk of suffering {e.g., pre-disposed such as genetically pre-disposed) from the diseases or conditions listed herein. [0028] The terms "subject", "individual", and "patient" may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals {e.g., canine or feline), laboratory mammals {e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals {e.g., equine, bovine, porcine, ovine). In various embodiments, the subject can be a human {e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker. [0029] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a TrkA inhibitor, e.g., ADDN-1351 or an analogue thereof, optionally in combination with one or more other pharmaceuticals, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the treatment to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a treatment are substantially absent or are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" refers to an amount of an active agent or composition comprising the same that is effective to "treat" a disease or disorder in a mammal {e.g., a patient). In one embodiment, a therapeutically effective amount is an amount sufficient to improve at least one symptom associated with a neurological disorder, improve neurological function, improve cognition, or one or more markers of a neurological disease, or to enhance the efficacy of one or more pharmaceuticals administered for the treatment or prophylaxis of a neurodegenerative pathology. In certain embodiments, an effective amount is an amount sufficient alone, or in combination with a pharmaceutical agent to prevent advancement or the disease, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by the disease,
[0030] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.
[0031] The terms "treatment", "treating", or "treat" as used herein, refer to actions that produce a desirable effect on the symptoms or pathology of a disease or condition, particularly those that can be effected utilizing the TrkA kinase inhibitors described herein, and may include, but are not limited to, even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. Treatments also refers to delaying the onset of, retarding or reversing the progress of, reducing the severity of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition. "Treatment", "treating", or "treat" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. In one embodiment, treatment comprises improvement of at least one symptom of a disease being treated. The improvement may be partial or complete. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.
[0032] The term "mitigating" refers to reduction or elimination of one or more symptoms of that pathology or disease, and/or a reduction in the rate or delay of onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease.
[0033] As used herein, the phrases "improve at least one symptom" or "improve one or more symptoms" or equivalents thereof, refer to the reduction, elimination, or prevention of one or more symptoms of pathology or disease. Illustrative symptoms of pathologies treated, ameliorated, or prevented by the compositions described herein (e.g., small molecule TrkA kinase inhibitors, including, but not limited to, ADDN-1351, ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN-1351d, ADDN-1351e, ADDN-1351f, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN-1351j, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, and ADDN-1351p) include, but are not limited to, reduction, elimination, or prevention of one or more markers that are characteristic of the pathology or disease (e.g., of total-Tau (tTau), phospho-Tau (pTau), APPneo, soluble Αβ40, pTau/Ap42 ratio and tTau/Ap42 ratio, and/or an increase in the CSF of levels of one or more
components selected from the group consisting of Αβ42/Αβ40 ratio, Αβ42/Αβ38 ratio, sAPPa, βΑΡΡα/βΑΡΡβ ratio, βΑΡΡα/Αβ40 ratio, βΑΡΡα/Αβ42 ratio, etc.) and/or reduction, stabilization or reversal of one or more diagnostic criteria (e.g., clinical dementia rating (CDR)). Illustrative measures for improved neurological function include, but are not limited to the use of the mini-mental state examination (MMSE) or Folstein test (a questionnaire test that is used to screen for cognitive impairment), the General Practitioner Assessment of Cognition (GPCOG), a brief screening test for cognitive impairment described by Brodaty et ah, (2002) Geriatrics Society 50(3): 530-534, and the like.
[0034] The phrase "cause to be administered" refers to the actions taken by a medical professional (e.g., a physician), or a person prescribing and/or controlling medical care of a subject, that control and/or determine, and/or permit the administration of the agent(s)/compound(s) at issue to the subject. Causing to be administered can involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing particular agent(s)/compounds for a subject. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like.
[0035] The term "co-administering" or "concurrent administration" or
"administering in conjunction with" when used, for example with respect to the TrkA kinase inhibitors and another active agent (e.g., NGF, NGF mimetics, etc.), refers to administration of the TrkA inhibitor(s) and the other active agent(s) such that both can simultaneously achieve a physiological effect. The TrkA inhibitor and the active agent composition, however, need not be administered together, either temporally or at the same site; moreover, the TrkA inhibitor and the other active agent(s) need not be administered by the same method, e.g., the TrkA inhibitor may be administered orally and the other active agent(s) may be administered intravenously or orally. In one particular embodiment, the TrkA inhibitor(s) and other the active agent(s) are administered at different times and by different methods of administration. In certain embodiments, administration of one can precede administration of the other. Simultaneous physiological effect need not necessarily require presence of the TrkA inhibitors and the other active agent in the circulation at the same time. However, in certain embodiments, co-administering typically results in both the TrkA inhibitor(s) and the other active agent(s) being simultaneously present in the body (e.g., in the plasma) at a significant fraction (e.g., 20% or greater, preferably 30% or 40% or greater, more preferably 50%> or 60%> or greater, most preferably 70%> or 80%> or 90%> or greater) of their maximum serum concentration for any given dose.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 illustrates induction of APPneo fragment produced by C-terminal cleavage of APP, upon co-transfection with TrkA. [0037] Figure 2 shows that APPneo fragment is not induced by a kinase-dead TrkA.
[0038] Figure 3 shows inhibition of TrkA induction by a TrkA inhibitor.
[0039] Figure 4 shows that ADDN- 1351 is a TrkA inhibitor.
[0040] Figure 5 illustrates pyrazole derivative TrkA inhibitors from AstraZeneca.
[0041] Figure 6 illustrates various TrkA inhibitors from Bayer and BMS. [0042] Figure 7 illustrates various TrkA inhibitors from Cephalon.
[0043] Figure 8 illustrates various TrkA inhibitors from GSK, Japan Tobacco, and J
7 J.
[0044] Figure 9 illustrates TrkA inhibitors from Nerviano, Novartis/IRM, Pfizer, and Plexxikon. [0045] Figure 10 illustrates various TrkA inhibitors (see, e.g., Raeppel et al. (2012)
Int. J. Med. Chem., Article ID 412614, doi: 10.1155/2012/412614, which is incorporated herein by reference for the TrkA inhibitors and synthesis methods described therein).
[0046] Figure 11 illustrates CEP-701 and analogues (see, e.g., Wang et al. (2008) 51 : 4672-4684).
[0047] Figures 12A-12D illustrate how APP-C31 cleavage inhibition screening identified a TrkA inhibitor. Figure 12A: Scattergraph of results from APP-C31 cleavage inhibition screen of a CNS-focused small molecule library. 5000 compounds were screened and 52 inhibited the APPneo signal (the neo epitope exposed after APP-C31 cleavage, APP 1-664) by over 70%. One of them, ADDN-1351, inhibited APPneo signal by over 90%. Figure 12B: ADDN-1351 is a TrkA inhibitor. At a low concentration of O.luM, ADDN-1351 was tested against a panel of 24 kinases. Only TrkA was inhibited by ADDN- 1351 in this kinase inhibition assay. Figure 12C: Chemical structures of TrkA inhibitors ADDN-1351, PHA-739358, and GW441756. Figure 12D: IC50-Data for TrkA inhibition: ADDN-1351, PHA-739358, and GW441756.
[0048] Figures 13A-13D show that TrkA induced APP-C31 cleavage is mediated through TrkA kinase activity. Figure 13 A: TrkA induced APP-C31 cleavage was blocked by the kinase dead TrkA(K538A) mutation. CHO cells stably over-expressing human APP was transiently transfected with empty vector, TrkA or TrkA(K538A) mutant constructs, and the level of APPneo was detected by Western blot. TrkA over-expression induced
APP-C31 cleavage, and this induction was absent when the kinase dead TrkA(K538A) was overexpressed. Figure 13B: TrkA induced APP-C31 cleavage was blocked by the TrkA inhibitor GW441756. CHO cells stably over-expressing human APP was transiently transfected with empty vector or TrkA, and treated with vehicle (DMSO) or GW441756 (ΙμΜ). The level of APPneo was detected by Western blot. TrkA over-expression induced APP-C31 cleavage, and this induction was abolished by GW441756 treatment. Figure 13C: NGF treatment enhanced TrkA induced APP-C31 cleavage. CHO cells stably over- expressing human APP was transiently transfected with empty vector, TrkA, TrkA+p75NTR or p75NTR, in the presence or absence of NGF (5nM). The level of APPneo was detected by Western blot. TrkA induced APP-C31 cleavage was increased by NGF treatment, in the absence or presence of TrkA co-receptor p75NTR. Figure 13D: TrkA over-expression induced cell death was blocked by the kinase dead TrkA(K538A) mutation. CHO cells stably over-expressing human APP was transiently transfected with empty vector, TrkA or TrkA(K538A) mutant constructs, and MTT assay was performed to evaluate the rate of cell survival. While TrkA over-expression induced significant cell death, TrkA(K538A) over- expression did not. **P<0.01, n=3, Student's t-test. Error bars indicate SD.
[0049] Figures 14A-14E show that TrkA interacts with APP. Figures 14 A, 14B:
TrkA co-immunoprecipitated with wild type APP (Fig. 14A), APP carrying the Swedish and Indiana FAD mutations (APPsi, Fig. 14A), APP carrying the D664A mutation (Fig. 14B) and APP with the C-terminal 31 amino acids deleted (Fig. 14B). 293T cells transiently expressing TrkA and wild type APP (Fig. 14A), APPsi (Fig. 14A), APP(D664A) (Fig. 14B) or APPAC31 (Fig. 14B) were lysed and the crude lysates were subjected to immunoprecipitation with anti-APP antibody followed by Protein G agarose beads. Cells expressing TrkA only were used as controls. Immunoprecipitated samples were subjected to Western blotting with anti-TrkA antibody. Total cell lysates were used as input controls. Figure 14C: TrkA co-immunoprecipitated with APP-C99 but not APP-C83. Figure 14D: TrkA(K538 A) mutant co-immunoprecipitated with APP. Figure 14E: TrkA co- immunoprecipitated with APP in the presence of NGF (5nM). [0050] Figures 15A-15D show that TrkA inhibits APP-AICD signaling. Figure
15 A: Potent transcriptional transactivation was achieved with APP fused to the Gal4 DNA binding domain when both Mint3 and YAP were present. This transactivation was abolished by TrkA. p75NTR also inhibited the transactivation, but to a lesser extent. Figure 15B: Transactivation of transcription was achieved with APP fused to the Gal4 DNA binding domain when Fe65 was present, and this transactivation was also abolished by TrkA. Figure 15C: YAP directed APP-Gal4/Mint3 transactivation was inhibited by both TrkA and TrkA(K538A) mutant. Figure 15D: YAP directed APP-Gal4/Mint3
transactivation was abolished by TrkA alone or TrkA plus p75NTR. This inhibition was not substantially affected by 5 nM NGF treatment. Diagrams exhibit representative experiments in which cells were co-transfected with a UAS-luciferase reporter plasmid (to measure transactivation), a β-galactosidase plasmid (to normalize for transfection efficiency), and the test plasmids identified below the bars. The normalized luciferase activity is expressed as fold induction over transcription by APP-Gal4 alone (Figs. 15 A, 15B), or as a percentage of the APP-Gal4/Mint3/YAP control (Figs. 15C, 15D). Error bars indicate SD. [0051] Figures 16A-16E show that TrkA modulates APP processing. Figures 16 A,
16B: Conditioned media were collected for Αβ40 and Αβ42 assays. Over-expression of TrkA inhibited the production of Αβ40 and Αβ42. NGF treatment (5 nM) increased Αβ40 and Αβ42 production in the presence of TrkA. TrkA(K538A) mutant also inhibited the production of Αβ42, but the NGF mediated Αβ42 increase was also abolished by the kinase dead TrkA(K538A) mutation. **P<0.01, n=3, Student's t-test. Error bars indicate SD. Figure 16C: TrkA over-expression inhibited the production of sAPPa, while increasing the level of full-length APP. 293T cells were co-transfected with different forms of APP and empty vector or TrkA. Conditioned media were collected for sAPPa Western blot, and cell lysates were collected for full length APP Western blot. For wild type APP, APPAC31 and APP(D664A), TrkA over-expression decreased sAPPa production and increased the levels of full length APP. Figure 16D: TrkA(K538 A) over-expression also inhibited the production of sAPPa. NGF treatment (5 nM) increased sAPPa production in the presence of TrkA but not TrkA(K538A). Figure 16E: TrkA over-expression increased the level of β- CTF (APP-C99).
[0052] Figures 17A-17E show that TrkA inhibitor GW441756 treatment decreases
Αβ (Figs. 17A, 17B) and increases sAPPa (Fig. 17C) and sAPPa/Ap42 ratio (Fig. 17D) in an AD transgenic mouse model (PDAPP J20). 6-6.5 months old J20 mice were treated with DMSO control or GW441756 at lOmg/kg/day for 5 days. **P<0.01, n=7, Student's t-test. Error bars indicate SEM. Figure 17E: Chronic hyper-activation of TrkA in the target regions of Basal Forebrain Cholinergic Neurons (BFCN) as an early mechanism in
Alzheimer's disease (AD). NGF is produced in the target regions of BFCN, and synapses of BFCN take up NGF and retrogradely transport it back to the cell bodies in the form of NGF - TrkA complexes. In non-AD controls, sufficient amounts of NGF trophic support maintain the normal function of BFCN. With aging, the retrograde transport system efficiency is reduced, which leads to lower levels of active NGF-TrkA complexes in the BFCN, and higher levels of NGF-TrkA complexes in the target regions of BFCN, including the entorhinal cortex, hippocampus and neo-cortex. Accumulation of active NGF-TrkA complexes in the target regions of BFCN leads to increased Αβ and APP-C31 production, which results in progression into the mild cognitive impairment stage (MCI). In the MCI stage, hyper-activation of TrkA in the target regions of BFCN continues to facilitate the progression of AD. In the mild AD stage, TrkA levels are decreased in the target regions of BFCN, but the cholinergic functions are still normal. In the severe AD stage, due to prolonged NGF-TrkA signaling deficit, BFCN degeneration and cholinergic deficits are prominent. Although total levels of TrkA are decreased in the target regions of BFCN,
NGF levels are not, and it is likely that local hyper-activation of TrkA in the target regions of BFCN still exists. Therefore anti-TrkA treatment will still have anti-AD effects in the target regions of BFCN. However, because of the prominent BFCN degeneration problems at this stage, a combination of anti-TrkA treatment and targeted BFCN NGF delivery may be ideal.
[0053] Figures 18 illustrates a synthesis scheme for ADDN-1351 and analogs thereof. DETAILED DESCRIPTION
[0054] The prevailing view of Alzheimer's disease (AD) is that amyloid-beta peptides cause toxicity through chemical and physical mechanisms, such as metal binding, ROS production, and membrane damage. Our data suggest an alternative view of AD as an imbalance in physiological signaling mediated by APP: in this model, Αβ functions physiologically as an anti-trophin, and Αβ binding to APP induces the formation of peptides mediating neurite retraction and cell death (see, e.g., Lu et al. (2000) Nat. Med., 6: 397- 404). Thus, preventing production of APP-C31 and Jcasp in transgenic mice renders them resistant to AD (Galvan et al. (2006) Proc. Natl. Acad. Sci. USA, 103:7130-7135).
Competing with Αβ is a novel APP ligand, netrin-1, which mediates neurite extension. A CNS-focused library was used to identify compounds that block APP-C31. One compound, ADDN-1351, reduced APP-C31 by 90%, reduced Αβ, and increased sAPPalpha.
ADDN1351 was identified as a TrkA kinase inhibitor. Complementing these results, TrkA co-expressed with APP increased APP-C31 production and cell death. APP-C31 cleavage did not occur with the kinase-dead TrkA (mutation of lysine 538 to alanine) mutant or in the presence of TrkA inhibitor GW441756, whereas induction was enhanced by NGF.
[0055] TrkA is a dependence receptor, that induces cell death in response to reduced
NGF concentration (Nikoletopoulou et al. (2010) Nature 467: 59-63). Previous work showed that p75NTR interacts with APP and modulates its signaling (Fombonne et al. (2009) Ann. Neurol., 65: 294-303). The present inventors discovered that TrkA interacts with APP. The intracellular domain of APP (AICD) interacts with several adaptor proteins. AICD/Fe65/Tip60 and AICD/Mint3 /Y AP complexes are transcriptionally active (Cao and Sudhof (2001) Scie/ice 293: 115-120; Swistowski et al. (2009) J. Neurosci., 29: 15703- 15712). TrkA co-immunoprecipitated with APP, and this did not require the C31 region of APP. TrkA also co-immunoprecipitated with APP-C99 but not C83. Thus, the first 16 amino acids of the Αβ region are important in TrkA- APP interaction. In APPGal4 transactivation assays, co-expression of TrkA inhibited the transactivation directed by Fe65 or Mint3/YAP by over 90%. [0056] Basal forebrain cholinergic neurons (BFCN), hippocampus and entorhinal cortex are the earliest targets of AD. BFCN provide cholinergic innervation to the hippocampus and cortex, while NGF produced in the hippocampus and cortex is retrogradely transported to BFCN. Without being bound to a particular theory, it is contemplated that disrupted retrograde transport of NGF-TrkA complexes underlies BFCN degeneration in AD. Thus, TrkA-mediated effects on APP represent a novel therapeutic target for AD: TrkA inhibition can alleviate AD associated damage in the cortex and hippocampus, where active NGF-TrkA complexes accumulate.
[0057] In various embodiments, methods of inhibiting the C-terminal cleavage of APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal are provided. C-terminal cleavage of full-length ΑΡΡ659 results in the proteolytic fragments APPneo (APPneo) and the APP-C31 terminal peptide. In addition, induction of the APPneo fragment correlates with increases in cell death in APP transfected cells, and cotransfection of TrkA in APP transfected cells results in increased APPneo production. In addition, a TrkA kinase dead mutant did not inhibit APPneo production (see, e.g., Figure 1). and cotransfection of a kinase dead TrkA kinase did induce APP cleavage to form APPneo (see, e.g., Figure 2). Moreover, inhibition of TrkA kinase by a potent small molecule inhibitor GW441756 (IC50 TrkA ~ 2 nM) inhibited APPneo production in cells transfected with TrkA kinase (see, e.g., Figure 3). [0058] Accordingly, methods provided herein typically involve administering to a mammal, a tropomyosin-related kinase A (TrkA) inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a C31 peptide and/or APPneo. Without being bound to a particular theory, it is contemplated that inhibition of expression and/or activity of a TrkA kinase, particularly in neurological tissue, will inhibit the processing of APP to APPneo and APP-C31 and thereby reduce the rate of onset, severity, or possibly reverse one or more symptoms of Alzheimer's disease, and potentially other disorders characterized by amyloid plaque formation. In certain embodiments, one or more TrkA kinase inhibitors are prophylactically administered to a mammal (e.g., a human) at risk for Alzheimer's disease, or are therapeutically administered to a mammal (e.g. , a human diagnosed as having pre-, early stage, mid stage, or late stage Alzheimer's disease.
[0059] In various other embodiments, the use of TrkA inhibitors is contemplated for mitigating in a mammal one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms, said method comprising and/or for reducing the risk, lessening the severity, or delaying the progression or onset of a disease (e.g., Alzheimer's disease, age-related macular
degeneration (AMD), Cerebrovascular dementia, Parkinson's disease, Huntington's disease, and Cerebral amyloid angiopathy, and the like) characterized by beta-amyloid deposits in the brain of a mammal. The TrkA kinase inhibitors are also contemplated for use in preventing or delaying the onset of a pre- Alzheimer's condition and/or cognitive
dysfunction, and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction. In particular embodiments, TrkA kinase inhibitors can be used to promote the processing of amyloid precursor protein (APP) by the non- amyloidogenic pathway as characterized, for example, by increasing sAPPa and/or the sAPPa/A 42 ratio in a mammal.
[0060] In certain embodiments, the methods comprise administering one or more
TrkA inhibitors, e.g. , a TrkA inhibitor as described herein, in an amount sufficient to produce the desired activity (e.g., mitigating one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms, and/or reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal, and/or promoting the processing of amyloid precursor protein (APP) by the non- amyloidogenic pathway).
[0061] In particular embodiments, the timing of administration of the TrkA kinase inhibitors can offer significant benefits. For example, during the stage of mild AD (or a pre- AD condition (e.g., MCI), there is no (or little) cholinergic deficit, suggesting that at least the function of neurotransmission is preserved in the basal forebrain cholinergic neurons (BFCN) at this stage. However, at this same stage, in the BFCN target regions, progressive Αβ accumulation and Tau pathology are developing and are associated with clinical symptoms. In certain embodiments, it may be preferred to use TrkA inhibitors at this stage of the disease, in order to slow or reverse AD progression at the target regions. Without being bound to a particular theory, the present invention contemplates, in part, that administration of TrkA inhibitors at mild or pre-AD stages protects BFCN target regions and prevents deficits in memory and other cognitive functions; potentially blocks the positive feedback loops at BFCN target regions; reduces the levels of Αβ and p-Tau;
prevents further deterioration of disrupted retrograde transport; and restores NGF support back to pre-AD levels.
[0062] However, at the severe AD stage, in which there is significant loss of BFCN function, the somal effects may predominate over the process effects, and therefore the application of a TrkA inhibitor may potentially exacerbate the AD symptoms. In support of this notion, targeted BFCN delivery of NGF has been found to be promising (Tuszynski et al. (2005) Nat. Med., 11 : 551-555), and is currently in a phase II clinical trial. Thus, in certain embodiments, TrkA inhibition can be a viable therapeutic strategy in a stage- dependent fashion and in combination therapy with NGF or NGF mimetics, or as part of a therapeutic cocktail that targets multiple mechanisms of AD pathogenesis (see, Example 1 herein for further discussion).
[0063] The present inventors discovered that ADDN-1351 (see, Figure 12C) is a potent TrkA inhibitor, easily crosses the blood barrier, and is effective to inhibit cleavage of APP to form APPneo and APP-C31. Accordingly, in various embodiments, pharmaceutical formulations comprising ADDN-1351 and various derivatives thereof are contemplated.
[0064] While the methods described herein are detailed primarily in the context of mild cognitive impairment (MCI) and Alzheimer's disease (AD) it is believed they can apply equally to other pathologies characterized by amyloidosis. An illustrative, but non- limiting list of conditions characterized by amyloid plaque formation is shown in Table 1.
Table 1. Illustrative, but non-limiting pathologies characterized by amyloid
formation/ deposition.
Figure imgf000031_0001
Figure imgf000032_0001
Subjects Who Can Benefit from the Present Methods
[0065] Subjects/patients amenable to treatment using the methods described herein include individuals at risk of disease (e.g., a pathology characterized by amyloid plaque formation) but not showing symptoms, as well as subjects presently showing symptoms. Accordingly, certain subjects include subjects at increased risk for the onset of a pre-
Alzheimer's condition and/or cognitive dysfunction (e.g., MCI) and/or subjects diagnosed as having a pre- Alzheimer's condition and/or cognitive dysfunction (e.g., MCI).
[0066] Accordingly, in various embodiments, therapeutic and/or prophylactic methods are provided that utilize the active agent(s) (e.g., TrkA kinase inhibitors) are provided. Typically the methods involve administering one or more active agent(s) (e.g., TrkA kinase inhibitors) to a subject (e.g., to a human in need thereof) in an amount sufficient to realize the desired therapeutic or prophylactic result.
Prophylaxis
[0067] In certain embodiments, active agent(s) (e.g., TrkA kinase inhibitors), alone or in conjunction with other active agent(s), are utilized in various prophylactic contexts. Thus, for example, in certain embodiments, TrkA inhibitors can be used to prevent or delay the onset of a pre -Alzheimer's cognitive dysfunction, and/or to ameliorate one more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or to prevent or delay the progression of a pre- Alzheimer's condition and/or cognitive dysfunction to Alzheimer's disease.
[0068] Accordingly in certain embodiments, the prophylactic methods described herein are contemplated for subjects identified as "at risk" and/or as having evidence of early Alzheimer's Disease (AD) pathological changes, but who do not meet clinical criteria for MCI or dementia. Without being bound to a particular theory, it is contemplated that even this "preclinical" stage of the disease represents a continuum from completely asymptomatic individuals with biomarker evidence suggestive of AD-pathophysiological process(es) (abbreviated as AD-P, see, e.g., Sperling et al, (201 1) Alzheimer 's & Dementia, 1-13) at risk for progression to AD dementia to biomarker-positive individuals who are already demonstrating very subtle decline but not yet meeting standardized criteria for MCI (see, e.g., Albert et al., (2011) Alzheimer's and Dementia, 1-10
(doi: 10.1016/j.jalz.2011.03.008)).
[0069] This latter group of individuals might be classified as "not normal, not MCI" but can be designated "pre-symptomatic" or "pre-clinical or "asymptomatic" or
"premanifest"). In various embodiments, this continuum of pre-symptomatic AD can also encompass (1) individuals who carry one or more apolipoprotein E (APOE) ε4 alleles who are known or believed to have an increased risk of developing AD dementia, at the point they are AD-P biomarker-positive, and (2) carriers of autosomal dominant mutations, who are in the presymptomatic biomarker-positive stage of their illness, and who will almost certainly manifest clinical symptoms and progress to dementia.
[0070] A biomarker model has been proposed in which the most widely validated biomarkers of AD-P become abnormal and likewise reach a ceiling in an ordered manner (see, e.g., Jack et al, (2010) Lancet Neurol, 9: 119-128.). This biomarker model parallels proposed pathophysiological sequence of (pre- AD/ AD), and is relevant to tracking the preclinical (asymptomatic) stages of AD (see, e.g., Figure 3 in Sperling et al, (2011) Alzheimer's & Dementia, 1-13). Biomarkers of brain amyloidosis include, but are not limited to reductions in CSF Αβ42 and increased amyloid tracer retention on positron emission tomography (PET) imaging. Elevated CSF tau is not specific to AD and is thought to be a biomarker of neuronal injury. Decreased fluorodeoxyglucose 18F (FDG) uptake on PET with a temporoparietal pattern of hypometabolism is a biomarker of AD- related synaptic dysfunction. Brain atrophy on structural magnetic resonance imaging (MRI) in a characteristic pattern involving the medial temporal lobes, paralimbic and temporoparietal cortices is a biomarker of AD-related neurodegeneration. Other markers include, but are not limited to volumetric MRI, FDG-PET, or plasma biomarkers (see, e.g., Vemuri et al, (2009) Neurology, 73: 294-301; Yaffe et al, (2011) JAMA 305: 261-266).
[0071] In certain embodiments, the subjects suitable for the prophylactic methods contemplated herein include, but are not limited to subject characterized as having asymptomatic cerebral amyloidosis. In various embodiments, these individuals have biomarker evidence of Αβ accumulation with elevated tracer retention on PET amyloid imaging and/or low Αβ42 in CSF assay, but typically no detectable evidence of additional brain alterations suggestive of neurodegeneration or subtle cognitive and/or behavioral symptomatology.
[0072] It is noted that currently available CSF and PET imaging biomarkers of Αβ primarily provide evidence of amyloid accumulation and deposition of fibrillar forms of amyloid. Data suggest that soluble or oligomeric forms of Αβ are likely in equilibrium with plaques, which may serve as reservoirs. In certain embodiments, it is contemplated that there is an identifiable preplaque stage in which only soluble forms of Αβ are present. In certain embodiments, it is contemplated that oligomeric forms of amyloid may be critical in the pathological cascade, and provide useful markers. In addition, early synaptic changes may be present before evidence of amyloid accumulation.
[0073] In certain embodiments, the subjects suitable for the prophylactic methods contemplated herein include, but are not limited to, subjects characterized as amyloid positive with evidence of synaptic dysfunction and/or early neurodegeneration. In various embodiments, these subjects have evidence of amyloid positivity and presence of one or more markers of "downstream" AD-P-related neuronal injury. Illustrative, but non- limiting markers of neuronal injury include, but are not limited to (1) elevated CSF tau or phospho- tau, (2) hypometabolism in an AD-like pattern {e.g., posterior cingulate, precuneus, and/or temporoparietal cortices) on FDG-PET, and (3) cortical thinning/gray matter loss in a specific anatomic distribution {e.g., lateral and medial parietal, posterior cingulate, and lateral temporal cortices) and/or hippocampal atrophy on volumetric MRI. Other markers include, but are not limited to fMRI measures of default network connectivity. In certain embodiments, early synaptic dysfunction, as assessed by functional imaging techniques such as FDG-PET and fMRI, can be detectable before volumetric loss. Without being bound to a particular theory, it is contemplated that amyloid-positive individuals with evidence of early neurodegeneration may be farther down the trajectory {e.g. , in later stages of preclinical (asymptomatic) AD).
[0074] In certain embodiments, the subjects suitable for the prophylactic methods contemplated herein include, but are not limited to, subjects characterized as amyloid positive with evidence of neurodegeneration and subtle cognitive decline. Without being bound to a particular theory, it is contemplated that those individuals with biomarker evidence of amyloid accumulation, early neurodegeneration, and evidence of subtle cognitive decline are in the last stage of preclinical (asymptomatic) AD, and are
approaching the border zone with clinical criteria for mild cognitive impairment (MCI). These individuals may demonstrate evidence of decline from their own baseline (particularly if proxies of cognitive reserve are taken into consideration), even if they still perform within the "normal" range on standard cognitive measures. Without being bound to a particular theory, it is contemplated that more sensitive cognitive measures, particularly with challenging episodic memory measures, may detect very subtle cognitive impairment in amyloid-positive individuals. In certain embodiments, criteria include, but are not limited to, self-complaint of memory decline or other subtle neurobehavioral changes.
[0075] As indicated above, subjects/patients amenable to prophylactic methods described herein include individuals at risk of disease (e.g. , a pathology characterized by amyloid plaque formation such as MCI) but not showing symptoms, as well as subjects presently showing certain symptoms or markers. It is known that the risk of MCI and later Alzheimer's disease generally increases with age. Accordingly, in asymptomatic subjects with no other known risk factors, in certain embodiments, prophylactic application is contemplated for subjects over 50 years of age, or subjects over 55 years of age, or subjects over 60 years of age, or subjects over 65 years of age, or subjects over 70 years of age, or subjects over 75 years of age, or subjects over 80 years of age, in particular to prevent or slow the onset or ultimate severity of mild cognitive impairment (MCI), and/or to slow or prevent the progression from MCI to early stage Alzheimer's disease (AD).
[0076] In certain embodiments, the methods described herein present methods are especially useful for individuals who do have a known genetic risk of Alzheimer's disease (or other amyloidogenic pathologies), whether they are asymptomatic or showing symptoms of disease. Such individuals include those having relatives who have experienced MCI or AD (e.g., a parent, a grandparent, a sibling), and those whose risk is determined by analysis of genetic or biochemical markers. Genetic markers of risk toward Alzheimer's disease include, for example, mutations in the APP gene, particularly mutations at position 717 and positions 670 and 671 referred to as the Hardy and Swedish mutations respectively (see, e.g., Hardy (1997) Trends. Neurosci., 20: 154-159). Other markers of risk include mutations in the presenilin genes (PS1 and PS2), family history of AD, having the familial Alzheimer's disease (FAD) mutation, the APOE ε4 allele, hypercholesterolemia or atherosclerosis. Further susceptibility genes for the development of Alzheimer's disease are reviewed, e.g., in Sleegers, et al, (2010) Trends Genet. 26(2): 84-93.
[0077] In some embodiments, the subject is asymptomatic but has familial and/or genetic risk factors for developing MCI or Alzheimer's disease. In asymptomatic patients, treatment can begin at any age (e.g., 20, 30, 40, 50 years of age). Usually, however, it is not necessary to begin treatment until a patient reaches at least about 40, 50, 60 or 70 years of age.
[0078] In some embodiments, the subject is exhibiting symptoms, for example, of mild cognitive impairment (MCI) or Alzheimer's disease (AD). Individuals presently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors described above. In addition, a number of diagnostic tests are available for identifying individuals who have AD. These include measurement of CSF Tau, phospho-tau (pTau), Αβ42 levels and C-terminally cleaved APP fragment (APPneo). Elevated total-Tau (tTau), phospho-Tau (pTau), APPneo, soluble Αβ40, pTau/Ap42 ratio and tTau/A|342 ratio, and decreased Αβ42 levels, Αβ42/Αβ40 ratio, Αβ42/Αβ38 ratio, sAPPa levels, βΑΡΡα/βΑΡΡβ ratio, 8ΑΡΡα/Αβ40 ratio, and 8ΑΡΡα/Αβ42 ratio signify the presence of AD. In some embodiments, the subject or patient is diagnosed as having MCI. Increased levels of neural thread protein (NTP) in urine and/or increased levels of a2- macroglobulin (a2M) and/or complement factor H (CFH) in plasma are also biomarkers of MCI and/or AD (see, e.g., Anoop et al., (2010) Int. J. Alzheimer's Dz's.2010:606802).
[0079] In certain embodiments, subjects amenable to treatment may have age- associated memory impairment (AAMI), or mild cognitive impairment (MCI). The methods described herein are particularly well-suited to the prophylaxis and/or treatment of MCI. In such instances, the methods can delay or prevent the onset of MCI, and or reduce one or more symptoms characteristic of MCI and/or delay or prevent the progression from MCI to early-, mid- or late- stage Alzheimer's disease or reduce the ultimate severity of the disease.
Mild Cognitive Impairment (MCI)
[0080] In various embodiments, TrkA inhibitors, alone or in conjunction with other active agents, are contemplated in the treatment and/or prophylaxis of age-related cognitive decline and/or in the treatment and/or prophylaxis of mild cognitive impairment (MCI). Mild cognitive impairment, also known as incipient dementia, or isolated memory impairment) is a diagnosis given to individuals who have cognitive impairments beyond that expected for their age and education, but that typically do not interfere significantly with their daily activities (see, e.g., Petersen et al, (1999) Arch. Neurol. 56(3): 303-308). It is considered in many instances to be a boundary or transitional stage between normal aging and dementia. Although MCI can present with a variety of symptoms, when memory loss is the predominant symptom it is termed "amnestic MCI" and is frequently seen as a risk factor for Alzheimer's disease (see, e.g., Grundman et al., (2004) Arch. Neurol. 61(1): 59- 66; and on the internet at en.wikipedia.org/wiki/Mild_cognitive_impairment - cite note- Grundman-1). When individuals have impairments in domains other than memory it is often classified as non-amnestic single- or multiple-domain MCI and these individuals are believed to be more likely to convert to other dementias (e.g. dementia with Lewy bodies). There is evidence suggesting that while amnestic MCI patients may not meet
neuropathologic criteria for Alzheimer's disease, patients may be in a transitional stage of evolving Alzheimer's disease; patients in this hypothesized transitional stage demonstrated diffuse amyloid in the neocortex and frequent neurofibrillary tangles in the medial temporal lobe (see, e.g., Petersen et al, (2006) Arch. Neurol, 63(5): 665-72).
[0081] The diagnosis of MCI typically involves a comprehensive clinical assessment including clinical observation, neuroimaging, blood tests and
neuropsychological testing. In certain embodiments, diagnostic criteria for MCI include, but are not limited to those described by Albert et al, (201 1) Alzheimer 's & Dementia. 1- 10. As described therein, diagnostic criteria include (1) core clinical criteria that could be used by healthcare providers without access to advanced imaging techniques or
cerebrospinal fluid analysis, and (2) research criteria that could be used in clinical research settings, including clinical trials. The second set of criteria incorporate the use of biomarkers based on imaging and cerebrospinal fluid measures. The final set of criteria for mild cognitive impairment due to AD has four levels of certainty, depending on the presence and nature of the biomarker findings.
[0082] In certain embodiments, clinical evaluation/diagnosis of MCI involves: (1) concern reflecting a change in cognition reported by patient or informant or clinician (e.g. , historical or observed evidence of decline over time); (2) objective evidence of impairment in one or more cognitive domains, typically including memory (e.g., formal or bedside testing to establish level of cognitive function in multiple domains); (3) preservation of independence in functional abilities; (4) not demented; and in certain embodiments, (5) an etiology of mci consistent with ad pathophysiological processes, typically vascular, traumatic, medical causes of cognitive decline are ruled out where possible, in certain embodiments, evidence of longitudinal decline in cognition is identified, when feasible. Diagnosis is reinforced by a history consistent with AD genetic factors, where relevant.
[0083] With respect to impairment in cognitive domain(s), there should be evidence of concern about a change in cognition, in comparison with the person's previous level. There should be evidence of lower performance in one or more cognitive domains that is greater than would be expected for the patient's age and educational background. If repeated assessments are available, then a decline in performance should be evident over time. This change can occur in a variety of cognitive domains, including memory, executive function, attention, language, and visuospatial skills. An impairment in episodic memory (e.g. , the ability to learn and retain new information) is seen most commonly in MCI patients who subsequently progress to a diagnosis of AD dementia.
[0084] With respect to preservation of independence in functional abilities, it is noted that persons with MCI commonly have mild problems performing complex functional tasks which they used to perform shopping. They may take more time, be less efficient, and make more errors at performing such activities than in the past. Nevertheless, they generally maintain their independence of function in daily life, with minimal aids or assistance.
[0085] With respect to dementia, the cognitive changes should be sufficiently mild that there is no evidence of a significant impairment in social or occupational functioning. If an individual has only been evaluated once, change will be inferred from the history and/or evidence that cognitive performance is impaired beyond what would have been expected for that individual.
[0086] Cognitive testing is optimal for objectively assessing the degree of cognitive impairment for an individual. Scores on cognitive tests for individuals with MCI are typically 1 to 1.5 standard deviations below the mean for their age and education matched peers on culturally appropriate normative data (e.g., for the impaired domain(s), when available).
[0087] Episodic memory (i.e., the ability to learn and retain new information) is most commonly seen in MCI patients who subsequently progress to a diagnosis of AD dementia. There are a variety of episodic memory tests that are useful for identifying those MCI patients who have a high likelihood of progressing to AD dementia within a few years. These tests typically assess both immediate and delayed recall, so that it is possible to determine retention over a delay. Many, although not all, of the tests that have proven useful in this regard are wordlist learning tests with multiple trials. Such tests reveal the rate of learning over time, as well as the maximum amount acquired over the course of the learning trials. They are also useful for demonstrating that the individual is, in fact, paying attention to the task on immediate recall, which then can be used as a baseline to assess the relative amount of material retained on delayed recall. Examples of such tests include (but are not limited to: the Free and Cued Selective Reminding Test, the Rey Auditory Verbal Learning Test, and the California Verbal Learning Test. Other episodic memory measures include, but are not limited to: immediate and delayed recall of a paragraph such as the Logical Memory I and II of the Wechsler Memory Scale Revised (or other versions) and immediate and delayed recall of nonverbal materials, such as the Visual Reproduction subtests of the Wechsler Memory Scale-Revised I and II.
[0088] Because other cognitive domains can be impaired among individuals with
MCI, it is desirable to examine domains in addition to memory. These include, but are not limited to executive functions (e.g., set-shifting, reasoning, problem-solving, planning), language (e.g., naming, fluency, expressive speech, and comprehension), visuospatial skills, and attentional control (e.g., simple and divided attention). Many clinical
neuropsychological measures are available to assess these cognitive domains, including (but not limited to the Trail Making Test (executive function), the Boston Naming Test, letter and category fluency (language), figure copying (spatial skills), and digit span forward (attention). [0089] As indicated above, genetic factors can be incorporated into the diagnosis of
MCI. If an autosomal dominant form of AD is known to be present (e.g., mutation in APP, PS1, PS2), then the development of MCI is most likely the precursor to AD dementia. The large majority of these cases develop early onset AD (e.g., onset below 65 years of age).
[0090] In addition, there are genetic influences on the development of late onset AD dementia. For example, the presence of one or two ε4 alleles in the apolipoprotein E (APOE) gene is a genetic variant broadly accepted as increasing risk for late -onset AD dementia. Evidence suggests that an individual who meets the clinical, cognitive, and etiologic criteria for MCI, and is also APOE ε4 positive, is more likely to progress to AD dementia within a few years than an individual without this genetic characteristic. It is believed that additional genes play an important, but smaller role than APOE and also confer changes in risk for progression to AD dementia (see, e.g., Bertram et al., (2010) Neuron, 21 : 270-281).
[0091] In certain embodiments, subjects suitable for the prophylactic methods described herein (e.g., administration of TrkA kinase inhibitors) include, but need not be limited to subjects identified having one or more of the core clinical criteria described above and/or subjects identified with one or more "research criteria" for MCI, e.g., as described below. [0092] "Research criteria" for the identification/prognosis of MCI include, but are not limited to biomarkers that increase the likelihood that MCI syndrome is due to the pathophysiological processes of AD. Without being bound to a particular theory, it is contemplated that the conjoint application of clinical criteria and biomarkers can result in various levels of certainty that the MCI syndrome is due to AD pathophysiological processes. In certain embodiments, two categories of biomarkers have been the most studied and applied to clinical outcomes are contemplated. These include "Αβ" (which includes CSF Αβ42 and/or PET amyloid imaging) and "biomarkers of neuronal injury" (which include, but are not limited to CSF tau/p-tau, hippocampal, or medial temporal lobe atrophy on MRI, and temporoparietal/ precuneus hypometabolism or hypoperfusion on PET or SPECT).
[0093] Without being bound to a particular theory, it is contemplated that evidence of both Αβ, and neuronal injury (either an increase in tau/p-tau or imaging biomarkers in a topographical pattern characteristic of AD), together confers the highest probability that the AD pathophysiological process is present. Conversely, if these biomarkers are negative, an alternate diagnosis may be appropriate. In some instances, biomarker findings may be contradictory. Accordingly, particular biomarker combinations may be indicative of a differential diagnosis, but not itself dispositive. It is recognized that varying severities of an abnormality may confer different likelihoods or prognoses that are difficult to quantify accurately for broad application.
[0094] For those potential MCI subjects whose clinical and cognitive MCI syndrome is consistent with AD as the etiology, the addition of biomarker analysis effects levels of certainty in the diagnosis. In the most typical example in which the clinical and cognitive syndrome of MCI has been established, including evidence of an episodic memory disorder and a presumed degenerative etiology, the most likely cause is the neurodegenerative process of AD. However, the eventual outcome still has variable degrees of certainty. The likelihood of progression to AD dementia will vary with the severity of the cognitive decline and the nature of the evidence suggesting that AD pathophysiology is the underlying cause. Without being bound to a particular theory it is contemplated that positive biomarkers reflecting neuronal injury increase the likelihood that progression to dementia will occur within a few years and that positive findings reflecting both Αβ accumulation and neuronal injury together confer the highest likelihood that the diagnosis is MCI due to AD. [0095] A positive Αβ biomarker and a positive biomarker of neuronal injury provide an indication that the MCI syndrome is due to AD processes and the subject is well suited for the methods described herein.
[0096] A positive Αβ biomarker in a situation in which neuronal injury biomarkers have not been or cannot be tested or a positive biomarker of neuronal injury in a situation in which Αβ biomarkers have not been or cannot be tested indicate an intermediate likelihood that the MCI syndrome is due to AD. Such subjects are believed to be is well suited for the methods described herein
[0097] Negative biomarkers for both Αβ and neuronal injury suggest that the MCI syndrome is not due to AD. In such instances the subjects may not be well suited for the methods described herein.
[0098] There is evidence that magnetic resonance imaging can observe
deterioration, including progressive loss of gray matter in the brain, from mild cognitive impairment to full-blown Alzheimer disease (see, e.g., Whitwell et ah, (2008) Neurology 70(7): 512-520). A technique known as PiB PET imaging is used to clearly show the sites and shapes of beta amyloid deposits in living subjects using a CI 1 tracer that binds selectively to such deposits (see, e.g., Jack et ah, (2008) Brain 131 (Pt 3): 665-680).
[0099] In certain embodiments, mci is typically diagnosed when there is 1) evidence of memory impairment; 2) preservation of general cognitive and functional abilities; and 3) absence of diagnosed dementia.
[0100] In certain embodiments, MCI and stages of Alzheimer's disease can be identified/categorized, in part by Clinical Dementia Rating (CDR) scores. The CDR is a five point scale used to characterize six domains of cognitive and functional performance applicable to Alzheimer disease and related dementias: 1) memory, orientation, 2) judgment & problem solving, 3) community affairs, 4) home & hobbies, and 5) personal care. The information to make each rating is obtained through a semi-structured interview of the patient and a reliable informant or collateral source (e.g., family member).
[0101] The CDR table provides descriptive anchors that guide the clinician in making appropriate ratings based on interview data and clinical judgment. In addition to ratings for each domain, an overall CDR score may be calculated through the use of an algorithm. This score is useful for characterizing and tracking a patient's level of impairment/dementia: 0 = Normal; 0.5 = Very Mild Dementia; 1 = Mild Dementia; 2 = Moderate Dementia; and 3 = Severe Dementia. An illustrative CDR table is shown in Table 2.
Table 2. Illustrative clinical dementia rating (CDR) table.
Impairment: None Questionable Mild Moderate Severe
CDR: 0 0.5 1 2 3
Memory No memory Consistent Moderate Severe Severe loss or slight slight memory loss; memory memory inconsistent forgetfulness; more marked loss; only loss; only forgetfulness partial for recent highly fragments recollection events; defect learned remain of events' interferes material
"benign" with retained;
forgetfulness everyday new material
activities rapidly lost
Orientation Fully Fully Moderate Severe Oriented to oriented oriented difficulty difficulty person only except for with time with time
slight relationships; relationships;
difficulty oriented for usually
with time place at disoriented
relationships examination; to time, often
may have to place.
geographic
disorientation
elsewhere
Judgment & Solves Slight Moderate Severely Unable to
Problem everyday impairment difficulty in impaired in make
Solving problems & in solving handling handling judgments handles problems, problems, problems, or solve business & similarities, similarities similarities problems financial and and and
affairs well; differences differences; differences; judgment social social
good in judgment judgment relation to usually usually
past maintained impaired
performance
Community Independent Slight Unable to No pretense of independent
Affairs function at impairment function function outside of home usual level in these independently Appears well Appears too in job, activities at these enough to be ill to be shopping, activities taken to taken to volunteer, although may functions functions and social still be outside a outside a groups engaged in family home family
some; home. appears
normal to casual
inspection
Home and Life at Life at home, Mild bit Only simple No
Hobbies home, hobbies, and definite chores significant hobbies, and intellectual impairment preserved; function in intellectual interests of function at very home interests slightly home; more restricted
well impaired difficult interests,
maintained chores poorly
abandoned; maintained
more
complicated
hobbies and
interests
abandoned
Personal Fully capable of self-care Needs Requires Requires Care prompting assistance in much help dressing, with hygiene, personal keeping of care;
personal frequent effects incontinence
[0102] A CDR rating of -0.5 or -0.5 to 1.0 is often considered clinically relevant
MCI. Higher CDR ratings can be indicative of progression into Alzheimer's disease.
[0103] In certain embodiments, administration of one or more TrkA kinase inhibitor(s), alone or in conjunction with other active agent(s), is deemed effective when there is a reduction in the CSF of levels of one or more components selected from the group consisting of Tau, phospho-Tau (pTau), APPneo, soluble Αβ40, soluble Αβ42, and/or Αβ42/Αβ40 ratio, and/or when there is a reduction of the plaque load in the brain of the subject, and/or when there is a reduction in the rate of plaque formation in the brain of the subject, and/or when there is an improvement in the cognitive abilities of the subject, and/or when there is a perceived improvement in quality of life by the subject, and/or when there is a significant reduction in clinical dementia rating (CDR), and/or when the rate of increase in clinical dementia rating is slowed or stopped and/or when the progression from MCI to early stage AD is slowed or stopped.
[0104] In some embodiments, a diagnosis of MCI can be determined by considering the results of several clinical tests. For example, Grundman, et al., (2004) Arch Neurol 61 : 59-66, report that a diagnosis of MCI can be established with clinical efficiency using a simple memory test (paragraph recall) to establish an objective memory deficit, a measure of general cognition (Mini-Mental State Exam (MMSE), discussed in greater detail below) to exclude a broader cognitive decline beyond memory, and a structured clinical interview (CDR) with patients and caregivers to verify the patient's memory complaint and memory loss and to ensure that the patient was not demented. Patients with MCI perform, on average, less than 1 standard deviation (SD) below normal on nonmemory cognitive measures included in the battery. Tests of learning, attention, perceptual speed, category fluency, and executive function may be impaired in patients with MCI, but these are far less prominent than the memory deficit.
Alzheimer's Disease (AD).
[0105] In certain embodiments, the TrkA kinase inhibitor(s) are contemplated for the treatment of Alzheimer's disease. In such instances the methods described herein are useful in preventing or slowing the onset of Alzheimer's disease (AD), in reducing the severity of AD when the subject has transitioned to clinical AD diagnosis, and/or in mitigating one or more symptoms of Alzheimer's disease.
[0106] In particular, where the Alzheimer's disease is early stage, the methods can reduce or eliminate one or more symptoms characteristic of AD and/or delay or prevent the progression from MCI to early or later stage Alzheimer's disease.
[0107] Individuals presently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors described above. In addition, a number of diagnostic tests are available for identifying individuals who have AD. Individuals presently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors described above. In addition, a number of diagnostic tests are available for identifying individuals who have AD. These include measurement of CSF Tau, phospho-tau (pTau), sAPPa, sAPPp, Αβ40, Αβ42 levels and/or C terminally cleaved APP fragment (APPneo). Elevated Tau, pTau, sAPPp and/or APPneo, and/or decreased sAPPa, soluble Αβ40 and/or soluble Αβ42 levels, particularly in the context of a differential diagnosis, can signify the presence of AD.
[0108] In certain embodiments, subjects amenable to treatment may have
Alzheimer's disease. Individuals suffering from Alzheimer's disease can also be diagnosed by Alzheimer's disease and Related Disorders Association (ADRDA) criteria. The
NINCDS-ADRDA Alzheimer's criteria were proposed in 1984 by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (now known as the Alzheimer's Association) and are among the most used in the diagnosis of Alzheimer's disease (AD). McKhann, et ah, (1984) Neurology 34(7): 939-944. According to these criteria, the presence of cognitive impairment and a suspected dementia syndrome should be confirmed by
neuropsychological testing for a clinical diagnosis of possible or probable AD. However, histopathologic confirmation (microscopic examination of brain tissue) is generally used for a dispositive diagnosis. The NINCDS-ADRDA Alzheimer's Criteria specify eight cognitive domains that may be impaired in AD: 1) memory, 2) language, 3) perceptual skills, 4) attention, 5) constructive abilities, 6) orientation, 7) problem solving, and 8) functional abilities. These criteria have shown good reliability and validity.
[0109] Baseline evaluations of patient function can made using classic psychometric measures, such as the Mini-Mental State Exam (MMSE) (Folstein et al., (1975) J.
Psychiatric Research 12 (3): 189-198), and the Alzheimer's Disease Assessment Scale (ADAS), which is a comprehensive scale for evaluating patients with Alzheimer's Disease status and function {see, e.g., Rosen, et al, (1984) Am. J. Psychiatr., 141 : 1356-1364). These psychometric scales provide a measure of progression of the Alzheimer's condition. Suitable qualitative life scales can also be used to monitor treatment. The extent of disease progression can be determined using a Mini-Mental State Exam (MMSE) (see, e.g., Folstein, et al., supra). Any score greater than or equal to 25 points (out of 30) is effectively normal (intact). Below this, scores can indicate severe (<9 points), moderate (10-20 points) or mild (21-24 points) Alzheimer's disease. [0110] Alzheimer's disease can be broken down into various stages including:
1) Moderate cognitive decline (Mild or early-stage Alzheimer's disease), 2) Moderately severe cognitive decline (Moderate or mid-stage Alzheimer's disease), 3) Severe cognitive decline (Moderately severe or mid-stage Alzheimer's disease), and 4) Very severe cognitive decline (Severe or late-stage Alzheimer's disease) as shown in Table 3. Table 3. Illustrative stages of Alzheimer's disease.
Figure imgf000045_0001
Moderately severe cognitive decline (Moderate or mid-stage Alzheimer's disease)
Major gaps in memory and deficits in cognitive function emerge. Some assistance with day-to-day activities becomes essential. At this stage, individuals may:
be unable during a medical interview to recall such important details as their current address, their telephone number, or the name of the college or high school from which they graduated;
become confused about where they are or about the date, day of the week or season;
have trouble with less challenging mental arithmetic; for example, counting backward from 40 by 4s or from 20 by 2s;
need help choosing proper clothing for the season or the occasion; usually retain substantial knowledge about themselves and know their own name and the names of their spouse or children; and
usually require no assistance with eating or using the toilet.
Severe cognitive decline (Moderately severe or mid-stage Alzheimer's disease)
Memory difficulties continue to worsen, significant personality changes may emerge, and affected individuals need extensive help with daily activities. At this stage, individuals may:
lose most awareness of recent experiences and events as well as of their surroundings;
recollect their personal history imperfectly, although they generally recall their own name;
occasionally forget the name of their spouse or primary caregiver but generally can distinguish familiar from unfamiliar faces;
need help getting dressed properly; without supervision, may make such errors as putting pajamas over daytime clothes or shoes on wrong feet;
experience disruption of their normal sleep/waking cycle;
need help with handling details of toileting (flushing toilet, wiping and disposing of tissue properly);
have increasing episodes of urinary or fecal incontinence;
experience significant personality changes and behavioral symptoms, including suspiciousness and delusions (for example, believing that their caregiver is an impostor); hallucinations (seeing or hearing things that are not really there); or compulsive, repetitive behaviors such as hand-wringing or tissue shredding; and
tend to wander and become lost.
Very severe cognitive decline (Severe or late-stage Alzheimer's disease)
This is the final stage of the disease when individuals lose the ability to respond to their environment, the ability to speak, and, ultimately, the ability to control movement;
frequently individuals lose their capacity for recognizable speech, although words or phrases may occasionally be uttered;
individuals need help with eating and toileting and there is general incontinence;
individuals lose the ability to walk without assistance, then the ability to sit without support, the ability to smile, and the ability to hold their head up; and
reflexes become abnormal and muscles grow rigid, swallowing is impaired.
[0111] In various embodiments, administration of one or more agents described herein to subjects diagnosed with Alzheimer's disease is deemed effective when the there is a reduction in the CSF of levels of one or more components selected from the group consisting of Tau, phospho-Tau (pTau), APPneo, soluble Αβ40, soluble Αβ42, and/or and Αβ42/Αβ40 ratio, and/or when there is a reduction of the plaque load in the brain of the subject, and/or when there is a reduction in the rate of plaque formation in the brain of the subject, and/or when there is an improvement in the cognitive abilities of the subject, and/or when there is a perceived improvement in quality of life by the subject, and/or when there is a significant reduction in clinical dementia rating (CDR) of the subject, and/or when the rate of increase in clinical dementia rating is slowed or stopped and/or when the progression of AD is slowed or stopped {e.g. , when the transition from one stage to another as listed in Table 3 is slowed or stopped).
[0112] In certain embodiments, subjects amenable to the present methods generally are free of a neurological disease or disorder other than Alzheimer's disease. For example, in certain embodiments, the subject does not have and is not at risk of developing a neurological disease or disorder such as Huntington's Disease, and/or Parkinson's disease, and/or schizophrenia, and/or psychosis.
[0113] In various embodiments, the effectiveness of treatment can be determined by comparing a baseline measure of a parameter of disease before administration of the TrkA kinase inhibitor(s) is commenced to the same parameter one or more time points after the formulation has been administered. One illustrative parameter that can be measured is a biomarker {e.g., a peptide oligomer) of APP processing. Such biomarkers include, but are not limited to increased levels of sAPPa, p3 (Αβ 17-42 or Αβ 17-40), βΑΡΡβ, soluble Αβ40, and/or soluble Αβ42 in the blood, plasma, serum, urine, mucous or cerebrospinal fluid (CSF). Detection of increased levels of sAPPa and/or p3, and decreased levels of βΑΡΡβ and/or APPneo is an indicator that the treatment is effective. Conversely, detection of decreased levels of sAPPa and/or p3, and/or increased levels of βΑΡΡβ, APPneo, Tau or phospho-Tau (pTau) is an indicator that the treatment is not effective.
[0114] Another parameter to determine effectiveness of treatment is the level of amyloid plaque deposits in the brain. Amyloid plaques can be determined using any method known in the art, e.g., as determined by CT, PET, PIB-PET and/or MRI. [0115] In various embodiments, administration of the active agent(s) described herein can result in a reduction in the rate of plaque formation, and even a retraction or reduction of plaque deposits in the brain. Effectiveness of treatment can also be determined by observing a stabilization and/or improvement of cognitive abilities of the subject.
Cognitive abilities can be evaluated using any art-accepted method, including for example, Clinical Dementia Rating (CDR), the mini-mental state examination (MMSE) or Folstein test, evaluative criteria listed in the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) or DSM-V, and the like.
[0116] In certain embodiments, the monitoring methods can entail determining a baseline value of a measurable biomarker or parameter (e.g., amyloid plaque load or cognitive abilities) in a subject before administering a dosage of the TrkA inhibitor described herein alone or in combination with one or more pharmaceuticals, and comparing this biomarker or parameter with a value for the same measurable biomarker or parameter after treatment. [0117] In other embodiments, a control value (e.g., a mean and standard deviation) of the measurable biomarker or parameter is determined for a control population. In certain embodiments, the individuals in the control population have not received prior treatment and do not have AD, MCI, nor are at risk of developing AD or MCI. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered efficacious. In other embodiments, the individuals in the control population have not received prior treatment and have been diagnosed with AD or MCI. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered inefficacious.
[0118] In yet other embodiments, a subject who is not presently receiving treatment but has undergone a previous course of treatment is monitored for one or more of the biomarkers or clinical parameters to determine whether a resumption of treatment is required. The measured value of one or more of the biomarkers or clinical parameters in the subject can be compared with a value previously achieved in the subject after a previous course of treatment. In particular embodiments, the value measured in the subject can be compared with a control value (mean plus standard deviation/ ANOVA) determined in population of subjects after undergoing a course of treatment. In one embodiment, the measured value in the subject can be compared with a control value in populations of prophylactically treated subjects who remain free of symptoms of disease, or populations of therapeutically treated subjects who show amelioration of disease characteristics. In various embodiments, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered efficacious and need not be resumed. In preferred embodiments, a significant difference relative to the control level (e.g. , more than a standard deviation) is an indicator that treatment should be resumed in the subject.
[0119] In various embodiments, the tissue sample for analysis is typically blood, plasma, serum, urine, mucous or cerebrospinal fluid from the subject.
TrkA Inhibitors.
[0120] As indicated above, it was discovered that TrkA provides a good target to inhibit APP processing to APP-C31 and APPneo. Moreover, it is contemplated that inhibition of TrkA kinase activity and/or expression can mitigate the severity or delay the onset or reverse certain symptoms of Alzheimer's disease. Numerous TrkA inhibitors are known to those of skilled in the art. Such inhibitors include, but are not limited to peptide inhibitors, anti-TrkA antibodies, TrkA siRNA, TrkA ribozymes, and small organic molecule inhibitors of TrkA kinase activity.
Small Organic Molecules.
ADDN-1351 and derivatives.
[0121] A number of TrkA kinase inhibitors (ADDN-1351) (IC50-700 nM) and analogues that rapidly cross the blood/brain barrier were identified. We have also demonstrated that these molecules inhibit APPneo production.
[0122] Illustrative ADDN-1351 type inhibitors include, but are not limited to molecules according to Formula I:
Figure imgf000049_0001
where R1 is alkyl (e.g., a Ci_6 alkyl, or substituted alkyl group); alkoxy or substituted alkoxy; alkenyl or substituted alkenyl; or alkynyl or substituted alkynyl; and R2 and R3 are independently aryl, substituted aryl, or heteroaryl and substituted heteroaryl. In certain embodiments the compounds encompassed by Formula I exclude ADDN-1351. [0123] ADDN 1351 and illustrative analogues (compounds ADDN- 1351 a-ADDN-
135 lp) are shown in Table 4.
Table 4. ADDN 1351 and analogues and IC50 for TrkA kinase.
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
-50-
Figure imgf000053_0001
-51-
Figure imgf000054_0001
-52-
Figure imgf000055_0001
[0124] Methods of synthesizing ADDN1351 and various analogs thereof are provided in Example 2. Using these methods numerous synthetic schemes will be available to one of skill to produce these and other analogs.
Other small organic molecules.
[0125] Other TrkA kinase inhibitors are well known to those of skill in the art. A number of such inhibitors are illustrated herein in Figures 7-11.
[0126] Illustrative TrkA inhibitors include for example 4- aminopyrazolylpyrimidines. Thus, for example, Wang et al. (2008) J. Med. Chem., 51 : 4672-4684 describe the synthesis and screening of a number of TrkA inhibitors based on the lead compound of Formula II:
Figure imgf000056_0001
In certain embodiments the compounds comprise compounds according to
Figure imgf000056_0002
where X is selected from the group consisting of Me, H, and halogen; R1 is selected from the group consisting of cyclopropyl, O'Pr, SMe, Me, OPr, H, and ¾u; R2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and halogen; R3 is selected from the group consisting of H, (S)-Me, (R)-Me, (S)-CH2OH, (R)-CH2OH, (S)-Me, (i?)-CH2OH, (5)-CH2CONMe2, and (5)-CH2CONHMe; Y1 and Y2 are independent selected from the group consisting of CH, and N; and R7 is selected from the group consisting of H, OH, CH3,
Figure imgf000056_0003
In certain embodiments R2 is F. In certain embodiments Y1 and Y2 are both N or Y1 and Y2 are both CH. Methods of synthesizing such compounds are described in Wang et al. (2008) supra., which is incorporated herein by reference for the compounds and synthesis protocols described therein. [0128] Illustrative compounds include, for example the compounds listed in Tables
5, 6, 7, and 8 (see below).
[0129] In certain embodiments particular compounds comprise substitutions at the pyrimidine 2-position (see, e.g., of Table 5.
Table 5. Illustrative substitutions at the pyrimidine 2-position (Formula IV) (see, e.g. Wang et al. (2008) supra.).
Figure imgf000057_0001
Alternative substitutions at the pyrimidine 5-position were also described (see, e.g., Table 6). Table 6. Illustrative substitutions at the pyrimidine 5 -position (ΙΟο-q) (Formula V (see, e.g. Wang et al. (2008) supra.).
Figure imgf000058_0001
A wide variety of functional groups were tolerated in the TrkA solvent channel including terminal basic rings (such as 15a) and neutral linear (15c) and branched hydroxyl side chains (15b, 15d-g) (see Table 7).
Table 7. Functional group substitutions in the TrkA solvent channel (Formula VI) (see, e.g. Wang et al. (2008) supra.).
Figure imgf000059_0001
[0130] Table 8 shows introduction of nitrogens at the 2 and/or 6 position of the phenyl ring. These substitutions are tolerated in terms of cellular potency.
Table 8. Introduction of nitrogens at the 2 and/or 6 position of the phenyl ring (Formula VII) (see, e.g. Wang et al. (2008) supra.).
Figure imgf000060_0001
Figure imgf000060_0002
[0131] Other illustrative TrkA kinase inhibitors include, but are not limited to isothiazoles such as the compound of Formula VIII (see e.g., Lippa et al. (2006) Bioorg. Med. Chem. Lett. 16: 3444-3448):
Figure imgf000061_0001
and related isothiazole compounds as shown below in Table 9 and aminoheterocycle isothiazole analogs shown in Table 10.
Table 9. TrkA kinase activity inhibition for isothiazoles 1, 5a-h, and 6 (Formula IX) (Lippa et al. (2006), supra).
Figure imgf000061_0002
Table 10. TrkA kinase activity inhibition for aminoheterocycle isothiazole analogs lOa-g (Formula X) (shown here as 13a-g) and 14a-c (Lippa et al. (2006), supra).
Figure imgf000062_0001
[0132] Illustrative, but non-limiting isothiazole TrkA kinase inhibitors with sulfur linked C3 bicycles are shown in Table 11. Table 11. TrkA kinase and cell inhibition for isothiazoles with sulfur linked C3 bicycles (Formula XI) (corresponding to compounds 5i-o in Lippa et al. (2006), supra).
Figure imgf000063_0001
Figure imgf000063_0003
aCompounds were tested as racemic mixtures unless otherwise noted.
bValues are means of at least two experiments, assay error is <2x.
CR enantiomer.
dS enantiomer.
[0133] Other illustrative TrkA kinase inhibitors include, but are not limited to (2E)-
3 - [3 ,5 -Bis( 1 , 1 -dimethylethyl)-4-hydroxyphenyl] -2-cyano-2-propenethioamide, also known as AG 879 or tyrphostin AG 879), see, e.g., Formula XII:
Figure imgf000063_0002
l,3-Dihydro-3-[(l-methyl-lH-indol-3-yl)methylene]-2H-pyrrolo[3,2-¾]pyridin-2-one, also known as GW 441756, see, e.g., Formula XIII:
and (9S, 1 OS, 12i?)-2,3 ,9, 10,11,12-hexahydro- 10-hydroxy- 10-(hydroxymethyl)-9-methyl- 9,12-epoxy-lH-diindolo[l,2,3-^:3',2', -^/]pyrrolo[3,4-z'][l,6]benzodiazocin-l-one, also known as CEP 701 or Lestaurtinib, see e.g., Formula XIV:
Figure imgf000064_0002
and the like.
[0134] Another illustrative, but non-limiting example of a TrkA kinase inhibitor is
GNF-5837 (see, e.g., Albaugh et al. (2012) ACS Med. Chem. Lett., 3(2): 140-145) shown below as Formula XV.
Figure imgf000064_0003
[0135] Suitable TrkA kinase inhibitors also include, but are not limited to disubstituted imidazo[4,5-£]pyridines and purines. The synthesis and characterization of these compounds is described in Wang et al. (2012) ACS Med. Chem. Lett., OI:
10.1021/ml300074j (see, e.g., Table 1 therein, and Table 12, below) Table 12. Illustrative, but non-limiting disubstituted imidazo[4,5-£]pyridines and purines as trkA kinase inhibitors (Formulas XVI-XVIII). XVII
Figure imgf000065_0001
[0136] The foregoing small organic molecules are illustrative and not intended to be limiting. Other small molecule TrkA inhibitors will be known to those of skill in the art.
Peptide TrkA inhibitors.
[0137] In certain embodiments TrkA antagonist polypeptides are contemplated.
Such peptides include any polypeptide which blocks, inhibits, interferes, or reduces the kinase activity of TrkA. In certain embodiments such peptides include TrkA peptide fragments that bind a TrkA receptor but lack kinase activity. Such peptides would act as competitive inhibitors of TrkA kinase.
Antisense approaches.
[0138] In certain embodiments TrkA expression can be downregulated or entirely inhibited by the use of antisense molecules. An "antisense sequence or antisense nucleic acid" is a nucleic acid that is complementary to the coding TrkA mRNA nucleic acid sequence or a subsequence thereof. Binding of the antisense molecule to the TrkA mRNA interferes with normal translation of the TrkA transcription factor.
[0139] Thus, in accordance with certain embodiments of this invention, antisense molecules include oligonucleotides and oligonucleotide analogs that are hybridizable with TrkA messenger RNA. This relationship is commonly denominated as "antisense." The oligonucleotides and oligonucleotide analogs are able to inhibit the function of the RNA, either its translation into protein, its translocation into the cytoplasm, or any other activity necessary to its overall biological function. The failure of the messenger RNA to perform all or part of its function results in a reduction or complete inhibition of expression of TrkA polypeptides.
[0140] In the context of this invention, the term "oligonucleotide" refers to a polynucleotide formed from naturally-occurring bases and/or cyclofuranosyl groups joined by native phosphodiester bonds. This term effectively refers to naturally-occurring species or synthetic species formed from naturally-occurring subunits or their close homo logs. The term "oligonucleotide" may also refer to moieties which function similarly to
oligonucleotides, but which have non naturally-occurring portions. Thus, oligonucleotides may have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species that are known for use in the art. In accordance with some preferred embodiments, at least one of the phosphodiester bonds of the oligonucleotide has been substituted with a structure which functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA whose activity is to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in the practice of the invention.
[0141] In one embodiment, the internucleotide phosphodiester linkage is replaced with a peptide linkage. Such peptide nucleic acids tend to show improved stability, penetrate the cell more easily, and show enhances affinity for their target. Methods of making peptide nucleic acids are known to those of skill in the art (see, e.g., U.S. Patent Nos: 6,015,887, 6,015,710, 5,986,053, 5,977,296, 5,902,786, 5,864,010, 5,786,461, 5,773,571, 5,766,855, 5,736,336, 5,719,262, and 5,714,331). [0142] Oligonucleotides may also include species that include at least some modified base forms. Thus purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the furanosyl portions of the nucleotide subunits may also be effected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2'-0-alkyl- and 2'-halogen-substituted nucleotides. Some specific examples of modifications at the 2' position of sugar moieties which are useful in the present invention are OH, SH, SCH3, F, OCH3, OCN, 0(CH2)„NH2 or 0(CH2)nCH3, where n is from 1 to about 10, and other substituents having similar properties. [0143] Such oligonucleotides are best described as being functionally
interchangeable with natural oligonucleotides or synthesized oligonucleotides along natural lines, but which have one or more differences from natural structure. All such analogs are comprehended by this invention so long as they function effectively to hybridize with messenger RNA of TrkA to inhibit the function of that RNA. [0144] The oligonucleotides in accordance with certain embodiments of this invention comprise from about 3 to about 50 subunits. It is more preferred that such oligonucleotides and analogs comprise from about 8 to about 25 subunits and still more preferred to have from about 12 to about 20 subunits. As will be appreciated, a subunit is a base and sugar combination suitably bound to adjacent subunits through phosphodiester or other bonds. The oligonucleotides used in accordance with this invention can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such syntheses is sold by several vendors (e.g. Applied
Biosystems). Any other means for such synthesis may also be employed; however, the actual synthesis of the oligonucleotides is well within the talents of those of skill in the art. Methods of preparing other oligonucleotides such as phosphorothioates and alkylated derivatives are also well known to those of skill in the art.
Ribozymes.
[0145] In another approach, TrkA expression can be inhibited by the use of ribozymes. As used herein, "ribozymes" include RNA molecules that contain antisense sequences for specific recognition, and an RNA-cleaving enzymatic activity. The catalytic strand cleaves a specific site in a target (TrkA) RNA, preferably at greater than
stoichiometric concentration. Two "types" of ribozymes are particularly useful in this invention, the hammerhead ribozyme (Rossi et al. (1991) Pharmac. Ther. 50: 245-254) and the hairpin ribozyme (Hampel et al. (1990) Nucl. Acids Res. 18: 299-304, and U.S. Pat. No. 5,254,678).
[0146] Because both hammerhead and hairpin ribozymes are catalytic molecules having antisense and endoribonucleotidase activity, ribozyme technology has emerged as a potentially powerful extension of the antisense approach to gene inactivation. The ribozymes of the invention typically consist of RNA, but such ribozymes may also be composed of nucleic acid molecules comprising chimeric nucleic acid sequences (such as DNA/RNA sequences) and/or nucleic acid analogs {e.g., phosphorothioates).
[0147] Accordingly, within one aspect of the present invention ribozymes have the ability to inhibit TrkA expression. Such ribozymes may be in the form of a "hammerhead" (for example, as described by Forster and Symons (1987) Cell 48: 211-220; Haseloff and Gerlach (1988) Nature 328: 596-600; Walbot and Bruening (1988) Nature 334: 196;
Haseloff and Gerlach (1988) Nature 334: 585) or a "hairpin" (see, e.g. U.S. Patent
5,254,678 and Hampel et al, European Patent Publication No. 0 360 257, published Mar. 26, 1990), and have the ability to specifically target, cleave and TrkA nucleic acids.
[0148] Ribozymes, as well as DNA encoding such ribozymes and other suitable nucleic acid molecules can be chemically synthesized using methods well known in the art for the synthesis of nucleic acid molecules. Alternatively, Promega, Madison, Wis., USA, provides a series of protocols suitable for the production of RNA molecules such as ribozymes. The ribozymes also can be prepared from a DNA molecule or other nucleic acid molecule (which, upon transcription, yields an RNA molecule) operably linked to an RNA polymerase promoter, e.g., the promoter for T7 RNA polymerase or SP6 RNA polymerase. Such a construct may be referred to as a vector. Accordingly, also provided by this invention are nucleic acid molecules, e.g., DNA or cDNA, coding for the ribozymes of this invention. When the vector also contains an RNA polymerase promoter operably linked to the DNA molecule, the ribozyme can be produced in vitro upon incubation with the RNA polymerase and appropriate nucleotides. In a separate embodiment, the DNA may be inserted into an expression cassette (see, e.g., Cotten and Birnstiel (1989) EMBO J 8(12): 3861-3866; Hempel et al. (1989) Biochem. 28: 4929-4933, etc.). [0149] After synthesis, the ribozyme can be modified by ligation to a DNA molecule having the ability to stabilize the ribozyme and make it resistant to RNase.
Alternatively, the ribozyme can be modified to the phosphothio analog for use in liposome delivery systems. This modification also renders the ribozyme resistant to endonuclease activity. [0150] The ribozyme molecule also can be in a host prokaryotic or eukaryotic cell in culture or in the cells of an organism/patient. Appropriate prokaryotic and eukaryotic cells can be transfected with an appropriate transfer vector containing the DNA molecule encoding a ribozyme of this invention. Alternatively, the ribozyme molecule, including nucleic acid molecules encoding the ribozyme, may be introduced into the host cell using traditional methods such as transformation using calcium phosphate precipitation
(Dubensky et al. (1984) Proc. Natl. Acad. Sci., USA, 81 : 7529-7533), direct microinjection of such nucleic acid molecules into intact target cells (Acsadi et al. (1991) Nature 352: 815- 818), and electroporation whereby cells suspended in a conducting solution are subjected to an intense electric field in order to transiently polarize the membrane, allowing entry of the nucleic acid molecules. Other procedures include the use of nucleic acid molecules linked to an inactive adenovirus (Cotton et al. (1990) Proc. Natl. Acad. Sci., USA, 89 :6094), lipofection (Feigner et al. (1989) Proc. Natl. Acad. Sci. USA 84: 7413-7417),
microprojectile bombardment (Williams et al. (1991) Proc. Natl. Acad. Sci., USA, 88: 2726- 2730), polycation compounds such as polylysine, receptor specific ligands, liposomes entrapping the nucleic acid molecules, spheroplast fusion whereby E coli containing the nucleic acid molecules are stripped of their outer cell walls and fused to animal cells using polyethylene glycol, viral transduction, (Cline et al, (1985) Pharmac. Ther. 29: 69; and Friedmann et al. (1989) Science 244: 1275), and DNA ligand (Wu et al (1989) J. Biol. Chem. 264: 16985-16987), as well as psoralen inactivated viruses such as Sendai or
Adenovirus. In one preferred embodiment, the ribozyme is introduced into the host cell utilizing a lipid, a liposome or a retroviral vector.
[0151] When the DNA molecule is operative ly linked to a promoter for RNA transcription, the RNA can be produced in the host cell when the host cell is grown under suitable conditions favoring transcription of the DNA molecule. The vector can be, but is not limited to, a plasmid, a virus, a retrotransposon or a cosmid. Examples of such vectors are disclosed in U.S. Pat. No. 5,166,320. Other representative vectors include, but are not limited to adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Kolls et al. (1994) PNAS 91(l):215-219; Kass-Eisler et a/., (1993) Proc. Natl. Acad. Sci., USA, 90(24): 11498-502, Guzman et al. (1993) Circulation 88(6): 2838-48, 1993; Guzman et al. (1993) Cir. Res. 73(6): 1202-1207, 1993; Zabner et al. (1993) Cell 75(2): 207-216; Li et al. (1993) Hum Gene Ther. 4(4): 403-409; CaiUaud et al. (1993) Eur. J Neurosci. 5(10): 1287-1291), adeno- associated vector type 1 ("AAV-1") or adeno-associated vector type 2 ("AAV-2") (see WO 95/13365; Flotte et al. (1993) Proc. Natl. Acad. Sci., USA, 90(22): 10613-10617), retroviral vectors (e.g., EP 0 415 731; WO 90/07936; WO 91/02805; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218) and herpes viral vectors (e.g., U.S. Pat. No. 5,288,641). Methods of utilizing such vectors in gene therapy are well known in the art, see, for example, Larrick and Burck (1991) Gene Therapy:
Application of Molecular Biology, Elsevier Science Publishing Co., Inc., New York, New York, and Kreigler (1990) Gene Transfer and Expression: A Laboratory Manual, W.H. Freeman and Company, New York.
[0152] To produce ribozymes in vivo utilizing vectors, the nucleotide sequences coding for ribozymes are preferably placed under the control of a strong promoter such as the lac, SV40 late, SV40 early, or lambda promoters. Ribozymes are then produced directly from the transfer vector in vivo
RNAi inhibition of TrkA.
[0153] Post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) refers to a mechanism by which double-stranded (sense strand) RNA (dsRNA) specifically blocks expression of its homologous gene when injected, or otherwise introduced into cells. The discovery of this incidence came with the observation that injection of antisense or sense RNA strands into Caenorhabditis elegans cells resulted in gene-specific inactivation (Guo and Kempheus (1995) Cell 81 : 611-620). While gene inactivation by the antisense strand was expected, gene silencing by the sense strand came as a surprise. Adding to the surprise was the finding that this gene-specific inactivation actually came from trace amounts of contaminating dsRNA (Fire et al. (1998) Nature 391 : 806-811).
[0154] Since then, this mode of post-transcriptional gene silencing has been tied to a wide variety of organisms: plants, flies, trypanosomes, planaria, hydra, zebrafish, and mice (Zamore et al. (2000). Cell 101 : 25-33; Gura (2000) Nature 404: 804-808). RNAi activity has been associated with functions as disparate as transposon-silencing, anti-viral defense mechanisms, and gene regulation (Grant (1999) Cell 96: 303-306).
[0155] By injecting dsRNA into tissues, one can inactivate specific genes not only in those tissues, but also during various stages of development. This is in contrast to tissue- specific knockouts or tissue-specific dominant-negative gene expressions, which do not allow for gene silencing during various stages of the developmental process (see, e.g., Gura (2000) Nature 404: 804-808). The double-stranded RNA is cut by a nuclease activity into 21-23 nucleotide fragments. These fragments, in turn, target the homologous region of their corresponding mRNA, hybridize, and result in a double-stranded substrate for a nuclease that degrades it into fragments of the same size (Hammond et al. (2000) N ' ature, 404: 293- 298; Zamore et al. (2000). Cell 101 : 25-33).
[0156] It has been shown that when short (18-30 bp) RNA duplexes are introduced into mammalian cells in culture, sequence-specific inhibition of target mRNA can be realized without inducing an interferon response. Certain of these short dsRNAs, referred to as small inhibitory RNAs ("siRNAs"), can act catalytically at sub-molar concentrations to cleave greater than 95% of the target mRNA in the cell. A description of the mechanisms for siRNA activity, as well as some of its applications are described in Provost et al. (2002) EMBO J., 21(21): 5864 -5874; Tabara et al. (2002) Cell 109(7):861-71; Martinez et al. (2002) Cell 110(5): 563; Hutvagner and Zamore (2002), Science 297: 2056, and the like.
[0157] Using the known nucleotide sequence for the TrkA gene and/or mRNA,
TrkA siRNAs can readily be produced. In various embodiments siRNA that inhibit TrkA can comprise partially purified RNA, substantially pure RNA, synthetic RNA,
recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include, for example, addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.
[0158] In various embodiments one or both strands of the siRNA can comprise a 3' overhang. As used herein, a "3' overhang" refers to at least one unpaired nucleotide extending from the 3 '-end of an RNA strand. Thus in one embodiment, the siRNA comprises at least one 3' overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, from 1 to about 5 nucleotides in length, from 1 to about 4 nucleotides in length, or about 2 to about 4 nucleotides in length. [0159] In an illustrative embodiment in which both strands of the siRNA molecule comprise a 3' overhang, the length of the overhangs can be the same or different for each strand. In certain embodiments the 3' overhang is present on both strands of the siRNA, and is one, two, or three nucleotides in length. For example, each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu"). [0160] In order to enhance the stability of the siRNA, the 3' overhangs can be also stabilized against degradation. In one embodiment, the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. In certain embodiments substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotides in the 3' overhangs with 2'-deoxythymidine, is tolerated and does not affect the efficiency of R Ai degradation. In particular, it is believed the absence of a 2' hydroxyl in the 2'-deoxythymidine can significantly enhance the nuclease resistance of the 3' overhang [0161] In certain embodiments, the siRNA comprises the sequence AA(N19)TT
(SEQ ID NO: l), AA(N21)TT (SEQ ID NO:2), NA(N21) (SEQ ID NO:3), and the like, where N is any nucleotide. In various embodiments these siRNA comprise approximately 30%-70% GC, and preferably comprise approximately 50% G/C. The sequence of the sense siRNA strand corresponds to (N19)TT or N21 (i.e., positions 3 to 23), respectively. In the latter case, the 3' end of the sense siRNA is converted to TT. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense strand 3' overhangs. The antisense RNA strand is then synthesized as the complement to positions 1 to 21 of the sense strand.
[0162] Because position 1 of the 23 -nt sense strand in these embodiments is not recognized in a sequence-specific manner by the antisense strand, the 3 '-most nucleotide residue of the antisense strand can be chosen deliberately. However, the penultimate nucleotide of the antisense strand (complementary to position 2 of the 23 -nt sense strand in either embodiment) is generally complementary to the targeted sequence.
[0163] In another illustrative embodiment, the siRNA comprises the sequence NAR(N 17)YNN (SEQ ID NO :4), where R is a purine (e.g. , A or G) and Y is a pyrimidine (e.g., C or U/T). The respective 21-nt sense and antisense RNA strands of this embodiment therefore generally begin with a purine nucleotide. Such siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine. [0164] In various embodiments the siRNA of the invention can be targeted to any stretch of approximately 10-30, or 15-25, or 19-25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence"). Techniques for selecting target sequences for siRNA are given, for example, in Tuschl et al, "The siRNA User Guide," revised May 6, 2004. The "siRNA User Guide" is available on the world wide web at a website maintained by Dr. Thomas Tuschl, and can be found by accessing the website of
Rockefeller University and searching with the keyword "siRNA." In addition, the "siRNA User Guide" can be located by performing a google search for "siRNA User Guide" and can also be found at "www.rockefeller.edu/labheads/tuschl/sirna.html. Techniques for selecting target sequences for siRNA and miRNA can also be found in Sioud (2008) siRNA and miRNA Gene Silencing: From Bench to Bedside (Methods in Molecular Biology) , Humana Press.
[0165] In certain embodiments, the sense strand of the present siRNA comprises a nucleotide sequence identical to any contiguous stretch of about 19 to about 25 nucleotides in the target tyrosine kinase receptor A (TrkA) mRNA. Generally, a target sequence on the target mRNA can be selected from a given cDNA sequence corresponding to the target mRNA, preferably beginning 50 to 100 nucleotides downstream {i.e., in the 3' direction) from the start codon. The target sequence can, however, be located in the 5 ' or 3' untranslated regions, or in the region nearby the start. [0166] The TrkA silencing siRNAs can be obtained using a number of techniques known to those of skill in the art. For example, the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. published application US 2002/0086356.
[0167] In certain embodiments the siRNAs are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. The siRNAs can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Commercial suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, III, USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK). Custom siRNA can be obtained from commercial suppliers {see, e.g., Thermo Fisher Scientific, Lafayette CO; Qiagen, Valencia, CA; Applied Biosystems, Foster City, CA; and the like).
[0168] In certain embodiments siRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing siRNA from a plasmid include, for example, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant plasmids can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
[0169] The siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly at or near the target area(s) in vivo. The use of recombinant plasmids to deliver siRNA to cells in vivo is discussed in more detail below.
[0170] siRNA can be expressed from a recombinant plasmid either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions. Selection of plasmids suitable for expressing siRNAs, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art {see, e.g., Tuschl (2002) Nat. Biotechnol, 20: 446-448; Brummelkamp et al. (2002) Science 296: 550 553; Miyagishi et al. (2002) Nat. Biotechnol. 20: 497-500; Paddison et al. (2002) Genes Dev. 16: 948-958; Lee et al. (2002) Nat. Biotechnol. 20: 500-505; Paul et al. (2002) Nat. Biotechnol. 20: 505-508, and the like).
[0171] In one illustrative embodiment, a plasmid comprising nucleic acid sequences for expressing an siRNA for inhibiting TrkA comprises a sense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter, and an antisense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter. The plasmid is ultimately intended for use in producing an recombinant adeno- associated viral vector comprising the same nucleic acid sequences for expressing the siRNA [0172] As used herein, "in operable connection with a polyT termination sequence" means that the nucleic acid sequences encoding the sense or antisense strands are adjacent to the polyT termination signal in the 5' direction or sufficiently close so that during transcription of the sense or antisense sequences from the plasmid, the polyT termination signals act to terminate transcription after the desired product is transcribed. [0173] As used herein, "under the control" of a promoter means that the nucleic acid sequences encoding the sense or antisense strands are located 3' of the promoter, so that the promoter can initiate transcription of the sense or antisense coding sequences.
[0174] In various embodiments the siRNA can be expressed from recombinant viral vectors intracellularly at or near the target site(s) in vivo. The recombinant viral vectors comprise sequences encoding the siRNA of the invention and any suitable promoter for expressing the siRNA sequences. Suitable promoters include, but are not limited to, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors can also comprise inducible or regulatable promoters for expression of the siR A in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver siRNA of the invention to cells in vivo is discussed in more detail below.
[0175] The siRNA can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
[0176] Any viral vector capable of accepting the coding sequences for the siRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses {e.g. lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of the viral vectors can also be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses. For example, an AAV vector can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
[0177] Selection of recombinant viral vectors suitable for use in methods for inserting nucleic acid sequences for expressing the siRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art {see, e.g., Domburg (1995) Gene Therap. 2: 301-310; Eglitis (1988) Biotechniques 6: 608-614; Miller (1990) Hum. Gene Therap. 1 : 5-14; Anderson (1998) Nature 392: 25-30, and the like).
[0178] In certain embodiments suitable viral vectors include those derived from AV and AAV. In one illustrative embodiment, the siRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter. A suitable AV vector for expressing the siRNA, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia et al. (2002) Nat. Biotech. 20: 1006 1010.
[0179] Suitable AAV vectors for expressing the siRNA, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are also described in Samulski et al. (1987) J. Virol. 61 : 3096-3101; Fisher et al. (1996) J. Virol, 70: 520-532; Samulski et al. (1989) J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479 and 5,139,941; International Patent Application Nos. WO 1994/013788; and WO 1993/024641, and the like.
[0180] The ability of an siRNA containing a given target sequence to cause RNAi- mediated degradation of the target mRNA can be evaluated using standard techniques for measuring the levels of R A or protein in cells. For example, siRNA can be delivered to cultured cells, and the levels of target mRNA can be measured by Northern blot or dot blotting techniques, or by quantitative RT-PCR. Alternatively, the levels of TrkA in cells can be measured by ELISA or Western blot. [0181] RNAi-mediated degradation of target TrkA mRNA by an siRNA containing a given target sequence can also be evaluated with suitable animal models of aging.
[0182] In certain embodiments the siRNA can be delivered as a small hairpin RNA or short hairpin RNA (shRNA). shRNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. In typical embodiments, shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA that is bound to it.
[0183] The shRNA/siRNA described herein target and cause the RNAi-mediated degradation of TrkA, or alternative splice forms, mutants or cognates thereof. Degradation of the target mRNA by the present siRNA reduces the production of a functional gene product from the TrkA gene. Thus, methods are provided for inhibiting expression of TrkA in a subject, comprising administering an effective amount of an TrkA siRNA to the subject, such that the target mRNA is degraded.
[0184] It is understood that the siRNA of described herein can degrade the target mRNA in substoichiometric amounts. Without wishing to be bound by any theory, contemplated that the siRNA described herein cause degradation of the target mRNA in a catalytic manner.
[0185] One skilled in the art can readily determine an effective amount of the siRNA of the invention to be administered to a given subject, by taking into account factors such as the size and weight of the subject; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic. Pharmaceutical formulations.
[0186] In certain embodiments, one or more active agents (e.g., the various TrkA inhibitors derivatives, prodrugs, etc. described herein) are administered to a mammal in need thereof, e.g., to a mammal at risk for or suffering from a pathology characterized by abnormal processing of amyloid precursor proteins, a mammal at risk for progression of MCI to Alzheimer's disease, and so forth.
[0187] The active agent(s) can be administered in the "native" form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically, e.g., effective in the present method(s). Salts, esters, amides, prodrugs and other derivatives of the active agents can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience.
[0188] Methods of formulating such derivatives are known to those of skill in the art. For example, a pharmaceutically acceptable salt can be prepared for any compound described herein having a functionality capable of forming a salt, such as the carboxylic acid or tetrazole functionality of the compounds described herein. A pharmaceutically acceptable salt is any salt which retains the activity of the parent compound and does not impart any deleterious or untoward effect on the subject to which it is administered and in the context in which it is administered.
[0189] In various embodiments, pharmaceutically acceptable salts may be derived from organic or inorganic bases. The salt may be a mono or polyvalent ion. Of particular interest are the inorganic ions, lithium, sodium, potassium, calcium, and magnesium.
Organic salts may be made with amines, particularly ammonium salts such as mono-, di- and trialkyl amines or ethanol amines. Salts may also be formed with caffeine,
tromethamine and similar molecules.
[0190] Methods of formulating pharmaceutically active agents as salts, esters, amide, prodrugs, and the like are well known to those of skill in the art. For example, salts can be prepared from the free base using conventional methodology that typically involves reaction with a suitable acid. Generally, the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto. The resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent.
Suitable acids for preparing acid addition salts include, but are not limited to both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. An acid addition salt can be reconverted to the free base by treatment with a suitable base. Certain particularly preferred acid addition salts of the active agents herein include halide salts, such as may be prepared using hydrochloric or hydrobromic acids. Conversely, preparation of basic salts of the active agents described herein (e.g.,TrkA inhibitors) are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like. Particularly preferred basic salts include alkali metal salts, e.g., the sodium salt, and copper salts. [0191] For the preparation of salt forms of basic drugs, the pKa of the counterion is preferably at least about 2 pH units lower than the pKa of the drug. Similarly, for the preparation of salt forms of acidic drugs, the pKa of the counterion is preferably at least about 2 pH units higher than the pKa of the drug. This permits the counterion to bring the solution's pH to a level lower than the pHmax to reach the salt plateau, at which the solubility of salt prevails over the solubility of free acid or base. The generalized rule of difference in pKa units of the ionizable group in the active pharmaceutical ingredient (API) and in the acid or base is meant to make the proton transfer energetically favorable. When the pKa of the API and counterion are not significantly different, a solid complex may form but may rapidly disproportionate {e.g., break down into the individual entities of drug and counterion) in an aqueous environment.
[0192] In various embodiments, the counterion is a pharmaceutically acceptable counterion. Suitable anionic salt forms include, but are not limited to acetate, benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate (embonate), phosphate and diphosphate, salicylate and disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like, while suitable cationic salt forms include, but are not limited to aluminum, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, zinc, and the like.
[0193] Preparation of esters typically involves functionalization of hydroxyl and/or carboxyl groups that are present within the molecular structure of the active agent. In certain embodiments, the esters are typically acyl-substituted derivatives of free alcohol groups, e.g., moieties that are derived from carboxylic acids of the formula RCOOH where R is alky, and preferably is lower alkyl. Esters can be reconverted to the free acids, if desired, by using conventional hydrogeno lysis or hydrolysis procedures.
[0194] Amides can also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine.
[0195] In various embodiments, the active agents identified herein are useful for parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment of one or more of the pathologies/indications described herein (e.g., amyloidogenic pathologies).
[0196] The active agents described herein (e.g., TrkA inhibitors) can also be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protection and uptake enhancers such as lipids, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers. [0197] Other physiologically acceptable compounds, particularly of use in the preparation of tablets, capsules, gel caps, and the like include, but are not limited to binders, diluent/fillers, disentegrants, lubricants, suspending agents, and the like.
[0198] In certain embodiments, to manufacture an oral dosage form (e.g., a tablet), an excipient (e.g., lactose, sucrose, starch, mannitol, etc.), an optional disintegrator (e.g., calcium carbonate, carboxymethylcellulose calcium, sodium starch glycollate, crospovidone etc.), a binder (e.g., alpha-starch, gum arabic, microcrystalline cellulose,
carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, cyclodextrin, etc.), and an optional lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.), for instance, are added to the active component or components (e.g., TrkA inhibitors described herein) and the resulting composition is compressed. Where necessary the compressed product is coated, e.g. , known methods for masking the taste or for enteric dissolution or sustained release. Suitable coating materials include, but are not limited to ethyl-cellulose, hydroxymethylcellulose, polyoxyethylene glycol, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, and Eudragit (Rohm & Haas, Germany;
methacrylic-acrylic copolymer).
[0199] Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of pharmaceutically acceptable carrier(s), including a physiologically acceptable compound depends, for example, on the route of administration of the active agent(s) and on the particular physio-chemical characteristics of the active agent(s). [0200] In certain embodiments, the excipients are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. For various oral dosage form excipients such as tablets and capsules sterility is not required. The USP/NF standard is usually sufficient.
[0201] The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectable, implantable sustained-release formulations, mucoadherent films, topical varnishes, lipid complexes, etc.
[0202] Pharmaceutical compositions comprising the TrkA inhibitors can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate processing of the active agents into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
[0203] For topical administration the active agents described herein can be formulated as solutions, gels, ointments, creams, suspensions, and the like as are well- known in the art. Systemic formulations include, but are not limited to, those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration. For injection, the active agents described herein can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer and/or in certain emulsion formulations. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In certain embodiments, the active agent(s) can be provided in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. For transmucosal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art.
[0204] For oral administration, the compounds can be readily formulated by combining the active agent(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds described herein {e.g., TrkA inhibitors) to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired,
disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
[0205] For oral liquid preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like can be added. For buccal administration, the compositions may take the form of tablets, lozenges, etc.
formulated in conventional manner.
[0206] For administration by inhalation, the active agent(s) are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[0207] In various embodiments, the active agent(s) can be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other glycerides. [0208] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations can be
administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[0209] Alternatively, other pharmaceutical delivery systems can be employed.
Liposomes and emulsions are well known examples of delivery vehicles that may be used to protect and deliver pharmaceutically active compounds. Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. Various uses of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
[0210] In certain embodiments, the active agents described herein are administered orally. This is readily accomplished by the use of tablets, caplets, lozenges, liquids, and the like.
[0211] In certain embodiments, the active agents described herein are administered systemically (e.g., orally, or as an injectable) in accordance with standard methods well known to those of skill in the art. In other embodiments, the agents can also be delivered through the skin using conventional transdermal drug delivery systems, e.g., transdermal "patches" wherein the active agent(s) are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is typically contained in a layer, or "reservoir," underlying an upper backing layer. It will be appreciated that the term "reservoir" in this context refers to a quantity of "active ingredient(s)" that is ultimately available for delivery to the surface of the skin.
Thus, for example, the "reservoir" may include the active ingredient(s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art. The patch may contain a single reservoir, or it may contain multiple reservoirs. [0212] In one illustrative embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. The backing layer in these laminates, which serves as the upper surface of the device, preferably functions as a primary structural element of the "patch" and provides the device with much of its flexibility. The material selected for the backing layer is preferably substantially impermeable to the active agent(s) and any other materials that are present.
[0213] In certain embodiments, one or more active agents described herein can be provided as a "concentrate", e.g., in a storage container {e.g., in a premeasured volume) ready for dilution, or in a soluble capsule ready for addition to a volume of water, alcohol, hydrogen peroxide, or other diluent.
[0214] In certain embodiments, the active agents described herein {e.g., one or more esters described above) are preferably suitable for oral administration. In various embodiments, the active agent(s) in the oral compositions can be either coated or non- coated. The preparation of enteric-coated particles is disclosed for example in U.S. Pat. Nos. 4,786,505 and 4,853,230.
[0215] In various embodiments, compositions contemplated herein typically comprise one or more of the various TrkA inhibitors described herein in an effective amount to achieve a pharmacological effect or therapeutic improvement without undue adverse side effects. Various effects deemed therapeutic are described above. Illustrative pharmacological effects or therapeutic improvements include, but are not limited to a reduction in the CSF of levels of one or more components selected from the group consisting of Tau, phospho-Tau (pTau), APPneo, soluble Αβ40 and soluble Αβ 42, and/or when a reduction of the plaque load in the brain of the subject, and/or a reduction in the rate of plaque formation in the brain of the subject, and/or an improvement in the cognitive abilities of the subject, and/or a perceived improvement in quality of life by the subject, and/or a significant reduction in clinical dementia rating (CDR) of the subject, and/or a slowing in the rate of increase in clinical dementia rating, and/or when a slowing or stopping in the progression of AD (e.g., when the transition from one stage to another as listed in Table 3 is slowed or stopped).
[0216] In various embodiments, the typical daily dose of compound(s) varies and will depend on various factors such as the individual requirements of the patients and the disease to be treated. In various embodiments, the daily dose of compounds can be in the range of 0.1 mg to about 5,000 mg or to about 2,500 mg, or to about 2,000 mg, or to about 1 ,500 mg, or about 1 mg, or about 5 mg, or about 10 mg, to about 1 ,000 mg, or about 1 mg, or about 5 mg, or about 10 mg, to about 800 mg, or about 1 mg, or about 5 mg, or about 10 mg to about 600 mg , or about 1 mg, or about 5 mg, or about 10 mg, to about 500 mg, or about 1-500 mg, or 1-400 mg, or 1-300 mg, or 1-200 mg, or 1-100 mg. In one illustrative standard approximate amount of the various TrkA inhibitor(s) described above present in the composition can be typically about 0.1 or about 1 to about lOOmg, or to about 400, or to about 1 ,000, or to about 2,000 mg, more preferably about 5 to 500 mg, and most preferably about 10 to 100 mg administered once a day, in certain embodiments, administered twice a day, in certain embodiments, administered 3 times/day, and in certain embodiments, administered 4, or 6, or 6 or 7, or 8 times/day. In certain embodiments the dosage ranges from about 0.01 or about 0.1 , or about 1 mg/kg to about 100 mg/kg, or to about 50 mg/kg, or to about 40 mg/kg, or to about 30 mg/kg, or to about 20 mg/kg, or to about 10 mg/kg, or to about 30 mg/kg, administered once a day, in certain embodiments, administered twice a day, in certain embodiments, administered 3 times/day, and in certain embodiments, administered 4, or 6, or 6 or 7, or 8 times/day.
[0217] The active ingredients of the are preferably formulated in a single oral dosage form containing all active ingredients. Such oral formulations include solid and liquid forms. It is noted that solid formulations typically provide improved stability as compared to liquid formulations and can often afford better patient compliance.
[0218] In one illustrative embodiment, the one or more of the various TrkA inhibitors described above are formulated in a single solid dosage form such as single- or multi-layered tablets, suspension tablets, effervescent tablets, powder, pellets, granules or capsules comprising multiple beads as well as a capsule within a capsule or a double chambered capsule. In another embodiment, the active agents may be formulated in a single liquid dosage form such as suspension containing all active ingredients or dry suspension to be reconstituted prior to use.
[0219] In certain embodiments, the compound(s) are formulated as enteric-coated delayed-release granules or as granules coated with non-enteric time-dependent release polymers in order to avoid contact with the gastric juice. Non-limiting examples of suitable pH-dependent enteric-coated polymers are: cellulose acetate phthalate,
hydroxypropylmethylcellulose phthalate, polyvinylacetate phthalate, methacrylic acid copolymer, shellac, hydroxypropylmethylcellulose succinate, cellulose acetate trimellitate, and mixtures of any of the foregoing. A suitable commercially available enteric material, for example, is sold under the trademark EUDRAGIT L 100-55®. This coating can be spray coated onto a substrate.
[0220] Illustrative non-enteric-coated time-dependent release polymers include, for example, one or more polymers that swell in the stomach via the absorption of water from the gastric fluid, thereby increasing the size of the particles to create thick coating layer. The time-dependent release coating generally possesses erosion and/or diffusion properties that are independent of the pH of the external aqueous medium. Thus, the active ingredient is slowly released from the particles by diffusion or following slow erosion of the particles in the stomach. [0221] Illustrative non-enteric time-dependent release coatings are for example: film-forming compounds such as cellulosic derivatives, such as methylcellulose, hydroxypropyl methylcellulose (HPMC), hydroxyethylcellulose, and/or acrylic polymers including the non-enteric forms of the EUDRAGIT® brand polymers. Other film-forming materials can be used alone or in combination with each other or with the ones listed above. These other film forming materials generally include, for example, poly(vinylpyrrolidone), Zein, poly(ethylene glycol), poly(ethylene oxide), poly( vinyl alcohol), poly( vinyl acetate), and ethyl cellulose, as well as other pharmaceutically acceptable hydrophilic and hydrophobic film-forming materials. These film-forming materials may be applied to the substrate cores using water as the vehicle or, alternatively, a solvent system. Hydro- alcoholic systems may also be employed to serve as a vehicle for film formation.
[0222] Other materials suitable for making the time-dependent release coating of the compounds described herein include, by way of example and without limitation, water soluble polysaccharide gums such as carrageenan, fucoidan, gum ghatti, tragacanth, arabinogalactan, pectin, and xanthan; water-soluble salts of polysaccharide gums such as sodium alginate, sodium tragacanthin, and sodium gum ghattate; water-soluble
hydroxyalkylcellulose wherein the alkyl member is straight or branched of 1 to 7 carbons such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose;
synthetic water-soluble cellulose-based lamina formers such as methyl cellulose and its hydroxyalkyl methylcellulose cellulose derivatives such as a member selected from the group consisting of hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, and hydroxybutyl methylcellulose; other cellulose polymers such as sodium
carboxymethylcellulose; and other materials known to those of ordinary skill in the art. Other lamina forming materials that can be used for this purpose include, but are not limited to poly(vinylpyrrolidone), polyvinylalcohol, polyethylene oxide, a blend of gelatin and polyvinyl-pyrrolidone, gelatin, glucose, saccharides, povidone, copovidone,
poly(vinylpyrrolidone)-poly( vinyl acetate) copolymer.
[0223] While the compositions and methods are described herein with respect to use in humans, they are also suitable for animal, e.g., veterinary use. Thus certain illustrative organisms include, but are not limited to humans, non-human primates, canines, equines, felines, porcines, ungulates, largomorphs, and the like.
[0224] The foregoing formulations and administration methods are intended to be illustrative and not limiting. It will be appreciated that, using the teaching provided herein, other suitable formulations and modes of administration can be readily devised. Combined treatment methods and combined formulations
[0225] In certain instances, one or more of the TrkA inhibitors described herein are administered in conjunction with one or more additional active agent that are known, or believed, to have utility in the treatment of neurodegenerative diseases including, but not limited to Alzheimer's disease, age-related cognitive impairment, MCI, and the like. The two agents {e.g., TrkA inhibitors and additional agent) can be administered simultaneously or sequentially. When administered sequentially the two agents are typically administered so that both achieve a physiologically relevant concentration and/or effect over a similar time period {e.g., so that both agents are active at some common time).
[0226] In certain instances, one or more of the TrkA inhibitors described herein are administered before the one or more additional active agents or they are administered after the one or more additional active agents. In certain embodiments one or more of the TrkA inhibitors described herein are administered simultaneously with one or more additional active agents and in such instances may be formulated as a compound formulation.
[0227] Suitable additional active agents include, but are not limited to, Donepezil {e.g. , Aricept), Rivastigmine {e.g. , EXELON®), Galantamine {e.g. , RAZADINE®), Tacrine {e.g., COGNEX®), Memantine {e.g., NAMENDA®), Solanezumab, Bapineuzmab, Alzemed, Flurizan, ELND005, Valproate, Semagacestat, Rosiglitazone, Phenserine, Cernezumab, Dimebon, EGCg, Gammagard, PBT2, PF04360365, NIC5-15, Bryostatin-1, AL-108, Nicotinamide, EHT-0202, BMS708163, NP12, Lithium, ACCOOl, AN1792, ABT089, NGF, CAD106, AZD3480, SB742457, AD02, Huperzine-A, EVP6124,
PRX03140, PUFA, HF02, MEM3454, TTP448, PF-04447943, Ent., GSK933776,
MABT5102A, Talsaclidine, UB311, Begacestat, R1450, PF3084014, V950, E2609, MK0752, CTS21166, AZD-3839, LY2886721, CHF5074, anti-inflammatories (e.g., Flurizan (Myriad Genetics), Dapsone, anti-TNF antibodies (e.g., etanercept
(Amgen/Pfizer)), and the like), statins (e.g., atorvastatin (LIPITOR®), simvastatin
(ZOCOR®, etc.), and the like. In certain embodiments, treatment methods comprising administration of one or more TrkA inhibitors in conjunction with any one of the foregoing additional active agents is contemplated. In certain embodiments, treatment methods comprising administration of one or more TrkA inhibitors in conjunction with any one or more of the foregoing additional active agents is contemplated.
[0228] In certain embodiments, combination formulations comprising one or more
TrkA inihibitors described herein, derivatives thereof, analogs thereof, polymorphs thereof, in combination with any one of the foregoing additional active agents is contemplated. In certain embodiments, combination formulations comprising one or more TrkA inhibitors with any one or more of the foregoing additional active agents is contemplated. In certain embodiments the additional agent comprises NGF or an NGF mimetic.
[0229] In certain embodiments, treatment methods comprising administration of one or more TrkA inhibitors derivatives thereof, analogs thereof, polymorphs thereof, and the like described herein in conjunction with additional therapeutic agents such as tropinol esters (e.g., as described in PCT Publication PCT/US2012/049223, which is incorporated herein by reference for the compounds described therein disulfiram and/or analogues thereof, honokiol and/or analogues thereof, tropisetron and/or analogues thereof, nimetazepam and/or analogues thereof (e.g., as described in USSN 13/213,960 (U.S. Patent Publication No: US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No: WO 2012/024616) which are incorporated herein by reference for the compounds described therein) are contemplated. In certain embodiments the treatment method comprises administration of tropisetron in conjunction with of one or more TrkA inhibitors. [0230] In certain embodiments, combination formulations comprising
administration of one or more TrkA inhibitors described herein derivatives thereof, analogs thereof, polymorphs thereof, and the like in combination with additional therapeutic agents such as tropinol esters (e.g., as described in PCT Publication PCT/US2012/049223), disulfiram and/or analogues thereof, honokiol and/or analogues thereof, tropisetron and/or analogues thereof, nimetazepam and/or analogues thereof (e.g., as described in USSN 13/213,960 (U.S. Patent Publication No: US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No: WO 2012/024616) which are incorporated herein by reference for the compounds described therein) are contemplated. In certain embodiments the combination formulation comprises tropisetron in combination with of one or more TrkA inhibitors.
Assay Systems to Evaluate APP processing
[0231] Without being bound to a particular theory, it is contemplated that, in certain embodiments, TrkA inhibitors (e.g., the inhibitors described herein) promote processing of APP by the nonamyloidogenic pathway and/or reduce or inhibit processing of APP by the amyloidogenic pathway. In the nonamyloidogeic pathway, APP is first cleaved by a- secretase within the Αβ sequence, releasing the APPsa ectodomain ("sAPPa"). In contrast, the amyloidogenic pathway is initiated when β-secretase cleaves APP at the amino terminus of the Αβ, thereby releasing the APPsP ectodomain ("sAPPP"). APP processing by the nonamyloidogenic and amyloidogenic pathways is known in the art and reviewed, e.g., by Xu (2009) J. Alzheimer's Dis., 16(2):211 -224 and De Strooper et al, (2010) Nat Rev Neurol, 6(2): 99-107.
[0232] One method to evaluate the efficacy of TrkA inhibitors described herein is to determine whether or not the compound(s) in question produce a reduction or elimination in the level of APP processing by the amyloidogenic pathway, e.g. , a reduction or elimination in the level of APP processing by β-secretase cleavage. Assays for determining the extent of APP cleavage at the β-secretase cleavage site are well known in the art. Illustrative assays, are described, for example, in U.S. Pat. Nos. 5,744,346 and 5,942,400. Kits for determining the presence and levels in a biological sample of sAPPa and sAPPp, as well as APPneo and Αβ commercially available, e.g., from PerkinElmer. Cell Free Assays
[0233] Illustrative assays that can be used to evaluate the inhibitory activity of TrkA inhibitors are described, for example, in PCT Publication Nos: WO 2000/017369, and WO 2000/003819, and in U.S. Patent Nos: 5,942,400 and 5,744,346. In certain embodiments, such assays can be performed in cell-free incubations or in cellular incubations using cells expressing an alpha-secretase and/or beta-secretase and an APP substrate having an alpha- secretase and beta-secretase cleavage sites. [0234] One illustrative assay, test the compound(s) of interest utilizing an APP substrate containing alpha-secretase and beta-secretase cleavage sites of APP, for example, a complete APP or variant, an APP fragment, or a recombinant or synthetic APP substrate containing the amino acid sequence: KM-DA or NL-DA, which is incubated in the presence of an a-secretase and/or β-secretase enzyme, a fragment thereof, or a synthetic or recombinant polypeptide variant having alpha-secretase or beta-secretase activity and effective to cleave the alpha-secretase or beta-secretase cleavage sites of APP, under incubation conditions suitable for the cleavage activity of the enzyme. Suitable substrates optionally include derivatives that may be fusion proteins or peptides that contain the substrate peptide and a modification useful to facilitate the purification or detection of the peptide or its a-secretase and/or β-secretase cleavage products. Useful modifications include the insertion of a known antigenic epitope for antibody binding; the linking of a label or detectable moiety, the linking of a binding substrate, and the like.
[0235] Suitable incubation conditions for a cell-free in vitro assay include, for example, approximately 200 nanomolar to 10 micromolar substrate, approximately 10 to 200 picomolar enzyme, and approximately 0.1 nanomolar to 10 micromolar TrkA inhibitor, in aqueous solution, at an approximate pH of 4-7, at approximately 37°C, for a time period of approximately 10 minutes to 3 hours. These incubation conditions are illustrative only, and can be varied as required for the particular assay components and/or desired
measurement system. Optimization of the incubation conditions for the particular assay components can account for the specific alpha-secretase and/or beta-secretase enzyme used and its pH optimum, any additional enzymes and/or markers that might be used in the assay, and the like. Such optimization is routine and does not require undue experimentation.
[0236] One useful assay utilizes a fusion peptide having maltose binding protein (MBP) fused to the C-terminal 125 amino acids of APP-SW. The MBP portion is captured on an assay substrate by anti-MBP capture antibody. Incubation of the captured fusion protein in the presence of alpha-secretase and/or beta-secretase results in cleavage of the substrate at the alpha-secretase and/or beta-secretase cleavage sites, respectively. Analysis of the cleavage activity can be, for example, by immunoassay of cleavage products. One such immunoassay detects a unique epitope exposed at the carboxy terminus of the cleaved fusion protein, for example, using the antibody SW192. This assay is described, for example, in U.S. Pat. No. 5,942,400. Cellular Assays
[0237] Numerous cell-based assays can be used to evaluate the effect of the compounds described herein on the ratio of relative alpha-secretase activity to beta- secretase activity and/or on the processing of APP to release amyloidogenic versus non- amyloidogenic Αβ oligomers. Contact of an APP substrate with an alpha-secretase and/or beta-secretase enzyme within the cell and in the presence or absence of compound(s) in question can be used to demonstrate a-secretase and/or β-secretase inhibitory activity of the compound(s). Preferably, the assay in the presence of compound(s) provides at least about 30%, most preferably at least about 50% inhibition of the enzymatic activity, as compared with a non-inhibited control.
[0238] In one illustrative embodiment, cells that naturally express alpha-secretase and/or beta-secretase are used. Alternatively, cells can be modified to express a
recombinant α-secretase and/or β-secretase or synthetic variant enzymes, as discussed above. In certain embodiments, the APP substrate can be added to the culture medium and in certain embodiments, the substrate is preferably expressed in the cells. Cells that naturally express APP, variant or mutant forms of APP, or cells transformed to express an isoform of APP, mutant or variant APP, recombinant or synthetic APP, APP fragment, or synthetic APP peptide or fusion protein containing the α-secretase and/or β-secretase APP cleavage sites can be used, provided that the expressed APP is permitted to contact the enzyme and enzymatic cleavage activity can be analyzed.
[0239] Human cell lines that normally process Αβ from APP provide a useful means to assay inhibitory activities of the compound(s) described herein. Production and release of Αβ and/or other cleavage products into the culture medium can be measured, for example by immunoassay, such as Western blot or enzyme-linked immunoassay (EIA) such as by ELISA.
[0240] In certain embodiments, cells expressing an APP substrate and an active a- secretase and/or β-secretase can be incubated in the presence of the compound(s) being tested to demonstrate the effect of the compound(s) on relative enzymatic activity of the a- secretase and/or β-secretase as compared with a control. Relative activity of the alpha- secretase to the beta-secretase can be measured by analysis of one or more cleavage products of the APP substrate. For example, inhibition of β-secretase activity against the substrate APP would be expected to decrease release of specific β-secretase induced APP cleavage products such as Αβ, βΑΡΡβ and APPneo. Promotion or enhancement of a- secretase activity against the substrate APP would be expected to increase release of specific a-secretase induced APP cleavage products such as sAPPa and p3 peptide.
[0241] Although both neural and non-neural cells process and release Αβ, levels of endogenous beta-secretase activity are low and often difficult to detect by EIA. The use of cell types known to have enhanced beta-secretase activity, enhanced processing of APP to Αβ, and/or enhanced production of Αβ are therefore preferred. For example, transfection of cells with the Swedish Mutant form of APP (APP-SW); with APP-KK (APP containing an ER retention signal (-K QN-, (SEQ ID NO:5)) appended to the C terminus of APP), or with APP-SW-K provides cells having enhanced beta-secretase activity and producing amounts of Αβ that can be readily measured.
[0242] In such assays, for example, the cells expressing APP, alpha-secretase and/or beta-secretase are incubated in a culture medium under conditions suitable for a-secretase and/or β-secretase enzymatic activity at its cleavage site on the APP substrate. On exposure of the cells to TrkA inhibitor, the amount of Αβ released into the medium and/or the amount of CTF99 fragments of APP in the cell lysates is reduced as compared with the control. The cleavage products of APP can be analyzed, for example, by immune reactions with specific antibodies, as discussed above.
[0243] In certain embodiments, preferred cells for analysis of α-secretase and/or β- secretase activity include primary human neuronal cells, primary transgenic animal neuronal cells where the transgene is APP, and other cells such as those of a stable 293 cell line expressing APP, for example, APP-SW.
In vivo Assays: Animal Models
[0244] Various animal models can be used to analyze the effect of a TrkA inhibitor alone or in combination with other therapeutic agents described herein on the relative alpha- secretase and/or beta-secretase activity and/or processing of APP to release Αβ. For example, transgenic animals expressing APP substrate, alpha-secretase and/or beta- secretase enzyme can be used to demonstrate inhibitory activity of the TrkA inhibitor.
Certain transgenic animal models have been described, for example, in U.S. Pat. Nos. 5,877,399; 5,612,486; 5,387,742; 5,720,936; 5,850,003; 5,877,015, and 5,811,633, and in Games et al., (1995) Nature 373: 523-527. Preferred are animals that exhibit characteristics associated with the pathophysiology of AD. Administration of the TrkA inhibitor to the transgenic mice described herein provides an alternative method for demonstrating the inhibitory activity of the compound(s) in question. In certain embodiments, administration of the TrkA inhibitor(s) in a pharmaceutically effective carrier and via an administrative route that reaches the target tissue in an appropriate therapeutic amount is preferred.
[0245] Inhibition of beta-secretase mediated cleavage of APP at the beta-secretase cleavage site and of Αβ release can be analyzed in these animals by measure of cleavage fragments in the animal's body fiuids such as cerebral fluid or tissues. Likewise, promotion or enhancement of alpha-secretase mediated cleavage of APP at the alpha-secretase cleavage site and of release of sAPPa can be analyzed in these animals by measure of cleavage fragments in the animal's body fluids such as cerebral fluid or tissues. In certain embodiments, analysis of brain tissues for Αβ deposits or plaques is preferred. [0246] In certain illustrative assays, an APP substrate is contacted with an alpha- secretase and/or beta-secretase enzyme in the presence of the TrkA inhibitor(s) under conditions sufficient to permit enzymatic mediated cleavage of APP and/or release of Αβ from the substrate. The TrkA inhibitor is deemed effective when it reduces beta-secretase- mediated cleavage of APP at the β-secretase cleavage site and/or reduces released amounts of Αβ. The TrkA inhibitor(s) are also deemed effective if they enhance a-secretase- mediated cleavage of APP at the a-secretase cleavage site and to increase released amounts of sAPPa and/or to reduce Αβ deposition in brain tissues of the animal, and to reduce the number and/or size of beta amyloid plaques.
Methods of Monitoring Clinical Efficacy
[0247] In certain embodiments, clinical efficacy can be monitored using any method known in the art. Measurable biomarkers to monitor efficacy include, but are not limited to, monitoring blood, plasma, serum, mucous or cerebrospinal fluid (CSF) levels of sAPPa, βΑΡΡβ, Αβ42, Αβ40, APPneo and p3 (e.g., Αβ17-42 or Αβ17-40). Detection of increased levels of sAPPa and/or p3 and decreased levels of βΑΡΡβ and APPneo are indicators that the treatment or prevention regime is efficacious. Conversely, detection of decreased levels of sAPPa and/or p3, Αβ42 and increased levels of βΑΡΡβ and APPneo are indicators that the treatment or prevention regime is not efficacious. Other biomarkers include Tau and phospho-Tau (pTau). Detection of decreased levels of Tau and pTau are indicators that the treatment or prevention regime is efficacious. [0248] Efficacy can also be determined by measuring amyloid plaque load in the brain. The treatment or prevention regime is considered efficacious when the amyloid plaque load in the brain does not increase or is reduced. Conversely, the treatment or prevention regime is considered inefficacious when the amyloid plaque load in the brain increases. Amyloid plaque load can be determined using any method known in the art, e.g., including magnetic resonance imaging (MRI).
[0249] Efficacy can also be determined by measuring the cognitive abilities of the subject. Cognitive abilities can be measured using any method known in the art. One test is the clinical dementia rating (CDR) described above, while another is the mini mental state examination (MMSE) (Folstein, et al., Journal of Psychiatric Research 12 (3): 189-98). In certain embodiments, subjects who maintain the same score or who achieve a higher score on a CDR and/or on an MMSE indicate that the treatment or prevention regime is efficacious. Conversely, subjects who score lower on a CDR and/or on an MMSE indicate that the treatment or prevention regime has not been efficacious.
[0250] In certain embodiments, the monitoring methods can entail determining a baseline value of a measurable biomarker or parameter (e.g., amyloid plaque load or cognitive ability) in a subject before administering a dosage of TrkA inhibitor, and comparing this with a value for the same measurable biomarker or parameter after treatment.
[0251] In other methods, a control value (e.g., a mean and standard deviation) of the measurable biomarker or parameter is determined for a control population. In certain embodiments, the individuals in the control population have not received prior treatment and do not have AD, MCI, nor are at risk of developing AD or MCI. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered efficacious. In other embodiments, the individuals in the control population have not received prior treatment and have been diagnosed with AD or MCI. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered inefficacious. [0252] In other methods, a subject who is not presently receiving treatment, but has undergone a previous course of treatment is monitored for one or more of the biomarkers or clinical parameters to determine whether a resumption of treatment is required. The measured value of one or more of the biomarkers or clinical parameters in the subject can be compared with a value previously achieved in the subject after a previous course of treatment. Alternatively, the value measured in the subject can be compared with a control value (mean plus standard deviation) determined in population of subjects after undergoing a course of treatment. Alternatively, the measured value in the subject can be compared with a control value in populations of prophylactically treated subjects who remain free of symptoms of disease, or populations of therapeutically treated subjects who show amelioration of disease characteristics. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered efficacious and a decision not to resume treatment can be considered/evaluated. In all of these cases, a significant difference relative to the control level (e.g., more than a standard deviation) is an indicator that resumption of the subject should be considered.
[0253] In certain embodiments, the tissue sample for analysis is typically blood, plasma, serum, mucous or cerebrospinal fluid from the subject.
EXAMPLES
[0254] The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Emerging relationship between development and degeneration: TrkA-APP interaction as a therapeutic target in Alzheimer's disease
[0255] An unbiased screen for compounds that block amyloid-β precursor protein (APP) caspase cleavage identified ADDN1351 , which reduced APP-C31 by 90%. Target identification studies showed that ADDN1351 is a TrkA inhibitor, and, in complementary studies, TrkA over-expression increased APP-C31 and cell death. TrkA was shown to interact with APP and suppress APP -mediated transcriptional activation. Moreover, treatment of PDAPP transgenic mice with the known TrkA inhibitor GW441756 not only decreased Αβ, but also increased sAPPa. Thus, TrkA inhibition, rather than NGF activation, can be used as a novel therapeutic approach, and may counteract the hyperactive signaling resulting from the accumulation of active NGF -TrkA complexes due to reduced retrograde transport. The optimal therapy for AD may involve a combination of the delivery of NGF or NGF mimetics to basal forebrain cholinergic neuron somata and a TrkA inhibitor that is active more distally.
Introduction
[0256] Alzheimer's disease (AD) is characterized by senile plaques, neurofibrillary tangles, and loss of synapses and neurons. The predominant components of senile plaques are the β-amyloid peptides (Αβ40 & Αβ42), peptides generated from the Αβ-precursor protein (APP). Although the accumulation of Αβ has been identified as an important mechanism underlying AD pathogenesis, neither the details underlying Αβ toxicity nor the physiological function(s) of Αβ has been fully elucidated.
[0257] A major neuroanatomical feature of AD is the selective degeneration of basal forebrain cholinergic neurons (BFCN). These neurons provide cholinergic innervation to the neocortex, hippocampus, and entorhinal cortex. BFCN express nerve growth factor (NGF) receptors p75NTR and TrkA, and NGF trophic support is required for the normal function and survival of BFCN. NGF is produced by target regions of the BFCN and retrogradely transported to the cell body in the form of NGF-TrkA complexes. It has been proposed that this retrograde transport deficit is one of the major causes of BFCN
degeneration in AD (Isacson et al. (2002) Trends Neurosci., 25: 79-84).
[0258] In addition to Αβ, APP generates another cytotoxic peptide, APP-C31 , by intracytoplasmic cleavage at D664 (Lu et al. (2000) Nat. Med., 6: 397-404).
[0259] A screen for small molecules that block the production of APP-C31 , identified a compound, ADDN-1351, that, surprisingly, was identified as a TrkA kinase inhibitor. This finding runs counter to the current notion that TrkA promotes anti-AD signaling: TrkA is reported to be reduced in BFCN in AD, and failure of NGF-TrkA signaling has been considered the major cause of BFCN degeneration. Therefore, NGF and TrkA agonists have been proposed as potential treatments for AD (Williams et al. (2006) Prog. Neurobiol, 80: 114-128; Mufson et a/. (2008) Expert Rev. Neurother., 8: 1703-1718). [0260] Thus, both TrkA hyper-activation (in neuronal processes) and TrkA hypo- activation (in neuronal somata) may feature in AD. Under these conditions, TrkA may promote pro-AD signaling through its kinase activity. Surprisingly, TrkA over-expression induces APP-C31 production, which could be prevented by a kinase-dead TrkA mutant or by TrkA inhibitor GW441756 (Wood et al. (2004) Bioorg. Med. Chem. Lett., 14: 953-957). TrkA also interacts with APP and modulates APP processing. In addition, activation of
TrkA by NGF induces increased Αβ production in vitro. Further confirmation of the role of TrkA in APP processing was provided by in vivo testing of GW441756. Treatment with this TrkA inhibitor resulted in decreased Αβ and increased sAPPa levels in the PDAPP AD transgenic mouse model. NGF and NGF mimetics may have detrimental as well as beneficial effects on AD pathophysiology, and thus, an optimal therapy for AD may comprise the combined delivery of NGF or NGF mimetics to BFCN somata, along with a TrkA inhibitor that is active more distally. Accordingly, selective pharmacological inhibition of TrkA kinase may prove to be part of an optimal therapeutic cocktail for AD, by negating the hyperactivation and resulting toxicity produced from accumulation of active NGF-TrkA complexes, as a result of the retrograde transport deficits that occur early in AD.
Materials and Methods Plasmids.
[0261] Constructs pCMV5-TrkA and pCMV5-TrkA(K538A) were kindly provided by Dr. Moses Chao. Construct pcDNA3-flag-rat-TrkA was a gift from Dr. Francis Lee. Constructs pCMV5-Mint3, pMst-APP, pG5ElB-luc, pCMV-LacZ, and pCMV-Fe65, were generously provided by Dr. Thomas Sudhof, Dr. Patrick Mehlen, and Dr. Veronique Corset. Construct pcDNA4-His-MaxB-hYAPl was a kind gift from Dr. Marius Sudol. Constructs pcDNA3-APP-C83, pcDNA3-APP-C99, pcDNA3-APP695, pcDNA3-APP-D664A, pcDNA3-APP-AC31, pcDNA3-APPsi and pcDNA3-fiag- p75OTR were described previously(Lu et al, 2000; Fombonne et al, 2009).
Antibodies and chemical compounds.
[0262] 6E10 anti-APP antibody was purchased from Covance. CT15 anti-APP C- terminus antibody was a kind gift from Dr. Edward Koo. Anti-APPneo antibody was described previously (Galvan et al. (2002) J. Neurochem., 82: 283-294). Anti-TrkA antibody was purchased from Santa Cruz Biotech. Nerve Growth Factor (NGF) was purchased from Sigma. GW441756 was purchased from Tocris Bioscience. PHA739358 was purchased from EMD. MTT was purchased from Sigma. Cell culture and co-immunoprecipitations.
[0263] The Chinese Hamster Ovary (CHO) cell line over-expressing human APP
(7W) was kindly provided by Dr. Edward Koo. The H4 neuroglioma cell line over- expressing human APP (H4APPwt) was a kind gift from Dr. Todd Golde. HEK293T and B103 cell lines were described previously (Lu et al. (2000) Nat. Med., 6: 397-404; Lu et al. (2003) Ann. Neurol, 54: 781-789). Plasmid constructs were transiently transfected into
HEK293T or 7W cells with Lipofectamine 2000 (Invitrogen). Coimmunoprecipitation and Western analysis were performed as previously described (Swistowski et al. (2009) J.
Neurosci., 29: 15703-15712). Briefly, 48 h after transfection, cells were harvested and lysed in NP-40 Cell Lysis Buffer (50 mM TrisHCl, pH 8.0, 150 mM NaCl and 1% NP-40), and then, after centrifugation, incubated overnight with anti-APP antibodies 6E10 or CT15. Protein G agarose beads (Santa Cruz Biotech, CA) were then added for 2 hours incubation at room temperature. The beads were subjected to five rounds of washing consisting of centrifugation, withdrawal of supernatant, and addition of fresh NP-40 Cell Lysis Buffer. During the final washing step, beads were resuspended in IX LDS loading buffer
(Invitrogen) with 50 mM DTT, and boiled at 100°C for 10 min. After SDS-PAGE and electrotransfer, Western blotting was performed using anti-TrkA antibody. Thirty minutes of TBS-Tween wash were followed by incubation with secondary antibodies.
Drug screening and kinase inhibition assay.
[0264] A high-throughput DELFIA screen was employed to identify small molecule modulators of the APPneo generation. The assay utilizes the DELFIA microtiter plates coated with the capture 6E10 antibody. After stimulation of the H4APPwt cells with staurosporine (STS), the assay measures the formation of the APPneo fragment using anti- APPneo polyclonal antibody and anti-rabbit-Europium (Eu) labeled reporter antibody. The screening of our CNS-focused 5000 compound library was done in a 96-well plate screen format, at a concentration of 10μΜ. The screening assay protocol involves: a) Use of H4APPwt cells to seed 96-well dishes with 40,000 cells/well; b) Preincubate with small molecules and treat with STS, lyse and assay cell lysates; c) Coat DELFIA microtiter plates with 6E10 mAb; d) Develop with lOOng/well anti-APPneo Ab and lOng/well anti-rabbit-Eu reporter antibody; e) Perform single data point screen of ADDN library; f) Analysis of the data is done as a function of percent activity of positive control (+ STS); Set positive control to 100%; g) Cherry pick 'hits' from daughter plates and then assay compounds at 10μΜ in triplicate. Kinase inhibition assays were performed by the Reaction Biology Corporation, Malvern, PA.
MTT cell viability assay.
[0265] 293T cells were transfected with pcDNA3, pCMV5-TrkA or pCMV5- TrkA(K538A) constructs. 48 hours after transfection, cell viability was measured by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, as previously described(Descamps et al). Briefly, 100 μg/ml of MTT was added to the transfected cells and the cells were incubated in dark for 2 hours. Then media were replaced with DMSO and the cells were incubated for 5 minutes with vigorous shaking. Optical densities were read at 570nm. Transactivation assay.
[0266] HEK293T cells were co-transfected with five or six plasmids: (1) pG5ElB- luc, O^g; (2) pCMV-LacZ, O.^g; (3) pMst-APP (APP-Gal4), 0.3μ§; (4) pCMV5-TrkA or pCMV5-TrkA(K538A), l g; (5) pcDNA4-His-MaxB-hYAPl or pCMV-Fe65, l .(^g. Where indicated, a sixth plasmid was co-transfected: pCMV5-Mint3, 1.0μ . For negative controls, the expression vector pcDNA3 was used without insert. Cells were harvested 48 hr after transfection in 0.2ml per well Cell Culture Lysis Buffer (Promega), and their luciferase and β-galactosidase activities were determined with the Promega luciferase assay kit and the Promega β-galactosidase assay kit, respectively. The luciferase activity was standardized by the β-galactosidase activity to control for transfection efficiency and general effects on transcription. Values shown are averages of transactivation assays performed in duplicate or triplicate and repeated at least three times each constructs. All constructs were assayed in HEK293T and B103 cell lines, and representative results from the HEK293T cell lines are shown. Transfections were performed at 80-90% confluency in six-well plates using Lipofectamine 2000 (Invitrogen).
Transgenic mice and in vivo GW441756 treatment.
[0267] The PDAPP (J20) mice were described previously (Galvan et al. (2006)
Proc. Natl. Acad. Sci. USA 103: 7130-7135). 6-6.5 months old J20 mice were treated with DMSO control or GW441756 at lOmg/kg/day (i.p.) for 5 days. 2 hours after the last injection, mice were sacrificed and hippocampi were dissected and homogenized. The levels of sAPPa were detected by AlphaLISA assay (PerkinElmer, Waltham, MA). Αβ40 and Αβ42 levels were quantified by ELISA (BioSource, Camarillo, CA).
Results
TrkA induces APP cleavage at Asp664 and cell death
[0268] A screen for small molecules that inhibit the production of APP-C31 , and identified 52 compounds from a 5000-compound CNS-focused library. One compound, ADDN-1351, reduced APP-C31 by over 90% (Fig. 12A). Since ADDN-1351 displays structural similarities to some kinase inhibitors, it was evaluated against a panel of 24 kinases at a low dose of 0.1 μΜ, to determine whether it interacts with any specific kinase from this panel. Using this approach TrkA was identified as a specific target for ADDN- 1351 (Fig. 12B). A dose-response analysis in the TrkA kinase assay showed that ADDN- 1351 is an inhibitor of TrkA kinase activity, with an IC50 of 358 nM (Fig. 12D). [0269] To complement this result, the effect of TrkA expression on the cleavage of
APP at Asp664, which leads to APP-C31 production, was assessed. 7W cells which stably overexpress APP (Koo and Squazzo (1994) J. Biol. Chem., 269: 17386-17389) were transiently transfected with TrkA. TrkA transfection induced a marked increase (more than 10-fold) in APP-C31 production, as detected by an antibody specific for the APPneo epitope (the epitope exposed after APP-C31 cleavage) (Galvan et al. (2002) J. Neurochem., 82: 283-294). This induction did not occur when a kinase dead TrkA (K538A) mutant construct was transfected (Fig. 13 A). Moreover, treatment with a potent and specific TrkA inhibitor, GW441756, abolished the TrkA-induced APP-C31 production from APP (Fig. 13B). These results indicate that TrkA, in a kinase activity dependent manner, activates APP cleavage to produce APP-C31.
[0270] The APP-C31 peptide is toxic, and blocking this cleavage by mutating the cleavage site in transgenic mice ameliorated the AD phenotype, without altering plaque load or Αβ concentration. Using an MTT assay, it was found that TrkA expression and TrkA induced APP-C31 cleavage significantly decreased cell survival, and this effect required the kinase activity of TrkA (Fig. 13D).
[0271] TrkA has been found to act as a dependence receptor and induce neuronal death in the absence of NGF, while transducing trophic signals in the presence of NGF (Nikoletopoulou et al. (2010) Nature 467: 59-63). Surprisingly, NGF treatment further enhanced TrkA induced APP-C31 cleavage, indicating that NGF-TrkA signaling positively regulates the production of APP-C31 (Fig. 13C).
TrkA interacts with APP
[0272] To explore potential mechanisms behind the induction of APP-C31 cleavage by TrkA, we asked whether TrkA interacts with APP. Indeed, TrkA not only co- immunoprecipitated with wild type APP (Fig. 14A), but also interacted with different mutant forms of APP, including the APP carrying the Swedish and Indiana familial Alzheimer's disease (FAD) mutations (Fig. 14A), APP carrying the D664A mutation that is resistant to caspase cleavage (Fig. 14B), and a fragment of APP with the C-terminal 31 amino acids deleted (Fig. 14B). In addition, the C-terminal APP fragment created after the β-secretase cleavage (C99) co-immunoprecipitated with TrkA, while the C-terminal fragment created after the a-secretase cleavage (C83) did not (Fig. 14C), indicating that the region corresponding to the first 1-16 amino acids of Αβ is required for this interaction. The effect of TrkA kinase activity modulation on the TrkA- APP interaction was evaluated in the presence of NGF or with the kinase dead TrkA(K538A) mutant. In both conditions TrkA and APP co-immunoprecipitated (Figs. 14D-14E), indicating that the interaction between APP and TrkA is kinase activity independent.
TrkA inhibits APP-Gal4 transactivation in a kinase independent manner
[0273] While the APP-C31 cleavage is associated with cell death, the APP intracellular domain (AICD) created following the γ-secretase cleavage has been implicated in various signaling pathways, and has been shown to modulate the expression of many genes including KAIl, Neprilysin, and APP itself (Muller et al. (2008) Prog. Neurobiol., 85: 393-406). An APP-Gal4/Mint3/YAP transactivation assay (Orcholski et al. (2011) J. Alzheimers Dis., 23: 689-699; Swistowski et al. (2009) J. Neurosci., 29: 15703-15712), showed that TrkA inhibited APP-Gal4 transactivation by over 95% (Fig. 15 A). This effect was confirmed using the APP-Gal4/Fe65 transactivation assay developed in the laboratory of Dr. Thomas Siidhof; potent inhibition of APP-Gal4 transactivation by TrkA (Fig. 15B) was observed. Inhibition of APP-Gal4 transactivation with the kinase dead TrkA(K538A) mutant (Fig. 15C), as well as in the presence of NGF (Fig. 15D) was also observed. These results indicate that the suppression of APP-Gal4 transactivation by TrkA is not dependent on the latter's kinase activity.
TrkA affects APP processing
[0274] APP is processed through two major pathways: the non-amyloidogenic pathway is initiated by a-secretase cleavage, generating sAPPa and a-CTF (C83), while the amyloidogenic pathway is initiated by β-secretase cleavage, producing sAPPp and β-CTF (C99). The β-CTF is then cleaved by the γ-secretase, which produces Αβ and AICD. Since TrkA interacts with APP, the effects of TrkA over-expression on APP processing were evaluated. [0275] TrkA and APP were co-expressed in 293T cells, and the levels of Αβ, full- length APP, sAPPa, and β-CTF were assayed. The level of full-length APP increased, while the levels of Αβ40, Αβ42 and sAPPa decreased (Figs. 16A-16C). These effects are similar to the effects of Mint 1, a protein that interacts with and stabilizes APP (Rogelj et al. (2006) Brain Res. Rev., 52: 305-315). In contrast to the effects of TrkA on Αβ and sAPPa, the level of β-CTF was increased (Fig. 16E). NGF signaling can enhance sAPPa
production through the PKC pathway (Rossner et al. (1998) Prog. Neurobiol, 56: 541-569), and NGF treatment increased the level of sAPPa, but the increase was not enough to return sAPPa to its level in the absence of TrkA expression (Fig. 16D). Surprisingly, NGF also increased the levels of both Αβ40 and Αβ42 (Figs. 16A-16B). The kinase-dead TrkA (K538A) mutant, did not substantially alter the inhibition of Αβ and sAPPa production. In addition, the effect of NGF on Αβ42 production was abolished in the presence of
TrkA(K538A) (Fig. 16B), indicating that the increase in Αβ production is mediated through TrkA kinase activity and the NGF -TrkA signaling pathways.
[0276] APP is processed through two major pathways: the non-amyloidogenic pathway is initiated by a-secretase cleavage, generating sAPPa and a-CTF (C83), while the amyloidogenic pathway is initiated by β-secretase cleavage, producing βΑΡΡβ and β-CTF (C99). The β-CTF is then cleaved by the γ-secretase, which produces Αβ and AICD. Since TrkA interacts with APP, the effects of TrkA over-expression on APP processing were evaluated.
[0277] TrkA and APP were co-expressed in 293T cells, and the levels of Αβ, full- length APP, sAPPa, and β-CTF were assayed. The level of full-length APP increased, while the levels of Αβ40, Αβ42 and sAPPa decreased (Figs. 16A-16C). These effects are similar to the effects of Mint 1, a protein that interacts with and stabilizes APP (Rogelj et al. (2006) Brain Res. Rev., 52: 305-315). In contrast to the effects of TrkA on Αβ and sAPPa, the level of β-CTF was increased (Fig. 16E). NGF signaling can enhance sAPPa
production through the PKC pathway (Rossner et al. (1998) Prog. Neurobiol, 56: 541-569), and NGF treatment increased the level of sAPPa, but the increase was not enough to return sAPPa to its level in the absence of TrkA expression (Fig. 16D). Surprisingly, NGF also increased the levels of both Αβ40 and Αβ42 (Figs. 16A-16B). The kinase-dead TrkA (K538A) mutant, did not substantially alter the inhibition of Αβ and sAPPa production. In addition, the effect of NGF on Αβ42 production was abolished in the presence of
TrkA(K538A) (Fig. 16B), indicating that the increase in Αβ production is mediated through TrkA kinase activity and the NGF -TrkA signaling pathways.
Discussion
[0278] Surprisingly, ADDN-1351, the potent APP-C31 cleavage inhibitor identified through library screening, was found to be a TrkA kinase inhibitor. This discovery is somewhat counter-intuitive because an NGF -TrkA signaling deficit has been considered as a potential cause of BFCN degeneration and cholinergic deficit, one of the major features of AD. However, in both in vitro and in vivo studies, TrkA hyper-activation not only induced APP-C31 cleavage, but also increased Αβ production, and anti-TrkA treatment in an AD transgenic mouse model decreased the levels of Αβ40 and Αβ42, while increasing sAPPa. Therefore, TrkA inhibition appears to represent a novel therapeutic approach to AD, and indicates that potent brain-permeable TrkA kinase inhibitors can be beneficial
therapeutically in AD. To the inventors knowledge, this is the first report directly indicating TrkA kinase inhibition as a potential part of the therapeutic armamentarium for AD.
[0279] These results indicate that, from the pre-symptomatic stage until the MCI stage, there is a sufficient amount of TrkA and NGF in the target regions of the BFCN; retrograde transport deficits during this period may counter-intuitively result in the accumulation of NGF-TrkA complexes and hyperactive TrkA signaling. Thus, at least in the initial stages of the disease, TrkA hyper-activation in synapses or processes of the
BFCN at target regions are contributors to the pathogenesis of AD. Once the Αβ and Tau pathology begin to appear, and the disease progresses(Frost and Diamond (2010) Nat. Rev. Neurosci., 11 : 155-159), a positive feedback loop arises that further impairs the retrograde transport of NGF-TrkA complexes. This would aggravate the hyperactivation of TrkA and cause more neuronal toxicity and cell death.
[0280] At the stage of mild AD, TrkA is decreased in target regions of BFCN, but the cholinergic markers, including acetylcholine (ACh) and choline acetyltransferase (ChAT), are not reduced(/<i). In severe AD, cholinergic function is significantly compromised and BFCN degeneration is prominent (Id.). Although TrkA levels are decreased in the BFCN target regions of these patients, at a single synapse, given that markers of TrkA downstream signaling like activated ERK1/2 and PLCy are increased in AD, it is likely that TrkA is locally hyper-activated, and in such a case, TrkA inhibitors may potentially retard the progression of AD at the target regions of the BFCN.
[0281] Thus, one optimal treatment for AD may include both TrkA inhibition in the BFCN processes and TrkA activation in the BFCN somata. Indeed, TrkA activation may be supportive for the BFCN, yet toxic for the target regions of BFCN, and since retrograde transport problems may develop with aging, this might contribute to the exponential increase in incidence of AD from age 65 to 85. One therapeutic consideration is whether TrkA inhibitors may be effective to treat AD without inducing significant side effects, and without augmenting BFCN degeneration. It is important to note that several TrkA inhibitors have entered advanced clinical testing for cancer, and these appear to be well tolerated (Wang et al. (2009) Expert Opin. Ther. Pat., 19: 305-319).
[0282] In particular embodiments, timing is important for the treatment of AD with
TrkA inhibitors: during the stage of mild AD, there is no cholinergic deficit, indicating that at least the function of neurotransmission is preserved in the BFCN at this stage. In addition, at this same stage, in the BFCN target regions, progressive Αβ accumulation and Tau pathology are developing and are associated with clinical symptoms. Thus, in particular embodiments, use of TrkA inhibitors at this stage of the disease may be preferred, in order to slow or reverse AD progression at the target regions.
[0283] TrkA inhibition may block the positive feedback loops at BFCN target regions, reduce the levels of Αβ and p-Tau, and prevent further deterioration of the disrupted retrograde transport, and therefore might bring NGF support back to pre- AD levels. Thus, use of TrkA inhibitors to treat mild stage AD is expected to result in protection of the BFCN target regions and prevention of deficits in memory and other cognitive functions.
[0284] At the severe AD stage, in which there is significant loss of BFCN function, the somal effects may predominate over the process effects, and therefore the application of a TrkA inhibitor may potentially exacerbate the AD symptoms. In support of this notion, targeted BFCN delivery of NGF has been found to be promising (Tuszynski et al. (2005) Nat. Med., 11 : 551-555), and is currently in a phase II clinical trial. Thus, TrkA inhibition is a viable therapeutic strategy in a stage-dependent fashion, optionally in combination therapy with NGF or NGF mimetics, or as part of a therapeutic cocktail that targets multiple mechanisms of AD pathogenesis (Mangialasche et al. (2010) Lancet Neurol., 9: 702-716). [0285] The data provided herein indicate that TrkA hyper-activation induces APP-
C31 cleavage and increases Αβ production. Thus, chronic hyper-activation of TrkA, due to the accumulation of active NGF -TrkA complexes in the target regions of BFCN, is an early mechanism in the pathogenesis of AD (Fig. 17E). Accordingly, potent and brain-permeable TrkA kinase inhibitors represent one component of an optimal treatment for AD. Example 2
Synthesis of ADDN-1351 and Analogs.
[0286] The following steps for the synthesis of ADDN-1351 and analogs thereof are illustrated in Figure 18.
[0287] In Step 1 of a synthesis scheme for ADDN-1351, 4-t-butyl-phenyl hydrazide (10 mmol) was mixed with ethanol (10 ml) and treated with the ethyl isothiocyanate (11 mmol). The mixture was stirred at reflux for approximately 7 hours, cooled to ambient temperature, concentrated in vacuo to one third of the original volume and diluted with two equivalents of deionised water to give an off white precipitate. The precipitate was collected by filtration through a sintered glass funnel and the structure and purity of the product were assessed using 1H-NMR spectroscopy and HPLC. The 4-t-butyl-phenyl hydrazinecarbothioamide was isolated in quantitative yield in greater than 90% purity (by HPLC) and no further purification was required. [0288] In Step 2 (see Fig. 18), the 4-t-butyl-phenyl hydrazinecarbothioamide (7 mmol) was mixed with sulfuric acid (10 mL) and stirred at 80 °C for 6 hours. The reaction was then cooled to ambient temperature and left overnight. The reaction mixture was poured onto ice. Aqueous ammonia (25%) was carefully added until the pH of the solutions was 8-9. The products precipitated and the solids were collected by filtration through sintered glass funnels. The 5-(4-(tert-butyl)phenyl)-N-ethyl-l,3,4-thiadiazol-2-amine was isolated in quantitative yield. The compound was characterized using H-NMR
spectroscopy and HPLC and no further purification was required.
[0289] In Step 2 (see Fig. 18), the nicotinic chloride (2.1 mmol) was prepared in situ by heating at 60°C in excess thionyl chloride for 60 minutes. The thionyl chloride was removed in vacuo and the relevant thiadiazole (0.7 mmol) in pyridine (2 ml) was carefully added. The solution was heated to 70°C and left stirring for 12 hours. The pyridine was removed in vacuo and the residue was rinsed with saturated sodium bicarbonate solution and extracted into DCM. The solvent was removed in vacuo and a portion of the residue was loaded onto a preparative TLC plate (50% EtOAc/hexane). The product band was scraped from the TLC plate, taken up in ethyl acetate and filtered to remove the silica
N-(5-(4-(tert-butyl)phenyl)-l,3,4-thiadiazol-2-yl)-N-ethylpyrimidine-5- carboxamide was isolated in 80% purity and was subsequently recrystallized from ethyl acetate and hexane to give 14 mg of the desired product in 95% purity (by HPLC and 1H-NMR) with a molecular ion ofm/z 368 [M +l]. [0290] The analogs were prepared using the same three step procedures with the appropriate hydrazides, and acid chlorides. The yields generally for the three steps were similar to ADDN-1351. The final compounds were isolated and recrystalized and analysed by 1HNMR and MS.
[0291] It is understood that the examples and embodiments, described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

What is claimed is:
1. A method of inhibiting the C-terminal cleavage of APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal, said method comprising:
administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a APP-C31 peptide and/or APPneo (APP664).
2. A method of mitigating in a mammal one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms in a mammal, said method comprising:
administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to mitigate said one or more symptoms.
3. A method of reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal, said method comprising:
administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to reduce the risk, lessen the severity, and/or delay the progression or onset of said disease. 4. The method of claim 3, wherein said disease is a disease selected from the group consisting of Alzheimer's disease, age-related macular degeneration
(AMD), Cerebrovascular dementia, Parkinson's disease, Huntington's disease, and Cerebral amyloid angiopathy.
5. A method of preventing or delaying the onset of a pre-Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre- Alzheimer's condition and/or cognitive dysfunction, and/or preventing or delaying the progression of a pre-Alzheimer's condition or cognitive dysfunction to Alzheimer's disease in a mammal, said method comprising:
administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway.
6. A method of promoting the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway as characterized by increasing sAPPa and/or the sAPPa/A 42 ratio in a mammal, said method comprising:
administering, or causing to be administered, to said mammal a TrkA kinase inhibitor in an amount sufficient to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway
7. A method comprising:
administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to reduce C-terminal cleavage of APP and production of a APP-C31 peptide and/or APPneo (APP664); and/or
administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to mitigate said one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms; and/or administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to reduce the risk, lessen the severity, and/or delay the progression or onset of a disease characterized by beta-amyloid deposits in the brain of the mammal; and/or
administering to a mammal, a TrkA kinase inhibitor in an amount sufficient for preventing or delaying the onset of a pre-Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre-Alzheimer's condition and/or cognitive dysfunction, and/or preventing or delaying the progression of a pre-Alzheimer's condition or cognitive dysfunction to Alzheimer's disease in the mammal; and/or
administering to a mammal, a TrkA kinase inhibitor in an amount sufficient to increase the processing of amyloid precursor protein (APP) by the non- amyloidogenic pathway, wherein the increase is characterized by increasing sAPPa and/or the sAPPa/A 42 ratio in the mammal.
8. The method of claim 7 wherein said disease is a disease selected from the group consisting of Alzheimer's disease, age-related macular degeneration (AMD), Cerebrovascular dementia, Parkinson's disease, Huntington's disease, and Cerebral amyloid angiopathy.
9. The method according to any one of claims 1-8, wherein said TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siR A.
10. The method of claim 9, wherein said TrkA inhibitor comprises a small organic molecule inhibitor of TrkA.
1 1. The method of claim 10, wherein said TrkA kinase inhibitor has the formula:
Figure imgf000108_0001
or is an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof, or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof; wherein
R1 is selected from the group consisting of substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alknyl, thioalkyl, and aminoalkyl; and
R2 and R3 are independently selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl.
12. The method of claim 1 wherein R1 is a Ci_6 alkyl.
13. The method of claim 1 wherein R1 is substituted alkyl.
14. The method of claim 1 wherein R1 is unsubstituted alkyl.
15. The method of claim 1 wherein R1 is substituted alkenyl.
16. The method of claim 1 wherein R1 is unsubstituted alkenyl.
17. The method of claim 1 wherein R1 is substituted alkynyl.
18. The method of claim 1 wherein R1 is unsubstituted alkynyl.
19. The method of claim 1 wherein R1 is substituted alkoxy.
20. The method of claim 1 wherein R1 is unsubstituted alkoxy.
21. The method of claim 1 wherein R1 is substituted thioalkyl.
22. The method of claim 1 wherein R1 is unsubstituted thioalkyl.
23. The method of claim 11, wherein R1 is substituted aminoalkyl.
24. The method of claim 11, wherein R1 is unsubstituted aminoalkyl.
25. The method according to any one of claims 11-24, wherein R2 is aryl
26. The method according to any one of claims 11-24, wherein R2 is substituted aryl.
27. The method according to any one of claims 11-24, wherein R2 is heteroaryl.
28. The method according to any one of claims 11-24, wherein R2 is substituted heteroaryl.
The method according to any one of claims 11-28, wherein R3 is aryl
30. The method according to any one of claims 11-28, wherein R3 is substituted aryl.
31. The method according to any one of claims 11-28, wherein R3 is heteroaryl. 32. The method according to any one of claims 11-28, wherein R3 is substituted heteroaryl.
33. The method of claim 11, wherein said TrkA kinase inhibitor is ADDN- 1351 or an analogue thereof.
34. The method of claim 33, wherein said TrkA kinase inhibitor is ADDN-1351.
35. The method of claim 33, wherein said TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN- 135 Id, ADDN-1351e, ADDN-1351f, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN- 135 lj, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN- 135 lp as shown in Table 4.
36. The method of claim 10, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000110_0001
wherein
X is selected from the group consisting of Me, H, and halogen;
R1 is selected from the group consisting of cyclopropyl, O'Pr, SMe, Me, OPr, H, and 'Bu
selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and halogen;
R3 is selected from the group consisting of H, (5)-Me, ( ?)-Me, (S)- CH2OH, ( ?)-CH2OH, (5)-Me, (i?)-CH2OH, (S)-CH2CONMe2, and (S)-CH2CONHMe;
Y1 and Y2 are independent selected from the group consisting of CH, and N; and
selected from the group consisting of H, OH CH 3
Figure imgf000110_0002
37. The method of claim 36, wherein R2 is F.
The method according to any one of claims 36-37, wherein Y1 is CH and Y2 is N.
The method according to any one of claims 36-37, wherein Y1 and Y 2 are both N.
2
The method according to any one of claims 36-37, wherein Y1 and Y are both CH.
41. The method of claim 36, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000111_0001
wherein
X is selected from the group consisting of Me, H, and halogen; selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe,
4-F, 4-Cl, and F; and
selected from the group consisting of H, (5)-Me, ( ?)-Me, (S)-
CH2OH, (i?)-CH2OH, (S)-MQ, (i?)-CH2OH, (S)-CH2CONMe2, and (S)-CH2CONHMe.
42. The method of claim 41 , wherein X is CI or Br.
43. The method of claim 41 , wherein said compound is a compound selected from the group consisting of compound 10a, compound 10b, compound 10c, compound lOd, compound lOe, compound lOf, compound lOg, compound lOh, compound lOi, compound lOj, compound 10k, compound 101, compound 10m, and compound 10η, as shown in Table 5.
44. The method of claim 36, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000112_0001
wherein
X is selected from the group consisting of Me, H, and halogen;
R10 is selected from the group consisting of H, and OH; and
R1 is selected from the group consisting of cyclopropyl, O'Pr, SMe,
Me, OPr, H, and ¾u.
45. The method of claim 44, wherein X is selected from the group consisting of Br, Me, H, F, and CI.
46. The method of claim 44, wherein said compound is a compound selected from the group consisting of compound 10ο, compound lOp, compound lOq, compound lOr, compound 10s, compound lOt, compound lOu, compound lOv, and compound lOw, as shown in Table 6.
47. The method of claim 36, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000112_0002
wherein X is selected from the group consisting of Me, H, and halogen;
R10 is selected from the group consisting of H, and OH;
R1 is selected from the group consisting of cyclopropyl, O'Pr, Me,
OPr, H, and ¾u; and
R7 is selected from the group consisting of H, OH, CH3,
Figure imgf000113_0001
O^ , HO''^^ H0-^^N -* , and HCT
48. The method of claim 47, wherein X is CI or H.
49. The method according to any one of claims 47-48, wherein R1 is selected from the group consisting of Cp, Me, and O'Pr.
50. The method according to any one of claims 47-49, wherein R7 is selected from the group consisting of
Figure imgf000113_0002
Oj , HO--^ N^ Ha-^-^ N- and HCT
51. The method of claim 47, wherein said compound is a compound selected from the group consisting of compound 15a, compound 15b, compound 15c, compound 15d, compound 15e, compound 15f, and compound 15g, as shown in Table 7.
52. The method of claim 36, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000114_0001
wherein
X is selected from the group consisting of Me, H, and halogen;
R1 is selected from the group consisting of Cp, and O'Pr.
Y1 and Y2 are independent selected from the group consisting of CH, and CH; and
selected from the group consisting of H, OH, CH 3,
Figure imgf000114_0002
53. The method of claim 52, wherein X is F or CI.
The method according to any one of claims 52-53, wherein Y1 is CH and Y2 is N.
2
55. The method according to any one of claims 52-53, wherein Y1 and Y are both N.
56. The method accordin to any one of claims 52-55, wherein R7 is
selected from the group consisting of H,
Figure imgf000114_0003
57. The method of claim 52, wherein said compound is a compound selected from the group consisting of compound lOx, compound lOy, compound lOz, compound 15h, compound 15i, and compound 1 la, as shown in Table 8.
58. The method of claim 10, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000115_0001
wherein
X is selected from the group consisting of CN, and CONH2; and R is selected from the group consisting of H, Me, Et, z'-Pr, n-Pr, Ph, and H.
59. The method of claim 58, wherein said TrkA inhibitor comprises a compound selected from the group consisting of compound 1 , compound 5a, compound 5b, compound 5c, compound 5d, compound 5e, compound 5f, compound 5g, compound 5h, and compound 6, as shown in Table 9.
60. The method of claim 10, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000115_0002
wherein
selected from the group consisting of /?-Chlorobenzyl,
CH(Me)/?-chlorobenzyl, CH2(cyclohexyl), and o-Chlorobenzyl; and
Pv2 is selected from the group consisting of H, 2-pyr, 3 -pyr, 3 -pyr, 3-
pyr, 4-pyr, 4-pyr, 4-pyramidine,
Figure imgf000115_0003
and
61. The method of claim 60 wherein said TrkA inhibitor comprises a compound selected from the group consisting of compoundl3a, compound 13b, compound 13c, compound 14a, compound 14b, compound 13d, compound 14c, compound 13e, compound 13f, and compound 13g, as shown in Table 10.
62. The method of claim 10, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000116_0001
wherein
X is selected from the group consisting of halogen, and H;
n is 1, 2, or 3; and
R is H or Me.
63. The method of claim 62, wherein X is H.
64. The method of claim 62, wherein X is CI.
65. The method according to any one of claims 62-64, wherein n is 1.
66. The method according to any one of claims 62-64, wherein n is 2.
67. The method according to any one of claims 62-64, wherein n is 3.
68. The method according to any one of claims 62-67, wherein R is H.
69. The method according to any one of claims 62-67, wherein R is Me.
70. The method of claim 62, wherein said TrkA inhibitor comprises a compound selected from the group consisting of compound 19i, compound 19j, compound 191, compound 191, compound 19m, compound 19n, and compound 19o, as shown in Table
XI.
71. The method of claim 10, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000116_0002
72. The method of claim 10, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000117_0001
73. The method of claim 10, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000117_0002
74. The method of claim 10, wherein said TrkA inhibitor comprises a compound accordin to the formula:
Figure imgf000117_0003
75. The method of claim 10, wherein said TrkA inhibitor comprises a compound according to the formulas:
Figure imgf000118_0001
wherein
R1 is selected from the group consisting of Cp, CH3, O'Pr, O'Pr, O'Pr, O'Pr, OEt, and OCH3; and
R2, when present, is selected from the group consisting of CH3, and
CH2OH.
76. The method of claim 75, wherein said TrkA inhibitor comprises a compound selected from the group consisting of compound 17a, compound 17b, compound 17c, compound 17d, compound 17e, compound 18a, compound 18b, and compound 18c, as shown in Table 12.
77. The method of claim 10, wherein said inhibitor is a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11.
78. The method according to any one of claims 1-77, wherein said mammal is a human. 79. The method according to any one of claims 1-78, wherein the mammal is diagnosed as a pre- Alzheimer's and/or a pre-MCI cognitive impairment.
80. The method of claim 79, wherein administration of said TrkA kinase inhibitor delays or prevents the progression of cognitive dysfunction to MCI.
81. The method according to any one of claims 1 -78, wherein the mammal is diagnosed as having mild cognitive impairment (MCI).
82. The method of claim 81 , wherein administration of said TrkA kinase inhibitor delays or prevents the progression of MCI to Alzheimer's disease.
83. The method according to any one of claims 1-78, wherein the mammal is diagnosed as having moderately severe cognitive decline (Moderate or mid- stage Alzheimer's disease).
84. The method according to any one of claims 1-78, wherein the mammal is diagnosed as having severe cognitive decline (moderately severe or mid-stage Alzheimer's disease).
85. The method according to any one of claims 1-78, wherein the mammal is diagnosed as having Alzheimer's disease.
86. The method according to any one of claims 1-78, wherein the mammal is at risk of developing Alzheimer's disease.
87. The method of claim 86, wherein the mammal has a familial risk for having Alzheimer's disease.
88. The method of claim 86, wherein the mammal has a familial Alzheimer's disease (FAD) mutation. 89. The method of claim 86, wherein the mammal has the APOE ε4 allele.
90. The method according to any one of claims 1-89, wherein the mammal is free of and does not have genetic risk factors of Parkinson's disease or schizophrenia. 91. The method according to any one of claims 1-89, wherein the mammal is not diagnosed as having or at risk for Parkinson's disease or schizophrenia.
92. The method according to any one of claims 1-89, wherein the mammal does not have a neurological disease or disorder other than Alzheimer's disease.
93. The method according to any one of claims 1-89, wherein the mammal is not diagnosed as having or at risk for a neurological disease or disorder other than Alzheimer's disease.
94. The method according to any one of claims 1-93, wherein the mammal is not diagnosed as having, and/or at risk for, and/or under treatment for one or more indications selected from the group consisting of cancer, pain, inflammation, rheumatoid arthritis, and an immunological disorder.
95. The method according to any one of claims 1-93, wherein said mammal is a mammal not diagnosed as having cancer and/or not being treated for cancer. 96. The method according to any one of claims 1-95, wherein said method produces a reduction in the CSF of levels of one or more additional components selected from the group consisting of Tau, phospho-Tau (pTau), soluble Αβ40 and soluble Αβ 42.
97. The method according to any one of claims 1-96, wherein said method results in a reduction of the plaque load in the brain of the mammal.
98. The method according to any one of claims 1-97, wherein said method results in a reduction in the rate of plaque formation or deposition in the brain of the mammal.
99. The method according to any one of claims 1-98, wherein said method results in an improvement in the cognitive abilities of the mammal.
100. The method according to any one of claims 1-99, wherein the mammal is a human and said method produces a perceived improvement in quality of life by said human.
101. The method according to any one of claims 1-100, wherein the TrkA kinase inhibitor is administered orally.
102. The method according to any one of claims 1-101, wherein the administering is over a period of at least three weeks.
103. The method according to any one of claims 1-101, wherein the administering is over a period of at least 6 months. 104. The method according to any one of claims 1-103, wherein the TrkA kinase inhibitor is formulated for administration via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration,
administration via inhalation, oral administration, intravenous administration, and rectal administration.
105. The method according to any one of claims 1-104, wherein the TrkA kinase inhibitor is administered via a route selected from the group consisting of isophoretic delivery, cannula, transdermal delivery, aerosol administration, administration via inhalation, oral administration, intravenous administration, and rectal administration. 106. The method according to any one of claims 1-105, wherein the TrkA inhibitor is administered before the onset of mild Alzheimer's disease.
107. The method of claim 106, wherein the TrkA inhibitor is administered to a subject diagnosed with MCI.
108. The method of claim 106, wherein the TrkA inhibitor is administered before the onset of MCI .
109. The method of claim 106, wherein the TrkA inhibitor is administered prophylactically to a healthy subject.
110. The method according to any one of claims 1-105, wherein the TrkA inhibitor is administered in conjunction with another neuropharmaceutical. 111. The method of claim 110, wherein said TrkA inhibitor is administered in conjunction with an agent selected from the group consisting of NGF, an NGF mimetic, a tropinol ester, tropisetron, a tropisetron analog, disulfiram, a disulfiram analog, honokiol, a honokiol analog, nimetazepam, a nimetazepam analog, donepezil, rivastigmine, galantamine, tacrine, memantine, solanezumab, bapineuzmab, alzemed, flurizan, ELND005, valproate, semagacestat, rosiglitazone, phenserine, cernezumab, dimebon, egcg, gammagard, PBT2, PF04360365, NIC5-15, bryostatin-1, AL-108, nicotinamide, EHT-0202, BMS708163, NP12, lithium, ACCOOl, AN 1792, ABT089, NGF, CAD106, AZD3480, SB742457, AD02, huperzine-A, EVP6124, PRX03140, PUFA, HF02, MEM3454, TTP448, PF-04447943, GSK933776, MABT5102A, talsaclidine, UB311, begacestat, R1450, PF3084014, V950, E2609, MK0752, CTS21166, AZD-3839,
LY2886721, CHF5074, an anti-inflammatory, dapsone, an anti-TNF antibody, and a statin.
112. The method according to any one of claims 1-105, wherein the subject is in severe stage Alzheimer's disease and the TrkA inhibitor is administered in conjunction with NGF and/or an NGF mimetic.
1 13. The method according to any one of claims 1-1 12, wherein said administering or causing to be administered is administering.
1 14. A pharmaceutical formulation comprising a TrkA kinase inhibitor having the formula:
Figure imgf000122_0001
or a derivative, an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof; or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof; wherein
R1 is selected from the group consisting of substituted and
unsubstituted alkyl, alkenyl and alkynyl ,alkoxy, thioalkyl, aminoalkyl; and
R2 and R3 are independently selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl; and
a pharmaceutically acceptable carrier or excipient.
1 15. The formulation of claim 1 14, wherein R1 is a Ci_6 alkyl. 1 16. The formulation of claim 1 14, wherein R1 is an unsubstituted alkyl.
1 17. The formulation of claim 1 14, wherein R1 is a substituted alkyl.
1 18. The formulation of claim 1 14, wherein R1 is an unsubstituted alkenyl.
1 19. The formulation of claim 1 14, wherein R1 is a substituted alkenyl.
120. The formulation of claim 1 14, wherein R1 is an unsubstituted alkynyl. 121. The formulation of claim 1 14, wherein R1 is a substituted alkynyl.
122. The formulation of claim 1 14, wherein R1 is an unsubstituted alkoxy.
123. The formulation of claim 1 14, wherein R1 is a substituted alkoxy.
124. The formulation of claim 1 14, wherein R1 is an unsubstituted thioalkyl.
125. The formulation of claim 114, wherein R1 is a substituted thioalkyl.
126. The formulation of claim 114, wherein R1 is an unsubstituted aminoalkyl.
127. The formulation of claim 114, wherein R1 is a substituted aminoalkyl.
128. The formulation according to any one of claims 114-127, wherein R is aryl.
129. The formulation according to any one of claims 114-127, wherein R2 is substituted aryl.
130. The formulation according to any one of claims 114-127, wherein R 2 is heteroaryl.
The formulation according to any one of claims 114-130, wherein R3 is aryl.
132. The formulation according to any one of claims 114-130, wherein R 3 is substituted aryl. 133. The formulation according to any one of claims 114-130, wherein R3 is heteroaryl.
134. The formulation of claim 114, wherein said Formula excludes
ADDN-1351.
135. The formulation of claim 114, wherein said TrkA kinase inhibitor is ADDN- 1351 or an analogue thereof.
136. The formulation of claim 114, wherein said TrkA kinase inhibitor is
ADDN-1351.
137. The formulation of claim 135, wherein said TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN- 135 Id, ADDN-1351e, ADDN- 135 If, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN- 135 lj, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN-1351p as shown in Table 4.
138. The formulation according to any one of claims 114-137, wherein said formulation is a unit dosage formulation.
139. The formulation according to any one of claims 114-138, wherein said formulation is a sterile formulation. 140. The formulation according to any one of claims 114-139, wherein the
TrkA kinase inhibitor is formulated for administration via a route selected from the group consisting of isophoretic delivery, transdermal delivery, aerosol administration,
administration via inhalation, oral administration, intravenous administration, and rectal administration. 141. A TrkA kinase inhibitor for use in:
inhibiting the C-terminal cleavage of APP resulting in the formation of APP-C31 peptide and APPneo (APP664) in a mammal; and/or
mitigating in a mammal one or more symptoms associated with a disease characterized by amyloid deposits in the brain, or delaying or preventing the onset of said symptoms; and/or
reducing the risk, lessening the severity, or delaying the progression or onset of a disease characterized by beta-amyloid deposits in the brain of a mammal; and/or
preventing or delaying the onset of a pre -Alzheimer's condition and/or cognitive dysfunction, and/or ameliorating one or more symptoms of a pre-
Alzheimer's condition and/or cognitive dysfunction, and/or preventing or delaying the progression of a pre -Alzheimer's condition or cognitive dysfunction to Alzheimer's disease in a mammal; and/or
for promoting the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway as characterized by increasing sAPPa and/or the sAPPa/A 42 ratio in a mammal.
142. The TrkA inhibitor of claim 141, wherein said TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siR A. 143. The TrkA inhibitor of claim 142, wherein said TrkA inhibitor comprises a small organic molecule inhibitor of TrkA.
144. The TrkA inhibitor of claim 143, wherein said inhibitor comprises a compound having the formula:
Figure imgf000125_0001
or is an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof, or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof; wherein
R1 is selected from the group consisting of substituted and unsubstituted alkyl, alkenyl and alkynyl ,alkoxy, thioalkyl, aminoalkyl; and
R2 and R3 are independently selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl.
145. The inhibitor of claim 144, wherein R1 is a Ci_6 alkyl.
146. The inhibitor of claim 144, wherein R1 is substituted alkyl.
147. The inhibitor of claim 144, wherein R1 is unsubstituted alkyl.
148. The inhibitor of claim 144, wherein R1 is substituted alkenyl.
149. The inhibitor of claim 144, wherein R1 is unsubstituted alkenyl.
150. The inhibitor of claim 144, wherein R1 is substituted alkynyl.
151. The inhibitor of claim 144, wherein R1 is unsubstituted alkynyl.
152. The inhibitor of claim 144, wherein R1 is substituted alkoxy.
153. The inhibitor of claim 144, wherein R1 is unsubstituted alkoxy.
154. The inhibitor of claim 144, wherein R1 is substituted thioalkyl.
155. The inhibitor of claim 144, wherein R1 is unsubstituted thioalkyl.
156. The inhibitor of claim 144, wherein R1 is substituted aminoalkyl.
157. The inhibitor of claim 144, wherein R1 is unsubstituted aminoalkyl.
158. The inhibitor according to any one of claims 144-157, wherein R2 is aryl.
159. The inhibitor according to any one of claims 144-157, wherein R2 is substituted aryl. 160. The inhibitor according to any one of claims 144-157, wherein R2 is heteroaryl.
161. The inhibitor according to any one of claims 144-157, wherein R2 is substituted heteroaryl.
162. The inhibitor according to any one of claims 144-161, wherein R3 is aryl.
163. The inhibitor according to any one of claims 144-161, wherein R3 is substituted aryl.
164. The inhibitor according to any one of claims 144-161, wherein R3 is heteroaryl. 165. The inhibitor according to any one of claims 144-161, wherein R3 is substituted heteroaryl.
166. The inhibitor of claim 144, wherein said TrkA kinase inhibitor is ADDN- 1351 or an analogue thereof.
167. The inhibitor of claim 166, wherein said TrkA kinase inhibitor is ADDN-1351.
168. The inhibitor of claim 166, wherein said TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN- 135 Id, ADDN-1351e, ADDN- 135 If, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN- 135 lj, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN- 135 lp as shown in Table 4.
169. The TrkA inhibitor of claim 143, wherein said inhibitor comprises a compound having the formula:
Figure imgf000127_0001
wherein
X is selected from the group consisting of Me, H, and halogen;
R1 is selected from the group consisting of cyclopropyl, O'Pr, SMe,
Me, OPr, H, and ¾u;
R2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and halogen;
R3 is selected from the group consisting of H, (5)-Me, ( ?)-Me, (S)- CH2OH, (R)-CH2OH, (S)-MQ, (R)-CH2OH, (5)-CH2CONMe2, and (5)-CH2CONHMe;
Y1 and Y2 are independent selected from the group consisting of CH, and N; and
selected from the group consisting of H, OH, CH3
Figure imgf000127_0002
170. The inhibitor of claim 169, wherein R is F.
171. The inhibitor according to any one of claims 169-170, wherein Y1 is
CH and Y is N.
The inhibitor according to any one of claims 169-170, wherein Y1 and Y are both N.
173. The inhibitor according to any one of claims 169-170, wherein Y1 and Y are both CH.
174. The inhibitor of claim 169, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000128_0001
wherein
X is selected from the group consisting of Me, H, and halogen;
R2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe,
4-F, 4-Cl, and F; and
selected from the group consisting of H, (5)-Me, ( ?)-Me, (S)-
CH2OH, ( ?)-CH2OH, (5)-Me, (i?)-CH2OH, (S)-CH2CONMe2, and (S)-CH2CONHMe.
175. The inhibitor of claim 174, wherein X is CI or Br.
The inhibitor of claim 174, wherein said compound is a compound selected from the group consisting of compound 10a, compound 10b, compound 10c, compound lOd, compound lOe, compound lOf, compound lOg, compound lOh, compound lOi, compound lOj, compound 10k, compound 101, compound 10m, and compound 10η, as shown in Table 5.
177. The inhibitor of claim 169, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000128_0002
wherein
X is selected from the group consisting of Me, H, and halogen;
R10 is selected from the group consisting of H, and OH; and
R1 is selected from the group consisting of cyclopropyl, O'Pr, SMe,
Me, OPr, H, and ¾u.
178. The inhibitor of claim 177, wherein X is selected from the group consisting of Br, Me, H, F, and CI.
179. The inhibitor of claim 177, wherein said compound is a compound selected from the group consisting of compound 10ο, compound lOp, compound lOq, compound lOr, compound 10s, compound lOt, compound lOu, compound lOv, and compound lOw, as shown in Table 6.
180. The inhibitor of claim 169, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000129_0001
wherein
X is selected from the group consisting of Me, H, and halogen;
R10 is selected from the group consisting of H, and OH;
R1 is selected from the group consisting of cyclopropyl, O'Pr, Me,
OPr, H, and ¾u; and
R7 is selected from the group consisting of H, OH, CH3,
Figure imgf000129_0002
181. The inhibitor of claim 180, wherein X is CI or H.
182. The inhibitor according to any one of claims 180-181 , wherein R1 is selected from the group consisting of Cp, Me, and O'Pr.
183. The inhibitor according to any one of claims 180-182, wherein R7 is selected from the group consisting of
Figure imgf000130_0001
184. The inhibitor of claim 180, wherein said compound is a compound selected from the group consisting of compound 15a, compound 15b, compound 15c, compound 15d, compound 15e, compound 15f, and compound 15g, as shown in Table 7.
185. The inhibitor of claim 169, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000130_0002
wherein
X is selected from the group consisting of Me, H, and halogen;
R1 is selected from the group consisting of Cp, and O'Pr.
Y1 and Y2 are independent selected from the group consisting of CH, and CH; and
R is selected from the group consisting of H, OH, CH3,
Figure imgf000131_0001
186. The inhibitor of claim 185, wherein X is F or CI.
187. The inhibitor according to any one of claims 185-186, wherein Y1 is
CH and Y2 is N.
The inhibitor according to any one of claims 185-186, wherein Y1 and Y2 are both N.
The inhibitor accordin to any one of claims 185-188 wherein R7
selected from the group consisting of H,
Figure imgf000131_0002
190. The inhibitor of claim 185, wherein said compound is a compound selected from the group consisting of compound lOx, compound lOy, compound lOz, compound 15h, compound 15i, and compound 1 la, as shown in Table 8.
191. The inhibitor of claim 143, wherein said inhibitor comprises a compound having the formula:
Figure imgf000131_0003
wherein
X is selected from the group consisting of CN, and CONH2; and R is selected from the group consisting of H, Me, Et, z'-Pr, n-Pr, Ph, and H.
192. The inhibitor of claim 191 , wherein said TrkA inhibitor comprises a compound selected from the group consisting of compound 1 , compound 5a, compound 5b, compound 5c, compound 5d, compound 5e, compound 5f, compound 5g, compound 5h, and compound 6, as shown in Table 9.
193. The inhibitor of claim 143, wherein said inhibitor comprises a compound having the formula::
Figure imgf000132_0001
wherein
selected from the group consisting of /?-chlorobenzyl,
CH(Me)/?-chlorobenzyl, CH2(cyclohexyl), and o-chlorobenzyl; and
H, 2-pyr, 3 -pyr, 3 -pyr, 3-
Figure imgf000132_0002
pyr, 4-pyr, 4-pyr, 4-pyramidine, , and
194. The inhibitor of claim 193 wherein said TrkA inhibitor comprises a compound selected from the group consisting of compoundl3a, compound 13b, compound 13c, compound 14a, compound 14b, compound 13d, compound 14c, compound 13e, compound 13f, and compound 13g, as shown in Table 10.
195. The inhibitor of claim 143, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000132_0003
wherein
X is selected from the group consisting of halogen, and H;
n is 1, 2, or 3; and
R is H or Me.
196. The inhibitor of claim 195, wherein X is H.
197. The inhibitor of claim 195, wherein X is CI.
198. The inhibitor according to any one of claims 195-197, wherein n is 1.
199. The inhibitor according to any one of claims 195-197, wherein n is 2.
200. The inhibitor according to any one of claims 195-197, wherein n is 3.
201. The inhibitor according to any one of claims 195-200, wherein R is
H.
202. The inhibitor according to any one of claims 195-200, wherein R is
Me.
203. The inhibitor of claim 195, wherein said TrkA inhibitor comprises a compound selected from the group consisting of compound 19i, compound 19j, compound 191, compound 191, compound 19m, compound 19n, and compound 19o, as shown in Table XI.
204. The TrkA inhibitor of claim 143, wherein said inhibitor comprises a compound having the formula:
Figure imgf000133_0001
205. The TrkA inhibitor of claim 143, wherein said inhibitor comprises a compound having the formu
Figure imgf000133_0002
206. The TrkA inhibitor of claim 143, wherein said inhibitor comprises a compound having the formula:
Figure imgf000134_0001
207. The TrkA inhibitor of claim 143, wherein said inhibitor comprises a compound havin the formula:
Figure imgf000134_0002
208. The TrkA inhibitor of claim 143, wherein said inhibitor comprises a compound having the formulas:
Figure imgf000134_0003
wherein
R is selected from the group consisting of Cp, CH3, O'Pr, O'Pr, O'Pr, O'Pr, OEt, and OCH3; and
R2, when present, is selected from the group consisting of CH3, and
CH2OH.
209. The inhibitor of claim 208, wherein said TrkA inhibitor comprise a compound selected from the group consisting of compound 17a, compound 17b, compound 17c, compound 17d, compound 17e, compound 18a, compound 18b, and compound 18c, as shown in Table 12.
210. The TrkA inhibitor of claim 143, wherein said inhibitor comprises a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 1 1.
21 1. A method for the treatment or prophylaxis of age-related macular degeneration (AMD), said method comprising:
administering, or causing to be administered, to a mammal thereof a TrkA kinase inhibitor in an amount sufficient to ameliorate one or more symptoms of AMD and/or to slow or prevent the progression of AMD.
212. The method of claim 21 1 , wherein said TrkA inhibitor comprises an agent selected from the group consisting of a small organic molecule inhibitor of TrkA, a TrkA inhibitory peptide, an anti-TrkA antibody, and a TrkA siR A.
213. The method of claim 212, wherein said TrkA inhibitor comprises a small organic molecule inhibitor of TrkA.
214. The method of claim 213, wherein said inhibitor comprises a compound having the f rmula:
Figure imgf000135_0001
or is an enantiomer, a mixture of enantiomers, or a mixture of two or more diastereomers thereof, or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate, or prodrug thereof; wherein
R1 is selected from the group consisting of substituted and unsubstituted alkyl, alkenyl and alkynyl ,alkoxy, thioalkyl, aminoalkyl; and
R2 and R3 are independently selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl.
215. The method of claim 214, wherein R1 is a Ci_6 alkyl.
216. The method of claim 214, wherein R1 is substituted alkyl.
217. The method of claim 214, wherein R1 is unsubstituted alkyl.
218. The method of claim 214, wherein R1 is substituted alkenyl.
219. The method of claim 214, wherein R1 is unsubstituted alkenyl.
220. The method of claim 214, wherein R1 is substituted alkynyl.
221. The method of claim 214, wherein R1 is unsubstituted alkynyl.
222. The method of claim 214, wherein R1 is substituted alkoxy.
223. The method of claim 214, wherein R1 is unsubstituted alkoxy.
224. The method of claim 214, wherein R1 is substituted thioalkyl.
225. The method of claim 214, wherein R1 is unsubstituted thioalkyl.
226. The method of claim 214, wherein R1 is substituted aminoalkyl.
227. The method of claim 214, wherein R1 is unsubstituted aminoalkyl.
228. The method according to any one of claims 214-227, wherein R2 is aryl.
229. The method according to any one of claims 214-227, wherein R2 is substituted aryl.
230. The method according to any one of claims 214-227, wherein R heteroaryl.
231. The method according to any one of claims 214-227, wherein R2 substituted heteroaryl.
232. The method according to any one of claims 214-231, wherein R3 aryl.
233. The method according to any one of claims 214-231, wherein R3 is substituted aryl.
234. The method according to any one of claims 214-231, wherein R3 is heteroaryl.
235. The method according to any one of claims 214-231, wherein R3 is substituted heteroaryl. 236. The method of claim 214, wherein said TrkA kinase inhibitor is
ADDN- 1351 or an analogue thereof.
237. The method of claim 236, wherein said TrkA kinase inhibitor is
ADDN-1351.
238. The method of claim 236, wherein said TrkA kinase inhibitor is selected from the group consisting of ADDN-1351a, ADDN-1351b, ADDN-1351c, ADDN- 135 Id, ADDN-1351e, ADDN- 135 If, ADDN-1351g, ADDN-1351h, ADDN-135 H, ADDN- 135 lj, ADDN-1351k, ADDN-13511, ADDN-1351m, ADDN-1351n, ADDN-1351o, AND ADDN-1351p as shown in Table 4.
239. The method of claim 213, wherein said inhibitor comprises compound having the formula:
Figure imgf000137_0001
wherein
X is selected from the group consisting of Me, H, and halogen;
R1 is selected from the group consisting of cyclopropyl, O'Pr, SMe, Me, OPr, H, and ¾u
R2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and halogen; R3 is selected from the group consisting of H, (5)-Me, ( ?)-Me, (S)- CH2OH, (R)-CH2OH, (S)-MQ, (R)-CH2OH, (S)-CH2CONMe2, and (S)-CH2CONHMe;
Y1 and Y2 are independent selected from the group consisting of CH, and N; and
R7 is selected from the group consisting of H, OH, CH3
Figure imgf000138_0001
240. The method of claim 239, wherein R2 is F.
241. The method according to any one of claims 239-240, wherein Y1 is
CH and Y2 is N.
242. The method according to any one of claims 239-240, wherein Y1 and
Y2 are both N.
243. The method according to any one of claims 239-240, wherein Y1 and Y2 are both CH.
244. The method of claim 239, wherein said TrkA inhibitor comprises compound according to the formu
Figure imgf000138_0002
wherein
X is selected from the group consisting of Me, H, and halogen;
R2 is selected from the group consisting of H, 3-OMe, 2-Cl, 2-OMe, 4-F, 4-Cl, and F; and
R3 is selected from the group consisting of H, (5)-Me, ( ?)-Me, (S)-
CH2OH, (R)-CH2OH, (S)-Me, (i?)-CH2OH, (5)-CH2CONMe2, and (5)-CH2CONHMe.
245. The inhibitor of claim 244, wherein X is CI or Br.
246. The inhibitor of claim 244, wherein said compound is a compound selected from the group consisting of compound 10a, compound 10b, compound 10c, compound lOd, compound lOe, compound lOf, compound lOg, compound lOh, compound lOi, compound lOj, compound 10k, compound 101, compound 10m, and compound 10η, as shown in Table 5.
247. The method of claim 239, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000139_0001
wherein
X is selected from the group consisting of Me, H, and halogen;
R10 is selected from the group consisting of H, and OH; and
R1 is selected from the group consisting of cyclopropyl, O'Pr, SMe,
Me, OPr, H, and ¾u.
248. The method of claim 247, wherein X is selected from the group consisting of Br, Me, H, F, and CI.
249. The method of claim 247, wherein said compound is a compound selected from the group consisting of compound 10ο, compound lOp, compound lOq, compound lOr, compound 10s, compound lOt, compound lOu, compound lOv, and compound lOw, as shown in Table 6.
250. The method of claim 239, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000140_0001
wherein
X is selected from the group consisting of Me, H, and halogen;
R is selected from the group consisting of H, and OH;
R1 is selected from the group consisting of cyclopropyl, O'Pr, Me,
OPr, H, and ¾u; and
selected from the group consisting of H, OH, CH3
Figure imgf000140_0002
251. The method of claim 250, wherein X is CI or H.
252. The method according to any one of claims 250-251 , wherein R1 is selected from the group consisting of Cp, Me, and O'Pr.
253. The method according to any one of claims 250-252, wherein R7 is selected from the group consisting of
Figure imgf000140_0003
254. The method of claim 250, wherein said compound is a compound selected from the group consisting of compound 15a, compound 15b, compound 15c, compound 15d, compound 15e, compound 15f, and compound 15g, as shown in Table 7.
255. The method of claim 239, wherein said TrkA inhibitor comprises a compound according to the formula:
Figure imgf000141_0001
X is selected from the group consisting of Me, H, and halogen;
R1 is selected from the group consisting of Cp, and O'Pr.
Y1 and Y2 are independent selected from the group consisting of CH, and CH; and
selected from the group consisting of H, OH, CH 3,
Figure imgf000141_0002
256. The method of claim 255, wherein X is F or CI.
257. The method according to any one of claims 255-256, wherein Y1 is
CH and Y2 is N.
258. The method according to any one of claims 255-256, wherein Y1 and Y2 are both N.
259. The method according to any one of claims 255-258, wherein R7 is
Figure imgf000141_0003
selected from the group consisting of H, HO \^ *, and wn
260. The method of claim 255, wherein said compound is a compound selected from the group consisting of compound lOx, compound lOy, compound lOz, compound 15h, compound 15i, and compound 1 la, as shown in Table 8.
261. The method of claim 213, wherein said inhibitor comprises a compound having the formula:
Figure imgf000142_0001
wherein
X is selected from the group consisting of CN, and CONH2; and R is selected from the group consisting of H, Me, Et, z'-Pr, n-Pr, Ph, and H.
262. The method of claim 261 , wherein said TrkA inhibitor comprises a compound selected from the group consisting of compound 1 , compound 5a, compound 5b, compound 5c, compound 5d, compound 5e, compound 5f, compound 5g, compound 5h, and compound 6, as shown in Table 9.
263. The method of claim 213, wherein said inhibitor comprises a compound having the formula:
Figure imgf000142_0002
wherein
R is selected from the group consisting of /?-chlorobenzyl,
CH(Me)/?-chlorobenzyl, CH2(cyclohexyl), and o-chlorobenzyl; and
R2 is selected from the group consisting of H, 2-pyr, 3 -pyr, 3 -pyr, 3-
pyr, 4-pyr, 4-pyr, 4-pyramidine,
Figure imgf000142_0003
, and
264. The method of claim 263, wherein said TrkA inhibitor comprises a compound selected from the group consisting of compoundl3a, compound 13b, compound 13c, compound 14a, compound 14b, compound 13d, compound 14c, compound 13e, compound 13f, and compound 13g, as shown in Table 10.
265. The method of claim 213, wherein said inhibitor comprises a compound having the formula:
Figure imgf000143_0001
wherein
X is selected from the group consisting of halogen, and H;
n is 1, 2, or 3; and
R is H or Me.
266. The inhibitor of claim 265, wherein X is H.
267. The inhibitor of claim 265, wherein X is CI. 268. The inhibitor according to any one of claims 265-267, wherein n is 1.
269. The inhibitor according to any one of claims 265-267, wherein n is 2.
270. The inhibitor according to any one of claims 265-267, wherein n is 3.
271. The inhibitor according to any one of claims 265-270, wherein R is
H. 272. The inhibitor according to any one of claims 265-270, wherein R is
Me.
273. The inhibitor of claim 265, wherein said TrkA inhibitor comprises a compound selected from the group consisting of compound 19i, compound 19j, compound 191, compound 191, compound 19m, compound 19n, and compound 19o, as shown in Table XI.
274. The method of claim 213, wherein said inhibitor comprises a compound having the formula:
Figure imgf000144_0001
275. The method of claim 213, wherein said inhibitor comprises a compound having the formula:
Figure imgf000144_0002
277. The method of claim 213, wherein said inhibitor comprises a compound having the formula:
Figure imgf000145_0001
278. The method of claim 213, wherein said inhibitor comprises a compound having the formula:
Figure imgf000145_0002
wherein
R is selected from the group consisting of Cp, CH3, O'Pr, O'Pr, O'Pr, O'Pr, OEt, and OCH3; and
R2, when present, is selected from the group consisting of CH3, and
CH2OH. 279. The method of claim 278, wherein said TrkA inhibitor comprise a compound selected from the group consisting of compound 17a, compound 17b, compound 17c, compound 17d, compound 17e, compound 18a, compound 18b, and compound 18c, as shown in Table 12.
280. The method of claim 213, wherein said inhibitor comprises a compound found in any one of Figures 5, 6, 7, 8, 9, 10, or 11.
281. The method according to any one of claims 211-280, wherein said administering or causing to be administered is administering.
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