Cross Reference to Related Applications
[0001] This application claims benefit of United States Provisional Application Serial No.: 60/819,129, filed July 6, 2006, the entire contents of which are incorporated herein by reference for all purposes.
Field of the Invention
[0002] The present invention relates to compounds and combinations thereof for the treatment of diseases associated with cerebral accumulation of Alzheimer's amyloid, such as Alzheimer's disease, screening methods for identifying the compounds, and methods of use of the compounds for the treatment and diagnosis of diseases associated with cerebral accumulation of Alzheimer's amyloid.
Description of Related Art
[0003] Alzheimer's disease (AD) is the most common neurodegenerative disorder of aging, afflicting approximately 1% of the population over the age of 65. Characteristic features of the disease include neurofibrillary tangles composed of abnormal tau protein, paired helical filaments, neuronal loss, and alteration in multiple neurotransmitter systems. The hyperphosphorylation of microtubule-associated tau protein is a known marker of the pathogenic neuronal pre -tangle stage in AD brain (Tan et al., "Microglial Activation Resulting from CD40R/CD40L Interaction after Beta- Amyloid Stimulation," Science (1999) 286:2352-55).
[0004] A significant pathological feature of AD is an overabundance of diffuse and compact senile plaques in association with limbic areas of the brain. Although these plaques contain multiple proteins, their cores are composed primarily of β-amyloid protein, a 39-43 amino acid proteolytic fragment that is proteolytically derived from amyloid precursor protein (APP), a transmembrane glycoprotein. Additionally, C-terminal fragments (CTF) of APP are known to accumulate intraneuronally in AD.
[0005] β-amyloid is derived from APP, a single-transmembrane protein with a 590 to 680 amino acid extracellular amino terminal domain and an approximately 55 amino acid cytoplasmic tail. Messenger RNA from the APP gene on chromosome 21 undergoes alternative splicing to yield eight possible isoforms, three of which (the 695, 751 and 770 amino acid isoforms) predominate in the brain. APP undergoes proteolytic processing via
three enzymatic activities, termed α-, β-and γ-secretase. Alpha-secretase cleaves APP at amino acid 17 of the β-amyloid domain, thus releasing the large soluble amino-terminal fragment α-APP for secretion. Because α-secretase cleaves within the β-amyloid domain, this cleavage precludes β-amyloid formation. Alternatively, APP can be cleaved by β- secretase to define the amino terminus of β-amyloid and to generate the soluble amino- terminal fragment β-APP. Subsequent cleavage of the intracellular carboxy-terminal domain of APP by γ-secretase results in the generation of multiple peptides, the two most common being a 40 amino acid β-amyloid (Aβl-40) and 42 amino acid β-amyloid (Aβl-42). Aβl-40 comprises 90-95% of the secreted β-amyloid and is the predominant species recovered from cerebrospinal fluid (Seubert et al, Nature, 359:325-7, 1992). In contrast, less than 10% of secreted β-amyloid is Aβl-42. Despite the relative paucity of Aβ 1-42 production, Aβl-42 is the predominant species found in plaques and is deposited initially, perhaps due to its ability to form insoluble amyloid aggregates more rapidly than Aβl-40 (Jarrett et al., Biochemistry, 32:4693-7, 1993). The abnormal accumulation of β-amyloid in the brain is believed to be due to decreased clearance of β-amyloid from the brain to the periphery or excessive production of β-amyloid. Various studies suggests excessive production of β-amyloid is due to either overexpression of APP or altered processing of APP, or mutation in the γ secretases or APP responsible for β-amyloid formation.
[0006] β- Amyloid peptides are thus believed to play a critical role in the pathobiology of AD, as all the mutations associated with the familial form of AD result in altered processing of these peptides from APP. Indeed, deposits of insoluble, or aggregated, fibrils of β-amyloid in the brain are a prominent neuropathological feature of all forms of AD, regardless of the genetic predisposition of the subject. It also has been suggested that AD pathogenesis is due to the neurotoxic properties of β-amyloid. The cytotoxicity of β-amyloid was first established in primary cell cultures from rodent brains and also in human cell cultures. The work of Mattson et al. (J. Neurosci., 12:376-389, 1992) indicates that β-amyloid, in the presence of the excitatory neurotransmitter glutamate, causes an immediate pathological increase in intracellular calcium, which is believed to be very toxic to the cell through its greatly increased second messenger activities.
[0007] Concomitant with β-amyloid production and β-amyloid deposition, there exists robust activation of inflammatory pathways in AD brain, including production of proinflammatory cytokines and acute-phase reactants in and around β-amyloid deposits (McGeer et al., J. Leukocyte Biol, 65:409-15, 1999). Activation of the brain's resident innate immune
cells, the microglia, is thought to be intimately involved in this inflammatory cascade. It has been demonstrated that reactive microglia produce pro-inflammatory cytokines, such as inflammatory proteins and acute phase reactants, such as alpha- 1-antichymotrypsin, transforming growth factor β, apolipoprotein E and complement factors, all of which have been shown to be localized to β-amyloid plaques and to promote β-amyloid plaque "condensation" or maturation (Nilsson et al., J. Neurosci. 21 :1444-5, 2001), and which at high levels promote neurodegeneration. Epidemiological studies have shown that patients using non-steroidal anti-inflammatory drugs (NSAIDS) have as much as a 50% reduced risk for AD (Rogers et al., Neurobiol. Aging 17:681-6, 1996), and post-mortem evaluation of AD patients who have undergone NSAID treatment has demonstrated that risk reduction is associated with diminished numbers of activated microglia (Mackenzie et al., Neurology 50:986-90, 1998). Further, when Tg APPsw mice, a mouse model for Alzheimer's disease, are given an NSAID (ibuprofen), these animals show reduction in β-amyloid deposits, astrocytosis, and dystrophic neurites correlating with decreased microglial activation (Lim et al., J. Neurosci. 20:5709-14, 2000).
[0008] At present, treatment for AD is limited. However, there are several drugs approved by the FDA to improve or stabilize symptoms of AD (Alzheimer's Disease Medications Fact Sheet: (July 2004) U.S. Department of Health and Human Services), including Aricept® (donepezil), Exelon® (rivastigmine), Reminyl® (galantamine) Cognex® (tacrine) and Namenda ® (memantine). The effects with many drugs currently in use is small (Tariot et al., JAMA (2004), 291 : 317-24). Treatments for AD remain a largely unmet clinical need. [0009] U.S. Patent Application No. 2005009885 (January 13, 2005) (Mullan et al.) discloses a method for reducing beta-amyloid deposition using nilvadipine, as wells as methods of diagnosing cerebral amyloidogenic diseases using nilvadipine. Nimodipine has been studied for the treatment of dementia. Fritze et al., J. Neural Transm. (1995) 46: 439- 453; and Forette et al. Lancet (1998) 352: 1347-1351).
[0010] Augmentation of capacitative calcium entry (CCE) through the identification of agonist of plasma membrane store-operated calcium channels that mediate CCE, has been suggested as a treatment for AD (Tanzi et al. Neuron (2000) 27: 561-572). U.S. Patent Application Publication No. 20020015941 (February 7, 2002) discloses a method for the treatment of a neurodegenerative disease such as AD involving administering an agent which is capable of potentiating CCE.
[0011] There continues to be a need to identify compounds that can treat the inexorable progression of brain degeneration which is a hallmark of AD, wherein the treatment addresses β-amyloid production and the concomitant β-amyloid deposition, β-amyloid neurotoxicity (including abnormal hyperphosphorylation of tau), microglial-activated inflammation, and altered or over expression of APP which is seen in AD patients.
SUMMARY
[0012] Compounds and combinations thereof are provided that can be used for the reduction of β-amyloid. The compound combinations can be used in a method of treating a disease associated with cerebral accumulation of β-amyloid in animals or humans afflicted with the disease, such as AD, the method comprising administering a therapeutically effective amount of a compound or combination thereof disclosed herein. Preferably the compound or combination opposes the pathophysiological effects of the cerebral accumulation of Alzheimer's amyloid, and may, for example, reduce β-amyloid production, β-amyloid deposition, β-amyloid neurotoxicity and/or microgliosis in animals and humans afflicted with the disease.
[0013] It has been found that certain compounds can be effectively used in combination to obtain beneficial results. Compounds that can be used in combination include dihydropyridine compounds such as those disclosed herein. In order to administer the combination of compounds, the compounds may be provided together in a pharmaceutical composition, or may be in separate pharmaceutical formulations, and can be administered together or sequentally. In certain embodiments, two or more compounds are used which can act to lower β-amyloid production synergistically.
[0014] Examples of compounds that can be used in combination include any one of the combinations 1-6 of compounds A and B shown below in Table Ia:
Table Ia:
[0015] Thus, SR 33805, nilvadipine, HTS 01512, and amlodipine, can each be used in combination with any other of the compounds listed in Table Ia or Ib or other compound described herein.
[0016] In another embodiment, compounds that can be used in combination include any one of the combinations 1-6 of compounds A and B shown below in Table Ib:
Table Ib:
[0017] Thus, RJC03403 and nitrendipine can each be used in combination with any of the compounds listed in Table Ia or Ib or other compound described herein. [0018] The compounds and combinations thereof also can be used in a method for treating head injury, and optionally reducing the risk of β-amyloid production, β-amyloid deposition, β-amyloid neurotoxicity (including abnormal hyperphosphorylation of tau) or microgliosis, in
animals or humans suffering from traumatic brain injury, the method comprising administering to the animal or human immediately after the head injury a therapeutically effective amount a compound disclosed herein or combination thereof, and then optionally continuing treatment for a prescribed period of time thereafter.
[0019] The compounds and combinations thereof also can be used in a method for diagnosing diseases associated with cerebral accumulation of Alzheimer's amyloid, such as AD, in an animal or human, or determining if the animal or human is at risk for developing cerebral accumulation of Alzheimer's amyloid, the method comprising: taking a first measurement of β-amyloid concentration in a body fluid such as plasma, serum, whole blood, urine or cerebral spinal fluid (CSF) of the animal or human; administering to the animal or human a diagnostically effective amount of a compound or combination thereof disclosed herein; taking a second measurement of β-amyloid concentration from plasma, serum, whole blood, urine or CSF of the animal or human at a later time; and calculating the difference between the first measurement and the second measurement. A change in the concentration of β-amyloid or fragment thereof in plasma, serum, whole blood, urine or CSF in the second measurement compared to the first measurement, such as an increase in concentration, indicates a risk of developing or a possible diagnosis of a disease associated with cerebral accumulation of Alzheimer's amyloid in the animal or human.
[0020] A variety of compounds disclosed herein can be used in combination in the methods disclosed herein for the treatment and diagnosis of diseases associated with cerebral accumulation of Alzheimer's amyloid.
[0021] In one embodiment, a dihydropyridine, such as nilvadipine, nimodipine or nitrendipine in used in combination with a second dihydropyridine.
[0022] In another embodiment, the compounds used in combination can be selected from any of the compounds disclosed herein, including one or more of: an imidazole compound, an isoquinoline alkaloid compound, a calmodulin-mediated enzyme activation inhibitor, an inhibitor of kinase activity of the platelet-derived growth factor (PDGF) receptor, an NF -kB activation inhibitor, a diterpene or triterpene compound, a quinazoline compound, a sesquiterpene lactone, or an inhibitor of IKK-2.
[0023] In one embodiment, the compounds decreases CCE, for example, by at least about 10% or more in the medium of cultured cells that for example overexpress APP or a fragment thereof, and/or optionally reduces β amyloid production, for example, by at least about 20% or more, in cells that overexpress APP or a fragment thereof.
[0024] In one embodiment, compounds and combinations thereof which can be used for the treatment and diagnosis of diseases associated with cerebral accumulation of Alzheimer's amyloid in the embodiments disclosed herein include, without limitation:
[0025] SKF96365 (l-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]- lH-imidazole hydrochloride), econazole, clotrimazole;
[0026] SR 33805 (3,4-dimethoxy-N-methyl-N-[3-[4-[[l-methyl-3-(l-methylethyl) - lH-in-dol-2-yl]sulfonyl]phenoxy]propyl]benzeneethanamine oxalate);
[0027] loperamide;
[0028] tetrandrine;
[0029] R24571 (l-[bis(p-chlorophenyl)methyl]-3-[2-(2,4-di-chloro-β-(2,4- dichlorobenzyl-oxy)phenethyl)]-imidazolium chloride);
[0030] amlodipine;
[0031] nitrendipine;
[0032] MRS 1845 (N-propargylnitrendipine);
[0033] tyrphostin A9;
[0034] BTB 14328 (diethyl 4-(4-chlorophenyl)-2,6-dimethyl- 1 ,4-dihydropyridine-
3,5-dicarboxylate);
[0035] CD 04170 (diethyl 4-{5-[3,5-di(trifluoromethyl)phenyl]-2-furyl}-2,6- dimethyl-l,4-dihydro-pyridine-3,5-dicarboxylate);
[0036] HTS 01512 (l-cyclohexyl-5-phenyl-l,6-dihydro-2,3-pyridinedione);
[0037] HTS 07578 (4-(l,3-diphenyl-lH-pyrazol-4-yl)-2-oxo-6-phenyl-l,2-dihydro -
3-pyridinecarbonitrile);
[0038] HTS 10306 (2-oxo-6-phenyl-4-(2-thienyl)-l,2-dihydro-3- pyridinecarbonitrile);
[0039] JFD 01209 (diethyl 4-(4-bromophenyl)-2,6-dimethyl- 1 ,4-dihydropyridine-
3,5-dicarboxylate);
[0040] JFD 03266 (diethyl 2,6-dimethyl-4-(4-nitrophenyl)- 1 ,4-dihydropyridine-
3,5-dicarboxylate;
[0041] JFD 03274 (diethyl 4-(3-chlorophenyl)-2,6-dimethyl-l, 4-dihydropyridine-
3,5-dicarboxylate);
[0042] JFD 03282 (diethyl 2,6-dimethyl-4-(4-methylphenyl)- 1 ,4-dihydropyridine-
3 ,5 -dicarboxylate) : ;
[0043] JFD 03292 (4-(3,4-dichlorophenyl)-2,6-dimethyl-l,4-dihydropyridine-3,5 - dicarbonitrile;
[0044] JFD 03293 (dimethyl 4-(3,4-dichlorophenyl)-2,6-dimethyl-l,4- dihydropyridine-3,5-dicarboxylate);
[0045] JFD 03294 (diethyl 4-(3,4-dichlorophenyl)-2,6-dimethyl-l ,4- dihydropyridine-3,5-dicarboxylate);
[0046] JFD 03305 (diethyl 4-(2-chlorophenyl)-2,6-dimethyl- 1 ,4-dihydropyridine-
3,5-dicarboxylate);
[0047] JFD 03311 (diethyl 2,6-dimethyl-4-(2-nitrophenyl)- 1 ,4-dihydropyridine-
3,5-dicarboxylate);
[0048] JFD 03318 (diethyl 4-(4-fluorophenyl)-2,6-dimethyl-l ,4-dihydropyridine-
3,5-dicarboxylate);
[0049] PD 00463 (l-[4-(4-chlorophenoxy)phenyl]-4-phenyldihydropyridine-
2,6(lH,3H)-dione);
[0050] RJC 03403 (diethyl 4-(2,4-dichlorophenyl)-2,6-dimethyl-l,4-dihydro-3,5- pyridinedicarboxylate);
[0051] RJC 03405 (diethyl 2,6-dimethyl-4- {5-[2-(frifluoromethyl)phenyl]-2-furyl} - l,4-dihydro-3,5-pyridinedicarboxylate);
[0052] RJC 03413 (diethyl 4-(2-chloro-4-methoxyphenyl)-2,6-dimethyl- 1 ,4- dihydro-3,5-pyridinedicarboxylate);
[0053] RJC 03423 (dimethyl 4-(2,4-dichlorophenyl)-2,6-dimethyl-l,4-dihydro-3,5- pyridinedicarboxylate);
[0054] SEW 02070 (dimethyl 4- {5-[2-(methoxycarbonyl)-3-thienyl]-2-furyl} -2,6- dimethyl-l,4-dihydropyridine-3,5-dicarboxylate);
[0055] XBX 00343 (diethyl 2,6-dimethyl-4-(3-nitrophenyl)- 1 ,4-dihydropyridine-
3,5-dicarboxylate);
[0056] R-niguldipine,
[0057] (S)-(+)-niguldipine,
[0058] artemisinin;
[0059] celastrol;
[0060] 6-amino-4-(4-phenoxyphenylethylamino)quinazoline;
[0061] isohelenin;
[0062] kamebakaurin;
[0063] parthenolide; and
[0064] IKK-2 Inhibitor IV;
[0065] or salts, esters, prodrugs, stereoisomers, or derivatives thereof.
[0066] In one embodiment, the compound is one of the following compounds: [0067] HTS 01512 (l-cyclohexyl-5-phenyl-l,6-dihydro-2,3-pyridinedione):
[0068] BTB 14328 (diethyl 4-(4-chlorophenyl)-2,6-dimethyl-l,4-dihydropyridine-3,5- dicarboxylate):
[0069] CD 04170 (diethyl 4-{5-[3,5-di(trifluoromethyl)phenyl]-2-furyl}-2,6-dimethyl-l ,4- dihydro-pyridine-3 ,5 -dicarboxylate) :
[0070] JFD 03292 (4-(3,4-dichlorophenyl)-2,6-dimethyl-l,4-dihydropyridine-3,5 - dicarbonitrile:
[0071] PD 00463 (l-[4-(4-chlorophenoxy)phenyl]-4-phenyldihydropyridine-2,6(l H,3H)- dione):
[0072] In another embodiment, the compound is one of the following compounds: [0073] Diethyl 4-(2-bromophenyl)-l ,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylate:
2-23
[0074] Diethyl 4-(2-fluorophenyl)-l ,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylate:
2-27
[0075] Di-tert-butyl 4-(2-fluorophenyl)-l,4-dihydro-2,6-dimethylpyridine-3,5- dicarboxylate:
2-28
[0076] Diethyl 1 ,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarb oxylate:
2-29
[0077] Di-tert-butyl 4-(2-bromophenyl)-l ,4-dihydro-2,6-dimethylpyridine-3,5- dicarboxylate:
2-32
[0078] Di-tert-butyl 1 ,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5- dicarboxylate:
2-33
[0079] Di-tert-butyl 4-(4-bromo-2-fluorophenyl)-l,4-dihydro-2,6-dimethylpyridine- 3,5- dicarboxylate:
[0080] Bis(2-methoxyethyl) 4-(4-bromophenyl)- 1 ,4-dihydro-2,6-dimethylpyridine-3 ,5 - dicarboxylate:
[0081] Diethyl 4-(5-bromo-2-fluorophenyl)-l,4-dihydro-2,6-dimethylpyridine- 3,5- dicarboxylate
3-41
[0082] In one embodiment, two or more compounds are used for the reduction of beta- amyloid in the methods and compositions disclosed herein, which are optionally selected from SKF96365, econazole, clotrimazole, SR33805, loperamide, tetrandrine, R24571, amlodipine, nitrendipine, MRS1845, tyrphostin A9, BTB 14328, CD 04170, HTS 01512, HTS 07578, HTS 10306, JFD 01209, JFD 03266, JFD 03274, JFD 03282, JFD 03292, JFD 03293, JFD 03294, JFD 03305, JFD 03311, JFD 03318, PD 00463, RJC 03403, RJC 03405, RJC 03413, RJC 03423, SEW 02070, XBX 00343, R-niguldipine, (S)-(+)-niguldipine,
artemisinin, celastrol, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline, isohelenin, kamebakaurin, parthenolide, IKK-2 Inhibitor IV, 2-23, 2-27, 2-28, 2-29, 2-32, 2-33, 3-34, 3- 38, 3-41, a compound as disclosed in Tables 1, 2 or 3 herein, or a compound of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, or XI or other compound disclosed herein, or salt, prodrug or derivative thereof.
[0083] In certain embodiments, the compounds and combinations thereof disclosed herein can be further combined with other treatments directed to reduction of β-amyloid or the treatment of a disease associated with the cerebral accumulation of β-amyloid, or treatment or alleviation of its symptoms or sequelae, in a patient in need thereof. Thus, compounds and combinations of the invention can be administered as part of a therapeutic regimen that includes co-administration of one or more standard or experimental therapeutics for the treatment or prophylaxis or a disease or symptom associated with the accumulation β- amyloid, e.g., acetylcholinesterase inhibitors, secretase inhibitors, anti-inflammatories and active or passive vaccines. In certain embodiments the combinations of the invention comprise ARICEPT® (donepezil), EXELON® (rivastigmine), REMINYL® (galantamine), COGNEX® (tacrine), NAMENDA® (memantine), RAZADYNE® (galantamine), LY2062430 (Eli Lilly), LY450139 (Eli Lily), FLURIZAN® (r-fiurbiprofen), ALZHEMED® (tramiprosate), PBT-2 (Prana Biotechnology), phenserine, TTP488 (Trans Tech Pharma), bapineuzumab (Elan Corp.), AAC-OOl (Elan Corp.), KETSYN® (AC-1202, Accera), AZD3480 (AstraZeneca), CX717(Cortex Pharmaceuticals), AC-3933 (Dainippon Sumitomo), Debio-9902 SR (Debiopharm), NERAMEXANE® (MRZ 21579, Forest Laboratories), AVANDIA® (rosiglitazone), dimebon, MEM 1003 (Memory Pharmaceuticals), MEM 3454 (Memory Pharmaceuticals), MK-0952 (Merk), huperzine A, SGS742 (Saegis Pharmaceuticals), XAPRILA® (xaliproden), SR 57667 (Sanofi-Aventis), AVE 1625 (Sanofi-Aventis), AZILECT® (rasagiline mesylate), ladostigil tartrate, VP 4896 (Voyager Pharmaceutical) or lecozotan (SRA-333), or combinations thereof. [0084] In one embodiment, one or more of the compounds decreases CCE by at least about 5%, 10%, 15%, 20% or more in cells, that for example overexpress APP or a fragment thereof, and/or optionally reduces β-amyloid production by at least about 5%, 10%, 15%, 20%, 25%, 30%, 50%, or more in cells that overexpress APP or a fragment thereof, as can be measured, for example in a culture medium comprising the cells. The method may in one embodiment include one or more of reducing β-amyloid production, β-amyloid deposition, β- amyloid neurotoxicity (including abnormal hyperphosphorylation of tau) and microgliosis.
Because most diseases having cerebral accumulation of Alzheimer's amyloid, such as AD, are chronic, progressive, intractable brain dementias, it is contemplated that the duration of treatment with at least one of the active agents can optionally last for up to the lifetime of the animal or human.
[0085] In one embodiment, one or more of the compounds decrease capacitative calcium entry, for example, by about 5%, 10%, 15%, 20%, 22%, 25%, 28%, 30%, 40%, 50%, 60% or more in cultured mammalian cells, for example cells which overexpress amyloid precursor protein (APP), wherein optionally the compounds also decrease β-amyloid production. Such compounds can be used in the methods disclosed herein.
[0086] In one embodiment one or more of the compounds has the property of decreasing CCE, for example, by 10% or more in cultured cells which for example overexpress APP or a fragment thereof, and optionally reduce β-amyloid production, e.g., production of total ABi_ 40 and ABi_42, by at least about 20% or more in cells that overexpress APP or a fragment thereof. [0087] The therapeutically effective amount of the one or more compounds that is administered, e.g., in unit dosage form to animals or humans afflicted with a cerebral amyloidogenic disease or suffering from a traumatic brain injury, as well as administered for the purpose of determining the risk of developing and/or a diagnosis of a cerebral amyloidogenic disease in an animal or human, according to the methods of the present invention, can range from for example from about 0.05 mg to 20 mg per day, about 2 mg to 15 mg per day about 4 mg to 12 mg per day, or about 8 mg per day. The daily dosage in one embodiment can be administered in a single unit dose or divided into two, three or four unit doses per day.
[0088] In one embodiment, a method for treating a disease associated with cerebral accumulation of Alzheimer amyloid is provided, comprising administering to an animal or human a therapeutically effective amount of at least two compounds that decrease capacitative calcium entry by at least about 10% or more in cells which optionally overexpress APP or a fragment thereof. Optionally, the cells are Chinese hamster ovary cells that overexpress APP751, or are selected from human neuronal precursor cells (HNPC); primary culture of human astrocytes; neuroblastoma cells; human brain microvascular endothelial primary culture; or human umbilical cord endothelial cells (HUVEC). [0089] In one embodiment, each compound is independently administered in an amount of about 0.02 to 1000 mg per unit dose; or about 0.5 to 500 mg per unit dose.
[0090] In one non-limiting embodiment, the compounds in combination are each other than as described in U.S. Pat. Publ. No. 2005/0009885, published January 13, 2005. In another non-limiting embodiment, the compounds are other than nilvadipine, nimodipine or nitrendipine, or a pharmaceutically acceptable salt, or free base thereof, or prodrug thereof. [0091] In yet another embodiment, two or more compounds are used for the treatment of a disorder associated with cerebral accumulation, wherein at least one of the compounds is nilvadipine, nimodipine or nitrendipine.
[0092] The disease associated with cerebral accumulation of Alzheimer's amyloid is for example, Alzheimer's disease, cerebral amyloid angiopathy, hereditary cerebral hemorrhage with amyloidosis Dutch-type, other forms of familial Alzheimer's disease and familial cerebral Alzheimer's amyloid angiopathy. Cerebral amyloidogenic diseases that can be treated or diagnosed include transmissible spongiform encephalopathy, scrapie, traumatic brain injury, cerebral amyloid angiopathy, and Gerstmann-Straussler-Scheinker syndrome. [0093] The invention thus provides a pharmaceutical composition comprising (i) a therapeutically effective amount of the compounds or combinations thereof disclosed herein; and (ii) a pharmaceutically acceptable carrier. The invention also provides combination therapy methods. The methods of the invention can be carried out in combination with any standard or experimental treatment for the particular indication, e.g., treatment or amelioration of a disease, or symptom thereof, associated with the accumulation of an amyloid protein, in particular, cerebral accumulation of β-amyloid protein. The compounds and combinations thereof of the invention may be administered with other therapies, e.g., acetylcholinesterase inhibitors, secretase inhibitors, anti-inflammatories and active or passive vaccines
[0094] In certain embodiments of the invention, pharmaceutical compositions are provided for use in accordance with the methods of the invention, said pharmaceutical compositions comprising the compounds and/or combinations thereof disclosed herein, in an amount effective to prevent, treat, manage, or ameliorate a disease associated with the accumulation of an amyloid protein (e.g., cerebral accumulation of β-amyloid (e.g., Alzheimer's disease)), or one or more symptoms thereof, and a pharmaceutically acceptable carrier. The invention also provides pharmaceutical compositions for use in accordance with the methods of the invention, said pharmaceutical compositions comprising the compounds and/or combinations disclosed herein, a standard or experimental prophylactic or therapeutic agent for the
treatment or amelioration of a disease associated with the accumulation of an amyloid protein (or symptom thereof), and a pharmaceutically acceptable carrier..
BRIEF DESCRIPTION OF THE DRAWINGS
[0095] Figures IA-D are bar graphs showing the effect of various calcium channel blockers, such as SKF 96365, nilvadipine, nitrendipine and amlodipine, on Aβl-40 production by 7W WT APP 751 Chinese hamster ovary (7W WT APP 751 CHO) cells. Fig.
IA shows the effect of calcium channel blocker treatment after 4 hours. Fig. IB shows the effect of calcium channel blocker treatment after 24 hours. Fig. 1C shows the effect of calcium channel blocker treatment plated at low density after 24 hours. Fig. ID shows the effect of calcium channel blocker treatment plated at low density after 48 hours.
[0096] Figure 2 is a bar graph showing the effect of three CCE inhibitors, SKF96365, econazole and tyrphostin A9, on Aβl-40, Aβl-42 and total β-amyloid production by 7W WT
APP751 CHO cells.
[0097] Figure 3 is a bar graph showing the effect of various dihydropyridine calcium channel blockers, such as nilvadipine, nitrendipine and MRS 1835, on Aβl-40, Aβl-42 and total β-amyloid production by 7W WT APP751 CHO cells.
[0098] Figure 4 is a bar graph showing the effect of various non-dihydropyridine and dihydropyridine calcium channel blockers, such as SR 33805, MRS 1845, loperamide, clotrimazole and tetrandine, on Aβl-40, Aβl-42 and total β-amyloid production by 7W WT
APP751 CHO cells.
[0099] Figure 5A-B are bar graphs showing the effect of treating 7W WT APP751 CHO cells for 24 hours with various dihydropyridine compounds (obtained from Maybridge;
England) on Aβl-40, Aβl-42 and total β-amyloid production.
[00100] Figure 6 is a bar graph showing the effect of various NF -kB activation inhibitors on Aβl-40, Aβl-42 and total β-amyloid production by 7W WT APP751 CHO cells.
[00101] Figure 7 A is a graph showing that compounds which inhibit CCE in CHO cells also inhibit total Aβ production.
[00102] Figure 7B is a list of compounds represented in Figure 7A.
[00103] Figure 8A is a graph showing that compounds which inhibit CCE in CHO cells also inhibit Aβ-40 production.
[00104] Figure 8B is a list of compounds represented in Figure 8A.
[00105] Figures 9-11 show compounds useful in the methods and compositions described herein.
[00106] Figure 12-14 are bar graphs showing the effect of various compounds on Aβl-40,
Aβl-42 and total (Aβl-40 plus Aβl-42) β-amyloid production.
[00107] Figure 15 is a bar graph showing the effect of various compounds on β-amyloid production.
[00108] Figures 16-21 show compounds useful in the methods and compositions disclosed herein.
[00109] Figures 22A, 22B, 23A and 23B are graphs showing the effect of various compounds on Aβl-40 and Aβl-42 production.
[00110] Figure 24 is a bar graph showing the effect of various compounds on Aβl-40 production.
[00111] Figure 25 is a bar graph showing the effect of various compound combinations on
Aβl-40 production.
[00112] Figure 26 is a bar graph showing the effect of various compound combinations on
Aβl-42 production.
[00113] Figure 27 is a bar graph showing the effect of various compound combinations on
Aβl-40 production.
DETAILED DESCRIPTION
[00114] Compounds are provided which can be used in combination for the treatment of disorders associated with the accumulation of cerebral β-amyloid. In one embodiment, compounds that decrease capacitative calcium entry in mammalian cells that overexpress amyloid precursor protein (APP) or a fragment thereof, and/or can decrease β-amyloid production in the mammalian cells can be used in combination in the diagnosis and treatment of diseases associated with the accumulation of β-amyloid in individuals. Compounds and pharmaceutical compositions comprising the compounds, are provided, that can be used in one embodiment to treat the inexorable progression of brain degeneration that is a hallmark of certain diseases associated with cerebral accumulation of Alzheimer's amyloid, such as Alzheimer's disease (AD), in animals and humans.
Definitions
[00115] As used herein, the term "Alzheimer's amyloid" is defined as a β-amyloid amino acid fragment that is for example proteolytically derived from amyloid precursor protein
(APP). A β-amyloid amino acid fragment may include, for example, about 5 to 43 or 5 to 47 consecutive amino acids of the β-amyloid sequence. As used herein, the terms "β-amyloid,"
"β-amyloid protein" and "Aβ" are used interchangeably with Alzheimer's amyloid that accumulates cerebrally in an animal or human.
[00116] As used herein the phrase a cell that "overexpresses APP or fragment thereof refers to a cell that overexpresses an amyloid precursor protein, or fragment thereof, that in one preferred embodiment, includes a β-amyloid sequence and β and γ secretase cleavage sites. The cell that overexpresses APP or a fragment thereof preferably expresses an APP or fragment thereof that produces β-amyloid in the cell in which it is expressed.
[00117] As used herein, the term "amyloidogenic disease" includes a disease associated with cerebral accumulation of Alzheimer's amyloid.
[00118] The term "alkyl", as used herein, unless otherwise specified, includes a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon, of Ci_ 22 and specifically includes methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, secbutyl, t- butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, heptyl, cycloheptyl, octyl, cyclo-octyl, dodecyl, tridecyl, pentadecyl, icosyl, hemicosyl, and decosyl. The alkyl group may be optionally substituted with, e.g., halogen (fluoro, chloro, bromo or iodo), hydroxy, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, heterocycle, phenyl, aryl, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al, Protective Groups in Organic
Synthesis, John Wiley and Sons, Second Edition, 1991, hereby incorporated by reference.
[00119] The term "lower alkyl", as used herein, and unless otherwise specified, includes a
Ci to C 4 saturated straight, branched, or if appropriate, a cyclic (for example, cyclopropyl) alkyl group, which is optionally substituted.
[00120] The term "aralkyl" as used herein unless otherwise specified, includes an aryl group linked to the molecule through an alkyl group.
[00121] The term "alkaryl" as used herein unless otherwise specified, includes an alkyl group linked to the molecule through an aryl group.
[00122] The term "aryl ether" as herein unless otherwise specified, includes an aryl group linked to the molecule through an ether group.
[00123] The term "alkyl ether" as herein unless otherwise specified, includes an alkyl group linked to the molecule through an ether group.
[00124] The term "aryl thioether" as herein unless otherwise specified, includes an aryl group linked to the molecule through a sulfur.
[00125] The term "alkyl thioether" as herein unless otherwise specified, includes an alkyl group linked to the molecule through a sulfur.
[00126] The term "amino" includes an "-N(R) 2 " group, and includes primary amines, and secondary and tertiary amines which is optionally substituted for example with alkyl, aryl, hetercycle, and or sulfonyl groups. Thus, (R) 2 may include, but is not limited to, two hydrogens, a hydrogen and an alkyl, a hydrogen and an aryl, a hydrogen and an alkenyl, two alkyls, two aryls, two alkenyls, one alkyl and one alkenyl, one alkyl and one aryl, or one aryl and one alkenyl.
[00127] Whenever a range of carbon atoms is referred to, it includes independently and separately every member of the range. As a nonlimiting example, the term "C 1 -C 10 alkyl" is considered to include, independently, each member of the group, such that, for example, C 1 -
Cio alkyl includes straight, branched and where appropriate cyclic C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 ,
Cg, Cg and C 10 alkyl functionalities.
[00128] The term "amido" includes a moiety represented by the structure "-C(O)N(R) 2 ", wherein R may independently include H, alkyl, alkenyl and aryl that is optionally substituted.
[00129] The term "protected" as used herein and unless otherwise defined includes a group that is added to an atom such as an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.
[00130] The term "aryl", as used herein, and unless otherwise specified, includes a stable monocyclic, bicyclic, or tricyclic carbon ring with up to 8 members in each ring, and at least one ring being aromatic. Examples include, but are not limited to, benzyl, phenyl, biphenyl, or naphthyl. The aryl group can be substituted with one or more moieties including halogen
(fluoro, chloro, bromo or iodo), hydroxy, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as
taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons,
Second Edition, 1991.
[00131] The term "halo", as used herein, includes chloro, bromo, iodo, and fluoro.
[00132] The term "alkenyl" includes a straight, branched, or cyclic hydrocarbon of C2-22 with at least one double bond. Examples include, but are not limited to, vinyl, allyl, and methyl-vinyl. The alkenyl group can be optionally substituted in the same manner as described above for the alkyl groups.
[00133] The term "alkynyl" includes a C2-22 straight or branched hydrocarbon with at least one triple bond. The alkynyl group can be optionally substituted in the same manner as described above for the alkyl groups.
[00134] The term "alkoxy" includes a moiety of the structure -O-alkyl.
[00135] The term "heterocycle" or "heterocyclic" includes a saturated, unsaturated, or aromatic stable 5 to 7 membered monocyclic or 8 to 11 membered bicyclic heterocyclic ring that consists of carbon atoms and from one to three heteroatoms including but not limited to
O, S, N, and P; and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and/or the nitrogen atoms quarternized and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure.
Nonlimiting examples or heterocyclic groups include pyrrolyl, pyrimidyl, pyridinyl, imidazolyl, pyridyl, furanyl, pyrazole, oxazolyl, oxirane, isooxazolyl, indolyl, isoindolyl, thiazolyl, isothiazolyl, quinolyl, tetrazolyl, bonzofuranyl, thiophrene, piperazine, and pyrrolidine.
[00136] The term "acyl" includes a group of the formula R'C(O), wherein R' is a H, or a straight, branched, or cyclic, substituted or unsubstituted alkyl or aryl.
[00137] The term "host", as used herein, unless otherwise specified, includes mammals
(e.g., cats, dogs, horses, mice, etc.), humans, or other organisms in need of treatment, all of which can be treated or diagnosed using the methods described herein.
[00138] The term "in combination," as used herein refers to the use of more than one prophylactic and/or therapeutic agents. The use of the term "in combination" does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with a disorder, e.g., amyloid disease or disorder, especially Alzheimer's disease. A first prophylactic or therapeutic agent can be administered prior to {e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48
hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject which had, has, or is susceptible to a disorder. The prophylactic or therapeutic agents are administered to a subject in a sequence and within a time interval such that the agent of the invention can act together with the other agent to provide an increased benefit than if they were administered otherwise. Any additional prophylactic or therapeutic agent can be administered in any order with the other additional prophylactic or therapeutic agents. [00139] The term, "therapeutically effective amount" and analgous terms/phrases, as used herein, refer to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder associated with the accumulation of β-amyloid in a subject, in particular, cerebral accumulation of β-amyloid. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease or symptoms thereof, e.g., delay the appearance or minimize the progression of symptoms of Alzheimer's disease. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention, e.g. , a compound or combination disclosed herein, means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease associated with the accumulation of β-amyloid in a subject. [00140] The term "treatment" as used herein includes any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. [00141] The term "pharmaceutically acceptable salt" as used herein, unless otherwise specified, includes those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of hosts without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio and effective for their intended use. The salts can be prepared in situ during the final isolation and purification of one or more compounds of the composition, or separately by reacting the free base function with a suitable organic acid. Non-pharmaceutically acceptable acids and bases also find use herein, as for example, in the synthesis and/or purification of the compounds of interest. Nonlimiting examples of such salts are (a) acid addition salts formed with inorganic
salts (for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic salts such as acetic acid, oxalic acid, tartaric acid, succinic acid, ascorbic acid, benzoic acid, tannic acid, and the like; (b) base addition salts formed with metal cations such as zinc, calcium, magnesium, aluminum, copper, nickel and the like; (c) combinations of (a) and (b).
[00142] The term "pharmaceutically acceptable esters" as used herein, unless otherwise specified, includes those esters of one or more compounds, which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of hosts without undue toxicity, irritation, allergic response and the like, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.
[00143] The term "pharmaceutically acceptable prodrugs" as used herein, unless otherwise specified, includes those prodrugs of one or more compounds of the composition which are, with the scope of sound medical judgment, suitable for use in contact with the tissues of hosts without undue toxicity, irritation, allergic response and the like, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use. Pharmaceutically acceptable prodrugs also include zwitterionic forms, where possible, of one or more compounds of the composition. The term "prodrug" includes compounds that are rapidly transformed in vivo to yield the parent compound, for example by hydrolysis in blood. [00144] The term "enantiomerically enriched", as used herein, refers to a compound that is a mixture of enantiomers in which one enantiomer is present in excess, and preferably present to the extent of 95% or more, and more preferably 98% or more, including 100%. [00145] The term "optionally substituted," as used herein, includes substituted and unsubstituted. Wherein a group is referenced as "optionally substituted" the group may be optionally substituted with e.g., halogen, hydroxyl, amino, alkylester, arylester, silylester, alkylamino, arylamino, alkylamido, arylamido, alkoxy, aryloxy, nitro, cyano, alkenyl, alkynyl, heterocycles, sulfonic acid, sulfate, phosphonic acid, phosphate, boronic acid, or borate.
In Vitro Assay Methods
[00146] Compounds which decrease capacitative calcium entry in mammalian cells that overexpress amyloid precursor protein (APP) can decrease β-amyloid production in the cells. Such compounds can be used in methods for the treatment of diseases associated with the
accumulation of β-amyloid, as described in U.S. Application No. 11/328,470, filed January 9, 2006, the disclosure of which is incorporated herein.
[00147] In one embodiment, an in vitro method is provided for screening for compounds which are useful in methods of treatment and diagnosis of diseases associated with β-amyloid accumulation, wherein the method comprises detecting a reduction in CCE measurement in the cells upon exposure to the test compound in comparison to the CCE measurement in the absence of the compound. Such compounds that reduce CCE are useful in decreasing β- amyloid production in mammalian cells overexpressing the protein, and are therapeutically and diagnostically useful in the treatment of diseases associated with β-amyloid production, such as Azheimer's disease.
[00148] Capacitative calcium entry (CCE) is one of the most prevalent mechanisms of cellular Ca 2+ signaling and, unlike the other calcium channels, CCE is ubiquitous in cells. Capacitative calcium entry involves the activation of plasma membrane calcium channels to cause the influx of extracellular calcium, in response to a fall in Ca 2+ concentration within the lumen of Ca 2+ storing organelles, most commonly components of the endoplasmic reticulum. The endoplasmic reticulum is believed to signal the plasma membrane calcium channels in the process of capacitative calcium entry. Capacitative calcium entry replenishes cellular Ca 2+ stores at a rapid rate, for example, as required following transient receptor activation by neurotransmitters. J. W. Putney, Jr., Molecular Inventions, 1 :84, June, 2001. Cells which overexpress APP or fragment thereof surprisingly can respond to CCE inhibitors by reducing β-amyloid production. Such CCE inhibitors are useful in reducing β-amyloid production and treating diseases associated with β-amyloid accumulation.
[00149] In one embodiment, the method comprises exposing cells to the test compound or compounds; measuring capacitative calcium entry (CCE) in the cells; and identifying a reduction in CCE, in comparison to control cells unexposed to the compound or compounds, as an indicator of the effectiveness of the compound in the treatment or diagnosis of a disease associated with the accumulation of β-amyloid. The cultured cells optionally are cells that overexpress amyloid precursor protein (APP) or a fragment thereof. In the assay, a measurement of CCE in cells unexposed to compound can be obtained as a control, to allow a comparison of the CCE measurement of exposed and unexposed cells. A decrease in CCE of, for example, about 5%, 10%, 15%, 20% or more in the exposed cultured cells in comparison to cells unexposed to compound indicates the potential therapeutic effectiveness
of the compound or compounds to treat animals or humans afflicted with a disease associated with cerebral accumulation of Alzheimer's amyloid.
[00150] Optionally or additionally, in an in vitro assay method to identify compounds useful in the treatment of diseases associated with the accumulation of β-amyloid, an assay to determine the compounds' ability to decrease β-amyloid production is conducted. For example, the test compound or compounds are exposed to cells that overexpress APP or a fragment thereof; β-amyloid production in the cells is measured; and a decrease in β-amyloid production of e.g., at least about 20% more in the cells that overexpress APP or a fragment thereof is detected as an indicator of the therapeutic usefulness of the compound or compounds to treat animals or humans afflicted with a disease associated with cerebral accumulation of Alzheimer's amyloid. The assay is conducted using cells that overexpress APP or a fragment thereof available in the art such as Chinese hamster ovary cells that overexpress APP751. The β-amyloid measured, is, e.g., Aβl-40, Aβl-42, or total Aβl-40 + Aβl-42. A decrease in the production of Aβl-40 and/or Aβl-42, and in particular, total ABl- 40 + AB1-42, of, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 50%, or more, indicates the therapeutic effectiveness of the compound(s) to treat animals or humans afflicted with a disease associated with cerebral accumulation of Alzheimer's amyloid. The β-amyloid concentrations can be measured for example, intracellularly or, e.g., extracellularly in the culture medium.
[00151] Cells which overexpress APP or a fragment thereof which can be used according to the methods disclosed herein include mammalian or non-mammalian cells including but not limited to 7W WT APP751 Chinese hamster ovary cells. APP which is overexpressed can include, without limitation, APP751. Cells which can be used to measure changes in CCE include non-mammalian and mammalian cells, such as epithelial or endothelial cells. [00152] The compounds which are tested for their ability to inhibit CCE as well as to reduce Aβ production are screened in a range of concentrations, for example, about 1 nM to 10 mM, about 500 nM to 50 μM, or about 5 μM to 30 μM.
[00153] The CCE assay for compounds is advantageous because it is a rapid assay. High volume assays can be conducted using arrays of samples. Rapid combinatorial methods known in the art can be used, such as the use of microarrays with 1000, 10,000 or more samples with the appropriate sample delivery devices and detectors. Advantageously, the assay can be completed, e.g., in about an hour.
[00154] By way of example, in one embodiment, a 96 well plate is used. Cells are washed to remove calcium ions, e.g. with EDTA, and incubated with a fluorescent Ca 2+ indicator, such as FluorPure, available from Molecular Probes, Eugene, OR. The cells are preferably washed and placed in a calcium ion free culture medium such as HBSS (Hank's balanced salt solution). A sample of cells in the culture medium and, e.g., 90 different compounds are combined in 96 wells on the plate, and control wells are included on the plate. The control is, for example, a sample of cells in culture combined with an equivalent unit volume of buffer or water as was used for the compound sample. The compound is allowed to incubate with the cells for an amount of time which can be determined with routine testing. Typically, about 15 minutes is sufficient. Baseline fluorescence measurements are taken. Thapsigargin (TG) is used to administered to deplete intracellular Ca 2+ . CaCl 2 is added in HBSS and then fluorescence is measured, as described in the Examples. The percentage of CCE inhibition is calculated as the difference between the compound treated cells and the control. [00155] Either separately or in combination with the measurement of CCE as described above, the cells also can be tested for a reduction in β-amyloid production in cells exposed to the test compound. In the method, the concentration of β-amyloid (e.g., Aβl-40 and/or Aβl- 42) in cells exposed to the compound can be measured and compared with a measurement of β-amyloid production in unexposed cells, for example, in a control run in parallel. A decrease in the production β-amyloid, alone or in combination, for example of about 5%, 10%, 15%, 20%, 25%, 30%, 50%, or more in the exposed cells compared to the control cells indicates the potential therapeutic effectiveness of the compound to treat animals or humans afflicted with a disease associated with cerebral accumulation of Alzheimer's amyloid. In one embodiment, total β-amyloid concentration (Aβl-40 + Aβl-42) is measured. The β- amyloid is measured, e.g. in the culture medium comprising the cells, or intracellularly. [00156] The method of measuring β-amyloid may include testing an array of compounds, e.g., in a 96 well plate, as well as one or more control samples. In the assay, the compound is often required to be incubated with the cells for about 4-48 hours, or e.g., 18-36 hours, β- amyloid can be detected using an ELISA sandwich assay using quantitatively commercially available enzymatically labeled (with horseradish peroxidase) antibodies to Aβl-40 and Aβl- 42 as described in the Examples. The labeled antibody ELISA assay also can require on the order of 24 hours to complete. Thus, the CCE assay is advantageously less time consuming and requires less reagents than the β-amyloid assay.
[00157] CCE, also referred to as store-operated calcium influx, serves as an important calcium-refilling mechanism in both electrically non-excitable and excitable cells, such as neurons. In particular, when calcium is released from its storage sites in the endoplasmic reticulum, calcium levels rise in the cytosol, which normally is followed by calcium influx from the extracellular space that refills the cytosol and then is stored in the endoplasmic reticulum.
[00158] Measurement of CCE in cultured cells is performed using the methods for assaying CCE described herein or any method known in the art. Any appropriate assay for measuring CCE in cultured cells can be used. Skilled artisans will appreciate the experimental variability associated with various testing protocols, which typically is corrected by standardization techniques commonly known to those skilled in the art. See, e.g. Putney J.W., Jr., Sci STKE, (243):37 (2004); and Putney J.W., Jr., MoI. Interv., l(2):84-94 (2001).
[00159] The compounds which are tested for their ability to inhibit CCE (and optionally reduce AB production) are screened in a range of concentrations, for example of about 1 nM to 10 mM, about 500 nM to 50 μM, or about 5 μM to 30 μM.
[00160] Cells which can be used in the assays described herein for measuring a reduction in β-amyloid production include mammalian or non-mammalian cells that overexpress APP or a fragment thereof, including but not limited to Chinese hamster ovary (CHO) cells, for example, 7W WT APP751 CHO cells. See, e.g., Koo and Squazzo, J. Biol. Chem., Vol. 269, Issue 26, 17386-17389, JuI, 1994. Cell lines transfected with APP have been described in the art and include 7W (wt APP751); 7W ΔC (APP751 with deletion of almost the entire cytoplasmic tail (residue 710-751); 7W SW (APP751 with the "Swedish" KM651/652NL double-mutation); and 7W VF (APP751 with the V698F mutation). See, e.g. Xia et al, Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 8208-8213, July 1997; and Perez, R. & Koo, E. (1997) in Processing of the β-Amyloid Precursor Protein: Effects of C-T erminal Mutations on Amyloid Production, eds. Iqbal, K., Winblad, B., Nishimura, T., Takeda, M. & Wisniewski, H. M. (J. Wiley & Sons, London), pp. 407-416. The APP which is overexpressed can include transcripts of APP, such as, without limitation, APP751.
[00161] Cells which can be used to measure changes in CCE include most non-mammalian and mammalian cells, such as epithelial or endothelial cells, and CHO cells, and in one embodiment, 7W WT APP 751 CHO cells. Cells may be used that overexpress APP or a fragment thereof, however cells with normal expression of APP also can be used. Thus, the
CCE assay is highly advantageous, since there is not a requirement for a specific cell type, or overexpression of APP. Other exemplary cells include cultured neurons, e.g., human neuronal precursor cells (HNPC), which are commercially available, for example, from QBM Cell Science (Canada); primary culture of human astrocytes; neuroblastoma cells, available e.g., from ATCC; endothelial cells, such as human brain microvascular endothelial primary culture; and human umbilical cord endothelial cells (HUVEC).
Methods of Treatment
[00162] In another embodiment, a method is provided for treating an animal or human afflicted with a disease associated with cerebral accumulation of Alzheimer's amyloid, such as Alzheimer's disease (AD), comprising administering a therapeutically effective amount of a compound or compounds disclosed herein. Administration of the compound(s) in one embodiment results in one or more of reducing β-amyloid production, β-amyloid deposition, β-amyloid neurotoxicity (including abnormal hyperphosphorylation of tau) or microgliosis, or combination thereof. In one embodiment, the one or more compounds have the property of decreasing CCE, for example, by at least about 5%, 10%, 15%, 20%, or more in cells. The compound may have the property that it decreases CCE measured in cells, such as CHO cells, that in one embodiment overexpress APP or a fragment thereof. Alternatively, or additionally, one or more of the compounds is characterized in that it reduces β-amyloid production for example by at least about 5%, 10%, 15%, 20%, 25%, 30%, 50%, or more in cells that overexpress APP or a fragment thereof, as measured, for example, in a culture medium comprising the cells or as measured intracellularly.
[00163] As used herein, reference to a compound that reduces CCE in cells, refers to a compound that reduces CCE in cells which may be 7W WT APP751 CHO cells that overexpress APP, or the cells may be selected from, e.g., cultured neurons, e.g., human neuronal precursor cells (HNPC); primary culture of human astrocytes; neuroblastoma cells, endothelial cells, such as human brain microvascular endothelial primary culture; and human umbilical cord endothelial cells (HUVEC).
[00164] As used herein, reference to a compound that reduces β-amyloid production, refers to a compound that reduces β-amyloid production in cells that overexpress APP or a fragment thereof, and the cells may be for example Chinese hamster ovary (CHO) cells that overexpress APP, for example, 7W WT APP751 CHO cells; 7W (wt APP751) cells; 7W Δ c cells; 7Wsw cells; or 7W V F cells.
[00165] It is noted that wherever the embodiments disclosed herein refer to a reduction in β-amyloid in cells that overexpress APP, alternatively, an increase in αCTF (α C-terminal APP fragment, also known as CTF-α) and/or APPSα soluble fragment can be measured for example, in the cell culture or intracellularly, when they are produced in increased amounts from APP as the compound causes the production of β-amyloid to decrease. [00166] It is further noted that wherever the embodiments disclosed herein refer to a reduction in β-amyloid in cells that overexpress APP, alternatively, a decrease in β CTF (β C- terminal APP fragment, also known as CTF-β) or APPSβ soluble fragment can be measured, e.g., in the cell culture media or intracellularly, when they are produced in decreased amounts from APP as the compound causes the production of β-amyloid to decrease. [00167] In a further embodiment, a method is provided for treating animals or humans suffering from traumatic brain injury (TBI). In one embodiment, β-amyloid production, β- amyloid deposition, β-amyloid neurotoxicity (including abnormal hyperphosphorylation of tau) and/or microgliosis is reduced. The method includes administering to the animal or human, for example, immediately after the TBI, a therapeutically effective amount of one, two or more compounds disclosed herein. In one embodiment, at least one compound is one that decreases CCE for example, by at least about 5%, 10%, 15%, 20% or more in cultured cells. The cultured cells optionally are mammalian or non-mammalian cells that overexpress APP or a fragment thereof. The method may include continuing treatment with the compound or compounds for a prescribed period of time thereafter. It has been shown that TBI increases the susceptibility to the development of AD, and thus it is believed, without being bound by the theory, that TBI accelerates brain β-amyloid accumulation and oxidative stress, which may work synergistically to promote the onset or drive the progression of AD. Alternatively or in addition to decreasing CCE in cells, the compound also may decrease β- amyloid production as disclosed herein. Treatment with the compound or compounds of animals or humans suffering from a TBI can continue, for example, for about one hour, 24 hours, a week, two weeks, 1-6 months, one year, two years or three years. [00168] Amyloidogenic diseases which can be treated according to the methods of the present invention can include, without limitation, Alzheimer's disease, cerebral amyloid angiopathy, hereditary cerebral hemorrhage with amyloidosis Dutch-type, or other forms of familial AD and familial cerebral Alzheimer's amyloid angiopathy.
[00169] The methods of the present invention can be used on transgenic animal models for AD, such as, without limitation, PDAPP and TgAPPsw mouse models, which can be useful
for treating, preventing and/or inhibiting conditions associated with β-amyloid production, β- amyloid deposition, β-amyloid neurotoxicity (including abnormal hyperphosphorylation of tau) and microgliosis in the central nervous system of such animals or in humans. Transgenic animal models for AD can be constructed using standard methods known in the art, as set forth for example, without limitation, in United States Patent Nos. 5,487,992; 5,464,764; 5,387,742; 5,360,735; 5,347,075; 5,298,422; 5,288,846; 5,221,778; 5,175,385; 5,175,384; 5,175,383; and 4,736,866.
[00170] Exemplary dosages of the one or more compounds that can be administered include 0.001-1.0 mg/kg body weight. An exemplary dose of compound is about 1 to 50 mg/kg body weight per day, 1 to 20 mg/kg body weight per day, or 0.1 to about 100 mg per kilogram body weight of the recipient per day. Lower doses may be preferable, for example doses of 0.5-100 mg, 0.5-50 mg, 0.5-10 mg, or 0.5-5 mg per kilogram body weight per day, or e.g., 0.01-0.5 mg per kilogram body weight per day. The effective dosage range can be calculated based on the activity of the compound and other factors known in the art of pharmacology.
[00171] The compound or compounds are conveniently administered in any suitable dosage form, including but not limited to 1 to 3000 mg, or 10 to 1000 mg of each active ingredient per unit dosage form. For each compound, an oral dosage of 50-1000 mg is possible. Lower doses may be preferable, for example from 10-100 or 1-50 mg, or 0.1-50 mg, or 0.1-20 mg or 0.01-10.0 mg. Furthermore, lower doses may be utilized in the case of administration by a non-oral route, as, for example, by injection or inhalation. [00172] In another embodiment, the dosage can range for each compound from about 0.05 mg to 20 mg per day, from between about 2 mg to 15 mg per day, about 4 mg to 12 mg per day, and or about 8 mg per day.
[00173] In another embodiment, the dosage ranges, e.g. from about one day to twelve months, from about one week to six months, or from about two weeks to four weeks. [00174] Because most diseases having cerebral accumulation of Alzheimer's amyloid, such as AD, are chronic, progressive, intractable brain dementias, it is contemplated that the duration of treatment with compounds disclosed herein can last for up to the lifetime of the animal or human.
[00175] The invention encompasses the use and/or administration of other standard and experimental therapeutics for the treatment of diseases associated with the accumulation of β- amyloid and/or the treatment or alleviation of one or more symptoms associated therewith
(e.g., the use of acetylcholinesterase inhibitors, secretase inhibitors, and/or active or passive vaccines). Examples of therapeutics that may be used in accordance with the methods described herein include, but are not limited to, ARICEPT® (donepezil), EXELON® (rivastigmine), REMINYL® (galantamine), COGNEX® (tacrine), NAMENDA® (memantine), RAZAD YNE® (galantamine), LY2062430 (Eli Lilly), LY450139 (Eli Lily), FLURIZAN® (r-flurbiprofen), ALZHEMED® (tramiprosate), PBT-2 (Prana Biotechnology), phenserine, TTP488 (Trans Tech Pharma), KETSYN® (AC-1202, Accera), AZD3480 (AstraZeneca), CX717(Cortex Pharmaceuticals), AC-3933 (Dainippon Sumitomo), Debio- 9902 SR (Debiopharm), NERAMEXANE® (MRZ 21579, Forest Laboratories), AVAND IA® (rosiglitazone), dimebon, MEM 1003 (Memory Pharmaceuticals), MEM 3454 (Memory Pharmaceuticals), MK-0952 (Merk), huperzine A, SGS742 (Saegis Pharmaceuticals), XAPRILA® (xaliproden), SR 57667 (Sanofi-Aventis), AVE1625 (Sanofϊ- Aventis), AZILECT® (rasagiline mesylate), ladostigil tartrate, VP 4896 (Voyager Pharmaceutical) or lecozotan (SRA-333), or combinations thereof.
[00176] The invention also encompasses the use of the compounds and combinations thereof of the invention in further combination with active and/or passive vaccines for β- amyloid diseases, in particular, Alzheimer's disease. The combinations of the invention encompass the administration and/or use of passive vaccines, i.e., antibodies or fragments thereof, including, but not limited to bapineuzumab and AAC-001 (both of Elan Corp.). In certain embodiments, the compositions and/or methods of the invention also encompass the use and/or administration of vaccines as disclosed, e.g., in U.S. Patent No.: 7,189,819; U.S. Patent No.: 7,179,892; U.S. Patent No.: 6,913,745; U.S. Patent No.: 6,761,888; U.S. Patent No.: 6,750,324; U.S. Patent No.:6,743,427; U.S. Patent Application Publication 2006/024007; U.S. Patent Application Publication 2002/0009445; U.S. Patent Application Publication 2007/0031416; U.S. Patent Application Publication 20060057702; U.S. Patent Application Publication 20050181460; and U.S. Patent Application Publication 2005/0090648 (each of which is hereby incorporated by reference in its entirety). In other embodiments, the compositions and/or methods of the invention encompass the use and/or administration of active vaccine as disclosed, e.g., in U.S. Patent No.: 6,962,707; U.S. Patent No.: 7,014,855; U.S. Patent No.: 6,982,084; U.S. Patent No.: 6,972,127; U.S. Patent No.: 6,905,686; U.S. Patent No.: 6,875,434; U.S. Patent No.: 6,866,850; U.S. Patent No.: 6,866,849; U.S. Patent No.: 6,818,218; U.S. Patent No.: 6,808,712; U.S. Patent No.: 6,787,637; U.S. Patent No.: 6,787,523; U.S. Patent No.: 6,787,144; U.S. Patent No.:
6,787,143; U.S. Patent No.:6,787,140; U.S. Patent No.: 6,787,139; U.S. Patent No.: 6,787,138, and U.S. Patent No.: 6,761,188 (each of which is hereby incorporated by reference in its entirety).
Methods of Diagnosis
[00177] In still a further embodiment, a method is provided for diagnosing or determining the risk for developing a disease associated with cerebral accumulation of Alzheimer's amyloid, such as AD, in an animal or human, by taking a first measurement of β-amyloid concentration from a peripheral body fluid such as plasma, serum, whole blood, urine or cerebral spinal fluid (CSF) of the animal or human. Subsequently the method includes administering to the animal or human a diagnostically effective amount of a compound as disclosed herein or combination thereof. In one embodiment, one or more of the compounds decreases CCE in the cell, for example, by at least about 5%, 10%, 15%, 20% or more. Alternatively, or in addition to decreasing CCE, one or more of the compounds decreases β amyloid production for example by at least about 5%, 10%, 15%, 20%, 25%, 30%, 50%, or more, as measured, for example, in the medium of cultured cells which overexpress APP or a fragment thereof, or as measured intracellularly. A second (selected endpoint) measurement of β-amyloid concentration is taken from plasma, serum, whole blood, urine or CSF of the animal or human at a later time, and the difference between the first measurement and the second measurement is determined. A change in the concentration of β-amyloid in plasma, serum, whole blood, urine or CSF in the second measurement compared to the first measurement indicates a risk of developing or a possible diagnosis of a disease associated with cerebral accumulation of Alzheimer's amyloid in the animal or human. In one embodiment, an increase in peripheral β-amyloid indicates the presence of an accumulation of cerebral β-amyloid, and therefore the risk of disease or the presence of the disease. [00178] It is believed, without being bound by any theory, that the compounds can cause an increase in β-amyloid concentration in plasma, urine, serum, whole blood or CSF by facilitating the clearance of already produced β-amyloid from the central nervous system into the periphery, thus increasing β-amyloid concentration in the peripheral fluid being assayed. [00179] The duration of time of administration of the one, two or more compounds after the first peripheral body fluid measurement, up until the second (selected endpoint) peripheral body fluid measurement, is, e.g., any suitable time period, e.g. about 1-12 hours, about 1-7 days, about 1-4 weeks; about 2-6 months, or more. The time length can be adjusted
as needed depending, for example, on the progression of the disease, and the patient. A suitable periodic (e.g., daily) dosage of the compound or compounds disclosed herein is administered, e.g. orally or intravenously, and the β-amyloid levels in the individual can be monitored periodically up until the endpoint. In one preferred embodiment, the compound or compounds are administered daily for about 3 days to 4 weeks from the start of administration to the endpoint measurement. The change in concentration indicative of the risk or presence of a disease associated with β-amyloid accumulation is, e.g. about 10-20% or more between the first and endpoint measurements.
[00180] Exemplary dosages of the one or more compounds disclosed herein that can be administered include 0.001-1.0 mg/kg body weight, for example daily. An exemplary dose of compound is about 1 to 50 mg/kg body weight per day, 1 to 20 mg/kg body weight per day, or 0.1 to about 100 mg per kilogram body weight of the recipient per day. Lower doses may be preferable, for example doses of 0.5-100 mg, 0.5-50 mg, 0.5-10 mg, or 0.5-5 mg per kilogram body weight per day, or e.g., 0.01-0.5 mg per kilogram body weight per day. The effective dosage range can be calculated based on the activity of the compound and other factors known in the art of pharmacology.
[00181] The one, two or more compounds disclosed herein are conveniently administered in any suitable dosage form, including but not limited to one containing 1 to 3000 mg, or 10 to 1000 mg of active ingredient per unit dosage form. An oral dosage of 50-1000 mg is possible. Lower doses may be preferable, for example from 10-100 or 1-50 mg, or 0.1-50 mg, or 0.1-20 mg or 0.01-10.0 mg. Furthermore, lower doses may be utilized in the case of administration by a non-oral route, as, for example, by injection or inhalation.
[00182] Compounds
[00183] A variety of compounds are provided as disclosed herein and below, which in one embodiment can be used in methods described herein, including the treatment or diagnosis of diseases associated with cerebral accumulation of Alzheimer's amyloid. Any of the compounds disclosed herein and below can be used in combination in the methods and compositions disclosed herein.
[00184] In one embodiment, one, two or more of the compounds decreases CCE, for example, by at least about 5%, 10%, 15% or 20% in cultured cells, wherein the cells optionally overexpress APP or a fragment thereof. Additionally, or alternatively, the selected
compound or compounds reduce β amyloid production, for example, by at least about 5%, 10%, 15%, 20% or more, in cells that overexpress APP or a fragment thereof. [00185] In one embodiment, one or more of the compounds is an imidazole compound, an isoquinoline alkaloid compound, a calmodulin-mediated enzyme activation an inhibitor of kinase activity of the platelet-derived growth factor (PDGF) receptor, an NF-kB activation inhibitor, a diterpene or triterpene compound, a quinazoline compound, a sesquiterpene lactone, or an inhibitor of IkappaB kinase 2 (IKK-2), that in one embodiment decreases CCE, for example, by at least about 10% or more in cultured cells that in one embodiment are cells that overexpress APP or a fragment thereof, and optionally, additionally or alternatively reduces β amyloid production, for example, by at least about 20% or more, in cells that overexpress APP or a fragment thereof.
[00186] In one embodiment, one, two or more of the compounds described below may be used. An example of a suitable compound is a compound of Formula I, or a salt, ester or prodrug thereof, including R and S isomers thereof:
I
wherein:
R 1 is H, alkyl (including straight chain, branched, and cyclic alkyl), optionally substituted aryl, optionally substituted heterocycle, alkyl or aryl ether;
R 2 and R 6 are independently alkyl, alkyl ether, aryl ether, halogen, or hydroxy;
R 3 and R 5 are independently optionally substituted alkyl ester, aryl ester, silyl ester, alkyl amide, aryl amide, cyano, or nitro;
R 2 and R 6 are independently H, alkyl, optionally substituted alkyl ether, optionally substituted aryl ether, halogen, hydroxy, nitro, carboxylic acid, boronic acid, haloalkyl, amine, optionally substituted alkyl amine, nitrile, optionally substituted alkyl thioether, optionally substituted aryl thioether, or optionally substituted heterocycle;
R 3 and R 5 are independently H, alkyl, optionally substituted alkyl ether, optionally substituted aryl ether, halogen, hydroxy, nitro, carboxylic acid, boronic acid, haloalkyl, amine, optionally substituted alkyl amine, nitrile, optionally substituted alkyl thioether, optionally substituted aryl thioether, or optionally substituted heterocycle;
R 4 is independently H, alkyl, optionally substituted alkyl ether, optionally substituted aryl ether, halogen, hydroxy, nitro, carboxylic acid, boronic acid, haloalkyl, amine, optionally substituted alkyl amine, nitrile, optionally substituted alkyl thioether, optionally substituted aryl thioether, or optionally substituted heterocycle; alternatively, R 2 and R 3 together can optionally form a 4, 5, 6 or 7 membered heterocycle containing 1, 2, or 3 heteratoms and can be optionally substituted with alkyl, optionally substituted alkyl ether, optionally substituted aryl ether, halogen, hydroxy, nitro, carboxylic acid, boronic acid, haloalkyl, amine, optionally substituted alkyl amine, nitrile, optionally substituted alkyl thioether, optionally substituted aryl thioether, or optionally substituted heterocycle; alternatively, R 3 and R 4 together can optionally form a 4, 5, 6 or 7 membered heterocycle containing 1, 2, or 3 heteratoms and can be optionally substituted with alkyl, optionally substituted alkyl ether, optionally substituted aryl ether, halogen, hydroxy, nitro, carboxylic acid, boronic acid, haloalkyl, amine, optionally substituted alkyl amine, nitrile, optionally substituted alkyl thioether, optionally substituted aryl thioether, or optionally substituted heterocycle; alternatively, R 4 and R 5 together can optionally form a 4, 5, 6 or 7 membered heterocycle containing 1, 2, or 3 heteratoms and can be optionally substituted with alkyl, optionally substituted alkyl ether, optionally substituted aryl ether, halogen, hydroxy, nitro, carboxylic acid, boronic acid, haloalkyl, amine, optionally substituted alkyl amine, nitrile, optionally substituted alkyl thioether, optionally substituted aryl thioether, or optionally substituted heterocycle; alternatively, R 5 and R 6 together can optionally form a 4, 5, 6 or 7 membered heterocycle containing 1, 2, or 3 heteratoms and can be optionally substituted
with alkyl, optionally substituted alkyl ether, optionally substituted aryl ether, halogen, hydroxy, nitro, carboxylic acid, boronic acid, haloalkyl, amine, optionally substituted alkyl amine, nitrile, optionally substituted alkyl thioether, optionally substituted aryl thioether, or optionally substituted heterocycle.
In another embodiment, the compounds is a compound of Formula I, or a salt, ester or prodrug thereof, including R and S isomers thereof, wherein:
I
R 1 is H, alkyl (including straight chain, branched, and cyclic alkyl), optionally substituted aryl, optionally substituted heterocycle, alkyl or aryl ether; R 2 and R 6 are independently alkyl, alkyl ether, aryl ether, halogen, or hydroxy; R 3 and R 5 are independently alkyl ester, aryl ester, silyl ester, alkyl amide, aryl amide, cyano, or nitro; R 2 and R 6 are independently H, optionally substituted alkyl, alkyl ether, aryl ether, halogen, hydroxy, nitro, or optionally substituted heterocycle; R 3 and R 5 are independently H, optionally substituted alkyl, alkyl ether, aryl ether, halogen, hydroxy, nitro, or optionally substituted heterocycle;
R 4 is in nddeeppeennddeennttllyy HH,, aallkkyyll,, aallkkyyll ether, aryl ether, halogen, hydroxy, nitro, or optionally substituted heterocycle.
[00187] In one embodiment, the compound comprises at least two nitro substituents.
[00188] In one embodiment, R 3 = R 5 and R 3 = alkyl ester, wherein the alkyl is optionally substituted with a group other than alkoxyl.
[00189] In one embodiment, R 3 = R 5 and R 3 = alkyl ester, wherein the alkyl is optionally substituted.
[00190] In one embodiment, R 3 = R 5 and R 3 is unsubstituted alkyl ester.
[00191] In another embodiment of a compound of Formula I or a salt, ester or prodrug thereof, including an R or S isomer thereof:
R 1 is H, alkyl (including straight chain, branched, and cyclic alkyl), optionally substituted aryl, optionally substituted heterocycle, alkyl or aryl ether; R 2 and R 6 are independently alkyl, alkyl ether, aryl ether, halogen, or hydroxy; R 3 and R 5 are independently alkyl ester, aryl ester, silyl ester, alkyl amide, aryl amide, cyano, or nitro; R 2 and R 6 are independently H, alkyl, alkyl ether, aryl ether, halogen, hydroxy, nitro, or optionally substituted heterocycle; R 3 and R 5 are independently H, optionally substituted alkyl, alkyl ether, aryl ether, halogen, hydroxy, or optionally substituted heterocycle; and
R 4 is independently H, optionally substituted alkyl, alkyl ether, aryl ether, halogen, hydroxy, nitro, or optionally substituted heterocycle.
[00192] In another embodiment one or more of the compounds is a compound of Formula I or a salt, ester or prodrug thereof, including an R or S isomer thereof, wherein:
R 1 is H, alkyl (including straight chain, branched, and cyclic alkyl), optionally substituted aryl, optionally substituted heterocycle, or alkyl; R 2 and R 6 are independently alkyl, alkyl ether, aryl ether, halogen, or hydroxy; R 3 and R 5 are independently alkyl ester, aryl ester, silyl ester, alkyl amide, aryl amide, cyano, or nitro; R 2 and R 6 are independently H, alkyl, alkyl ether, aryl ether, halogen, hydroxy, nitro, or optionally substituted heterocycle; R 3 and R 5 are independently H, optionally substituted alkyl, alkyl ether, aryl ether, halogen, hydroxy, or optionally substituted heterocycle; and
R 4 is in nddeeppeennddeennttllyy HH,, ooppttiioonnaallllyy ssuubbssttiittuutteedd aallkkyyll,, aallkkyyll eetthher, aryl ether, halogen, hydroxy, nitro, or optionally substituted heterocycle.
[00193] In another embodiment, in the compound of Formula I, or a salt, ester or prodrug there of, including an R or S isomer thereof:
R 1 is H, alkyl including straight chain, e.g., methyl; branched alkyl, e.g., isopropyl; cyclic alkyl, e.g., cyclohexyl; substituted aryl, e.g., o-chlorophenyl; substituted heterocycle, e.g., 2-methyl furyl; alkyl ether, e.g., methoxy; or aryl ether, e.g., phenoxy;
R 2 =R 6 and each are alkyl, e.g. methyl; alkyl ether, e.g., ethoxy; or halogen, e.g., F; R 3 =R 5 and each are alkyl ester, e.g., ethyl ester; aryl ester, e.g., benzoate; silyl ester; alkyl amide, e.g., methyl amide; aryl amide, e.g., phenyl amide; cyano; or nitro; R 2 and R 6 are independently H, alkyl, e.g. methyl; alkyl ether e.g. ethoxy; aryl ether e.g. phenoxy; halogen, e.g. F; hydroxy; nitro; or heterocycle, e.g., 2-methyl furyl; R 3 and R 5 are independently H, alkyl, e.g., methyl; alkyl ether, e.g. ethoxy; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; nitro; or heterocycle, e.g., 2- methyl furyl; and R 4 is H, alkyl, e.g., methyl; alkyl ether, e.g. ethoxy; aryl ether, e.g., phenoxy, halogen, e.g., F; hydroxy; nitro; or heterocycle, e.g., 2-methyl furyl.
[00194] In one embodiment, the compound is a compound of Formula I, or a salt, ester or prodrug thereof, including R and S isomers thereof, wherein:
R 1 is H;
R 2 and R 6 are independently alkyl, e.g. methyl or ethyl;
R 3 and R 5 are independently cyano or alkyl ester;
R 2 and R 6 are independently H, halo, or nitro;
R 3 and R 5 are independently H or halo; and
R 4 is independently H, alkyl, alkyl ether, halo, or nitro.
[00195] In another embodiment, the compound is a compound of Formula I, or a salt, ester or prodrug thereof, including R and S isomers thereof, wherein:
R 1 is H;
R 2 and R 6 are independently alkyl;
R 3 and R 5 are independently alkyl ester, wherein, in at least one of R 2 and R3 the alkyl of the alkyl ester comprises at least 10, 20 or 30 carbon atoms, e.g. 10 to 30 carbon atoms;
R 2' , R 3' , R 4' , R 5' , and R 6' are independently H, halo, or nitro.
[00196] In another embodiment, the compound is a compound of Formula I, or a salt, ester or prodrug thereof, including R and S isomers thereof, wherein:
R 1 is H;
R 2 and R 6 each are alkyl, e.g. methyl;
R 3 and R 5 are independently C(O)OCH 2 CH 2 Oalkyl, wherein the alkyl is, e.g. methyl and is optionally substituted;
R 2' , R 3' , R 4' , R 5' , and R 6' are independently H, halo, or nitro.
[00197] In another embodiment, the compound is a compound of Formula I, or a salt, ester or prodrug thereof, including R and S isomers thereof, wherein:
R 1 is H;
R 2 and R 6 each are alkyl, e.g. methyl;
R 3 and R 5 are independently C(O)Oalkyl, wherein the alkyl is substituted with alkenyl or alkynyl, e.g. R 3 and R 5 are C(O)OCH 2 CHCH 2 ;
R 2' , R 3' , R 4' , R 5' , and R 6' are independently H, halo, or nitro.
[00198] In another embodiment, the compound is a compound of Formula I, or a salt, ester or prodrug thereof, including R and S isomers thereof, wherein:
R 1 is H;
R 2 and R 6 each are CH 2 Oalkyl, e.g. CH 2 OCH 3 ;
R 3 and R 5 are independently C(O)Oalkyl, e.g. C(O)OCH 3 ;
R 2' , R 3' , R 4' , R 5' , and R 6' are independently H, halo, or nitro.
[00199] In another embodiment, the compound is a compound of Formula I, or a salt, ester or prodrug thereof, including R and S isomers thereof, wherein:
R 1 is H;
R 2 and R 6 each are alkyl, e.g. methyl;
R 3 and R 5 are independently C(O)Oalkyl, e.g. C(O)OCH 2 CH 3 , or C(O)OCH 2 C(CH 3 ) 3 ;
R 2 and R 6 are independently H, F, Br, or nitro.
R 3' and R 5' each are H.
R 4' is H or halo.
[00200] In another embodiment, the compound is a compound of Formula I, or a salt, ester or prodrug thereof, including R and S isomers thereof, wherein:
R 1 is H;
R 2 and R 6 each are alkyl, e.g. methyl;
R 3 and R 5 are independently C(O)Oalkyl, e.g. C(O)OCH 2 CH 3 , or C(O)OCH 2 C(CHs) 3 ;
R 2 and R 6 each are H or F and not the same;
R 3' , R 4' , R 5' are independently H, or Br.
[00201] In another embodiment, the compound useful in the methods and compositions disclosed herein is a compound of formula II, or a salt, ester or prodrug there of, including an R or S isomer thereof, wherein:
II
R 1 is heterocycle, optionally substituted with one or more of alkyl, alkyl ether, aryl ether, alkylaryl, arylalkyl, halogen, hydroxy, optionally substituted alkyl ester, optionally substituted aryl ester, alkyl amide, aryl amide, or nitro;
R 2 and R 6 are independently optionally substituted alkyl, heteroalkyl, alkyl ether, aryl ether, halogen, hydroxy, nitro, cyano, or heterocycle; and
R 3 and R 5 are independently H, alkyl, alkyl ether, aryl ether, halogen, hydroxy, nitro, or heterocycle;
R 4 is H, alkyl, alkyl ether, aryl ether, halogen, hydroxy, nitro, cyano, or heterocycle; wherein, in one embodiment, at least two of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are nitro.
[00202] In another embodiment, the compound is a compound of formula II, or a salt, ester or prodrug there of, including an R or S isomer thereof, wherein:
R 1 is heterocycle, optionally substituted with one or more of alkyl, alkyl ether, aryl ether, alkylaryl, arylalkyl, halogen, hydroxy, optionally substituted alkyl ester, optionally substituted aryl ester, alkyl amide, aryl amide;
R 2 and R 6 are independently optionally substituted alkyl, heteroalkyl, alkyl ether, aryl ether, halogen, hydroxy, cyano, or heterocycle; R 3 and R 5 are independently H, alkyl, alkyl ether, aryl ether, halogen, hydroxy, or heterocycle; and R 4 is H, alkyl, alkyl ether, aryl ether, halogen, hydroxy, nitro, cyano, or heterocycle.
[00203] In another embodiment, the compound is a compound of formula II, or a salt, ester or prodrug there of, including an R or S isomer thereof, wherein:
R 1 is unsubstituted heterocycle, e.g., furyl, or is optionally heterocycle substituted with alkyl, e.g., methyl; alkyl ether, e.g. methoxy; aryl ether, e.g. phenoxy; halogen, e.g., F; hydroxy; alkyl ester, e.g. ethyl ester; aryl ester, e.g. benzoate; alkyl amide, e.g., methyl amide; aryl amide, e.g., phenyl amide; nitro; or cyano; R 2 =R 6 and are each H, optionally substituted alkyl, e.g. methyl; alkyl ether, e.g. methoxy; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; nitro; cyano; or heterocycle, e.g., pyrazole; R 3 =R 5 and are each H, alkyl, e.g., methyl; alkyl ether, e.g., methoxy; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; nitro; cyano, or heterocycle, e.g., pyrazole; R 4 is H, alkyl, e.g., methyl; alkyl ether, e.g., methoxy; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; nitro; cyano; or heterocycle, e.g. pyrazole.
[00204] In another embodiment, the compound useful in the methods and compositions disclosed herein is a compound of formula III, or a salt, ester or prodrug there of, including an R or S isomer thereof, wherein:
III
R 1 is alkyl including straight chain, branched, or cyclic alkyl; optionally substituted aryl; optionally substituted heterocycle; alkyl; aryl ether; or aryl-O-(optionally substituted aryl);
R 2 and R 6 are independently alkyl, alkyl ether, aryl ether, halogen, or hydroxy;
R 3 and R 5 are independently alkyl ester, aryl ester, silyl ester, alkyl amide, aryl amide, cyano, or nitro;
R 4 is alkyl (including straight chain, branched, and cyclic) or heterocycle optionally substituted e.g. with one or more of alkyl, alkyl ether, aryl ether, halogen, hydroxy, alkyl ester, aryl ester, alkyl amide, aryl amide, or nitro;
[00205] In another embodiment of the compound of formula III, or a salt, ester or prodrug there of, including an R or S isomer thereof, wherein:
R 1 is H, alkyl (including straight chain, e.g., methyl; branched, e.g. isopropyl; cyclic, e.g. cyclohexyl); optionally substituted aryl, e.g., o-chlorophenyl; substituted heterocycle (substituted at one or more positions by alkyl, e.g., methyl; alkyl ether, e.g., methoxy; aryl ether, e.g., phenoxy; halogen, e.g. F; hydroxy; alkyl ester, e.g., ethyl; aryl ester, e.g., benzoate; alkyl amide, e.g., methyl amide; aryl amide, e.g., phenyl amide; nitro; or cyano) unsubstituted heterocycle, e.g., furyl; alkyl ether, e.g., methoxy; or aryl ether, e.g., phenoxy;
R 2 =R 6 and each are alkyl, e.g., methyl; alkyl ether, e.g. ethoxy; halogen, e.g., F; or hydroxy;
R 3 =R 5 and each are alkyl ester, e.g., ethyl; aryl ester, e.g., benzoate; silyl ester; alkyl amide, e.g. methyl; aryl amide, e.g., phenyl; cyano; or nitro; and
R 4 is alkyl (including straight chain, e.g., methyl; branched, e.g., isopropyl; cyclic e.g., cyclohexyl); optionally substituted aryl, e.g., o-chlorophenyl; substituted heterocycle (substituted at one or more positions by alkyl, e.g., methyl; alkyl ether, e.g., methoxy; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; alkyl ester, e.g., ethyl ester; aryl ester, e.g., benzoate; alkyl amide e.g. methyl amide; aryl amide, e.g., phenyl amide; nitro; or cyano); or unsubstituted heterocycle, e.g. furyl.
[00206] In another embodiment, the compound useful in the methods and compositions disclosed herein is a compound of formula IV, or a salt, ester or prodrug there of, including an R or S isomer thereof, wherein:
IV
R 1 is H, alkyl (including straight chain, branched, and cyclic); optionally substituted aryl; or optionally substituted heterocycle;
R 3 is cyano, nitro, alkyl ester, aryl ester, silyl ester, alkyl amide, or aryl amide;
R 4 is alkyl, haloalkyl, cyano, unsubstituted aryl, substituted aryl (substituted at one more positions by, e.g., cyano, nitro, halo, ester, carboxylic or carbonyl); unsubstituted heterocycle, substituted heterocycle (substituted at one more positions by e.g. alkyl, alkyl ether, aryl, aryl ether, halogen, hydroxy, ester, alkyl ester, aryl ester, alkyl amide, aryl amide, nitro, or cyano); and
R 5 and R 6 each are independently H, alkyl ester, aryl ester, silyl ester, alkyl amide, aryl amide, cyano, nitro, alkyl ether, aryl ether, halogen, hydroxy, alkyl (including straight chain, branched, and cyclic), or optionally substituted aryl.
[00207] In another embodiment, the compound is a compound of formula IV, or a salt, ester or prodrug there of, including an R or S isomer thereof wherein:
R 1 is H, alkyl (including straight chain, e.g., methyl; branched, e.g., isopropyl; cyclic, e.g., cyclohexyl; substituted aryl, e.g., o-chlorophenyl); substituted heterocycle (substituted at one or more positions by alkyl, e.g., methyl; alkyl ether, e.g., methoxy; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; alkyl ester, e.g., ethyl; aryl ester, e.g., benzoate; alkyl amide, e.g., methyl; aryl amide, e.g., phenyl; nitro, or cyano) or unsubstituted heterocycle, e.g., furyl;
R 3 =R 5 =R 6 and are H, alkyl ester, e.g., ethyl; aryl ester, e.g., benzoate; silyl ester; alkyl amide, e.g., methyl amide; aryl amide, e.g., phenyl amide; cyano; nitro; alkyl ether, e.g., methoxy; and aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; alkyl (including straight chain, e.g., methyl; branched, e.g., isopropyl; and cyclic, e.g., cyclohexyl); optionally substituted aryl, e.g., o-chlorophenyl; or unsubstituted aryl, e.g., naphthyl; and
R 4 is substituted heterocycle (substituted at one or more positions by alkyl, e.g., methyl; alkyl ether, e.g., methoxy; aryl; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; alkyl ester, e.g., ethyl ester; aryl ester, e.g., benzoate; alkyl
amide, e.g., methyl amide; aryl amide, e.g. phenyl amide; nitro; or cyano) or unsubstituted heterocycle, e.g., furyl.
[00208] In another embodiment, the compound useful in the methods and compositions disclosed herein is a compound of formula V, or a salt, ester or prodrug there of, including an R or S isomer thereof, wherein:
V
R 1 is substituted or unsubstituted aryl, alkyl, alkyl ether, substituted or unsubstituted aryl ether (e.g., 4(4-chlorophenoxy)phenyl), substituted heterocycle (substituted at different positions by alkyl, alkyl ether, aryl ether, halogen, hydroxy, alkyl ester, aryl ester, alkyl amide, aryl amide, nitro, or cyano), unsubstituted heterocycle, or halogen;
R 3 , R 4 and R 5 are independently H, alkyl ester, aryl ester, silyl ester, alkyl amide, aryl amide, cyano, nitro, alkyl ether, aryl ether, halogen, hydroxy, alkyl (including straight chain, branched, or cyclic), substituted aryl, unsubstituted aryl, or heterocycle.
[00209] In another embodiment, the compound is a compound of formula V, or a salt, ester or prodrug there of, including an R or S isomer thereof wherein:
R 1 is substituted aryl, e.g., o-chlorophenyl; unsubstituted aryl, e.g., naphthyl; alkyl, e.g., methyl; alkyl ether, e.g., methoxy; substituted aryl ether, e.g., 4(4- chlorophenoxy)phenyl; unsubstituted aryl ether, e.g., phenoxyphenyl; substituted (at one or more positions by alkyl, e.g., methyl; alkyl ether, e.g., methoxy; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; alkyl ester, e.g., ethyl; aryl ester, e.g., benzoate; alkyl amide, e.g., methyl amide; aryl amide, e.g., phenyl amide; nitro; or cyano) or unsubstituted heterocycle, e.g., piperidine; and
R 3 =R 4 =R 5 and each are H, alkyl ester, e.g., ethyl; aryl ester, e.g., benzoate; silyl ester; alkyl amide, e.g., methyl amide; aryl amide, e.g., phenyl amide; cyano; nitro;
alkyl ether, e.g., methoxy; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; alkyl (including straight chain, e.g., methyl; branched, e.g., isopropyl; and cyclic, e.g., cyclohexyl); substituted aryl, e.g., o-chlorophenyl; or unsubstituted aryl, e.g., phenyl.
[00210] In another embodiment of a compound of Formula V: R 1 is halo substituted phenoxyphenyl; R 3 =R 5 =H; and R 4 is optionally substituted phenyl, substituted e.g. with OH or halo.
[00211] In another embodiment, the compound useful in the methods and compositions disclosed herein is a compound of formula VI, or a salt, ester or prodrug there of, including an R or S isomer thereof, wherein:
VI
R 1 and R 3 are independently alkyl ether, aryl ether, halogen, hydroxy, alkyl (including straight chain, branched, or cyclic), substituted aryl or unsubstituted aryl; and
R 2 and R 4 are independently H, alkyl ether, substituted and unsubstituted aryl ether, substituted heterocycle (substituted at one or more positions,e.g., by alkyl, alkyl ether, aryl ether, halogen, hydroxy, alkyl ester, aryl ester, alkyl amide, aryl amide, nitro, or cyano), unsubstituted heterocycle, halogen, hydroxy, alkyl ester, aryl ester, silyl ester, alkyl amide, aryl amide, cyano, or nitro.
[00212] In another embodiment, the compound a compound of formula VI, or a salt, ester or prodrug there of, including an R or S isomer thereof wherein:
R 1 = R 3 and is alkyl ether, e.g., methoxy; substituted aryl ether, e.g., 4(4- chlorophenoxy)phenyl; unsubstituted aryl ether, e.g., methoxy phenyl; halogen, e.g., F; hydroxy; alkyl, including straight chain, e.g., methyl; branched, e.g., isopropyl; or cyclic, e.g., cyclohexyl); substituted aryl, e.g., o-chlorophenyl; or unsubstituted aryl, e.g., phenyl; and
R 2 = R 4 is H, alkyl ether, e.g., methoxy; substituted aryl ether, e.g., 4(4- chlorophenoxy)phenyl; unsubstituted aryl ether, e.g., methoxy phenyl; substituted heterocycle (substituted, e.g., at one or more positions by alkyl, e.g., methyl; alkyl ether, e.g., methoxy; aryl ether, e.g., phenoxy; halogen, e.g., F; hydroxy; alkyl ester, e.g., ethyl; aryl ester, e.g., benzoate; alkyl amide, e.g., methyl; aryl amide, e.g., phenyl; nitro; or cyano); unsubstituted heterocycle, e.g., pyrazole; halogen, e.g., F; hydroxy; alkyl ester, e.g., ethyl; aryl ester, e.g., benzoate; silyl ester; alkyl amide, e.g., methyl amide; aryl amide, e.g., phenyl amide; cyano; or nitro.
[00213] In another embodiment of the compound of Formula VI, or a salt, ester or prodrug there of, including an R or S isomer thereof wherein:
R 1 is alkyl, which in one embodiment is C3-12 alkyl, e.g., cycloalkyl, including cyclohexyl or cyclopentyl; R 2 and R 4 are independently H or halo; and
R 3 is unsubstituted or substituted aryl, e.g., phenyl substituted for example with halo.
[00214] In another embodiment, a compound of Formula VII, or a salt, ester or prodrug thereof, including an R or S isomer thereof, is provided:
VII wherein:
R 4 is H, halo, alkyl, or aryl;
R 3 and R 5 are independently H, halo, alkyloxy, hydroxy, or aryl; and
R 2 and R 6 are independently H, halo, alkyl, or aryl.
[00215] In another embodiment, a compound of Formula VII, or a salt, ester or prodrug thereof, including an R or S isomer thereof, is provided:
VIII wherein:
R 4 is optionally substituted aryl, e.g., phenyl optionally substituted with halo; and
R 1 is alkyl, e.g., cycloalkyl.
[00216] In another embodiment, the compound is a compound of Formula IX, or a prodrug, or salt thereof, including an R or S isomer:
IX wherein:
R 1 is alkyl, hydrogen, substituted aryl (e.g., with halogen, ether, alkyl, haloalkyl, or hydroxy) or unsubstituted aryl;
R 2 , R 3 , and R 4 are independently, alkyl, haloalkyl, thioalkyl, hydroxy, hydrogen, substituted aryl (substituted e.g., with halogen, ether, haloether, alkyl, haloalkyl, or hydroxy), unsubstituted aryl, substituted heterocycle (substituted e.g., with alkyl, halogen, haloalkyl, or amide) or unsubstituted hetrocyclic;
R 5 is alkyl, haloalkyl, hydroxy, hydrogen, ether, haloether
R 6 is nitro, cyano, hydrogen, ester, amide, carboxylic, or carbonyl.
[00217] In another embodiment, the compound is a compound of Formula X, or a prodrug, or salt thereof, including an R or S isomer:
X wherein R 1 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are independently, alkyl, haloalkyl, hydroxy, hydrogen, ether, haloether, thioalkyl, halogen; and
R 2 and R 4 are independently amide, ester, carboxylic, or nitro.
[00218] In another embodiment, the compound is a compound of Formula XI, or a prodrug, or salt thereof, including an R or S isomer:
Formula XI
wherein R 2 is alkyl ester, aryl ester, alkyl amide, aryl amide, hydrogen, carboxylic, nitro, or cyano; and
R 3 , R 1 ,R 4 , R 5 , R 6 , R 7 , R 8 are independently alkyl, haloalkyl, hydroxy, H, ether, haloether, thioalkyl, or halogen.
[00219] Other examples of a compound or any combination of two, three or more of such compounds, that are useful in the methods and compositions disclosed herein are listed below. In one embodiment, the compound can decrease CCE, for example, by at least about 10% or more in cells that, e.g, overexpress APP or a fragment thereof, and optionally reduce β amyloid production, for example, by at least about 20% or more, in cultured cells which overexpress APP or a fragment thereof.
[00220] SKF 96365, l-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-lH- imidazole hydrochloride:
[00221] econazole, (RS)-I -[2,4-dichloro-beta-(p-chlorobenzyl-oxy)phen-ethyl]imidazole nitrate:
[00222] clotrimazole, l-[(2-chlorophenyl)diphenylmethyl]-lH-imidazole (and other imidazole-based cytochrome P-450 inhibitors of divalent cation uptake that are mediated by depletion of intracellular stores induced by depletion of the intracellular calcium pool, such as by exposure to calcium- free solutions):
[00223] SR33805, 3,4-dimethoxy-N-methyl-N-[3-[4-[[l-methyl-3-(l-methylethyl)- lH-in- dol-2-yl]sulfonyl]phenoxy]propyl]benzeneethanamine oxalate, and other potent calcium antagonists that binds allosterically to the αi-subunit of L-type calcium channels:
[00224] loperamide, 4-(4-chlorophenyl)-4-hydroxy-N,N-dimethyl-α,α-diphenyl- 1 -peper- idinebutanamide, a calcium channel blocker as well as an antidiarrheal agent with high affinity for both peripheral and central opioid receptors (at low micromolar concentrations), loperamide blocks broad spectrum neuronal high voltage-activated (HVA) calcium channels and at high concentrations it reduces calcium flux through N-methyl-D-aspartate (NMDA) receptor operated channels:
[00225] tetrandrine (T et), a bis-benzylisoquinoline alkaloid isolated from the Chinese medicinal herb-root of Stephania tetrandra:
[00226] calmidazolium chloride (R24571), l-[bis(p-chlorophenyl)methyl]-3-[2-(2,4-di- chloro-β-(2,4-dichlorobenzyl-oxy)phenethyl)]-imidazolium chloride, which binds reversibly to calmodulin, thus inhibiting calmodulin-mediated enzyme activation, and other calmodulin- mediated enzyme activation inhibitors (R24571 also blocks sodium channel and voltage- gated calcium channels, inhibits the calcium/calmodulin-induced activation of myosin light chain kinase in a concentration dependent manner, and inhibits calmodulin N- methyltransferase) :
[00227] amlodipine, (R,S) 3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)- l,4-dihydro-6-methyl-3,5-pyridinedicarboxylate benzenesulfonate, a dihydropyridine calcium antagonist that inhibits the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle (amlodipine binds to both dihydropyridine and nondihydropyridine binding sites and inhibits calcium ion influx across cell membranes selectively, having a greater effect on vascular smooth muscle cells than on cardiac muscle cells):
[00228] nitrendipine (1 ,4-Dihydro-2,6-dimethyl-4-(meta-nitrophenyl)-3,5-pyridine- dicarboxylic acid, ethyl methyl ester (ethyl methyl l,4-dihydro-2,6-dimethyl-4-(meta- nitrophenyl)-3,5- pyridine dicarboxylate), and other dihydropyridine calcium channel blockers:
[00229] N-propargylnitrendipine (MRS 1845), 1 ,4-dihydro-2,6-dimethyl-4-(3-nitro-phenyl)- l-(2-propynyl)-3,5-pyridinedicarboxylic acid, ethyl, methyl ester, a dihydropyridine compound calcium channel blocker:
[00230] tyrphostin A9 ([[3,5-bis(l,l-dimethylethyl)-4-hydroxy-phenyl]methyl- ene]propane-dinitrile), and other selective inhibitors of kinase activity of the platelet-derived growth factor (PDGF) receptor, or derivatives thereof:
[00231] Various other dihydropyridine compounds can be used according to the treatment and diagnostic methods herein, including, without limitation, the following compounds and derivatives, salts and prodrugs thereof. Particularly preferred are those compounds which that can decrease CCE, for example, by at least about 10% or more in cells overexpressing β-
amyloid, and optionally may reduce β amyloid production, for example, by at least about 20% or more in the cells.
[00232] Examples of compounds are provided in Table 1 , which may be obtained from Maybridge (England):
TABLE 1
Compound Chemical Name Structure Designation
BTB 03160 4-(4-chlorophenyl)-6-methoxy-2-oxo- 1 ,2-dihydropyridine -3,5-dicarbonitrile
BTB 03173 6-methoxy-2-oxo-4-(3,4,5- trimethoxyphenyl)-l ,2- dihydropyridine-3,5-dicarbonitrile
BTB 09160 6-methyl-2-oxo-5 -(2 -phenyl- 1 ,3- thiazol-4-yl)-l,2-dihydropyridine-3- carbonitrile
BTB 09214 6-methyl-5-(2-methyl- 1 ,3-thiazol-4- yl)-2-oxo- 1 ,2-dihydropyridine-3- carbonitrile
BTB 09261 5-{2-[(3-fiuorophenyl)thio]acetyl}-6- methyl-2-oxo- 1 ,2-dihydropyridine-3- carbonitrile
RH 02165 2-oxo-6-pyridin-3-yl-4- (trifluoromethyl)- 1 ,2-dihydropyridine- 3-carbonitrile
RH 02186 1 -amino-2-oxo-6-phenyl-4- (trifluoromethyl)- 1 ,2-dihydropyridine- 3-carbonitrile
RJC 03342 4-hydroxy-2-methyl-6-oxo-5-phenyl- 1 ,6-dihydropyridine-3 -carbonitrile
RJC 03403 diethyl 4-(2,4-dichlorophenyl)-2,6- dimethyl-l,4-dihydro-3,5- pyridinedicarboxylate
RJC 03405 diethyl 2,6-dimethyl-4-{5-[2- (trifluoromethyl)phenyl]-2-furyl}-l,4- dihydro-3 ,5 -pyridinedicarboxylate
RJC 03410 diethyl 2,6-dimethyl-4-(6-methyl-2- pyridyl)-l ,4-dihydro-3,5- pyridinedicarboxylate
[00233] In another embodiment, the following compounds are provided, listed in Table 2, which can be used in the methods described herein:
TABLE 2
[00234]
