(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization {,4
`lntemational Bureau
`,1;
`
`‘t
`
`
`
`
`(43) International Publication Date
`14 August 2008 (14.08.2008)
`
`PCT
`
`(51) International Patent Classification:
`GOIN 33/50 (2006.01)
`
`(81)
`
`(21) International Application Number:
`PCT/U82008/001602
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`(22) International Filing Date: 7 February 2008 (07.02.2008)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/888,921
`
`8 February 2007 (08.02.2007)
`
`US
`
`(84)
`
`(71) Applicant (for all designated States except US): BIOGEN
`[DEC MA INC. [US/US]; 14 Cambridge Center, Cant-
`bridge, MA 02142 (US).
`
`(72) Inventor; and
`LUKASHEV,
`(for US only):
`(75) Inventor/Applicant
`Matvey, E.
`[US/US]; 3 Louis Road. Tewksbury, MA
`01876 (US).
`
`(74) Agent: GARRETT, Arthur, 8.; Finnegan, Henderson,
`Fambow, Garrett & Dunner L.L.P., 901 New York Avenue,
`NW. Washington, DC 0001-4413 (US).
`
`(10) International Publication Number
`
`W0 2008/097596 A2
`
`Designated States (unless otherwise indicated. for every
`kind of national protection available): AE, AG, AL, AM,
`A0, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ. DE, DK, DM, DO, DZ, EC. BE.
`BG, ES, 1‘1, GB, GD, (iii, (iH, GM, GT, HN, HR, HU, 11),
`IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA. LC.
`LK, LR, LS, LT, LU. LY, MA, MD, ME, MG, MK, MN,
`MW. MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG. PH,
`PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV,
`SY, ’1‘.l, TM, TN, TR, ’l"l‘, ’17., UA, UG, US, 117., VC. VN.
`ZA, ZM, ZW.
`
`Designated Statts (unless otherwise indicated. for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, Kli. LS, MW. MZ, NA, SD. SL, S'/.. '17., UG. ZM.
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CII, CY, CZ, DE, DK, 1313, ES, Fl,
`FR, GB. GR. HR, HU, IE, IS, IT, LT, LU. LV, MC, MT, NL.
`NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG,
`CI. CM. GA, GN, GQ, GW, ML, MR. NE, SN, TD. TG).
`
`Published:
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`without international search report and to be republished
`upon receipt of that report
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`
`
`008/097596A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`(54) Title: NRFZ SCREENING ASSAYS AND RELATED METHODS AND COMPOSITIONS
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`N (57) Abstract: Provided are certain methods of screening, identifying, and evaluating neuroprotective compounds useful for treat-
`menl of neurological diseases, such as, e.g., multiple sclerosis (MS). The compounds described upregulate the cellular cytoprotective
`pathway regulated by Ner. Also provided are certain methods of utilizing such compounds in therapy for neurological disease. par-
`ticularly, for slowing or reducing dcmyelination, axonal loss, or neuronal and oligodcndrocytc death.
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`W0
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`Nrf2 SCREENING ASSAYS
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`AND RELATED METHODS AND COMPOSITIONS
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`[0001]
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`Provided are certain compounds for treating neurological diseases,
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`including demyelinating neurological diseases, such as, e.g., multiple sclerosis.
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`[0002]
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`Multiple sclerosis (MS) is an autoimmune disease with the autoimmune
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`activity directed against central nervous system (CNS) antigens. The disease is
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`characterized by inflammation in parts of the CNS, leading to the loss of the myelin
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`sheathing around neuronal axons (demyelination), loss of axons, and the eventual
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`death of neurons, oligodenrocytes and glial cells.
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`[0003] An estimated 2,500,000 people in the world suffer from MS.
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`it is one of
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`the most common diseases of the CNS in young adults. MS is a chronic, progressing,
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`disabling disease, which generally strikes its victims some time after adolescence, with
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`diagnosis generally made between 20 and 40 years of age, although onset may occur
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`earlier. The disease is not directly hereditary, although genetic susceptibility plays a
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`part in its development. Relapsing-remitting MS presents in the form of recurrent
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`attacks of focal or multifocal neurologic dysfunction. Attacks may occur, remit, and
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`recur, seemingly randomly over many years. Remission is often incomplete and as
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`one attack follows another, a stepwise downward progression ensues with increasing
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`permanent neurological deficit.
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`[0004] Although various immunotherapeutic drugs can provide relief in
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`patients with MS, none is capable of reversing disease progression, and some can
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`cause serious adverse effects. Most current therapies for MS are aimed at the
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`reduction of inflammation and suppression or modulation of the immune system. As of
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`2006, the available treatments for MS reduce inflammation and the number of new
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`episodes but not all have an effect on disease progression. A number of clinical trials
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`have shown that the suppression of inflammation in chronic MS rarely significantly
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`limits the accumulation of disability through sustained disease progression, suggesting
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`that neuronal damage and inflammation are independent pathologies. Promoting CNS
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`remyelination as a repair mechanism and othenrvise preventing axonal loss and
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`neuronal death are some of the important goals for the treatment of MS. For a
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`comprehensive review of MS and its current therapies, see, e.g., McAIpine’s Multiple
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`Sclerosis, by Alastair Compston et al., 4th edition, Churchill Livingstone Elsevier,
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`2006.
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`[0005]
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`“Phase 2 enzymes" serve as a protection mechanism in mammalian
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`cells against oxygen/nitrogen species (ROS/RNS), electrophiles and xenobiotics.
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`These enzymes are not normally expressed at their maximal levels and, their
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`expression can be induced by a variety of natural and synthetic agents. Nuclear factor
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`E2-related factor 2 (Nrf2) is a transcription factor responsible for the induction of a
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`variety of important antioxidant and detoxification enzymes that coordinate a protective
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`cellular response to metabolic and toxic stress.
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`[0006] ROS/RNS are most damaging in the brain and neuronal tissue, where
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`they attack post-mitotic (i.e., non-dividing) cells such as glial cells, oligodendocytes,
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`and neurons, which are particularly sensitive to free radicals. This process leads to
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`neuronal damage. Oxidative stress has been implicated in the pathogenesis of a
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`variety of neurodegenerative diseases, including ALS. Alzheimer's disease (AD), and
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`Parkinson’s disease (PD). For review, see, e.g., van Muiswinkel et al., Curr. Drug
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`Targets CNS—-Neurol. Disord., 2005, 42267-281. An anti-oxidative enzyme under
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`control of Nrf2, NQO1 (NAD(P)H dehydrogenase, quinone (1), was recently reported
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`to be substantially upregulated in the brain tissues of AD and PD subjects (Muiswinkel
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`et al., Neurobiol. Aging, 2004, 25: 1253). Similarly, increased expression of NQO1
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`was reported in the ALS subjects’ spinal cord (Muiswinkel et al., Curr. Drug
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`Targets--CNS. Neurol. Disord., 2005, 4267-281) and in active and chronic lesions in
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`the brains of patients suffering from MS (van Horssen et al., Free Radical Biol. & Med.,
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`2006, 41 311-311). These observations indicate that the Nrf2 pathway may be
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`activated in neurodegenerative and neuroinflammatory diseases as an endogenous
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`protective mechanism. Indeed, most recently, it has been reported that induced
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`activation of Nrf2-dependent genes by certain cyclopenanone-based compounds
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`(NEPP) counters the toxic effects of metabolic inhibition and ROS/RNS production in
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`the brain and protects neurons from death in vitro and in vivo (see Satoh et al., PNAS,
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`2006, 103(3):768—773).
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`[0007] Additionally, many publications have reported neuroprotective effects of
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`compounds in natural plant-derived compounds (“phytochemicals”), including
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`d-tocopherol (vitamin E), lycopene (tomatoes), resveratrol (red grapes), sulforaphane
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`(broccoli), EGCG (green tea), etc. For review, see Mattson and Cheng, Trends in
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`Neurosci., 2006, 29(11):632-639. Originally, the action of these compounds was
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`attributed to their anti-oxidant properties. However, while most anti-oxidants are
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`effective only at high concentrations, at least some of these compounds appear to
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`exert neuroprotective effects at much lower doses. Emerging evidence suggests that
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`these compounds may exert their neuroprotective effects by activating cellular
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`stress-response pathways, including the Ner pathway, resulting in the upregulation of
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`neuroprotective genes. However, the exact mechanism of action of these compounds
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`remains poorly understood.
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`[0008] To date, more than 10 different chemical classes of inducers of Ner
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`pathway have been identified including isothiocyanates and their thiol addition
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`products, dithiocarbamates, as well as 1,2-dithiole-3-thiones, trivalent arsenic
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`derivatives (e.g., phenyl arsenoxide), heavy metals, certain conjugated cyclic and
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`acyclic polyenes (including porphyrins, chlorophyllins, and chlorophyll), and vicinal
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`dimercaptans. These inducers have few structural similarities. They are mostly
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`electrophiles, and all can react chemically with thiol groups by alkylation, oxidation, or
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`reduction, suggesting that the intracellular sensor for inducers is likely to contain very
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`highly reactive (cysteine) thiols. The inducers can modify thiol groups by a variety of
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`mechanisms including: alkylation (Michael addition acceptors, isothiocyanates,
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`quinones); oxidation (e.g., peroxides and hydroperoxides); and direct reaction with
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`thiol/disulfide linkages (e.g., vicinal dithiols such as 1 ,2-dimercaptopropanol, lipoic
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`acid). These diverse response mechanisms provide plasticity for cellular responses to
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`a variety of electrophilic and oxidant stressors.
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`[0009] Provided are methods that comprise at least one of the following
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`methods:
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`1) methods of screening for at least one new candidate compound for
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`treating a neurological disease;
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`2) methods of evaluating neuroprotective properties of at least one drug
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`candidate for treating a neurological disease;
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`3) methods of comparing (e.g., for bioequivalence) at least two
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`pharmaceutical compositions which comprise fumaric acid derivatives;
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`4) methods of treating a neurological disease by administering to the subject
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`in need thereof at least one compound that is partially structurally similar to
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`DMF or MMF; and
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`5) methods of treating a neurological disease by a combination therapy that
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`comprises administration of at least one first compound that upregulates
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`the Ner pathway and at least one second compound that does not
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`upregulate the Ner pathway.
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`[0010]
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`In some embodiments, the neurological disease is a neurodegenerative
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`disease such as, for example, ALS, Parkinson’s disease, Alzheimer’s disease, and
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`Huntington’s disease.
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`In some embodiments the neurological disease is MS or
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`another demyelinating neurological disease.
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`[0011]
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`In some embodiments, the methods 1-3 further comprise:
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`a) contacting a cell with the test compound, and
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`b) determining whether the Ner pathway is upregulated in the cell.
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`In some embodiments, the methods may further comprise:
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`c) determining whether the test compound slows or prevents demyelination,
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`axonal loss, and/or neuronal death, and/or
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`d) selecting the test compound as a candidate for treating neurodegeneration
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`in a neurological disease if 1) the Ner pathway is upregulated and 2)
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`demyelination, axonal loss, and/or neuronal death are/is prevented or
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`slowed.
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`[0012]
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`In some embodiments, the methods 1-3 comprise contacting a cell with
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`at least one test compound and determining whether the Ner pathway is upregulated
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`in the cell.
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`In such methods, an upregulation of the Ner pathway above a threshold
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`(e.g., by at least 30% over a control) indicates that the at least one compound has at
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`least one biological property beneficial in treating a neurological disease (e.g.,
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`In some embodiments, the upregulation of the Ner
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`pathway is assessed (in vivo and/or in vitro) by at least one of the following:
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`i) expression levels of endogenously produced and/or exogenously
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`ii) subcellular localization and/or nuclear translocation of Nrf2;
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`iii) expression levels and/or activity of one or more genes under control of
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`Nrf2 (e.g., endogenous NQO1) or an Nrf2-regulated reporter gene in an
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`iv)
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`levels of Ner binding to the Nrf2-binding DNA element ARE;
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`v) stability of Nrf2/Keap1 complexes; and
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`vi) modification (e.g., alkylation) levels of Keap1 and/or at least one other
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`[0013]
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`In some embodiments of methods 1-3, the compounds that are being
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`screened, evaluated, or compared comprise at least one member of at least one of the
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`In some embodiments, the Michael addition acceptor has the structure of
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`Formula I, ll, Ill, or IV set forth below.
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`[0014]
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`In some embodiments method 1 comprises:
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`a) contacting a cell with a plurality of test compounds,
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`b) determining whether the Ner pathway is upregulated in the cell, and
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`c) selecting from the plurality of compounds at least one compound that
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`wherein an upregulation of the Ner pathway by the selected at least one compound
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`indicates that the selected at least one compound may be useful for treating a
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`neurological disease. The plurality of compounds may be represented, e.g., by a
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`combinatorial chemical library, and the method may be performed, e.g., by
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`PCT/US2008/001602
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`[0015]
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`In some embodiments method 2 comprises:
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`a) contacting a cell with the at least one drug or drug candidate, and
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`wherein an upregulation of the Ner pathway by the at least one drug or drug
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`candidate indicates that the at least one drug or drug candidate is useful for
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`neuroprotection in treating a human having a neurological disease.
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`[0016]
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`In some embodiments method 3 comprises:
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`a) contacting a cell with a first composition comprising at least one test
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`compound, and
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`b) comparing the level of Ner pathway upregulation in the cell by the at least
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`one test compound to the corresponding level of the Ner pathway
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`upregulation in a control cell treated with a second composition comprising
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`at least one of DMF and MMF.
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`[0017]
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`In some embodiments of method 3, the test compound is fumaric acid,
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`a salt thereof, or a fumaric acid derivative.
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`In some embodiments, the first
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`composition comprises DMF, MMF, or both.
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`In some embodiments, the dose and/or
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`the formulation of the first composition differs from the dose and/or the formulation of
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`[0018]
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`In some embodiments, method 3 further comprises:
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`c) comparing at least one pharmacokinetic parameter (e.g., serum-half—Iife) of
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`the first and the second compositions.
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`[0019]
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`In some embodiments method 4 comprises administering to the
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`mammal a therapeutically effective amount of at least one neuroprotective compound
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`having Formula I, II, III, or IV, e.g., a fumaric acid derivative (e.g., DMF or MMF).
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`[0020]
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`In some embodiments method 4 provides a method of slowing or
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`preventing neurodegeneration in a patient in need thereof, by administering the
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`compound in an amount and for a period of time sufficient to slow or prevent
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`demyelination, axonal loss, and/or neuronal death, e.g., by at least 30% relative to a
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`control.
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`[0021]
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`In some embodiments method 5 comprises:
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`Page 7 of 42
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`Page 7 of 42
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`a) administering to the mammal a therapeutically effective amount of at least
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`one first compound that upregulates the Nrf2 pathway, and
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`b) administering a therapeutically effective amount of at least one second
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`compound that does not upregulate the Nrf2 pathway.
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`[0022]
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`In some embodiments of method 5, the at least one first compound,
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`used in step (a), is a compound of Formula I, II, III, or IV, e.g., a fumaric acid derivative
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`(e.g., DMF or MMF); and the at least one second compound, which is used in step (b),
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`is an immunosuppressive or an immunomodulatory compound that does not
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`upregulate the Nrf2 pathway (e.g., by more than 30% over a control).
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`[0023]
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`In some embodiments method 5 comprises administering to the
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`mammal a therapeutically effective amount of a compound of Formula I, II, III, or IV.
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`[0024]
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`In some embodiments of methods 1-5, the at least onecompound being
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`screened, identified, evaluated, or used for treating a neurological disorder is not
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`fumaric acid or its salt, or a fumaric acid derivative (e.g., DMF or MMF).
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`[0025] Other features and embodiments of the invention will be apparent from
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`the following description and the claims.
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`BRIEF DESCRIPTION OF THE FIGURES
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`[0026] Figure 1 demonstrates that DMF and MMF are activators of Nrf2 at
`concentrations within clinical exposure range (cells in culture).
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`[0027] Figure 2 shows results of RNAi experiments.
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`[0028] Figure 3 shows evidence of Nrf2 activation by DMF and MMF In vivo.
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`[0029] Figure 4 shows evidence of Nrf2 activation by DMF and MMF In vivo.
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`[0030] Fumaric acid esters, such as DMF, have been proposed fortreatment
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`of MS (see, e.g., Schimrigk et al., Eur. J. Neurol., 2006, 13(6):604-10; Drugs R&D,
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`2005, 6(4):229-30).
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`[0031] Provided are, among other things, means for identifying compounds
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`with a new therapeutic modality useful in at least one of multiple neUrological
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`indications and, optionally, complementary to other drugs for the treatment of a
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`neurological disease, including a number of currently used immunomodulators.
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`Page 8 of 42
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`Page 8 of 42
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`PCT/US2008/001602
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`[0032] DMF is a member of a large group of anti-oxidant molecules known for
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`their cytoprotective and anti-inflammatory properties. These molecules also share the
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`property of the Ner pathway activation. Thus, the finding that DMF activates the Ner
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`pathway in conjunction with the neuroprotective effects of DMF further offers a
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`rationale for identification of structurally and/or mechanistically related molecules that
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`would be expected to be therapeutically effective for the treatment of neurological
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`disorders, such as, e.g., MS.
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`[0033] Certain terms are defined in this section; additional definitions are
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`provided throughout the description.
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`[0034] The terms “activation” and “upregulation,” when used in reference to
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`the Ner pathway, are used interchangeably herein.
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`[0035] The terms “disease” and “disorder" are used interchangeably herein.
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`[0036] The term “a drug for treating a neurological disease" refers to a
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`compound that has a therapeutic benefit in a specified neurological disease as shown
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`in at least one animal model of a neurological disease or in human clinical trials for the
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`treatment of a neurological disease.
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`[0037] The term “neuroprotection” and its cognates refer to prevention or a
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`slowing in neuronal degeneration, including, for example, demyelination and/or axonal
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`loss, and/or, neuronal and/or oligodendrocyte death. Neuroprotection may occur
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`through several mechanisms, e.g., through reducing inflammation, providing
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`neurotrophic factors, scavenging free radicals, etc. As used herein, a compound is
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`considered neuroprotective if it (1) upregulates the Ner pathway above a certain
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`threshold and (2) provides neuroprotection, regardless of possible other mechanisms
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`of action.
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`[0038] The terms “treatment," “therapeutic method,
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`therapeutic benefits," and
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`the like refer to therapeutic as well as prophylactic/preventative measures. Thus,
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`those in need of treatment may include individuals already having a specified disease
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`and those who are at risk for acquiring that disease.
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`[0039] The terms “therapeutically effective dose” and “therapeutically effective
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`amount" refer to that amount of a compound which results in at least one of prevention
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`Page 9 of 42
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`Page 9 of 42
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`WO 2008/097596
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`PCT/U82008/001602
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`or delay of onset or amelioration of symptoms of a neurological disorder in a subject or
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`an attainment of a desired biological outcome, such as reduced neurodegeneration
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`(e.g., demyelination, axonal loss, and neuronal death) or reduced inflammation of the
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`cells of the CNS.
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`[0040]
`In one aspect, provided are methods of evaluating neuroprotective
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`properties of test compounds, including the following methods:
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`1) methods of screening for new candidate compounds that may be
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`useful for treating a neurological disease;
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`2) methods of evaluating neuroprotective properties of drugs and
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`candidates that are used or proposed for treating a neurological
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`disease;
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`3) methods of comparing (e.g., for bioequivalence) two or more
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`pharmaceutical compositions which contain fumaric acid
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`derivatives;
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`[0041]
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`In some embodiments, methods 1-3 may comprise:
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`a) contacting a cell with the test compound,
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`b) determining whether the Ner pathway is upregulated in the cell,
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`and, in some embodiments, additionally performing the following step(s):
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`c) determining whether the test compound slows or prevents demyelination,
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`axonal loss, and/or neuronal death, and/or
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`d) selecting the test compound as a candidate for treating neurodegeneration
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`in a neurological disease if 1)the Nrf2 pathway is upregulated and 2)
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`demyelination, axonal loss, and/or neuronal death are/is prevented or
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`slowed.
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`Method 1
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`[0042]
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`In some embodiments the methods of screening for a candidate
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`compound for treating a neurological disease comprise:
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`a) contacting a cell with a plurality of test compounds,
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`b) determining whether the Ner pathway is upregulated in the cell, and
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`c) selecting from the plurality of compounds at least one compound that
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`upregulates the Ner pathway,
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`PCT/US2008/001602
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`wherein an upregulation of the Ner pathway by the selected at least one compound
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`indicates that the selected at least one compound may be useful for treating a
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`neurological disease. For example, the plurality of compounds may be represented by
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`a combinatorial chemical library, and the screening method may be performed by a
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`high-throughput screening as described in, e.g., High-Throughput Screening in Drug
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`Discovery (Methods and Principles in Medicinal Chemistry), by Jorg HUser (ed.), John
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`Wiley & Sons (2006).
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`[0043] Combinatorial libraries of compounds are also described in, e.g.,
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`Solid—Supported Combinatorial and Parallel Synthesis of Small-Molecular—Weight
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`Compound Libraries (Tetrahedron Organic Chemistry) Ian Salusbury (ed.), Elsevier
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`(1998); Combinatorial Libraries: Synthesis, Screening and Application Potential
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`(Library Binding), by Riccardo Cortese (ed.), Walter de Gruyter (1995). The libraries of
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`compounds may be, for example, quinone libraries and other libraries as described in
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`Mittoo, Comb. Chem. & High Throughput Screen, 2006, 9:421-423.
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`[0044]
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`In some embodiments, the at least one compound or plurality of
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`compounds being screened and/or selected comprises at least one compound
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`selected from at least one of the following groups of compounds: mild alkylating
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`agents, Michael addition acceptors or compounds that are metabolized to Michael
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`addition acceptors, including compounds of Formulas I, II, III, or IV.
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`[0045]
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`In some of the embodiments, the at least one compound is selected
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`from fumaric acid, its salts, and fumaric acid derivatives.
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`Method 2
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`[0046] Also provided are methods of evaluating neuroprotective properties of
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`at least one drug or drug candidate for treating at least one neurological disease.
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`Such methods comprise:
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`a) contacting a cell with the at least one drug or drug candidate, and
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`b) determining whether the Ner pathway is upregulated in the cell,
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`wherein the upregulation of the Ner pathway by the at least one drug or drug
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`candidate indicates that the at least one drug or drug candidate is neuroprotective in
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`treating a human having a neurological disease.
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`[0047]
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`In some embodiments, the upregulation of the Nrf2 pathway by the at
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`least one drug or drug candidate indicates that the at least one drug or drug candidate
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`has at least one activity selected from slowing demyelination, slowing the loss of
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`axons, and slowing the rate of neuronal death.
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`[0048]
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`In some embodiments, the method of evaluating at least one drug or
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`drug candidate comprises an additional step:
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`c) evaluating demyelination, loss of axons, and/or neuronal death.
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`In some embodiments, steps a) and c) are performed in vivo in at least
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`[0049]
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`one model of a neurological disease, e.g., as described below.
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`[0050]
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`In other embodiments, particularly those in which the neurological
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`disease is multiple sclerosis or another demyelinating disease, the evaluated at least
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`one drug or drug candidate for a neurological disease is chosen from the following:
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`FTY720 (2-(4-octylphenethyl)-2-aminopropane-1,3-diol; Novartis); anti-IL12 antibody
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`(e.g., ABT-874; Abbott Laboratories); GSK683699 (GSK/Tanabe); NeuroVax (Immune
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`Response Corp.; Darlington, Curr. Opin. Mol. Ther., 2005, 7(6):598-603); anti-CCR2
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`antibody (e.g., MLN 1202; Millennium); interferon [3-1a (e.g., Avonex®; Biogen Idec);
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`anti-a4-integrin antibody (e.g., Tysabri®; Biogen ldec/Elan); anti-CD20 antibody (e.g.,
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`Rituxan® (Biogen Idec/Genentech); TV 5010 (Teva); NBI-788 (Neurocrine); MBP8298
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`(BioMS (see Warren et al., Eur. J. Neurol., 2006, 13(8):887-95); Mylinax (Oral
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`Cladribine; 2-chlorodeoxyadenosine; Serono/IVAX); Teriflunomide
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