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`Nrf2 SCREENING ASSAYS AND RELATED METHODS AND COMPOSITIONS
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`BIOGEN IDECI FINNEGAN HENDERSON, LLP
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`Customer Number 65,779
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`ENCLOSED APPLICATION PARTS (check all that apply)
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`The invention was made by an agency of the United States Government or under a contract with an
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`[Text]____—__—_,__—__—.__—_.————————-——--—
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`Respectfully submitted on b
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`SIGNATURE Kg2%a? 5
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`Date February 8, 2007
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`alf of the patent practitioners associated with Customer Number 65,779.
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`TYPED OR PRINTED NAME Konstantin M. Linnik
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`REGISTRATION NO. 56,309
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`[:| Additional inventors are being named on separately numbered sheets attached hereto.
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`PROVISIONAL APPLICATION FILING ONLY
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`Page 1 of 68
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`Biogen Exhibit 2104
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`Mylan v. Biogen
`IPR 2018-01403
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`Page 1 of 68
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`Biogen Exhibit 2104
`Mylan v. Biogen
`IPR 2018-01403
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`Attorney Docket No. 03201 13042-00000
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`Nrf2 SCREENING ASSAYS
`AND RELATED METHODS AND COMPOSITIONS
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`[0001]
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`The invention relates to the field of cell and molecular biology and
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`to the development and use of therapeutic compounds, more particularly,
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`compounds for treating neurological diseases, including demyelinating
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`neurological diseases, such as, e.g., multiple sclerosis.
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`[0002] Multiple sclerosis (MS) is an autoimmune disease with the
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`autoimmune activity directed against central nervous system (CNS) antigens.
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`The disease is characterized by inflammation in parts of the CNS, leading to the
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`loss of the myeiin sheathing around neuronal axons (demyelination), loss of
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`axons, and the eventual death of neurons, oligodenrocytes and glial cells.
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`[0003]
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`An estimated 2,500,000 people in the world suffer from MS.
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`It is
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`one of the most common diseases of the CNS in young adults. MS is a chronic,
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`progressing, disabling disease, which generally strikes its victims some time after
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`adolescence, with diagnosis generally made between 20 and 40 years of age,
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`although onset may occur earlier. The disease is not directly hereditary,
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`although genetic susceptibility plays a part in its development.
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`Relapsing—remitting MS presents in the form of recurrent attacks of focal or
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`multifocal neurologic dysfunction. Attacks may occur, remit, and recur,
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`seemingly randomly over many years. Remission is often incomplete and as one
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`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
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`can cause serious adverse effects. Most current therapies for MS are aimed at
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`the reduction of inflammation and suppression or modulation of the immune
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`system. As of 2006, the available treatments for MS reduce inflammation and
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`the number of new episodes but not all have an effect on disease progression. A
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`number of clinical trials have shown that the suppression of inflammation in
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`chronic MS rarely significantly limits the accumulation of disability through
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`Attorney Docket No. 08201.6042—00000
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`sustained disease progression, suggesting that neuronal damage and
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`inflammation are independent pathologies. Promoting CNS remyelination as a
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`repair mechanism and otherwise preventing axonal loss and neuronal death are
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`some of the important goals for the treatment of MS. For a comprehensive
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`review of MS and its current therapies, see, e.g., McAlpine's Multiple Sclerosis,
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`by Alastair Compston et at, 4th edition, Churchill Livingstone Elsevier, 2006.
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`[0005]
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`“Phase 2 enzymes" serve as a protection mechanism in
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`mammalian cells against oxygen/nitrogen species (ROS/HMS), electrophiles and
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`xenobiotics. These enzymes are not normally expressed at their maximal levels
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`and, their expression can be induced by a variety of natural and synthetic agents.
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`Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor responsible for
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`the induction of a variety of important antioxidant and detoxification enzymes that
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`coordinate a protective cellular response to metabolic and toxic stress.
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`[0006] FtOS/FlNS are most damaging in the brain and neuronal tissue,
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`where they attack post-mitotic (i.e., non-dividing) cells such as glial cells,
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`oligodendocytes, and neurons, which are particularly sensitive to free radicals.
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`This process leads to neuronal damage. Oxidative stress has been implicated in
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`the pathogenesis of a variety of neurodegenerative diseases, including ALS,
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`Alzheimer‘s disease (AD), and Parkinson's disease (PD). For review, see, e.g.,
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`van Mulswinkel et al., Curr. Drug Targets CNS--Neurol. Disord., 2005, 4:267-281.
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`An anti-oxidative enzyme under control of Nrf2, N001 (NADlPlH
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`dehydrogenase, quinone 1), was recently reported to be substantially
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`upregulated in the brain tissues of AD and PD subjects (Muiswinkel et al.,
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`Neurobiol. Aging, 2004, 25: 1253). Similarly, increased expression of N001 was
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`reported in the ALS subjects” spinal cord (Muiswinkel et al., Curr. Drug
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`Targets-CNS. Neurol. Disord, 2005, 4:267-281) and in active and chronic
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`lesions in the brains of patients suffering from MS (van Horssen et al., Free
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`Radical Biol. & Med, 2006, 41 311-311). These observations indicate that the
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`Ner pathway may be activated in neurodegenerative and neuroinflammatory
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`diseases as an endogenous protective mechanism.
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`Indeed, most recently, it has
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`Attorney Docket No. 08201 .6042—00000
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`been reported that induced activation of Nrf2-dependent genes by certain
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`cyclopenanone—based compounds (NEPP) counters the toxic effects of metabolic
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`inhibition and ROS/ENS production in the brain and protects neurons from death
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`in vitro and in vivo (see Satoh et al., PNAS, 2006, 103(3):?68-773).
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`[0007] Additionally, many publications have reported neuroprotective
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`effects of compounds in natural plant-derived compounds (“phytochemicals”),
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`including cr—tocopherol (vitamin E), lycopene (tomatoes), resveratrol (red grapes),
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`sulforaphane (broccoli), EGCG (green tea), etc. For review, see Mattson and
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`Cheng, Trends in Neurosci., 2006, 29(11):632-639. Originally, the action of
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`these compounds was attributed to their anti—oxidant properties. However, while
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`most anti-oxidants are effective only at high concentrations, at least some of
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`these compounds appear to exert neuroprotective effects at much lower doses.
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`Emerging evidence suggests that these compounds may exert their
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`neuroprotective effects by activating cellular stress-response pathways, including
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`the Nrf2 pathway, resulting in the upregulation of neuroprotective genes.
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`However, the exact mechanism of action of these compounds remains poorly
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`understood.
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`[0008] To date, more than 10 different chemical classes of inducers of
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`Nrf2 pathway have been identified including isothiocyanates and their thiol
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`addition products, dithiocarbamates, as well as 1,2«dithiole-3-thiones, trivalent
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`arsenic derivatives (e.g., phenyl arsenoxide), heavy metals, certain conjugated
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`cyclic and acyclic polyenes (including porphyrins, chlorophyllins, and chlor0phyll),
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`and vicinal dimercaptans. These inducers have few structural similarities. They
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`are mostly electrophiles, and all can react chemically with thiol groups by
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`alkylation, oxidation, or reduction, suggesting that the intracellular sensor for
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`inducers is likely to contain very highly reactive (cysteine) thiols. The inducers
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`can modify thiol groups by a variety of mechanisms including: alkylation (Michael
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`addition acceptors, isothiocyanates, quinones); oxidation (e.g., peroxides and
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`hydroperoxides); and direct reaction with thiolldisulfide linkages (e.g., vicinal
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`dithiols such as 1,2-dimercaptopropanol, Iipoic acid). These diverse response
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`Attorney Docket No. 08201 .6042—00000
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`mechanisms provide plasticity for cellular responses to a variety of electrophilic
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`and oxidant stressors.
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`[0009]
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`There is a need to develop new treatments, and in particular,
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`compounds for treating MS that provide neuroprotection. The development of
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`cell-based and in vitro assays to identify and characterize existing drug
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`candidates serves this goal.
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`[0010]
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`In some embodiments, the invention provides the following
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`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 drug
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`candidates for treating a neurological 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 derivatives;
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`4) methods of treating a neurological disease by administering to the
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`subject in need thereof compounds that are partially structurally
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`similar to DMF or MMF; and
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`5) methods of treating a neurological disorder by a combination therapy
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`that includes administration of a first compound that upregulates the
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`Nrf2 pathway and a second compound that does not upregulate the
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`Nrf2 pathway.
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`[0011] A neurological disease in methods 1-5 above is preferrably a
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`neurodegenerative disease such as, for example, ALS, Parkinson’s disease,
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`Alzheimer’s disease, and Huntington’s disease. More preferably, the
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`neurological disease is MS or another demyelinating neurological disease.
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`[0012] Methods 1-3 of the invention may comprise:
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`a) contacting a cell with the test compound, and
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`b) determining whether the Nrf2 pathway is upregulated in the cell.
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`In some embodiments, the methods may further comprise:
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`Attorney Docket No. 08201 .6042—00000
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`c) determining whether the test compound slows or prevents
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`demyelination, axonal loss, and/or neuronal death, and/or
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`d) selecting the test compound as a candidate for treating
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`neurodegeneration in a neurological disease it 1) the Nrf2 pathway is
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`upregulated and 2) demyelination, axonal loss, and/or neuronal death
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`are!is prevented or slowed.
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`[0013] Methods 1-3 of the invention comprise contacting a cell with test
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`compound(s) and determining whether the Ner pathway is upregulated in the
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`cell.
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`In such methods, an upregulation of the Nrf2 pathway above a threshold
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`(9.9., by at least 30% over a control) indicates that the compoundts) has/have
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`certain biological properties beneficial in treating a neurological disease (e.g.,
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`neuroprotective properties).
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`In some embodiments, the upregulation of the Nrf2
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`pathway is assessed (in vivo andior in vitro) by one or more of the following:
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`i) expression levels of endogenously produced or exogenously
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`introduced Nr12;
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`ii)
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`subcellular localization and/or nuclear translocation of Nrf2;
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`iii) expression levels andlor activity of one or more genes under control
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`of Nrf2 (e.g., endogenous N001) or an Nrf2-regulated reporter gene
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`in an artificial reporter construct;
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`iv)
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`levels of Nrf2 binding to the Nrf2-binding DNA element ARE;
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`v)
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`stability of Nrf2fKeap1 complexes; and
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`vi) modification (e.g., alkylation) levels of Keap‘l and other
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`NerIKeapt associated proteins.
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`[0014]
`
`In some embodiments of methods 1-3, the compounds that are
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`being screened, evaluated, or compared are mild alkylating agents, and more
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`specifically, Michael addition acceptors, or compounds that are metabolized upon
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`administration to Michael addition acceptors.
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`In some embodiments, such
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`Michael addition acceptors have the structure of Formula I, II, III, or IV set forth in
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`the Detailed Description.
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`[0015]
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`_In certain embodiments of method 1, the method of screening for
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`a candidate compound for treating a neurological disease cemprises:
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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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`wherein an upregulation of the M12 pathway by the selected compound(s)
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`indicates that the selected compound(s) may be useful for treating a neurological
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`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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`high-throughput screening.
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`[0016]
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`in certain embodiments of method 2, the method of evaluating
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`neuroprotective properties of a drug or drug candidate for treating a neurological
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`disease comprises:
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`a) contacting a cell with the drug or drug candidate, and
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`b) determining whether the Nrf2 pathway is upregulated in the cell,
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`wherein an upregulation of the Ner pathway by the drug or drug candidate
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`indicates that the drug or drug candidate is useful for neuroprotection in treating
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`a human having a neurological disease.
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`[001?]
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`In certain embodiments of method 3, a method of comparing two
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`Or more pharmaceutical compositions (e.g., for bioequivalence) comprises:
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`a) contacting a cell with a first composition comprising a test compound,
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`and
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`b) comparing the level of Nrf2 pathway upregulation in the cell by the
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`test compound to the corresponding level of the M12 pathway
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`upregulation in a control cetl treated with a second composition
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`comprising DMF, MMF, or both.
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`[0018]
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`In some embodiments of method 3, the test compound is fumaric
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`acid, 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
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`Attorney Docket No. 08201 .6042—00000
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`andfor the formulation of the first composition differs from the dose andfor the
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`formulation of the second composition.
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`[0019]
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`In some embodiments. method 3 further comprises:
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`c) comparing at least one pharmacokinetic parameter (e.g.,
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`serum—haIf—Iife) of the first and the second compositions.
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`[0020]
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`In some embodiments of method 4, the method of treating a
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`mammal having a neurological disease comprises administering to the mammal
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`a therapeutically effective amount of a neuroprotective compound having
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`Formula I, II, III, or IV, e.g., a fumaric acid derivative (e.g., DMF or MMF).
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`[0021]
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`In some embodiments of method 4, the invention provides a
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`method of slowing or preventing neurodegeneration (more specifically, e.g.,
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`demyelination, axonal loss, andi’or neuronal death) in a patient in need thereof,
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`by administering the compound in an amount and for a period of time sufficient to
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`slow or prevent demyelination, axonal loss, and/or neuronal death, e.g., by at
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`least 30% relative to a control.
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`[0022]
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`In certain embodiments of method 5, the method of treating a
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`mammal having a neurological disease by combination therapy comprises:
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`a) administering to the mammal a therapeutically effective amount of a
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`first compound that upregulates the Nrf2 pathway, and
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`b) administering a therapeutically effective amount of a second
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`compound that does not upregulate the Ner pathway.
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`[0023]
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`In some of embodiments of method 5, the first compound, used in
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`step (a), is a compound of Formula I, ll, ill, or IV, e.g., a fumaric acid derivative
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`(e.g., DMF or MMF); and the second compound, which is used in step (b). is an
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`immunosuppressive or an immunomodulatory compound that does not
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`upregulate the Ner pathway (e.g., by more than 30% over a control).
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`[0024]
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`In some embodiments of method 5, the method of treating a
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`neurological disease in a mammal comprises administering to the mammal a
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`therapeutically effective amount of a compound of Formula I, II, III, or IV.
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`[0025]
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`In some of the embodiments of methods 1-5, the compound 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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`[0026] Other features and embodiments of the invention will be apparent
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`from the following description and the claims.
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`BRIEF DESCRIPTION OF THE FIGURES
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`[0027] Figure 1 demonstrates that DMF and MMF are potent activators
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`of Nrf2 at concentrations within clinical exposure range (cells in culture).
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`[0028] Figure 2 shows results of RNAi experiments.
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`DETAILED DESCRIPTION
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`[0029] The present invention is based, in part, on the discovery that
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`dimethyl fumarate (DMF) and monomethyl fumarate (MMF) are potent activators
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`of the Nrf2 pathway, a major neuroprotective and anti-inflammatory mechanism.
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`The invention is further based, at least in part, on the finding that DMF and MMF
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`are neuroprotective (myelinoprotective and axonoprotective) in a mouse model of
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`autoimmune neurodegenerative disease.
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`[0030] Due to the involvement of Nri2 in the regulation of cellular
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`response to metabolic stress, survival and inflammation, DMF, MMF, and other
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`Nrf2 activators may be useful for therapeutic management of a variety of
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`inflammatory, ischemic, and neurodegenerative processes.
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`[0031] Fumaric acid esters, such as DMF, have been proposed for
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`treatment of MS (see, e.g., Schimrigk et al., Eur. J. Neurol., 2006, 13(6):604-10;
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`Drugs Fl&D, 2005, 6(4):229—30).
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`[0032] DMF activates a major cytoprotective (neuroprotective) and
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`anti-inflammatory mechanism not targeted by current therapies. Thus, the
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`findings that DMF activates the Nrt2 pathway and has a neuroprotective effect,
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`offer a rationale for the use of DMF in combination with immunosuppressive or
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`Attorney Docket No. 08201 .6042—00000
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`immunodulatory therapeutics that do not upregulate the Nrf2 pathway. The
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`invention further provides means for identifying compounds with a new
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`therapeutic modality useful in multiple neurological indications and
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`complementary to other drugs for the treatment of a neurological disease,
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`including a number of currently used immunomodulators.
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`[0033] DMF is a member of a large group of anti~oxidant molecules
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`known for their cytoprotective and anti-inflammatory properties. These
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`molecules also share the property of the Nrf2 pathway activation. Thus, the
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`finding that DMF activates the Nrf2 pathway in conjunction with the
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`neuroprotective effects of DMF further offers a rationale for identification of
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`structurally and/or mechanistically related molecules that would be expected to
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`be therapeutically effective for the treatment of neurological disorders, such as,
`
`e.g., MS.
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`I. Definitions
`
`[0034] Certain terms are defined in this section: additional definitions are
`
`provided throughout the description.
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`[0035] The terms “activation” and "upregulation,” when used in reference
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`to the Nrf2 pathway, are used interchangeably herein.
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`[0036] The terms “disease” and “disorder” are used interchangeably
`
`herein.
`
`[0037] 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
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`shown in at least one animal model of a neurological disease or in human clinical
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`trials for the treatment of a neurological disease.
`
`[0038] The term “neuroprotection” and its cognates refer to prevention or
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`a slowing in neuronal degeneration, including, for example, demyelination and/or
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`axonal loss, and optionally, neuronal and oligodendrocyte death. Neuroprotection
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`may occur through several mechanisms, e.g., through reducing inflammation,
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`providing neurotrophic factors, scavenging free radicals. etc. As used herein, a
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`compound is considered neuroprotective if it (1) upregulates the Nrf2 pathway
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`Attorney Docket No. 08201.6042-00000
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`above a certain threshold and (2) provides neuroprotection, regardless of
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`possible other mechanisms of action.
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`[0039] The terms “treatment,” “therapeutic method,” “therapeutic
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`benefits,” and the like refer to therapeutic as well as prophylactic/preventative
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`measures. Thus, those in need of treatment may include individuals already
`
`having a specified disease and those who are at risk for acquiring that disease.
`
`[0040] The terms “therapeutically effective dose” and "therapeutically
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`effective amount" refer to that amount of a compound which results in prevention
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`or delay of onset or amelioration of symptoms of a neurological disorder in a
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`subject or an attainment of a desired biological outcome, such as reduced
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`neurodegeneration (e.g., demyelination, axonal loss, and neuronal death) or
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`reduced inflammation of the cells of the CNS.
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`II.
`
`Methods of Evaluating Compounds
`
`[0041]
`
`In one aspect, the invention provides method of evaluating
`
`neuroprotective properties of test compounds, including the following methods:
`
`1) methods of screening for new candidate compounds that may be
`
`useful for treating a neurological disease;
`
`2) methods of evaluating neuroprotective properties of drugs and
`
`candidates that are used or proposed for treating a neurological
`
`disease;
`
`3) methods of comparing (e.g., for bioequivalence) two or more
`
`pharmaceutical compositions which contain fumaric acid
`
`derivatives;
`
`[0042] Methods 1-3 of the invention may comprise:
`
`'
`
`a) contacting a cell with the test compound,
`
`b) determining whether the Nrf2 pathway is upregulated in the cell,
`
`and, in some embodiments, additionally performing the following stepis):
`
`c) determining whether the test compound slows or prevents
`
`demyelination, axonal toss, andfor neuronal death, andfor
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`d) selecting the test compound as a candidate for treating
`
`neurodegeneration in a neurological disease if 1) the Nrf2 pathway is
`
`upregulated and 2) demyelination, axonal loss, and/or neuronal death
`
`are/is prevented or slowed.
`
`[0043] Methods 1-3 are described in detail below.
`
`Method 1: Methods of screening
`
`[0044] The invention provides methods of screening for a candidate
`
`compound for treating a neurological disease. Such methods comprise:
`
`a) contacting a cell with a plurality of test compounds,
`
`b) determining whether the Ner pathway is upregulated in the cell, and
`
`c) selecting from the plurality of compounds at least one compound that
`
`upregulates the Nrf2 pathway,
`
`wherein an upregulation of the Nrf2 pathway by the selected compound(s)
`
`indicates that the selected compound(s) may be useful for treating a neurological
`
`disease. For example, the plurality of compounds may be represented by a
`
`combinatorial chemical library, and the screening method may be performed by a
`
`high-throughput screening as described in, e.g., High-Throughput Screening in
`
`Drug Discovery (Methods and Principles in Medicinal Chemistry), by Jorg Hiiser
`
`(ed), John Wiley & Sons (2006).
`
`[0045] Combinatorial libraries of compounds are also described in, e.g.,
`
`Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight
`
`Compound Libraries (Tetrahedron Organic Chemistry) Ian Salusbury (ed),
`
`Elsevier (1998); Combinatorial Libraries: Synthesis, Screening and Application
`
`Potential (Library Binding), by Riccardo Cortese (ed.), Walter de Gruyter (1995).
`
`The libraries of compounds may be, for example, quinone libraries and other
`
`libraries as described in Mittoo, Comb. Chem. & High Throughput Screen, 2006,
`
`9:421 -423.
`
`[0046]
`
`In some embodiments, the compounds that are being screened
`
`and/or selected comprise at least one or a plurality of mild alkylating agents, and
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`Attorney Docket No. 08201 .6042-00000
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`more particularly, Michael addition acceptors or compounds that are metabolized
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`to Michael addition acceptors, including compounds of Formulas I, ll, ill, or IV.
`
`[0047]
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`In some of the embodiments, the compounds comprise fumaric
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`acid, its salt(s), andfor fumaric acid derivative(s).
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`Methods 2: Methods of evaluating drugs and drug candidates
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`[0048] The invention further provides methods of evaluating
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`neuroprotective properties of a drug or drug candidate for treating a neurological
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`disease. Such methods comprise:
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`a) contacting a cell with the drug or drug candidate, and
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`b) determining whether the Nri2 pathway is upreguiated in the cell,
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`wherein the upregulation of the Nri2 pathway by the drug or drug candidate
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`indicates that the drug or drug candidate is neuroprotective in treating a human
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`having a neurological disease.
`
`[0049]
`
`in some embodiments, the upregulation of the Nrt2 pathway by
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`the drug or drug candidate indicates that the drug or drug candidate has the
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`ability to slow demyelination, the loss of axons, and/or neuronal death.
`
`[0050]
`
`In some embodiments, the method of evaluating a drug or drug
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`candidate comprise an additional step:
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`c) evaluating demyelination, loss of axons, and/or neuronal death.
`
`[0051]
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`In some embodiments, steps a) and c) are performed in vivo in at
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`least one model of a neurological disease, e.g., as described below.
`
`[0052]
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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
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`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-dioi; Novartis); anti-IL12
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`antibody (e.g., ABT-874; Abbott Laboratories); GSK683699 (GSK/Tanabe);
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`NeuroVax (Immune Response Corp.; Darlington, Curr. Opin. Moi. Ther., 2005,
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`7(6):598-603); anti-CCRZ antibody (e.g., MLN 1202; Millennium); interferon B—1a
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`(e.g., Avonex®; Biogen Idec); anti-a4-integrin antibody (e.g., Tysabri®; Biogen
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`ldecfElan); anti-CD20 antibody (e.g., Flituxan® (Biogen ldechenentech);
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`TV 5010 (Teva); MEI-788 (Neurocrine); MBP8298 (BioMS (see Warren et al.,
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`Eur. J. Neurol, 2006, 13(8):887-95); Mylinax (Oral Cladribine;
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`2-chlorodeoxyadenosine; Serono/IVAX); Teriflunomide
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`((Z)—2-cyano-N-(4—(trifluoromethyl)phenyl)-3-hydroxybut-2-enamide;
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`Sanofi-Aventis); Temsirolimus (Wyeth); Laquinimod
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`(5~chloro-N-ethyi-1,2-dihydro~4-hydroxy-1-methyl-2-oxo-N-phenquuinoline-3-car
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`boxamide; Active Biotech/Teva); and interferon tau (Tauferon; Pepgen).
`
`[0053]
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`In some embodiments, the drug or drug candidate being
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`evaluated is a mild alkylating agent, and more specifically, a Michael addition
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`acceptor, or a compound that is metabolized to a Michael addition acceptor,
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`including compounds of Formulas I, II, III, or IV.
`
`[0054]
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`In some of the embodiments, the compound is fumaric acid, its
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`salt, or a fumaric acid derivative.
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`Method 3: Methods of methods of evaluating neurogrotective properties
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`[0055] The invention further provides methods of comparing (e.g., for
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`bioequivalence) two or more pharmaceutical compositions. Such methods
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`comprise:
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`a) contacting a cell with a first composition comprising a test compound,
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`and
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`b) comparing the level of the Nrf2 pathway upregulation in the cell by
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`the test compound to the corresponding level of the Nrf2 pathway
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`upregulation in a cell treated with a second composition ("comparator
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`composition”) comprising DM F, MMF, or both.
`
`[0056]
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`In some embodiments, substantially dissimilar levels of
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`upregulation by the first and second compositions indicate that the compositions
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`are not bioequivalent.
`
`[0057]
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`In some embodiments, the test compound is fumaric acid, its salt
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`thereof, a fumaric acid derivative, or mixtures thereof.
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`In some embodiments, the
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`first composition comprises DMF, MMF, or both.
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`In some embodiments, the
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`dose andfor the formulation of the first composition differs from the dose andfor
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`the formulation of the second composition. The first composition may be a
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`controlled release composition such as, e.g., compositions described in
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`WO 2006/037342.
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`[0058]
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`In some embodiments, the method further comprises and
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`additional step:
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`c) comparing at least one pharmacokinetic parameter of the first and
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`the second compositions.
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`[0059] Pharmacokinetic parameters and methods for evaluating the
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`same are well known and are described in, e.g., Pharmacokinetics, Second
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`Edition (Drugs and the Pharmaceutical Sciences} by Milo Gibaldi et al. (eds),
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`Informa Healthcare (1982). Examples of such pharmacokinetic parameters that
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`can be evaluated include serum half-life, clearance, and volume distribution.
`
`[0060]
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`In some embodiments, substantially dissimilar pharmacokinetic
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`parameter(s) of the first and second compositions indicate that the compositions
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`are not bioequivalent.
`
`[0061]
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`In some embodiments, the test compound being evaluated is a
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`mild alkylating agent, and more specifically, a Michael addition acceptor, or a
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`compound that is metabolized to a Michael addition acceptor.
`
`[0062]
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`In some of the embodiments, the test compound is fumaric acid or
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`its salt, or a fumaric acid derivative.
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`Ill. Methods of Treatment
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`[0063] The invention provides methods of treating a mammal who has or
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`is at risk for developing a neurological disease, including the following methods:
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`4) methods of treating a neurological disease by administering to the
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`subject in need thereof compounds that are partially structurally
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`similar to DMF or MMF (including compounds selected using
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`methods 1-3 described above) ; and
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`5) methods of treating a neurological disorder by a combination
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`therapy that includes administration of a first compound that does
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`Attorney Docket No. 08201.6042-00000
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`not upregulate the Nrf2 pathway and a second compound that
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`upregulates the Nrf2 pathway.
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`[0064] Methods 4-5 are described in detail below.
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`Method 4: Treatment methods
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`[0065] The invention further provides methods of treating a neurological
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`disease by administering to the subject in need thereof compounds that are
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`partially structurally similar to DMF or MMF.
`
`[0066]
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`In some embodiments of method 4, the invention provides a
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`method of treating a mammal who has or is at risk for a neurological disease.
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`The method comprises administering to a mammal a therapeutically effective
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`amount of a neuroprotective compound which has Formula I, II, III, or IV, e.g., a
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`fumaric acid derivative (e.g., DMF or MMF).
`
`[0067]
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`In some embodiments of method 4, the invention provides a
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`method of slowing or preventing neurodegeneration (more specifically, e.g.,
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`demyelination, axonal loss, and/or neuronal death) in a subject in need thereof,
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`by administering the compound in an amount and for a period of time sufficient to
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`slow or prevent demyelination, axonal loss, and/or neuronal death, e.g., by at
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`least 30%, 50%, 100% or higher over a control over a period of at least 5, 10, 12,
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`20, 40, 52, 100, or 200 weeks.
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`Method 5: Combination therapy
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`[0068] The methods of treatment further include methods of treating a
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`mammal having a neurological disease by combination therapy. Such methods
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`comprise:
`
`.
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`a) administering to the mammal a therapeutically effective amount of a
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`first compound that upregulates the Nrf2 pathway, and
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`b) administering a therapeutically effective amount of a second
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`compound that does not upregulate the Nrf2 pathway.
`
`[0069]
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`In some of embodiments of method 5, the first compound, used in
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`step (a), is a compound of Formula I, II, III, or IV, e.g., DMF or MMF; and the
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`second compound, which is used in step (b), is an immunosuppressive or an
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`immunomodulatory compound that does not upregulate the Ner pathway (e.g.,
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`by more than 30%, 50%, 100% over a control).
`
`[0070]
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`In some embodiments of method 5, the method of treating a
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`neurological disease in a mammal, comprises administering to the mammal a
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`therapeutically effective amount of a compound of Formula I, ll, III, or IV.
`
`[0071]
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`In method 5, the first compound and the second compoun