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WO2015172083A1 - Diméthylfumarate et promédicaments pour le traitement de la sclérose en plaques - Google Patents

Diméthylfumarate et promédicaments pour le traitement de la sclérose en plaques Download PDF

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Publication number
WO2015172083A1
WO2015172083A1 PCT/US2015/029993 US2015029993W WO2015172083A1 WO 2015172083 A1 WO2015172083 A1 WO 2015172083A1 US 2015029993 W US2015029993 W US 2015029993W WO 2015172083 A1 WO2015172083 A1 WO 2015172083A1
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Prior art keywords
mmf
dmf
gene
prodrug
subject
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Robert H. SCANNEVIN
Kenneth J. Rhodes
Melanie BRENNAN
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Biogen MA Inc
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Biogen MA Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7004—Stress
    • G01N2800/7009—Oxidative stress

Definitions

  • the invention relates, inter alia, to the use and activity of drugs and their metabolites, e.g., dimethyl fumarate (DMF) and monomethyl fumarate (MMF), e.g., in the treatment of multiple sclerosis (MS) and other disorders.
  • drugs and their metabolites e.g., dimethyl fumarate (DMF) and monomethyl fumarate (MMF)
  • MS multiple sclerosis
  • Tecfidera® (BG-12, dimethyl fumarate, DMF) is a methyl ester of fumaric acid.
  • MS is an oral therapeutic approved in the U.S. for relapsing multiple sclerosis (MS).
  • MS is an inflammatory disease of the brain and spinal cord characterized by recurrent foci of inflammation that lead to destruction of the myelin sheath. In many areas, nerve fibers are also damaged.
  • the present invention provides, at least in part, methods, devices, reaction mixtures and kits for evaluating, identifying, and/or treating a subject, e.g., a subject having multiple sclerosis (MS) (e.g. , a subject with relapsing MS).
  • a subject having multiple sclerosis e.g., a subject with relapsing MS.
  • responsiveness of a subject to a treatment e.g., an MS therapy that includes dimethyl fumarate is evaluated by detecting a differential expression (e.g., level and/or expression), of a gene (e.g., a gene or a gene product) in response to a treatment that includes DMF and/or monomethyl fumarate (MMF).
  • the gene is oxidative stress induced growth inhibitor 1 (OSGIN1).
  • Applicants have identified both specific and common responses to DMF treatment and to MMF treatment in selected tissues and blood, e.g., whole blood, in a subject.
  • specific responses e.g., transcriptional signatures
  • induced by DMF and MMF indicate that not all the DMF in vivo effects are mediated through MMF, thus suggesting that DMF can directly mediate unique biological responses, not captured by MMF alone.
  • the invention can, therefore, be used, for example: To evaluate responsiveness to, or monitor, a therapy or treatment that includes DMF; identify a subject as likely to benefit from a therapy or treatment that includes DMF; stratify a subject or a patient populations (e.g., stratify a subject or patients as being likely or unlikely to respond to a therapy or treatment that includes DMF); and/or more effectively monitor, treat a disorder, e.g., MS, or prevent worsening of disease and/or relapse.
  • a disorder e.g., MS
  • Many of the methods, devices, reaction mixtures and other inventions provided herein are described for use with DMF and its active metabolite MMF.
  • the methods, devices, reaction mixtures and other inventions can be used with, or apply generically to, dialkyl fumarate prodrugs, e.g. , as shown in Formula A below, and other prodrugs, e.g., as shown in Formulas I-X, and their active metabolites (e.g., MMF).
  • the invention features a method of evaluating, monitoring, stratifying, or treating, a subject, e.g., a subject having or at risk for MS. The method includes: a) acquiring a value for the expression of the OSGINl gene;
  • classifying said subject e.g., classifying said subject as in need of treatment with DMF, MMF, or a prodrug of MMF,
  • the method comprises one of treating the subject, directly acquiring the value, or directly acquiring a sample from which the value is acquired.
  • the method comprises classifying said subject as in need of treatment with DMF, MMF, or a prodrug of MMF. In other embodiments, the method comprises selecting said subject for treatment with DMF, MMF, or a prodrug of MMF, or with a treatment other than DMF, MMF, or a prodrug of MMF. In certain embodiments, the method comprises administering DMF, MMF, or a prodrug of MMF, or a treatment other than DMF, MMF, or a prodrug of MMF, to said subject.
  • acquiring a value for the expression of the OSGINl gene comprises hybridization of a probe specific for OSGINl with an OSGINl mRNA or an OSGINl cDNA. In other embodiments, acquiring a value for the expression of the OSGINl gene comprises amplifying, e.g., with PCR amplification, an OSGINl mRNA or an OSGINl cDNA.
  • the value for expression of the gene comprises a value for a transcriptional parameter, e.g., the level of an mRNA encoded by the gene.
  • acquiring a value for the expression of the OSGINl gene comprises normalizing a value for an OSGINl mRNA or an OSGINl cDNA with the level of a second gene, e.g., gene the expression of which is not affected by MS.
  • the value for expression of the gene comprises a value for a translational parameter, e.g., the level of a protein encoded by the gene, e.g., by an antibody based measurement.
  • the subject e.g., a human subject, has an autoimmune disorder, e.g., MS.
  • the subject with MS has a relapsing form of MS.
  • the subject has been administered DMF, MMF, or a prodrug of MMF, e.g., prior to, or at the time of, acquiring the value.
  • a tissue of the subject e.g., the peripheral blood, comprises, greater than background levels, e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof, e.g., prior to, or at the time of, acquiring the value.
  • background levels e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof, e.g., prior to, or at the time of, acquiring the value.
  • the value for expression of the gene is for cerebral spinal fluid or blood, e.g., whole blood.
  • the value for expression of the gene comprises a value for a translational parameter, e.g., the level of a protein encoded by the gene, in cerebral spinal fluid or blood, e.g., whole blood.
  • the value for expression of the gene is for a cerebral spinal fluid sample, a blood sample, or a blood derived sample, e.g., serum, or an NK- cell containing fraction, from the subject.
  • the sample is blood, and comprises, greater than background levels, e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof.
  • the invention features a method of evaluating, or monitoring, an MS treatment, e.g., an MS treatment with DMF, MMF, or a prodrug of MMF, in a subject having MS, or at risk for developing MS.
  • an MS treatment e.g., an MS treatment with DMF, MMF, or a prodrug of MMF
  • the method includes:
  • a change in the gene expression is indicative of a differential response to DMF, MMF, or a prodrug of MMF.
  • the method further comprises, responsive to said value, treating, selecting and/or altering one or more of: the course of the MS treatment, the dosing of the MS treatment, the schedule or time course of the MS treatment, or administration of a treatment other than DMF, MMF, or a prodrug of MMF.
  • the invention features a method of treating a subject having, or at risk of having, MS, said method comprising:
  • the subject e.g., a human subject
  • has an autoimmune disorder e.g., MS.
  • the subject with MS has a relapsing form of MS.
  • the subject has been administered DMF, MMF, or a prodrug of MMF, e.g., prior to, or at the time of, acquiring the value.
  • a tissue of the subject e.g., the peripheral blood, comprises, greater than background levels, e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof, e.g., prior to, or at the time of, acquiring the value.
  • background levels e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof, e.g., prior to, or at the time of, acquiring the value.
  • the value for expression of the gene comprises a value for a transcriptional parameter, e.g., the level of an mRNA encoded by the gene.
  • the value for expression of the gene comprises a value for a translational parameter, e.g., the level of a protein encoded by the gene.
  • the value for expression of the gene is for cerebral spinal fluid or blood, e.g., whole blood.
  • the value for expression of the gene comprises a value for a translational parameter, e.g., the level of a protein encoded by the gene, in cerebral spinal fluid or blood, e.g., whole blood.
  • the value for expression of the gene is for a cerebral spinal fluid sample, a blood sample, or a blood derived sample, e.g., serum, or an NK-cell containing fraction, from the subject.
  • the sample is blood, and comprises, greater than background levels, e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof.
  • the invention features a method of evaluating, monitoring, stratifying, or treating, a subject.
  • the method includes: a) acquiring a value for the expression of a gene (e.g., a gene or a gene product), wherein said gene is chosen from one, two or all of:
  • DMF dimethyl fumarate
  • a monomethyl fumarate (MMF) -differentially expressed gene ii) a monomethyl fumarate (MMF) -differentially expressed gene, or iii) a DMF/MMF-differentially expressed gene;
  • administering DMF, or a treatment other than DMF, to said subject provided that the method comprises one of treating the subject, directly acquiring the value, or directly acquiring a sample from which the value is acquired.
  • the invention features a method of evaluating, or monitoring, a treatment (e.g., an MS treatment, e.g. , an MS treatment with a DMF) in a subject (e.g., a subject, a patient, a patient group or population, having MS, or at risk for developing MS).
  • a treatment e.g., an MS treatment, e.g. , an MS treatment with a DMF
  • a subject e.g., a subject, a patient, a patient group or population, having MS, or at risk for developing MS.
  • the method includes:
  • a subject in need of treatment e.g. , an MS treatment
  • a value for the expression of a gene (e.g. , a gene or a gene product), wherein said gene is chosen from one, two or all of:
  • DMF dimethyl fumarate
  • a monomethyl fumarate (MMF) -differentially expressed gene ii) a monomethyl fumarate (MMF) -differentially expressed gene, or iii) a DMF/MMF-differentially expressed gene,
  • a change in (i) or (ii) is indicative of a differential response to DMF or MMF, respectively, and a change in (iii) is indicative of a response to both DMF and MMF.
  • the method further comprises, responsive to said value, treating, selecting and/or altering one or more of: the course of the treatment (e.g. , MS treatment), the dosing of the treatment (e.g. , MS treatment), the schedule or time course of the treatment (e.g. , MS treatment), or administration of a second, alternative treatment (e.g., a treatment other than DMF).
  • the course of the treatment e.g. , MS treatment
  • the dosing of the treatment e.g. , MS treatment
  • the schedule or time course of the treatment e.g. , MS treatment
  • administration of a second, alternative treatment e.g., a treatment other than DMF
  • the invention features a method of treating a subject, e.g., a subject having, or at risk of having, MS.
  • the method includes:
  • a DMF in an amount sufficient to treat MS, provided that the subject is identified for treatment with the DMF on the basis of a value for the expression of a gene, wherein said gene is chosen from one, two or all of:
  • DMF dimethyl fumarate
  • ii) a monomethyl fumarate (MMF) -differentially expressed gene, or iii) a DMF/MMF-differentially expressed gene.
  • MMF monomethyl fumarate
  • the method includes acquiring a value for the expression of OSGIN1.
  • the method includes acquiring a value for the expression of PADI4. In other embodiments, the method includes acquiring a value for the expression of p53.
  • the method comprises acquiring a value for the expression of a plurality, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, genes, and, optionally, any step responsive thereto can be responsive to one, some, or all, of the acquired values.
  • the gene used in acquiring the value is chosen from one, two or all of: a DMF-differentially expressed gene, an MMF-differentially expressed gene, or a gene expressed in response to both DMF and MMF (e.g., a DMF/MMF-differentially expressed gene).
  • the plurality of genes comprise OSGIN1. In other embodiments, the plurality of genes comprise PADI4. In certain embodiments, the plurality of genes comprise p53.
  • the value for expression of the gene includes a value for a
  • the value for expression of the gene includes a value for a translational parameter, e.g. , the level of a protein encoded by the gene.
  • the method includes acquiring a value for the expression of a plurality of genes.
  • said plurality includes two, three, four or more of: a) a plurality, e.g. , 2, 3, 4, 5, 6, 7, 8, 9 or 10, or more, DMF-differentially expressed genes;
  • a plurality e.g. , 2, 3, 4, 5, 6, 7, 8, 9 or 10, or more, MMF-differentially expressed genes
  • the value for expression of the gene acquired is from blood, e.g. , whole blood (e.g. , a gene expressed in blood or a blood sample).
  • the value for expression of the gene includes a value for a transcriptional parameter, e.g. , the level of an mRNA encoded by the gene, in blood, e.g. , whole blood.
  • the gene is selected from one or more of the genes in Table 1 or Table 9.
  • the gene is a gene from Table 9 that shows differential expression as measured by mRNA levels.
  • the differential expression is detected prior to or after (e.g. , 2, 3, 5, 7, 10, 12, 15 or 24 hours after) administration of a treatment (e.g. , a DMF or an MMF).
  • the gene is chosen from one, two, three, four or all of: Granzyme A (Gzma), Natural cytotoxicity triggering receptor 1 (Ncrl), Killer cell lectin-like receptor subfamily C member 1 (Klrcl), Killer cell lectin-like receptor subfamily B member IB (Klrblb), or Killer cell lectin-like receptor family E member 1 (Klrel).
  • Gzma Granzyme A
  • Ncrl Natural cytotoxicity triggering receptor 1
  • Klrcl Killer cell lectin-like receptor subfamily C member 1
  • Klrblb Killer cell lectin-like receptor subfamily B member IB
  • Klrel Killer cell lectin-like receptor family E member 1
  • the gene is chosen from one, two, three or all of: Granzyme A (Gzma), Natural cytotoxicity triggering receptor 1 (Ncrl), Killer cell lectin-like receptor subfamily C member 1 (Klrcl), or Killer cell lectin-like receptor subfamily B member IB (Klrblb).
  • the value for expression of the gene comprises a value for a transcriptional parameter, e.g., the level of an mRNA encoded by the gene, in blood, for 1, 2, 3, 4, or all of, Gzma, Ncrl, Klrcl, Klrblb, and Klrel.
  • the value for expression of the gene comprises a value for a transcriptional parameter, e.g., the level of an mRNA encoded by the gene, in blood, for 1, 2, 3, or all of, Gzma, Ncrl, Klrcl, and Klrblb.
  • the value for expression of the gene comprises a value for a translational parameter, e.g., the level of a protein encoded by the gene, in blood, e.g., whole blood.
  • the gene is selected from one or more of the genes in Table 1 or Table 9.
  • the gene is a gene from Table 9 that shows differential expression as measured by protein levels.
  • the differential expression is detected prior to or after (e.g., 2, 3, 5, 7, 10, 12, 15 or 24 hours after) administration of a treatment (e.g., a DMF or an MMF).
  • the gene is chosen from one, two, three, or all of: Killer cell lectin-like receptor subfamily C member 1 (Klrcl), Killer cell lectin-like receptor subfamily B member IB (Klrblb), NKKG2d (Klrkl), or Natural killer cells (CD94) (Klrdl).
  • Klrcl Killer cell lectin-like receptor subfamily C member 1
  • Klrblb Killer cell lectin-like receptor subfamily B member IB
  • Klrkl NKKG2d
  • CD94 Natural killer cells
  • a value for expression of the gene comprises a value for a translational parameter, e.g., the level of a protein encoded by the gene, in blood, for 1, 2, 3, or all of, Klrcl, Klrblb, Klrkl, and Klrdl.
  • the value for expression of the gene is for a blood sample, or a blood derived sample, e.g., serum or plasma, or an NK-cell containing fraction, from the subject.
  • the blood comprises, greater than background levels, e.g., therapeutic levels, of DMF, MMF, or both.
  • the value for expression of the gene is for a tissue selected from cortical tissue, hippocampus, striatum, jejunum, kidney, liver, or spleen.
  • the value for expression of the gene is for cerebral spinal fluid.
  • said gene is selected from the genes in Table 2, Table 3, Table 4, Table 5a, Table 5b, Table 6, Table 7, Table 8, or Tables 18-26.
  • the value is acquired at one or more of the following periods: prior to beginning of treatment; during the treatment; or after the treatment has been administered.
  • the treatment is an MS treatment (e.g. , a treatment that includes a DMF).
  • the subject has been administered the treatment, e.g. , the DMF, e.g. , prior to, at the time of, or after, acquiring the value.
  • the value is acquired after (e.g. , 2, 3, 5, 7, 10, 12, 15 or 24 hours after) administration of a treatment (e.g. , a DMF).
  • the methods described herein include the step of comparing the value (e.g., level) of one or more genes described herein to a specified parameter (e.g., a reference value or sample; a sample obtained from a healthy subject; a sample obtained from the subject at different treatment intervals).
  • a specified parameter e.g., a reference value or sample; a sample obtained from a healthy subject; a sample obtained from the subject at different treatment intervals.
  • a sample can be analyzed at any stage of treatment, but preferably, prior to, during, or after terminating, administration of the therapy, e.g. , the MS therapy.
  • the methods include the step of detecting the level of one or more genes in the subject, prior to, or after, administering the therapy (e.g. , MS therapy), to the subject.
  • a change in gene expression indicates that the subject from whom the sample was obtained is responding to the therapy, e.g. , the MS therapy.
  • a tissue (e.g., cerebral spinal fluid) or blood (e.g. , a tissue or blood sample) of the subject, e.g. , the peripheral blood comprises, greater than background levels, e.g. , therapeutic levels, of DMF, MMF, or both, e.g. , prior to, or at the time of, acquiring the value.
  • the sample is chosen from a non-cellular body fluid; or a cellular or tissue fraction.
  • the non-cellular fraction is chosen from blood, e.g. , whole blood, plasma or serum.
  • the cellular fraction comprises one or more of: T cells, B cells or myeloid cells.
  • the cellular fraction can include one or more of: natural killer (NK) cells, peripheral blood mononuclear cells (PBMC), CD8+ T cells, or
  • the methods described herein further includes the step of acquiring the sample, e.g., a biological sample, from the subject.
  • a sample can include any material obtained and/or derived from a biological sample, including a polypeptide, and nucleic acid (e.g., genomic DNA, cDNA, RNA) purified or processed from the sample.
  • the subject treated, or the subject from which the value or sample is acquired is a subject having, or at risk of having MS at any stage of treatment.
  • the MS patient is chosen from a patient having one or more of: Benign MS, relapsing MS, e.g. , relapsing-remitting MS (RRMS) (e.g., quiescent RRMS, active RRMS), primary progressive MS, or secondary progressive MS.
  • RRMS relapsing-remitting MS
  • the subject has MS-like symptoms, such as those having clinically isolated syndrome (CIS) or clinically defined MS (CDMS).
  • the subject is an MS patient (e.g.
  • a patient with relapsing MS prior to administration of an MS therapy described herein (e.g., prior to administration of a DMF).
  • the subject is an MS patient (e.g., a relapsing MS patient) after administration of an MS therapy described herein (e.g., a DMF).
  • the subject is an MS patient after administration of the MS therapy for one, two, five, ten, twenty, twenty four hours; one week, two weeks, one month, two months, three months, four months, six months, one year or more.
  • the subject has a relapsing form of MS, e.g. , RRMS.
  • the invention features a method of treating a subject having one or more symptoms associated with MS.
  • the subject is identified as responding or not responding to a therapy, using the methods, devices, or kits described herein.
  • the method comprises treating the subject with DMF, MMF, or a combination thereof.
  • the treatment includes reducing, retarding or preventing, a relapse, or the worsening of a disability, in the MS patients.
  • the method includes administering to a subject (e.g. , a subject described herein) a therapy for MS (e.g. , a DMF), in an amount sufficient to reduce one or more symptoms associated with MS.
  • a subject e.g. , a subject described herein
  • a therapy for MS e.g. , a DMF
  • an alternative or other MS therapy can be chosen.
  • exemplary other therapies include, but are not limited to, an IFN-b agent (e.g., an IFN-b la molecule or an IFN-b lb molecule, including analogues and derivatives thereof (e.g., pegylated variants thereof)).
  • the other MS therapy includes an IFN-b la agent (e.g., Avonex®, Rebif®).
  • the other MS therapy includes an INF-b lb agent (e.g., Betaseron®, Betaferon®).
  • the other MS therapy includes a polymer of four amino acids found in myelin basic protein, e.g. , a polymer of glutamic acid, lysine, alanine and tyrosine (e.g., glatiramer (Copaxone®)); an antibody or fragment thereof against alpha-4 integrin (e.g., natalizumab (Tysabri®)); an anthracenedione molecule (e.g., mitoxantrone (Novantrone®)); fingolimod (FTY720; Gilenya®); Daclizumab; alemtuzumab (Lemtrada®)); or an anti-LINGO- 1 antibody.
  • the methods include the use of one or more symptom management therapies, such as antidepressants, analgesics, anti-tremor agents, among others.
  • the gene or gene product detected is, e.g., nucleic acid, cDNA, RNA (e.g., mRNA), or a polypeptide.
  • a nucleic acid can be detected, or the level determined, by any means of nucleic acid detection, or detection of the expression level of the nucleic acids, including but not limited to, nucleic acid hybridization assay, amplification-based assays (e.g., polymerase chain reaction), sequencing, and/or in situ hybridization.
  • nucleic acid hybridization assay e.g., amplification-based assays (e.g., polymerase chain reaction), sequencing, and/or in situ hybridization.
  • a probe is a nucleic acid that specifically hybridizes with a transcription product of the gene or genes.
  • the detection includes amplification of a transcription product of the gene or genes.
  • the detection includes reverse transcription and amplification of a transcription product of the gene or genes.
  • a translation product of the gene or genes e.g. , a polypeptide
  • the polypeptide can be detected, or the level determined, by any means of polypeptide detection, or detection of the expression level of the polypeptides.
  • the polypeptide can be detected using a probe or reagent which specifically binds with the polypeptides.
  • the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment, e.g., a labeled antibody (e.g., a fluorescent or a radioactive label), or fragment thereof, that specifically binds with a translation product of the gene or genes.
  • the polypeptide is detected using antibody-based detection techniques, such as enzyme-based immunoabsorbent assay, immunofluorescence cell sorting (FACS), immunohistochemistry, immunofluorescence (IF), antigen retrieval and/or microarray detection methods.
  • antibody-based detection techniques such as enzyme-based immunoabsorbent assay, immunofluorescence cell sorting (FACS), immunohistochemistry, immunofluorescence (IF), antigen retrieval and/or microarray detection methods.
  • FACS immunofluorescence cell sorting
  • IF immunofluorescence
  • Polypeptide detection methods can be performed in any other assay format, including but not limited to, ELISA, RIA, and mass spectrometry.
  • the probe is an antibody.
  • the method of detection includes a sandwich-based detection, e.g., ELISA based sandwich assay detection, of a translation product of the gene or genes.
  • the methods of the invention can further include the step of monitoring the subject, e.g., for a change (e.g., an increase or decrease) in one or more of: levels of one or more MS biomarkers; the rate of appearance of new lesions, e.g., in an MRI scan; the appearance of new disease-related symptoms; a change in EDSS score; a change in quality of life; or any other parameter related to clinical outcome.
  • the subject can be monitored in one or more of the following periods: prior to beginning of treatment; during the treatment; or after the treatment has been administered. Monitoring can be used to evaluate the need for further treatment with the same MS therapy, or for additional MS treatment. Generally, a decrease in one or more of the parameters described above is indicative of the improved condition of the subject.
  • the methods described herein further include: performing a
  • EDSS Expanded Disability Status Scale
  • the invention features a device comprising: one, or a plurality of, probes, each probe being specific for a product, e.g., a translational product or transcriptional product, of the OSGINl gene, wherein the device includes less than 10, 25, 50, 100, 200, 250, 300, or 500 probes specific for products, e.g., a translational product or transcriptional product, of a gene other than OSGINl .
  • the device further comprises, a sample from a tissue of a subject, e.g., the peripheral blood, which comprises greater than background levels, e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof.
  • a sample from a tissue of a subject e.g., the peripheral blood
  • background levels e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof.
  • the invention features a device comprising:
  • probes each probe being specific for a product, e.g., a translational product or transcriptional product, of a gene selected independently from:
  • DMF dimethyl fumarate
  • MMF monomethyl fumarate
  • the gene is OSGINl .
  • the gene is PADI4. In other embodiments, the gene is p53.
  • the device includes one, or a plurality of, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10, or more, probes, each probe being specific for a product, e.g., a translational product or transcriptional product, of a dimethyl fumarate (DMF)-differentially expressed gene.
  • a product e.g., a translational product or transcriptional product, of a dimethyl fumarate (DMF)-differentially expressed gene.
  • DMF dimethyl fumarate
  • the plurality of genes comprise OSGINl .
  • the plurality of genes comprise PADI4. In other embodiments, the plurality of genes comprise p53.
  • the probe or probes of the device are specific for a gene or genes selected from the genes in Table 9.
  • the probe or probes of the device are specific for a gene or genes selected from the genes in Table 9 that shows differential expression as measured by mRNA levels.
  • the device includes a probe specific for a transcriptional product of 1, 2, 3, 4, or all of, Gzma, Ncrl, Klrcl, Klrblb, and Klrel. In yet other embodiments, the device includes a probe specific for a transcriptional product of 1, 2, 3, or all of, Gzma, Ncrl, Klrcl, and Klrblb.
  • the device includes a probe specific for a gene or genes from the genes in Table 9 that shows differential expression as measured by protein levels.
  • the device includes a probe specific for a translational product of 1, 2, 3, or all of, Klrcl, Klrblb, Klrkl, and Klrdl .
  • the device further comprises a sample, e.g. , a sample as described herein.
  • the sample is from a subject having an autoimmune disorder, e.g. , MS, relapsing MS. In other embodiments, the sample is from a subject that has been
  • the sample is from a tissue of the subject, e.g. , the peripheral blood, which comprises greater than background levels, e.g. , therapeutic levels, of DMF, MMF, or both.
  • the device further comprises a sample, e.g. , a blood sample, or a substance derived from blood, e.g. , serum, or an NK-cell containing fraction.
  • a sample e.g. , a blood sample, or a substance derived from blood, e.g. , serum, or an NK-cell containing fraction.
  • the probe or probes of the device are specific for a gene or genes that are selected independently from the genes in Table 2, Table 3, Table 4, Table 5 a, Table 5b, Table 6, Table 7, Table 8, or Tables 18-26
  • the probe is a nucleic acid that specifically hybridizes with a transcription product of the gene or genes.
  • the device is configured to allow amplification of a transcription product of the gene or genes.
  • the device is configured to allow reverse transcription and amplification of a transcription product of the gene or genes.
  • a probe is an antibody, e.g. , a labeled antibody, or fragment thereof, that specifically binds with a translation product of the gene or genes.
  • the device is configured to allow sandwich-based detection, e.g. , ELISA based sandwich assay detection, of a translation product of the gene or genes.
  • sandwich-based detection e.g. , ELISA based sandwich assay detection
  • the device has less than 10, 25, 50, 100, 200, 250, 300, or 500 probes specific for products, e.g. , a translational product or transcriptional product, of genes that are not i) a dimethyl fumarate (DMF)-differentially expressed gene,
  • a translational product or transcriptional product of genes that are not i) a dimethyl fumarate (DMF)-differentially expressed gene
  • MMF monomethyl fumarate
  • the device has less than 10, 25, 50, 100, 200, 250, 300, or 500 probes specific for products, e.g. , a translational product or transcriptional product, of genes that are not a dimethyl fumarate (DMF)-differentially expressed gene.
  • a translational product or transcriptional product e.g. , a translational product or transcriptional product
  • the device has less than 10, 25, 50, 100, 200, 250, 300, or 500 probes specific for products, e.g. , a translational product or transcriptional product, of genes that are not listed in Table 9.
  • the device has at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 % of the probes of the device are specific for a product, e.g. , a translational product or transcriptional product, of:
  • DMF dimethyl fumarate
  • MMF monomethyl fumarate
  • the device has at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 % of the probes of the device are specific for a product, e.g. , a translational product or transcriptional product, of a dimethyl fumarate (DMF)-differentially expressed gene.
  • a product e.g. , a translational product or transcriptional product, of a dimethyl fumarate (DMF)-differentially expressed gene.
  • DMF dimethyl fumarate
  • the device has at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 % of the probes of the device are specific for a product, e.g. , a translational product or transcriptional product, of a gene listed in Table 9.
  • a product e.g. , a translational product or transcriptional product, of a gene listed in Table 9.
  • the probe or probes are disposed on a surface of the device.
  • the invention features a method of using a device described herein.
  • the method includes:
  • the method includes a step of capturing a signal, e.g. , an electronic, or visual signal, to evaluate the sample.
  • a signal e.g. , an electronic, or visual signal
  • the invention features a reaction mixture comprising:
  • a sample from a tissue of a subject e.g., the peripheral blood, e.g., tissue which comprises greater than background levels, e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof; and
  • each probe being specific for a product, e.g., a translational product or transcriptional product, of the OSGIN1 gene,
  • reaction mixture includes less than 10, 25, 50, 100, 200, 250, 300, or 500 probes specific for products, e.g., a translational product or transcriptional product, of genes other than OSGIN1.
  • reaction mixture comprising:
  • probes each probe being specific for a product, e.g., a translational product or transcriptional product, of a gene selected independently from:
  • DMF dimethyl fumarate
  • MMF monomethyl fumarate
  • the gene is OSGIN1.
  • the gene is PADI4. In other embodiments, the gene is p53.
  • the reaction mixture comprises one, or a plurality of, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10, or more, probes, each probe being specific for a product, e.g., a translational product or transcriptional product, of a dimethyl fumarate (DMF)-differentially expressed gene.
  • a product e.g., a translational product or transcriptional product
  • DMF dimethyl fumarate
  • the probe or probes are specific for a gene or genes selected from the genes in Table 9.
  • the probe or probes are specific for a gene or genes selected from the genes in Table 9 that shows differential expression as measured by mRNA levels.
  • the reaction mixture comprises probes specific for a transcriptional product of 1, 2, 3, 4, or all of, Gzma, Ncrl, Klrcl, Klrblb, and Klrel. In one embodiment, the reaction mixture comprises probes specific for a transcriptional product of 1, 2, 3, or all of, Gzma, Ncrl, Klrcl, and Klrblb.
  • the probe or probes are specific for a gene or genes from the genes in Table 9 that shows differential expression as measured by protein levels.
  • the reaction mixture comprises probes specific for a translational product of 1, 2, 3, or all of, Klrcl, Klrblb, Klrkl, and Klrdl .
  • said sample is from a subject having an autoimmune disorder, e.g. , MS, e.g. , relapsing MS.
  • an autoimmune disorder e.g. , MS, e.g. , relapsing MS.
  • said sample is from a subject that has been administered DMF.
  • said sample is from a tissue of the subject, e.g. , the peripheral blood, which comprises greater than background levels, e.g. , therapeutic levels, of DMF, MMF, or both.
  • said sample comprises blood, or a substance derived from blood, e.g. , serum, or an NK-cell containing fraction.
  • the probe or probes are specific for a gene or genes that are selected independently from the genes in Table 2, Table 3, Table 4, Table 5a, Table 5b, Table 6, Table 7, Table 8, or Tables 18-26.
  • a probe is a nucleic acid that specifically hybridizes with a
  • the reaction mixture further comprises reagents to allow for amplification of a transcription product of the gene or genes.
  • the reaction mixture further comprises reagents to allow for reverse transcription and amplification of a transcription product of the gene or genes.
  • a probe is an antibody, e.g. , a labeled antibody, or fragment thereof, that specifically binds with a translation product of the gene or genes.
  • the reaction mixture comprises reagents to allow sandwich-based detection, e.g. , ELISA based sandwich assay detection, of a translation product of the gene or genes.
  • the reaction mixture has less than 10, 25, 50, 100, 200, 250, 300, or 500 probes specific for products, e.g. , a translational product or transcriptional product, of genes that are not
  • DMF dimethyl fumarate
  • MMF monomethyl fumarate
  • the reaction mixture has less than 10, 25, 50, 100, 200, 250, 300, or 500 probes specific for products, e.g., a translational product or transcriptional product, of genes that are not a dimethyl fumarate (DMF)-differentially expressed gene.
  • a translational product or transcriptional product e.g., a translational product or transcriptional product
  • the reaction mixture has less than 10, 25, 50, 100, 200, 250, 300, or 500 probes specific for products, e.g., a translational product or transcriptional product, of genes that are not listed in Table 9.
  • the reaction mixture has at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 % of the probes of the device are specific for a product, e.g., a translational product or transcriptional product, of:
  • DMF dimethyl fumarate
  • MMF monomethyl fumarate
  • At least 10, 20, 30, 40, 50, 60, 70, 80, or 90 % of the probes are specific for a product, e.g., a translational product or transcriptional product, of a dimethyl fumarate (DMF)-differentially expressed gene.
  • a product e.g., a translational product or transcriptional product, of a dimethyl fumarate (DMF)-differentially expressed gene.
  • DMF dimethyl fumarate
  • At least 10, 20, 30, 40, 50, 60, 70, 80, or 90 % of the probes of the device are specific for a product, e.g., a translational product or transcriptional product, of a gene listed in Table 9.
  • the reaction mixture of can comprise a surface on which the probe or probes are disposed.
  • the invention features a method of making a reaction mixture comprising:
  • tissue of a subject e.g., the peripheral blood, e.g., tissue which comprises greater than background levels, e.g., therapeutic levels, of DMF, MMF, or a prodrug of MMF, or a combination thereof;
  • the reaction mixture includes less than 10, 25, 50, 100, 200, 250, 300, or 500 probes specific for products, e.g., a translational product or transcriptional product, of genes other than OSGINl,
  • the invention features a method of making a reaction mixture comprising:
  • the method of making includes capturing a signal, e.g., an electronic, or visual signal, to evaluate the sample.
  • a signal e.g., an electronic, or visual signal
  • kits for evaluating a sample e.g., a sample from an MS patient, to detect or determine the level of one or more genes as described herein.
  • the one or more genes comprise OSGINl .
  • the one or more genes comprise PADI4.
  • the one or more genes comprise p53.
  • the kit includes a means for detection of (e.g., a reagent that specifically detects) one or more genes as described herein.
  • the kit includes an MS therapy.
  • the kit comprises an antibody, an antibody derivative, or an antibody fragment to a protein produce of the gene.
  • the kit includes an antibody- based detection technique, such as immunofluorescence cell sorting (FACS),
  • kits are provided with a detectable label (e.g., a fluorescent or a radioactive label).
  • a detectable label e.g., a fluorescent or a radioactive label.
  • the kit is an ELISA or an immunohistochemistry (IHC) assay for detection of the gene.
  • the methods, devices, reaction mixtures, kits, and other inventions described herein can further include providing or generating, and/or transmitting information, e.g. , a report, containing data of the evaluation or treatment determined by the methods, assays, and/or kits as described herein.
  • the information can be transmitted to a report-receiving party or entity (e.g., a patient, a health care provider, a diagnostic provider, and/or a regulatory agency, e.g., the FDA), or otherwise submitting information about the methods, assays and kits disclosed herein to another party.
  • the method can relate to compliance with a regulatory requirement, e.g., a pre- or post approval requirement of a regulatory agency, e.g., the FDA.
  • the report- receiving party or entity can determine if a predetermined requirement or reference value is met by the data, and, optionally, a response from the report-receiving entity or party is received, e.g., by a physician, patient, diagnostic provider.
  • the invention features a method of evaluating, or monitoring, a prodrug, in a subject, e.g. , a human or a non-human mammal.
  • the method includes:
  • a value for the expression of a gene (e.g. , a gene or a gene product), wherein said gene is chosen from one, two or all of:
  • the gene is OSGIN1.
  • the gene is PADI4. In other embodiments, the gene is p53. In certain embodiments, the method further comprises comparing the value with a reference value.
  • the invention features a method of evaluating, or monitoring, a drug, in a subject, e.g. , a human or a non-human mammal.
  • the method includes:
  • a gene e.g. , a gene or a gene product
  • said gene is chosen from one, two or all of:
  • a change in (i) or (ii) is indicative of a differential response to drug or drug metabolite, respectively, and a change in (iii) is indicative of a response to both drug and drug metabolite.
  • the gene is OSGIN1.
  • the gene is PADI4. In other embodiments, the gene is p53. In certain embodiments, the method further comprises comparing the value with a reference value.
  • the drug is DMF and the drug metabolite is MMF.
  • the present invention contemplates treatment with DMF and its active metabolite MMF.
  • the methods and other inventions can be used with, or apply generically to, dialkyl fumarate prodrugs, e.g. , as shown in Formula A below, and other prodrugs, e.g., as shown in Formulas I-X, and their active metabolites (e.g., MMF), and monoalkyl fumarate drugs, e.g., as shown in Formula B below.
  • the drug metabolite is MMF and the drug is DMF.
  • the drug metabolite is MMF and the drug or prodrug is a compound of Formula I:
  • R la and R 2a are independently chosen from hydrogen, C 1-6 alkyl, and substituted C 1-6 alkyl;
  • R 3a and R 4a are independently chosen from hydrogen, C 1-6 alkyl, substituted C 1-6 alkyl, Ci-6 heteroalkyl, substituted C 1-6 heteroalkyl, C4_ 12 cycloalkylalkyl, substituted C 4-12 cycloalkylalkyl, C 7 _ 12 arylalkyl, and substituted C 7 _ 12 arylalkyl; or R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from a Cs-io heteroaryl, substituted C 5-1 o heteroaryl, C 5-1 o heterocycloalkyl, and substituted C 5-1 o heterocycloalkyl; and
  • R 5a is chosen from methyl, ethyl, and C 3 _ 6 alkyl; wherein each substituent group is independently chosen from halogen, -OH,
  • each R l la is independently chosen from hydrogen and C 1-4 alkyl; with the proviso that when R 5a is ethyl; then R 3a and R 4a are independently chosen from hydrogen, C 1-6 alkyl, and substituted Ci_6 alkyl.
  • each substituent group is independently chosen from halogen, -OH, -CN, -CF 3 , -R l la , -OR l la , and
  • each R l la is independently chosen from hydrogen and C 1-4 alkyl.
  • each substituent group is independently chosen from -OH, and
  • each of R la and R 2a is hydrogen.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is C 1-4 alkyl.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is methyl.
  • R 3a and R 4a are independently chosen from hydrogen and C 1-6 alkyl.
  • R 3a and R 4a are independently chosen from hydrogen and C 1-4 alkyl.
  • R 3a and R 4a are independently chosen from hydrogen, methyl, and ethyl.
  • each of R 3a and R 4a is hydrogen; in certain embodiments, each of R 3a and R 4a is methyl; and in certain embodiments, each of R 3a and R 4a is ethyl.
  • R 3a is hydrogen; and R 4a is chosen from Ci_4 alkyl, benzyl, 2-methoxyethyl, carboxymethyl, carboxypropyl, 1,2,4-thiadoxolyl, methoxy, 2-methoxycarbonyl, 2-oxo(l,3-oxazolidinyl), 2-(methylethoxy)ethyl, 2-ethoxyethyl, (tert- butyloxycarbonyl)methyl, (ethoxycarbonyl)methyl, carboxymethyl,
  • R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from a C 5 -6 heterocycloalkyl, substituted C 5 -6 heterocycloalkyl, C 5 -6 heteroaryl, and substituted C 5 -6 heteroaryl ring.
  • R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from a C 5 heterocycloalkyl, substituted C 5 heterocycloalkyl, C 5 heteroaryl, and substituted C 5 heteroaryl ring.
  • R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from a C 6 heterocycloalkyl, substituted C 6 heterocycloalkyl, C 6 heteroaryl, and substituted C 6 heteroaryl ring.
  • R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from piperazine, 1,3-oxazolidinyl, pyrrolidine, and morpholine ring.
  • R 3a and R 4a together with the nitrogen to which they are bonded form a Cs-io heterocycloalkyl ring.
  • R 5a is methyl
  • R 5a is ethyl
  • R 5a is C 3 _ 6 alkyl.
  • R 5a is chosen from methyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.
  • R 5a is chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is C 1-6 alkyl;
  • R 3a is hydrogen;
  • R 4a is chosen from hydrogen, C 1-6 alkyl, and benzyl.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is C 1-6 alkyl;
  • R 3a is hydrogen;
  • R 4a is chosen from hydrogen, C 1-6 alkyl, and benzyl; and
  • R 5a is methyl.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is chosen from hydrogen and Ci_6 alkyl; and each of R 3a and R 4a is Ci_6 alkyl.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is chosen from hydrogen and Ci_6 alkyl; each of R 3a and R 4a is Ci_6 alkyl; and R 5a is methyl.
  • each of R la and R 2a is hydrogen; each of R 3a and R 4a is Ci_ 6 alkyl; and R 5a is methyl.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is chosen from hydrogen and Ci_ 4 alkyl;
  • R 3a is hydrogen;
  • R 5a is methyl.
  • R la and R 2a are hydrogen and the other of R la and R 2a is methyl;
  • R 3a is hydrogen;
  • R 3a and R 4a together with the nitrogen to which they are bonded form a Cs-io heterocycloalkyl ring.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is chosen from hydrogen and C 1-6 alkyl; R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from a C 5 _6 heterocycloalkyl, substituted C 5 -6 heterocycloalkyl, C 5 -6 heteroaryl, and substituted C 5 -6 heteroaryl ring; and R a is methyl.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is methyl; R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from a C 5 _6 heterocycloalkyl, substituted C 5 _6 heterocycloalkyl, C 5 _6 heteroaryl, and substituted C 5 -6 heteroaryl ring; and R 5a is methyl.
  • each of R la and R 2a is hydrogen; R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from a C 5 _6 heterocycloalkyl, substituted C 5 _6 heterocycloalkyl, C 5 _6 heteroaryl, and substituted C 5 _6 heteroaryl ring; and R 5a is methyl.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is chosen from hydrogen and C 1-6 alkyl; and R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from morpholine, piperazine, and N- substituted piperazine.
  • one of R la and R 2a is hydro gen and the other of R la and R 2a is chosen from hydrogen and C 1-6 alkyl; R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from morpholine, piperazine, and N- substituted piperazine; and R 5a is methyl.
  • R 5a is not methyl.
  • R la is hydrogen
  • R 2a is hydrogen
  • the compound is chosen from: (N,N-diethylcarbamoyl)methyl methyl(2E)but-2-ene- 1 ,4-dioate; methyl[N- benzylcarbamoyl]methyl(2E)but-2-ene- 1 ,4-dioate; methyl 2-morpholin-4-yl-2-oxoethyl(2E)but- 2-ene-l,4-dioate; (N-butylcarbamoyl)methyl methyl(2E)but-2-ene-l,4-dioate; [N-(2- methoxyethyl)carbamoyl] methyl methyl(2E)but-2-ene- 1 ,4-dioate; 2- ⁇ 2- [(2E)-3- (methoxycarbonyl)prop-2-enoyloxy]acetylamino ⁇ acetic acid; 4- ⁇ 2-[
  • the compound is chosen from: (N,N-diethylcarbamoyl)methyl methyl(2E)but-2-ene- 1 ,4-dioate; methyl[N- benzylcarbamoyl]methyl(2E)but-2-ene- 1 ,4-dioate; methyl 2-morpholin-4-yl-2-oxoethyl(2E)but-
  • R 3a and R 4a are independently chosen from hydrogen, Ci_6 alkyl, substituted C 1-6 alkyl, C 6-10 aryl, substituted C 6 -io aryl, C4_ 12 cycloalkylalkyl, substituted C 4 - 12 cycloalkylalkyl, C 7-12 arylalkyl, substituted C 7-12 arylalkyl, C 1-6 heteroalkyl, substituted C 1-6 heteroalkyl, C 6 -io heteroaryl, substituted C 6 -io heteroaryl, C 4 _ 12 heterocycloalkylalkyl, substituted C 4 _ 12 heterocycloalkylalkyl, C7_ 12 heteroarylalkyl, substituted C 7 _ 12 heteroarylalkyl; or R 3a and R 4a together with the nitrogen to which they are bonded form a ring chosen from a C 5-1 o heteroaryl, substituted C 5-1 o heteroaryl, substituted C 5-1 o heteroary
  • the compound that metabolizes to MMF is a compound of Formula II:
  • R 6b is chosen from C 1-6 alkyl, substituted C 1-6 alkyl, C 1-6 heteroalkyl, substituted C 1-6 heteroalkyl, C 3 _8 cycloalkyl, substituted C 3 _8 cycloalkyl, C 6 -8 aryl, substituted C 6 -8 aryl, and -OR 10b wherein R 10b is chosen from Ci_6 alkyl, substituted C 1-6 alkyl, C 3-1 o cycloalkyl, substituted C 3-1 o cycloalkyl, C 6 -io aryl, and substituted C 6 -io aryl;
  • R 7b and R 8b are independently chosen from hydrogen, C 1-6 alkyl, and substituted C 1-6 alkyl;
  • each substituent group is independently chosen from halogen, -OH, -CN, -CF 3 , -R l lb , -OR l lb , and -NR l lb 2 wherein each
  • R l lb is independently chosen from hydrogen and C 1-4 alkyl.
  • one of R 7b and R 8b is hydro gen and the other of R 7b and R 8b is C 1-6 alkyl. In certain embodiments of a compound of Formula (II), one of R 7b and R 8b is hydrogen and the other of R 7b and R 8b is Ci_4 alkyl.
  • one of R 7b and R 8b is hydrogen and the other of R 7b and R 8b is chosen from methyl, ethyl, n-propyl, and isopropyl.
  • each of R 7b and R 8b is hydrogen.
  • R 9b is chosen from substituted Ci-6 alkyl and -OR l lb wherein R l lb is independently C 1-4 alkyl.
  • R 9b is C 1-6 alkyl, in certain embodiments, R 9b is C 1-3 alkyl; and in certain embodiments, R 9b is chosen from methyl and ethyl.
  • R 9b is methyl
  • R 9b is chosen from ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.
  • R 9b is chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.
  • R 6b is C 1-6 alkyl; one of R 7b and R 8b is hydrogen and the other of R 7b and R 8b is C 1-6 alkyl; and R 9b is chosen from C 1-6 alkyl and substituted Ci_6 alkyl.
  • R 6b is -OR 10b .
  • R 10b is chosen from
  • R is chosen from methyl, ethyl, n-propyl, and isopropyl; one of R 7b and R 8b is hydrogen and the other of R 7b and R 8b is chosen from methyl, ethyl, n-propyl, and isopropyl.
  • R 6b is substituted C 1-2 alkyl, wherein each of the one or more substituent groups are chosen from -COOH,
  • R 6b is chosen from ethoxy, methylethoxy, isopropyl, phenyl, cyclohexyl, cyclohexyloxy,
  • R 9b is chosen from methyl and ethyl; one of R 7b and R 8b is hydrogen and the other of R 7b and R 8b is chosen from hydrogen, methyl, ethyl, n-propyl, and isopropyl; and R 6b is chosen from C 1-3 alkyl, substituted C 1-2 alkyl wherein each of the one or more substituent groups are chosen -COOH, -NHC(0)CH 2 NH 2 , and - NH 2 , -OR 10b wherein R 10b is chosen from C 1-3 alkyl and cyclohexyl, phenyl, and cyclohexyl.
  • the compound is chosen from:
  • the compound is chosen from: methyl(2-methylpropanoyloxy)ethyl(2E)but-2-ene- 1 ,4-dioate; methyl
  • the compound is chosen from: ethoxycarbonyloxyethyl methyl(2E)but-2-ene- 1 ,4-dioate;
  • silicon-containing compounds which like DMF and the compounds of Formulae (I)-(II), can metabolize into MMF upon administration.
  • the compound that metabolizes to MMF is a compound of Formula (III):
  • R 2c is Ci-Cio alkyl, C 5 -C 15 aryl, hydroxyl, -O-CrCio alkyl, or -0-C 5 -C 15 aryl; each of R 3c , R 4c , and R 5c , independently, is C Cio alkyl, Cs-Qs aryl, hydroxyl, - O-C Cio alkyl, -0-C 5 -C 15 aryl, or
  • R lc is Q-C24 alkyl or C 5 -C 50 aryl; each of which can be optionally substituted;
  • each of m, n, and r, independently, is 0-4;
  • R 3c , R 4c , and R 5c is
  • Another group of compounds of Formula III include compounds wherein R c is optionally substituted Ci-C24 alkyl. Another group of compounds of Formula III include compounds wherein R lc is optionally substituted C]_-C alkyl. Another group of compounds of Formula III include compounds wherein R lc is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula III include compounds wherein R lc is optionally substituted C 5 - C 50 aryl. Another group of compounds of Formula III include compounds wherein R lc is optionally substituted Cs-Cio aryl. Another group of compounds of Formula III include compounds wherein R 2c is Q-Qo alkyl.
  • Another group of compounds of Formula III include compounds wherein R 2c is optionally substituted CrC 6 alkyl. Another group of compounds of Formula III include compounds wherein R 2c is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula III include compounds wherein R 2c is optionally substituted Cs-Cis aryl. Another group of compounds of Formula III include compounds wherein R 2c is optionally substituted Cs-Qo aryl.
  • the compound that metabolizes to MMF is a compound of Formula (HI):
  • R 2c is C 1 -C 10 alkyl, C 6 -C 10 aryl, hydroxyl, -O-Ci-Cio alkyl, or -O-C 6 -C 10 aryl;
  • each of R 3c , R 4c , and R 5c is C Cio alkyl, C6-C 10 aryl, hydroxyl,
  • R lc is Q-C24 alkyl or C 6 -C 10 aryl; each of which can be optionally substituted;
  • each of m, n, and r, independently, is 0-4;
  • R 3c , R 4c , and R 5c is
  • the compound that metabolizes to MMF is chosen from
  • the compound that metabolizes to MMF is a compound of Formula (IV):
  • each R LD is independently optionally substituted Ci-C 24 alkyl or C 5 -C 50 aryl;
  • each of, independently, R 2D and R 3D is Q-CK) alkyl or Cs-Cis aryl.
  • R 2D and R 3D can be the same or different, can be optionally substituted, and independently can be selected from the group consisting of C Cio alkyl or Cs-Qs aryl.
  • compounds of Formula IV include compounds wherein each R LD is independently optionally substituted Ci-C 24 alkyl or C 6 -Cio aryl.
  • compounds of Formula IV include compounds wherein R LD is optionally substituted C C ⁇ alkyl.
  • Another group of compounds of Formula IV include compounds wherein R LD is optionally substituted Ci-C alkyl.
  • Another group of compounds of Formula IV include compounds wherein R LD is optionally substituted methyl, ethyl, or isopropyl.
  • Another group of compounds of Formula IV include compounds wherein R LD is optionally substituted C 5 -C 50 aryl.
  • Another group of compounds of Formula IV include compounds wherein R LD is optionally substituted C 5 - Cio aryl.
  • Another group of compounds of Formula IV include compounds wherein each of R 2D and R is, independently, optionally substituted Ci-Cw alkyl. Another group of compounds of Formula IV include compounds wherein each of R 2d and R 3d is, independently, optionally substituted C -C alkyl. Another group of compounds of Formula IV include compounds wherein each of R 2d and R 3d is, independently, optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula IV include compounds wherein each of R 2d and R 3d is, independently, optionally substituted Cs-Qs aryl. Another group of compounds of Formula IV include compounds wherein each of R 2d and R 3d is, independently, optionally substituted Cs-Cio aryl.
  • the compound that metabolizes to MMF is a compound of Formula (IV):
  • R ld is C 1 -C 2 4 alkyl or C 6 -C 10 aryl
  • each of, independently, R 2d and R 3d is Ci-Cw alkyl or C 6 -C 10 aryl.
  • the compound that metabolizes to MMF is a compound of Formula (V): or a pharmaceutically acceptable salt thereof, wherein:
  • R le is Ci-C 24 alkyl or C5-C50 aryl; each of R 2e , R 3e , and R 5e , independently, is hydroxyl, C Qo alkyl, Cs-C ⁇ aryl, -O-C Cio alkyl, or -O-C 5 -Q5 aryl; and n is 1 or 2.
  • compounds of Formula V include compounds wherein R 6 is optionally substituted CrC 24 alkyl.
  • Another group of compounds of Formula V include compounds wherein R le is optionally substituted C]_-C alkyl.
  • Another group of compounds of Formula V include compounds wherein R le is optionally substituted methyl, ethyl, or isopropyl.
  • Another group of compounds of Formula V include compounds wherein R le is optionally substituted C 5 -C 50 aryl. Another group of compounds of Formula V include compounds wherein R le is optionally substituted Cs-Cio aryl. Another group of compounds of Formula V include compounds wherein each of R 2e , R 3e , and R 5e is, independently, hydroxyl. Another group of compounds of Formula V include compounds wherein each of R 2e , R 3e , and R 5e is,
  • Another group of compounds of Formula V include compounds wherein each of R 2e , R 3e , and R 5e is, independently, optionally substituted C -C alkyl.
  • Another group of compounds of Formula V include compounds wherein each of R 2e , R 3e , and R 5e is, independently, optionally substituted methyl, ethyl, or isopropyl.
  • Another group of compounds of Formula V include compounds wherein each of R 2e , R 3e , and R 5e is, independently, optionally substituted Cs-Cis aryl.
  • Another group of compounds of Formula V include compounds wherein each of R 2e , R 3e , and R 5e is, independently, optionally substituted C 5 -C 10 aryl.
  • the compound that metabolizes to MMF is a compound of Formula (V):
  • R le is C 1 -C 24 alkyl or C 6 -C 10 aryl; each of R 2e , R 3e , and R 5e , independently, is hydroxyl, C Cio alkyl, C 6 -C 10 aryl, -O-C Cio alkyl, or -0-C 6 -Cio aryl; and n is 1 or 2.
  • the compound that metabolizes to MMF is a compound of Formula (VI):
  • R is Q-C24 alkyl or C 5 -C 50 aryl; and R 2f is C 1 -C 10 alkyl.
  • compounds of Formula VI include compounds wherein R is optionally substituted Ci-C24 alkyl.
  • Another group of compounds of Formula VI include
  • Formula VI include compounds wherein R is optionally substituted methyl, ethyl, or isopropyl.
  • Another group of compounds of Formula VI include compounds wherein R is optionally substituted C 5 -C 50 aryl.
  • Another group of compounds of Formula VI include compounds wherein R is optionally substituted C 5 -C 50 aryl.
  • Another group of compounds of Formula VI include compounds
  • compounds of Formula VI include compounds wherein R is optionally substituted methyl, ethyl, or isopropyl.
  • the compound that metabolizes to MMF is a compound of Formula (VI): or a pharmaceutically acceptable salt thereof, wherein:
  • R is Q-C24 alkyl or C6-C 10 aryl
  • R 2f is C1-C10 alkyl.
  • dialkyl fumarate is:
  • R lg and R 2g which may be the same or different, independently represent a linear, branched or cyclic, saturated or unsaturated Ci_2o alkyl radical which may be optionally substituted with halogen (CI, F, I, Br), hydroxy, Ci_4 alkoxy, nitro or cyano.
  • R lg and R 2g which may be the same or different, independently are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, pentyl, cyclopentyl, 2-ethyl hexyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, vinyl, allyl, 2-hydroxy ethyl, 2 or 3-hydroxy propyl, 2-methoxy ethyl, methoxy methyl or 2- or 3-methoxy propyl.
  • R lg and R 2g are identical and are methyl or ethyl.
  • R lg and R 2g are methyl.
  • the compound is a monoalkyl fumarate.
  • the monoalkyl fumarate is:
  • R lh represents a linear, branched or cyclic, saturated or unsaturated C 1-2 o alkyl radical which may be optionally substituted with halogen (CI, F, I, Br), hydroxy, C 1-4 alkoxy, nitro or cyano;
  • R lh is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec -butyl, t-butyl, pentyl, cyclopentyl, 2-ethyl hexyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, vinyl, allyl, 2- hydroxy ethyl, 2 or 3-hydroxy propyl, 2-methoxy ethyl, methoxy methyl or 2- or 3-methoxy propyl.
  • R lh is methyl or ethyl.
  • R lh is methyl
  • the compound that metabolizes to MMF is a compound of Formula (VII):
  • R 1 is unsubstituted C -C alkyl
  • L a is substituted or unsubstituted Q-C 6 alkyl linker, substituted or unsubstituted C3-Q 0 carbocycle, substituted or unsubstituted C 6 -Q 0 aryl, substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; and
  • R 2 i and R 3i are each, independently, H, substituted or unsubstituted Ci-C 6 alkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted C 3 -C 10 carbocycle, substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; or alternatively, R 2 i and R 3i , together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S or a substituted or un
  • the compound of Formula (VII) is selected from a compound of Formula
  • R 1 is unsubstituted C -C alkyl
  • L a is substituted or unsubstituted CrC 6 alkyl linker, substituted or unsubstituted C 3 -C 10
  • carbocycle substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; and
  • R 2 i is H, substituted or unsubstituted C -C alkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C6-C 10 aryl, substituted or unsubstituted C 3 -C 10 carbocycle, substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S.
  • the compound of Formula (VII) is selected from a compound of Formula (Vllb):
  • a " is a pharmaceutically acceptable anion; R 1; is unsubstituted C -C alkyl;
  • L a is substituted or unsubstituted CrC 6 alkyl linker, substituted or unsubstituted C 3 -C 10 carbocycle, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S;
  • R 3i ' is substituted or unsubstituted C -C alkyl
  • R 2 i and R 3 i are each, independently, H, substituted or unsubstituted CrC 6 alkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted C3-Q0 carbocycle, substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S; or alternatively, R 2i and R i , together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or a substituted or unsub
  • Rn is unsubstituted CrC 6 alkyl
  • R4 1 and R5 1 are each, independently, H, substituted or unsubstituted C -C alkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted C3-C 10 carbocycle, substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or substituted or unsubstituted heteroaryl comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S;
  • R 6 i, R 7 i, Rsi and R91 are each, independently, H, substituted or unsubstituted CrC 6 alkyl, substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl or C(0)OR a ;
  • R a is H or substituted or unsubstituted CrC 6 alkyl.
  • the compound of Formula (VII) is selected from a compound of Formula (IX):
  • R 1 is unsubstituted C -C alkyl
  • X is N, O, S or S0 2 ;
  • R 6 i, R 7 i, Rgi and R 9i are each, independently, H, substituted or unsubstituted C C 6 alkyl, substituted or unsubstituted C 2 -C6 alkenyl, substituted or unsubstituted C 2 -C6 alkynyl or C(0)OR a ; and R a is H or substituted or unsubstituted CrC 6 alkyl; and each R 10 i is, independently, H, halogen, substituted or unsubstituted C -C alkyl, substituted or unsubstituted C 2 -C6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl, substituted or unsubstituted C 3 -Q0 carbocycle, substituted or unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S, or substituted or unsubstituted heteroaryl comprising
  • the compound is a compound listed in Table A herein.
  • Representative compounds of the present invention include compounds listed in Table A.
  • the compound of Formula (VII) is the compound (X):
  • the disorder or condition is cancer.
  • the disorder or condition is a hematological malignancy.
  • the hematological malignancy is selected from lymphocytic leukemia, chronic lymphocytic leukemia, and lymphoma.
  • the disorder or condition is a solid tumor.
  • the solid tumor is selected from gastrointestinal sarcoma,
  • the disorder or condition is a viral infection.
  • the invention features, a method of treating a disorder or condition described herein by administering to the subject: a dialkyl fumarate, e.g., DMF, MMF; or a combination thereof.
  • a dialkyl fumarate e.g., DMF, MMF
  • the invention features a method of modulating an immune response in a subject, comprising antagonizing expression of OSGINI in the subject, thereby promoting an immune response in the subject.
  • the level and/or activity of TNF-a is increased by antagonizing expression of OSGINI in the subject.
  • antagonizing expression of OSGINI comprises administering to the subject an antagonist selected from the group consisting of an anti-OSGINl antibody or OSGINI -binding fragment thereof, siRNA, shRNA, antisense RNA, miRNA, and combinations thereof.
  • the invention features a method of modulating an immune response in a subject, comprising agonizing expression of OSGINI in the subject, thereby inhibiting an immune response in the subject.
  • the level and/or activity of TNF-a is decreased by agonizing expression of OSGINI in the subject.
  • agonizing expression of OSGINI comprises administering to the subject an agonist selected from the group consisting of an expression vector encoding OSGINI (e.g., retroviral, lentiviral, among other expression vectors), mRNA encoding OSGIN1, OSGIN1 translation product, and combinations thereof.
  • FIGURE 1 depicts exemplary monomethyl fumarate (MMF) exposures after oral dimethyl fumarate (DMF) or MMF dosing. Satellite 5 animals per group were dosed orally with DMF or MMF (100 mg/kg) and sacrificed 30 minutes post-dosing. MMF exposures were determined and compared in various compartments. MMF levels were highly comparable between DMF and MMF dosing, suggesting any subsequent differences in pharmacodynamic responses would not simply be due to different exposures.
  • MMF monomethyl fumarate
  • FIGURE 2A depicts an exemplary overview of Venn diagrams comparing differentially expressed genes in each tissue. Data are presented as an aggregation of three time points for each tissue.
  • FIGURE 2B depicts exemplary Venn diagrams comparing differentially expressed genes in whole blood, cortex, hippocampus, striatum, jejunum, kidney, liver and spleen.
  • FIGURE 3 depicts exemplary analysis of whole blood DMF differentially expressed genes (DEGs) in various pathways. Common effects were observed on NK cell function.
  • DEGs DMF differentially expressed genes
  • FIGURE 4 depicts exemplary analysis of cortical MMF DEGs in various pathways.
  • FIGURE 5 depicts exemplary analysis of hippocampal MMF DEGs in various pathways.
  • FIGURE 6 depicts exemplary analysis of striatal MMF DEGs in various pathways.
  • FIGURES 7A and 7B depict exemplary analysis of jejunum DEGs in various pathways.
  • Figure 7A depicts DMF and MMF common DEGs.
  • Figure 7B depicts MMF DEGs.
  • FIGURES 8A-8C depict exemplary analysis of kidney DEGs in various pathways.
  • Figure 8A depicts DMF and MMF common DEGs.
  • DMF and MMF common DEGs showed good representation of Nrf2 pathway activation and xenobiotic metabolism, with particular emphasis on glutathione biosynthesis.
  • Figure 8B depicts DMF specific DEGs.
  • Figure 8C depicts MMF specific DEGs.
  • FIGURE 9 depicts exemplary analysis of liver DMF and MMF common DEGs in various pathways.
  • FIGURE 10 depicts an exemplary flow cytometry gating strategy for comparative analysis of DMF and MMF on Natural Killer (NK) cell phenotype. Protein expression is quantified by mean fluorescent intensity (MFI).
  • MFI mean fluorescent intensity
  • FIGURE 11 depicts an exemplary comparative analysis of DMF and MMF on NK cell phenotype using markers: NKl.l (klrblb), Nkg2d (klrkl), NKp46 (ncrl), Nkg2a (klrcl), and CD94 (klrdl).
  • Study design Naive C57B1/6 mice were dosed PO with 100 mpk dimethyl fumarate (DMF) or molar equivalency of monomethyl fumarate (MMF). 12-hours post-dose, mice were sacrificed and blood, spleen, and inguinal lymph node were collected for analysis by flow cytometry.
  • DMF dimethyl fumarate
  • MMF monomethyl fumarate
  • FIGURE 12 depicts an exemplary immunophenotyping panel for analysis of immune cell subsets in EAE mice treated with DMF or MMF.
  • FIGURE 13 depicts exemplary NK cell analysis in blood and spleen and EAE clinical score analysis for a chronic dosing experiment in EAE mice.
  • FIGURE 14 depicts exemplary NK cell protein expression in spleen and blood for a chronic dosing experiment in EAE mice.
  • FIGURE 15 depicts exemplary NK cell subset and protein expression analysis in spleen and blood for a chronic dosing experiment in EAE mice.
  • FIGURE 16 depicts exemplary NK cell analysis in spleen, iLN, and blood for a single dose experiment in EAE mice.
  • FIGURE 17 depicts exemplary NK cell protein expression in spleen, iLN and blood for a single dose experiment in EAE mice.
  • FIGURE 18 depicts exemplary NK cell subset and protein expression analysis in spleen, iLN and blood for a single dose experiment in EAE mice.
  • FIGURE 19 depicts an exemplary flow cytometry gating strategy for comparative analysis of DMF and MMF on T cell phenotype. Protein expression is quantified by mean fluorescent intensity (MFI).
  • MFI mean fluorescent intensity
  • FIGURE 20 depicts exemplary T regulatory cell analysis in spleen, iLN and blood for a chronic dosing experiment in EAE mice.
  • FIGURE 21 depicts exemplary CD4+ cell subset and protein expression analysis in spleen, iLN and blood for a chronic dosing experiment in EAE mice.
  • FIGURE 22 depicts exemplary CD8+ cell subset and protein expression analysis in spleen, iLN and blood for a chronic dosing experiment in EAE mice.
  • FIGURE 23 depicts exemplary CD4+ T cell subset vs. EAE clinical score analysis in spleen for a chronic dosing experiment in EAE mice.
  • FIGURE 24 depicts exemplary CD8+ T cell subset vs. EAE clinical score analysis in spleen for a chronic dosing experiment in EAE mice.
  • FIGURE 25 depicts exemplary B cell analysis in naive, vehicle, MMF or DMF treated EAE mice.
  • FIGURE 26 depicts an exemplary myeloid cell gating strategy for comparative analysis of DMF and MMF on myeloid cell phenotype.
  • FIGURE 27 depicts exemplary myeloid cell subset analysis in spleen and iLN for a chronic dosing experiment in EAE mice.
  • FIGURE 28 depicts the changes in OSGINl expression in mouse cortex, cerebellum, hippocampus and striatum after DMF administration.
  • the levels of OSGINl gene expression were measured by qtPCR.
  • the fold changes were normalized to vehicle control.
  • FIGURE 29A depicts the changes in OSGINl expression in hippocampus from wild- type mice and Nrf2-/- mice after DMF administration. The levels of OSGINl gene expression were measured by qtPCR. The fold changes were normalized to wild-type control.
  • FIGURE 29B depicts the changes in OSGINl expression in striatum from wild- type mice and Nrf2-/- mice after DMF administration. The levels of OSGINl gene expression were measured by qtPCR. The fold changes were normalized to wild-type control.
  • FIGURE 30 depicts the levels of OSGINI1 expression in human spinal cord astrocytes treated with MMF (0, 10 or 30 ⁇ ) and OSGINl siRNA (10 ⁇ ) or scrambled siRNA (10 ⁇ ).
  • the levels of OSGINl gene expression were measured by qtPCR. The fold changes were normalized to scrambled siRNA control. Left bars: scrambled siRNA; right bars: OSGINl siRNA.
  • FIGURE 31A depicts the number of live human spinal cord astrocytes after treatment with MMF (0, 10 or 30 ⁇ ), H 2 0 2 (200 ⁇ ), and OSGINl siRNA (10 ⁇ ) or scrambled siRNA (10 ⁇ ). The number of live cells was quantified by Calcein AM fluorescence intensity.
  • FIGURE 31B depicts the percentage of viable nuclear count in human spinal cord astrocytes treated with MMF (0, 10 or 30 ⁇ ), H 2 0 2 (200 ⁇ ), and OSGINl siRNA (10 ⁇ ) or scrambled siRNA (10 ⁇ ). The percentage of viable nuclear count was measured by DAPI staining and normalized to 0 ⁇ H 2 0 2 control.
  • FIGURE 31 C depicts exemplary images of live and dead human spinal cord astrocytes treated with MMF (0 or 30 ⁇ ), H 2 0 2 (200 ⁇ ), and OSGINl siRNA (10 ⁇ ) or scrambled siRNA (10 ⁇ ).
  • the live and dead cells are shown by calcein AM staining and ethidium homodimer staining, respectively.
  • FIGURE 32A depicts the levels of PADI4 gene expression in human spinal cord astrocytes treated with MMF (0, 10 or 30 ⁇ ), H 2 0 2 (200 ⁇ ), and OSGINl siRNA (10 ⁇ ), PADI4 siRNA (10 ⁇ ) or scrambled siRNA (10 ⁇ ).
  • the levels of PADI4 expression were measured by qtPCR and normalized to scramble control. Left bars: scrambled siRNA; middle bars:
  • FIGURE 32B depicts the levels of OSGINl gene expression in human spinal cord astrocytes treated with MMF (0, 10 or 30 ⁇ ), H 2 0 2 (200 ⁇ ), and OSGINl siRNA (10 ⁇ ), PADI4 siRNA (10 ⁇ ) or scrambled siRNA (10 ⁇ ). The levels of OSGINl expression were measured by qtPCR and normalized to scramble control. Left bars: scrambled siRNA; middle bars: OSGINl siRNA; right bars: PADI4 siRNA.
  • FIGURE 33A depicts the percentage of viable nuclear count in human spinal cord astrocytes treated with MMF (0, 10 or 30 ⁇ ), H 2 0 2 (300 ⁇ ), and PADI4 siRNA (10 ⁇ ) or scrambled siRNA (10 ⁇ ). The percentage of viable nuclear count was measured by DAPI staining and normalized to 0 ⁇ H 2 0 2 control.
  • FIGURE 33B depicts exemplary images of live and dead human spinal cord astrocytes treated with MMF (0 or 30 ⁇ ), H 2 0 2 (200 ⁇ ), and PADI4 siRNA (10 ⁇ ) or scrambled siRNA (10 ⁇ ).
  • the live and dead cells are shown by calcein AM staining and ethidium homodimer staining, respectively.
  • FIGURE 34 depicts the percentage of viable nuclear count in human spinal cord astrocytes treated with MMF (0, 10 or 30 ⁇ ), H 2 0 2 (300 ⁇ ), and p53 siRNA or scrambled siRNA. The percentage of live cells was measured by nuclear counting and normalized to 0 ⁇ H 2 0 2 control.
  • FIGURE 35 depicts gene selections for Fluidigm real-time PCR as described in Example 4.
  • FIGURE 36 depicts epitope locations of OSGINl isoform-specific antibodies. Boxes represent translational start sites of each OSGINl isoform labeled with the corresponding amino acid length protein; 560aa (OSGINl ⁇ 61kDa), 477aa (OSGINl ⁇ 52kDa) and 375aa (OSGINl ⁇ 38kDa). Dashed underlined amino acids indicate region of peptide generation for antibody creation; *:OSGINl ⁇ 61kDa, *:OSGINl ⁇ 52kDa and ***:OSGINl ⁇ 38kDa.
  • FIGURE 37A depicts the structure and basic Properties of dimethyl fumarate (DMF) and monomethyl fumarate (MMF).
  • FIGURE 37B depicts MMF exposure in peripheral and CNS tissues following a single dose of DMF. Drug exposure levels were measured in mouse tissue samples collected 30 minutes post- dose of DMF and were analyzed for levels of MMF, the active metabolite of DMF.
  • FIGURES 38A and 38B depict DMF transcriptional profiling time course in peripheral tissues: kidney/jejunum. Transcriptional changes following single lOOmg/kg oral dose of DMF in kidney (Figure 38A) and jejunum (Figure 38B), graphed as changes in selected genes over time. Data are graphed as fold change values normalized to vehicle treated animals, with the starting value of 1 equivalent to the normalized baseline of vehicle animals. Graphed genes represent only those that were found to be regulated in peripheral tissues, with Vegfa as a baseline control gene.
  • FIGURES 39A and 39B depict DMF transcriptional profiling time course in peripheral tissues: spleen/liver. Transcriptional changes following single lOOmg/kg oral dose of DMF in spleen ( Figure 39A) and liver ( Figure 39B), graphed as changes in selected genes over time. Data are graphed as fold change values normalized to vehicle treated animals, with the starting value of 1 equivalent to the normalized baseline of vehicle animals. Graphed genes represent only those that were found to be regulated in peripheral tissues, with Vegfa as a baseline control gene.
  • FIGURES 40A and 40B depict DMF transcriptional profiling time course in CNS tissues:
  • OSGIN1/BDNF Transcriptional changes following single lOOmg/kg oral dose of DMF for two genes found to be regulated in the brain: Osginl ( Figure 40A) and Bdnf ( Figure 40B). Data are graphed as fold change values normalized to vehicle treated animals, with the dashed line at 1 equivalent to the normalized baseline of vehicle animals. Later time points are not included since regulation of the graphed genes is consistent with the graphed 16.5 hour time point.
  • FIGURE 41 depicts DMF transcriptional profiling time course in CNS tissues: NQOl.
  • FIGURES 42A and 42B depict DMF transcriptional profiling dose-response in peripheral tissues: kidney/jejunum. Transcriptional changes following multiple doses of DMF in kidney ( Figure 42A) and jejunum ( Figure 42B), graphed as changes in selected genes across doses. Data are graphed as fold change values normalized to vehicle treated animals, with the starting value of 0 equivalent to the normalized baseline of vehicle animals. Graphed genes represent only those that were found to be regulated in peripheral tissues, with Vegfa as a baseline control gene.
  • FIGURES 43A and 43B depict DMF transcriptional profiling dose-response in peripheral tissues: spleen/liver. Transcriptional changes following multiple doses of DMF in spleen (Figure 43A) and liver ( Figure 43B), graphed as changes in selected genes across doses. Data are graphed as fold change values normalized to vehicle treated animals, with the starting value of 0 equivalent to the normalized baseline of vehicle animals. Graphed genes represent only those that were found to be regulated in peripheral tissues, with Vegfa as a baseline control gene.
  • FIGURES 44A and 44B depict DMF transcriptional profiling dose-response in CNS tissues: cortex/cerebellum. Transcriptional changes following multiple doses of DMF in Cortex ( Figure 44A) and Cerebellum (Figure 44B), graphed as changes in selected genes across doses. Data are graphed as fold change values normalized to vehicle treated animals, with the starting value of 0 equivalent to the normalized baseline of vehicle animals. Graphed genes represent only those that were found to be regulated in CNS tissues, with Vegfa as a baseline control gene.
  • FIGURES 45A and 45B depict DMF transcriptional profiling dose-response in CNS tissues: hippocampus/striatum. Transcriptional changes following multiple doses of DMF in Hippocampus ( Figure 45A), Striatum ( Figure 45B), graphed as changes in selected genes across doses. Data are graphed as fold change values normalized to vehicle treated animals, with the starting value of 0 equivalent to the normalized baseline of vehicle animals. Graphed genes represent only those that were found to be regulated in CNS tissues, with Vegfa as a baseline control gene.
  • FIGURES 46A and 46B depict DMF transcriptional profiling dose -response in whole blood. Transcriptional changes following a single dose of lOOmg/kg DMF across 8 time points in whole blood samples (Figure 46A). Data are graphed as fold change values normalized to vehicle treated animals, with the dashed line at 1 equivalent to the normalized baseline of vehicle animals. Transcriptional changes following multiple doses of DMF in whole blood ( Figure 46B). Data are graphed as fold change values normalized to vehicle treated animals, with the starting value of 0 equivalent to the normalized baseline of vehicle animals. Graphed genes represent only those that were found to be regulated in whole blood.
  • FIGURES 47A and 47B depict transcriptional time course with lOOmg/kg DMF in Nrf2-/- and wild type mice: peripheral tissues. Transcriptional changes following single lOOmg/kg oral dose of DMF in peripheral tissues: Jejunum ( Figure 47A) and Kidney (Figure 47B), graphed as changes in selected genes over time. Data are graphed as fold change values normalized to vehicle treated animals, with the starting value of 1 equivalent to the normalized baseline of vehicle animals. Graphed genes represent a select panel determined based on gene regulation from the time course study.
  • FIGURES 48A and 48B depict transcriptional time course with lOOmg/kg DMF in Nrf2-/- and wild type mice: CNS tissues. Transcriptional changes following single lOOmg/kg oral dose of DMF in CNS tissues: Hippocampus ( Figure 48A) and Striatum ( Figure 48B), graphed as changes in selected genes over time. Data are graphed as fold change values normalized to vehicle treated animals, with the starting value of 1 equivalent to the normalized baseline of vehicle animals. Graphed genes represent a select panel determined based on gene regulation from the time course study.
  • FIGURES 49A and 49B depict DMF-induced Nrf2-dependent protein expression in peripheral tissues. Nrf2-/- and wild type mice were treated with vehicle or lOOmg/kg DMF in triplicate and tissues harvested at 6 h after compound addition. Relative protein levels of Txnrdl, Sqstml, Nqol and GCLC were assessed and actin control levels measured as an internal control. Protein expression following a single lOOmg/kg oral dose of DMF in Kidney ( Figure 49A) and Jejunum ( Figure 49B).
  • FIGURES 50A and 50B depicts DMF-induced protein expression in CNS tissues. Nrf2-/- and wild type mice were treated with vehicle or lOOmg/kg DMF in triplicate and tissues harvested at 6 h after compound addition. Relative protein levels of Nqol and BDNF were assessed and actin control levels measured as an internal control. Protein expression following a single lOOmg/kg oral dose of DMF in Striatum ( Figure 50A) and Cortex ( Figure 50B).
  • FIGURES 51A and 51B depict MMF-induced Nrf2 target gene expression in human astrocytes.
  • Human astrocytes were treated with either DMSO or a titration of MMF for 24 hours before RNA extraction and q-PCR analysis. The graph represents triplicate samples; error bars indicate S.D.
  • Figure 51A q-PCR analysis of classical Nrf2 target genes.
  • Figure 51B OSGINl q-PCR analysis.
  • FIGURES 52A-52D depict MMF protection of human astrocytes from oxidative challenge.
  • Human astrocytes were treated with DMSO or 30uM MMF for 24 hours followed by oxidative challenge.
  • Figures 52A- Figure 52C show live imaging of LIVE/DEAD labeled cells pretreated with MMF and then challenged with H 2 O 2 .
  • Figure 52A shows positive control cells treated with DMSO.
  • Figure 52B shows control DMSO-treated and 300uM H 2 0 2 -challenged cells.
  • Figure 52C shows astrocytes pretreated with 30uM MMF and challenged with 300uM H 2 0 2 .
  • Figure 52D shows quantification of calcein AM fluorescence intensity in LIVE/DEAD labeled cells from A-C.
  • FIGURES 53A and 53B depict Nrf2 knockdown experiments.
  • Figure 53A shows q-PCR analysis of human astrocytes treated with ⁇ of control or Nrf2 siRNA and either DMSO or 30uM MMF. *, /? ⁇ .001 as compared to control siRNA using two-way ANOVA and Tukey's multiple comparison test.
  • Figure 53B shows western blot data correlating with Figure 53A. Actin is shown as a loading control.
  • FIGURE 54 depicts loss of Nrf2 and reduction in OSGIN1 expression.
  • Human astrocytes were transfected with scrambled (control) or Nrf2 siRNA followed by treatment with a titration of MMF.
  • q-PCR analysis was conducted for OSGINltranscription. Error bars represent SD and p values based on two-way ANOVA with Tukey's post-test for multiple-samples comparisons.
  • FIGURES 55A and 55B depict the abolishment of MMF-mediated cytoprotection with the loss of Nrf2.
  • Human astrocytes were transfected with scrambled (control) or Nrf2 siRNA and treated with either DMSO or MMF for 24 hours then challenged with H 2 0 2 .
  • A cells fixed and stained with DAPI nuclear dye.
  • Figure 55A shows a graph representing average cell nuclei counts in 20 fields per well (2 wells averaged in graph). Error bars represent SD and p values based on two- way ANOVA with Sidak's post-test for multiple-samples comparisons.
  • Figure 55B shows live imaging of LIVE/DEAD labeled cells pretreated with MMF and then challenged with H 2 0 2 . LIVE calcein AM (green) and DEAD ethidium homodimer (red) labeling.
  • FIGURES 56A and 56B depict OSGIN1 siRNA knockdown experiments.
  • Human astrocytes were transfected with scrambled (control) or OSGIN1 siRNA and treated with either DMSO, lOuM MMF or 30uM MMF for 24 hours before RNA extraction and q-PCR analysis.
  • Figure 56A shows q-PCR analysis for OSGIN1 transcriptional regulation.
  • Figure 56B shows q-PCR analysis for Nrf2 transcriptional regulation.
  • the graphs represent duplicate samples; error bars indicate S.D. p values based on two-way ANOVA with Tukey's post-test for multiple-samples comparisons.
  • FIGURES 57A-57C depict that OSGINl siRNA knockdown inhibits MMF-mediated cytoprotection.
  • Human astrocytes were transfected with scrambled (control) or OSGINl siRNA and treated with MMF for 24 hours then challenged with ⁇ 2 0 2 .
  • Figure 57A imaging of LIVE/DEAD labeled cells pretreated with MMF and then challenged with H 2 0 2 .
  • Figure 57B quantification of calcein AM fluorescence intensity in LIVE/DEAD labeled cells from Figure 57A.
  • Figure 57C replicate plates as in Figure 57A fixed and stained with DAPI nuclear dye. Graph represents average cell nuclei counts in 20 fields per well (2 wells averaged in graph). Error bars represent SD and p value based on one-way ANOVA with Tukey's post-test for multiple-samples comparisons.
  • FIGURES 58A and 58B depict optimization of OSGINl -52kDa and OSGINl -6 lkDa antibodies.
  • Human astrocyte cell lysates were probed with three antibody condition: antibody alone (NP), antibody pre-incubated with epitope specific peptide (P) or non-specific control peptide (CP).
  • Figure 58A OSGINl-52kDa antibody conditions.
  • Figure 58B OSGINl-61kDa antibody conditions.
  • FIGURE 59 depicts that OSGINl knockdown depletes OSGINl isoform specific
  • Human astrocytes were transfected with ⁇ of control or OSGINl specific siRNA and cell lysates were probed with OSGINl isoform- specific antibodies, ⁇ -actin is included as a loading control.
  • FIGURES 60A-60C depict the regulation of OSGINl 61kDa encoding isoform via MMF and Nrf2.
  • Human astrocytes were transfected with either control or OSGINl specific siRNA followed by treatment with 30uM MMF for 24 hours.
  • Figure 60A transfected cell lysates probed with OSGINl -52kDa antibody.
  • Figure 60B transfected cell lysates probed with OSGINl -6 lkDa antibody.
  • Figure 60C human astrocytes transfected with either control or Nrf2 specific siRNA and probed with OSGINl -6 lkDa antibody, ⁇ -actin is included in all figures as a loading control.
  • FIGURE 61 depicts immunocytochemical analysis of OSGINl -52kDa and OSGINl -6 lkDa antibodies.
  • Human astrocytes were treated with MMF for 24 hours, fixed and then probed with antibodies against OSGINl -52kDa and OSGINl -6 lkDa isoforms of OSGINl .
  • Total fluorescent spot count was accomplished using Thermo HCS Arrayscan technology, p values based on oneway ANOVA with Tukey' s post-test for multiple sample comparisons.
  • FIGURES 62A and 62B depict 3' and 5' RACE of OSGINl transcript.
  • RNA extracted from human astrocytes treated with a titration of MMF were subjected to 3' and 5' RACE.
  • Figure 62A shows resulting RACE products analyzed via gel electrophoresis.
  • Figure 62B sequencing of 5' RACE products identified two transcript variants of OSGINl within the 5' UTR of the OSGINl -52kDa transcript that differed in two nucleotide substitutions (indicated by arrows).
  • FIGURE 63 depicts the q-PCR analysis of identified 5 'RACE transcripts.
  • RNA was extracted from human astrocytes treated with DMSO or MMF for 24 hours and subjected to q-PCR using primer/probe sets specific to identified 5'RACE transcripts.
  • WT canonical sequence;
  • ALT identified 5' RACE sequence with nucleotide substitutions.
  • *, /? ⁇ .05 and /? ⁇ .0001 based on one-way ANOVA with Tukey' s post-test for multiple comparisons.
  • FIGURES 64A-64C depict q-PCR of p53 siRNA knockdown experiments.
  • Figure 64A q-PCR analysis of p53 mRNA levels. *, p ⁇ .01 based on two- way ANOVA with Tukey' s post-test for multiple comparisons.
  • Figure 64B human astrocytes transfected with either control (scrambled), p53 or Nrf2 siRNA and analyzed for Nrf2 mRNA levels.
  • Figure 64C same as Figure 64B but q-PCR directed against OSGINl transcript levels. B/C, * , /? ⁇ .01 based on one-way ANOVA with Dunnett' s post- test for multiple comparisons.
  • FIGURES 65A-65C depict p53 protein regulation by MMF in an Nrf2- and OSGINl -dependent manner.
  • Human astrocytes were transfected with ⁇ control (scrambled), p53, OSGINl or Nrf2 siRNA followed by treatment with DMSO or MMF for 24 hours.
  • Figure 65A shows p53 protein measure in scrambled (control), p53 and OSGINl siRNA knockdown samples treated with MMF.
  • Figure 65B shows Nrf2 protein measure in scrambled (control) and p53 siRNA knockdown samples treated with MMF.
  • Figure 65C shows Scrambled (control) and Nrf2 siRNA knockdown samples treated with MMF and probed for p53 protein, ⁇ -actin is included in all figures as a loading control.
  • FIGURES 66A and 66B depict MMF induction of nuclear translocation of p53: ICC. Human astrocytes were treated with a titration of MMF for 24 hours followed by fixation and ICC analysis of p53.
  • Figure 66A immunocytochemistry images acquired using the Thermo HCS Arrayscan and algorithm analysis overlaid .
  • FIGURES 67A and 67B depict MMF induction of nuclear translocation of p53: p53 TransAM assay.
  • Human astrocytes were treated with a titration of MMF for 24 hours followed by analysis of nuclear and cytoplasmic cell extracts for p53 expression.
  • Figure 67A nuclear extract quatification.
  • Figure 67B cytoplasmic extract quantification.
  • Statistical analysis was performed using one-way ANOVA with Dunett's multiple comparison post-test (*, /? ⁇ .05)
  • FIGURES 68A and 68B depict that p53 contributes to MMF-mediated cytoprotection.
  • Human astrocytes were transfected with scrambled (control) or p53 siRNA and treated with MMF for 24 hours then challenged with H 2 0 2 .
  • Figure 68A imaging of LIVE/DEAD labeled cells pretreated with MMF and then challenged with H 2 0 2 .
  • Figure 68B quantification of calcein AM fluorescence intensity in LIVE/DEAD labeled cells from Figure 68A. Error bars represent SD and p value based on oneway ANOVA with Tukey' s post-test for multiple-samples comparisons.
  • FIGURES 69A and 69B depict the q-PCR time course of NQOl and OSGIN1.
  • Human astrocytes were treated with 0, 10 or 30uM of MMF and analyzed for transcript regulation of OSGIN1 and NQOl over various time points.
  • Figure 69A OSGIN1 time course.
  • Figure 69B NQOl time course.
  • FIGURE 70 depicts the protein time course of MMF-regulated proteins.
  • Human astrocytes were treated with 0, 10 or 30uM of MMF and analyzed for protein regulation of OSGIN1, Nrf2, p53 and NQOl over various time points, ⁇ -actin is included as a loading control.
  • FIGURES 71A and 71B depict MMF inhibition of cell proliferation. Human astrocytes were transfected with scrambled (control) or OSGINl siRNA followed by treatment with 0, 10 or 30uM MMF and pulse incorporation of EdU. Cells were fixed and EdU incorporated cells were imaged and quantified using a Thermo HCS Arrayscan and HCS Studio software, as shown in Figure 71B.
  • Figure 71A shows quantification of four wells are averaged in grpahs (25 fields/well). * p ⁇ .05; * * p ⁇ .0l; *** /? ⁇ .001 based on two-way ANOVA with Tukey's post-test for multiple comparisons.
  • FIGURES 72A and 72B depicts the loss of OSGINl and PADI4 does not significantly induce apoptosis.
  • Figure 72A siRNA transfection alone.
  • Figure 72B siRNA transfection in the presence of MMF treatment for 24 hours.
  • FIGURE 73 depicts MMF-mediation of PADI4 in a similar manner to NQOl.
  • Human astrocytes were treated with 0, 10 or 30uM of MMF and analyzed for transcript regulation of PAD 14 over various time points.
  • FIGURES 74A and 74B depict that PADI4 is regulated by OSGINl and p53.
  • Human astrocytes were transfected with either scrambled (control), PADI4, OSGINl, Nrf2 or p53 siRNA followed by q-PCR analysis.
  • Figure 74A shows PADI4 q-PCR. *, /? ⁇ .0001 based on one-way ANOVA with Dunnett's post-test for multiple comparisons.
  • Figure 74B shows transfected cells treated with MMF for 24 hours followed by q-PCR for PADI4. p values based on two-way ANOVA with Tukey's post-test for multiple comparisons.
  • FIGURES 75A and 75B depict that PADI4 does not regulate OSGINl or p53.
  • Human astrocytes were transfected with either scrambled (control), PADI4, OSGINl or p53 siRNA followed by q- PCR analysis.
  • Figure 75A shows transfected cells treated with MMF for 24 hours followed by q- PCR for OSGINl.
  • Figure 75B shows q-PCR for p53. p values based on two-way ANOVA with Tukey's post-test for multiple comparisons.
  • FIGURE 76 depicts that OSGINl knockdown induces expression of TNF-alpha.
  • Human astrocytes were transfected with scrambled (control) or OSGINl specific siRNA. Samples were analyzed for alterations in TNF-a transcript levels. * , /? ⁇ .05 based on student' s t test.
  • FIGURES 77A and 77B depict a potential mechanism of action of OSGINl mediated cellular protection. Interaction of MMF with cysteine residues on Keapl results in allosteric conformational change in the Keapl protein, resulting in the inability of Nrf2 to be targeted for ubiquitination and subsequent proteosomal degradation. This allows Nrf2 to accumulate in the cytoplasm and translocate to the nucleus where it can regulate the transcription of various genes including OSGINl ( Figure 77A). OSGINl transcript expression is then translated to a 61kDa protein that can induce the accumulation of p53 in an unknown mechanism. p53 protein can then translocate to the nucleus and induce gene transcription Figure 77B).
  • the invention is based, at least in part, on the discovery that the DMF makes a contribution to pharmacologic effect that is distinct from that of its metabolite, MMF.
  • the articles “a” and “an” refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.
  • “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent ( ), typically, within 10%, and more typically, within 5% of a given value or range of values.
  • Directly acquiring means performing a physical process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value.
  • Indirectly acquiring refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value).
  • Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting material.
  • Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, .combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non covalent bond.
  • Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a
  • substance e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical
  • an analytical method e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non covalent bond, between a first and a second atom of the reagent.
  • a substance e.g., an analyte, or a fragment or other derivative thereof, from another substance
  • another substance e.g., a buffer,
  • a sample refers to obtaining possession of a sample, e.g., a tissue sample or nucleic acid sample, by “directly acquiring” or “indirectly acquiring” the sample.
  • Directly acquiring a sample means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample.
  • Indirectly acquiring a sample refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample).
  • Directly acquiring a sample includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue, e.g., a tissue in a human patient or a tissue that has was previously isolated from a patient.
  • a starting material such as a tissue
  • Exemplary changes include making a physical entity from a starting material, dissecting or scraping a tissue; separating or purifying a substance (e.g., a sample tissue or a nucleic acid sample); combining two or more separate entities into a mixture; performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond.
  • Directly acquiring a sample includes performing a process that includes a physical change in a sample or another substance, e.g., as described above.
  • a dimethyl fumarate (DMF)-differentially expressed gene is a gene, the expression of which differs in a subject that has been administered DMF, as compared to a subject not administered DMF. Differential expression can be manifest at the transcriptional or translation level, e.g. , at the level of mRNA or protein, or at both.
  • a gene is DMF-differentially expressed if the levels of the RNA or protein product, or both, of the gene are higher, in a subject administered DMF, as compared to a subject not administered DMF.
  • a DMF-differentially expressed gene can be characterized by differential expression at one or both of the transcriptional and translational levels. In an embodiment a DMF-differentially expressed gene will not also be MMF-differentially expressed, or the differential expression for DMF will differ from the differential expression seen for MMF.
  • a prodrug (PD)-differentially expressed gene is a gene, the expression of which differs in a subject that has been administered a prodrug, as compared to a subject not administered a prodrug. Differential expression can be manifest at the transcriptional or translation level, e.g. , at the level of mRNA or protein, or at both.
  • a gene is PD-differentially expressed if the levels of the RNA or protein product, or both, of the gene are higher, in a subject administered prodrug, as compared to a subject not administered prodrug, e.g., DMF.
  • a PD-differentially expressed gene can be characterized by differential expression at one or both of the transcriptional and translational levels.
  • a PD-differentially expressed gene will not also be drug, e.g., MMF-differentially expressed, or the differential expression for PD will differ from the differential expression seen for drug, e.g., MMF.
  • a monomethyl fumarate (MMF)-differentially expressed gene is a gene, the expression of which differs in a subject that has been administered MMF, as compared to a subject not administered MMF. Differential expression can be manifest at the transcriptional or translation level, e.g. , at the level of mRNA or protein, or at both.
  • a gene is MMF-differentially expressed if the levels of the RNA or protein product, or both, of the gene are higher, in a subject administered MMF, as compared to a subject not administered MMF.
  • An MMF-differentially expressed gene can be characterized by differential expression at one or both of the transcriptional and translational levels.
  • an MMF-differentially expressed gene will not also be DMF-differentially expressed, or the differential expression for MMF will differ from the differential expression seen for DMF.
  • a DMF/MMF-differentially expressed gene is a gene that is differentially expressed for both DMF and MMF.
  • a drug-differentially expressed gene is a gene, the expression of which differs in a subject that has been administered drug, e.g., MMF, as compared to a subject not administered the drug. Differential expression can be manifest at the transcriptional or translation level, e.g. , at the level of mRNA or protein, or at both.
  • a gene is drug-differentially expressed if the levels of the RNA or protein product, or both, of the gene are higher, in a subject administered drug, as compared to a subject not administered drug.
  • a drug-differentially expressed gene can be characterized by differential expression at one or both of the transcriptional and translational levels. In an embodiment a drug-differentially expressed gene will not also be PD-differentially expressed, or the differential expression for drug will differ from the differential expression seen for prodrug.
  • a Drug/PD-differentially expressed gene is a gene that is differentially expressed for both prodrug and drug.
  • EDSS Extended Disability Status Scale
  • EDSS is a rating system that is frequently used for classifying and standardizing MS.
  • the accepted scores range from 0 (normal) to 10 (death due to MS).
  • patients having an EDSS score of about 6 will have moderate disability (e.g., walk with a cane), whereas patients having an EDSS score of about 7 or 8 will have severe disability (e.g., will require a wheelchair).
  • EDSS scores in the range of 1-3 refer to an MS patient who is fully ambulatory, but has some signs in one or more functional systems; EDSS scores in the range higher than 3 to 4.5 show moderate to relatively severe disability; an EDSS score of 5 to 5.5 refers to a disability impairing or precluding full daily activities; EDSS scores of 6 to 6.5 refer to an MS patient requiring intermittent to constant, or unilateral to bilateral constant assistance (cane, crutch or brace) to walk; EDSS scores of 7 to 7.5 means that the MS patient is unable to walk beyond five meters even with aid, and is essentially restricted to a wheelchair; EDSS scores of 8 to 8.5 refer to patients that are restricted to bed; and EDSS scores of 9 to 10 mean that the MS patient is confined to bed, and progressively is unable to communicate effectively or eat and swallow, until death due to MS.
  • probe refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a transcription product, e.g. , an mRNA or cDNA, or a translation product, e.g., a polypeptide or protein. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes can be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic monomers.
  • prodrug refers to a compound that is processed, in the body of a subject, into a drug.
  • processing comprises the breaking or formation of a bond, e.g. , a covalent bond.
  • breakage of a covalent bond releases the drug.
  • tissue sample each refers to a biological sample obtained from a tissue, e.g. , a bodily fluid, of a subject or patient.
  • the source of the tissue sample can be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents (e.g., serum, plasma); bodily fluids such as cerebral spinal fluid, whole blood, plasma and serum.
  • the sample can include a non-cellular fraction (e.g., plasma, serum, or other non-cellular body fluid). In one embodiment, the sample is a serum sample.
  • the body fluid from which the sample is obtained from an individual comprises blood (e.g., whole blood).
  • the blood can be further processed to obtain plasma or serum.
  • the sample contains a tissue, cells (e.g., peripheral blood mononuclear cells (PBMC)).
  • PBMC peripheral blood mononuclear cells
  • the sample includes NK cells.
  • the sample can be a fine needle biopsy sample, an archival sample (e.g., an archived sample with a known diagnosis and/or treatment history), a histological section (e.g., a frozen or formalin-fixed section, e.g., after long term storage), among others.
  • sample includes any material obtained and/or derived from a biological sample, including a polypeptide, and nucleic acid (e.g., genomic DNA, cDNA, RNA) purified or processed from the sample. Purification and/or processing of the sample can involve one or more of extraction, concentration, antibody isolation, sorting, concentration, fixation, addition of reagents and the like.
  • the sample can contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like.
  • alkyl as employed herein by itself or as part of another group refers to both straight and branched chain radicals of up to 24 carbons.
  • Alkyl groups include straight-chained and branched Q-C 24 alkyl groups, e.g., Q-Qo alkyl groups.
  • Q-Qo alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, heptyl, 1-methylhexyl, 2- ethylhexyl, 1,4-dimethylpentyl, octyl, nonyl, and decyl. Unless otherwise indicated, all alkyl groups described herein include both unsubstituted and substituted alkyl groups. Further, each alkyl group can include its deuterated counterparts.
  • heteroalkyl is an alkyl group in which one to five carbons in the alkyl chain are replace by an independently selected oxygen, nitrogen or sulfur atom.
  • aryl as employed herein by itself or as part of another group refers to monocyclic, bicyclic, or tricyclic aromatic hydrocarbon containing from 5 to 50 carbons in the ring portion.
  • Aryl groups include Cs-is aryl, e.g., phenyl, p-tolyl,
  • arylalkyl refers to an alkyl group which is attached to another moiety through an alkyl group.
  • Halogen or “halo” may be fluoro, chloro, bromo or iodo.
  • cycloalkyl refers to completely saturated monocyclic, bicyclic or tricyclic hydrocarbon groups of 3- 12 carbon atoms, preferably 3-9, or more preferably 3-8 carbon atoms.
  • exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • Exemplary bicyclic cycloalkyl groups include bornyl, decahydronaphthyl, bicyclo[2.1. l]hexyl, bicyclo[2.2. l]heptyl, 6,6-dimethylbicyclo[3.1. l]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, or bicyclo[2.2.2]octyl.
  • Exemplary tricyclic carbocyclyl groups include adamantyl.
  • cycloalkylalkyl refers to a cycloalkyl group which is attached to another moiety through an alkyl group.
  • heterocycloalkyl refers to completely saturated monocyclic, bicyclic or tricyclic heterocyclyl comprising 3-15 ring members, at least one of which is a heteroatom, and up to 10 of which may be heteroatoms, wherein the heteroatoms are independently selected from O, S and N, and wherein N and S can be optionally oxidized to various oxidation states.
  • heterocycloalkyl groups include [l,3]dioxolane, 1,4-dioxane, 1,4-dithiane, piperazinyl, 1,3-dioxolane, imidazolidinyl, imidazolinyl, pyrrolidine, dihydropyran, oxathiolane, dithiolane, 1,3-dioxane, 1,3-dithianyl, oxathianyl, thiomorpholinyl, oxiranyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, and piperazinyl.
  • heteroaryl refers to a 5-14 membered monocyclic-, bicyclic-, or tricyclic-ring system, having 1 to 10 heteroatoms independently selected from N, O or S, wherein N and S can be optionally oxidized to various oxidation states, and wherein at least one ring in the ring system is aromatic.
  • the heteroaryl is monocyclic and has 5 or 6 ring members.
  • heteroaryl groups examples include pyridyl, thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl and tetrazolyl.
  • the heteroaryl is bicyclic and has from 8 to 10 ring members.
  • bicyclic heteroaryl groups include indolyl, benzofuranyl, quinolyl, isoquinolyl indazolyl, indolinyl, isoindolyl, indolizinyl, benzamidazolyl, quinolinyl, 5,6,7, 8-tetrahydroquinoline and 6,7-dihydro-5H-pyrrolo[3,2-d]pyrimidine.
  • heteroarylalkyl refers to an alkyl group which is attached to another moiety through an alkyl group.
  • MS Multiple sclerosis
  • Patients having MS can be identified by clinical criteria establishing a diagnosis of clinically definite MS as defined by Poser et al., Ann. Neurol. 13:227, 1983. Briefly, an individual with clinically definite MS has had two attacks and clinical evidence of either two lesions or clinical evidence of one lesion and paraclinical evidence of another, separate lesion. Definite MS may also be diagnosed by evidence of two attacks and oligoclonal bands of IgG in cerebrospinal fluid or by combination of an attack, clinical evidence of two lesions and oligoclonal band of IgG in cerebrospinal fluid. The McDonald criteria can also be used to diagnose MS.
  • the McDonald criteria include the use of MRI evidence of CNS impairment over time to be used in diagnosis of MS, in the absence of multiple clinical attacks. Effective treatment of multiple sclerosis may be evaluated in several different ways. The following parameters can be used to gauge effectiveness of treatment. Two exemplary criteria include: EDSS (extended disability status scale), and appearance of exacerbations on MRI (magnetic resonance imaging).
  • the EDSS is a means to grade clinical impairment due to MS (Kurtzke, Neurology 33: 1444, 1983). Eight functional systems are evaluated for the type and severity of neurologic impairment. Briefly, prior to treatment, patients are evaluated for impairment in the following systems: pyramidal, cerebella, brainstem, sensory, bowel and bladder, visual, cerebral, and other. Following-ups are conducted at defined intervals. The scale ranges from 0 (normal) to 10 (death due to MS). A decrease of one full step indicates an effective treatment (Kurtzke, Ann. Neurol.
  • Exacerbations are defined as the appearance of a new symptom that is attributable to MS and accompanied by an appropriate new neurologic abnormality (IFNB MS Study Group, supra). In addition, the exacerbation must last at least 24 hours and be preceded by stability or improvement for at least 30 days. Briefly, patients are given a standard neurological examination by clinicians. Exacerbations are mild, moderate, or severe according to changes in a
  • Treatment can be deemed to be effective using a clinical measure if there is a statistically significant difference in the rate or proportion of exacerbation-free or relapse-free patients between the treated group and the placebo group for either of these measurements.
  • time to first exacerbation and exacerbation duration and severity may also be measured.
  • a measure of effectiveness as therapy in this regard is a statistically significant difference in the time to first exacerbation or duration and severity in the treated group compared to control group.
  • An exacerbation-free or relapse-free period of greater than one year, 18 months, or 20 months is particularly noteworthy.
  • Clinical measurements include the relapse rate in one and two-year intervals, and a change in EDSS, including time to progression from baseline of 1.0 unit on the EDSS that persists for six months. On a Kaplan-Meier curve, a delay in sustained progression of disability shows efficacy. Other criteria include a change in area and volume of T2 images on MRI, and the number and volume of lesions determined by gadolinium enhanced images.
  • MRI can be used to measure active lesions using gadolinium-DTPA-enhanced imaging (McDonald et al., Ann. Neurol. 36: 14, 1994) or the location and extent of lesions using T2- weighted techniques. Briefly, baseline MRIs are obtained. The same imaging plane and patient position are used for each subsequent study. Positioning and imaging sequences can be chosen to maximize lesion detection and facilitate lesion tracing. The same positioning and imaging sequences can be used on subsequent studies. The presence, location and extent of MS lesions can be determined by radiologists. Areas of lesions can be outlined and summed slice by slice for total lesion area. Three analyses may be done: evidence of new lesions, rate of appearance of active lesions, percentage change in lesion area (Paty et al., Neurology 43:665, 1993).
  • Improvement due to therapy can be established by a statistically significant improvement in an individual patient compared to baseline or in a treated group versus a placebo group.
  • Exemplary symptoms associated with multiple sclerosis include: optic neuritis, diplopia, nystagmus, ocular dysmetria, internuclear opthalmoplegia, movement and sound phosphenes, afferent pupillary defect, paresis, monoparesis, paraparesis, hemiparesis, quadraparesis, plegia, paraplegia, hemiplegia, tetraplegia, quadraplegia, spasticity, dysarthria, muscle atrophy, spasms, cramps, hypotonia, clonus, myoclonus, myokymia, restless leg syndrome, footdrop, dysfunctional reflexes, paraesthesia, anaesthesia, neuralgia, neuropathic and neurogenic
  • MS relap sing-remitting MS
  • PPMS Primary-progressive MS
  • SPMS Secondary-progressive MS
  • PRMS progressive-relapsing
  • mice C57BL/6 mice that were administered vehicle, DMF or MMF (100 mg/kg). Treated mice were sacrificed at 2, 7, or 12 hours. Tissues (liver, spleen, kidney, jejunum, cortex, hippocampus,
  • mice striatum and whole blood were collected and analyzed by transcriptional profiling on mouse
  • Affymetrix GeneChips Differentially expressed genes were identified by comparing DMF or

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Abstract

L'invention concerne des procédés et des systèmes pour évaluer des promédicaments, des médicaments ou des métabolites de médicaments.
PCT/US2015/029993 2014-05-08 2015-05-08 Diméthylfumarate et promédicaments pour le traitement de la sclérose en plaques Ceased WO2015172083A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3697407A4 (fr) * 2017-10-16 2021-08-25 Dynamic Biologics Inc. Compositions polymères de fumarate de monométhyle dans le traitement de la sclérose en plaques récurrente-rémittente et du psoriasis
JP2023518205A (ja) * 2020-03-13 2023-04-28 ザ ユニバーシティー オブ アデレード 酸化ストレスと関連した障害の処置およびこの処置のための化合物

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3697407A4 (fr) * 2017-10-16 2021-08-25 Dynamic Biologics Inc. Compositions polymères de fumarate de monométhyle dans le traitement de la sclérose en plaques récurrente-rémittente et du psoriasis
EP4400170A3 (fr) * 2017-10-16 2024-09-04 Dynamic Biologics Inc. Compositions polymères de fumarate de monométhyle dans le traitement de la sclérose en plaques récurrente-rémittente et du psoriasis
JP2023518205A (ja) * 2020-03-13 2023-04-28 ザ ユニバーシティー オブ アデレード 酸化ストレスと関連した障害の処置およびこの処置のための化合物
EP4118066A4 (fr) * 2020-03-13 2023-09-06 The University of Adelaide Traitement de troubles associés au stress oxydatif et composés correspondants

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