Patent Application
20160115540
The invention relates, inter alia, to the use and activity of prodrugs and their drugs, e.g., dimethyl fumarate (DMF) and monomethyl fumarate (MMF), e.g., in the treatment of multiple sclerosis (MS) and other disorders.
The relationship between a drug and its metabolite and their contribution to overall pharmacologic effect is often poorly understood.
Tecfidera® (BG-12, dimethyl fumarate, DMF) is a methyl ester of fumaric acid. Tecfidera® 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.
Preclinical studies indicate that activation of the nuclear factor (erythroid-derived 2)-like 2(Nrf2) pathway is thought to be involved in the clinical effects of Tecfidera®. In vivo, DMF is rapidly metabolized to monomethyl fumarate (MMF), and both compounds are pharmacologically active. In vitro, DMF and MMF share some common effects, but also have divergent pharmacological properties.
Given the destructive effects of inflammatory MS lesions and the distinct effects of therapies such as DMF and MMF, the need exists for evaluating or monitoring a subject undergoing an MS therapy, or identifying a subject that would benefit from an MS therapy.
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). In certain embodiments, 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). 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. Without being bound by theory, the 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. Thus, 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. Many of the methods, devices, reaction mixtures and other inventions provided herein are described for use with DMF and its active metabolite MMF. However, it should be understood that 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).
Accordingly, in one aspect, 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:
b) responsive to said value, performing one, two or all of:
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.
In a related aspect, 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). The method includes:
administering to the subject, e.g., a subject in need of treatment (e.g., an MS treatment), a DMF;
acquiring from said subject 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:
In certain embodiments, 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).
In another related aspect, the invention features a method of treating a subject, e.g., a subject having, or at risk of having, MS. The method includes:
administering to the subject 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:
Additional embodiments or features of any of the above aspects are as follows:
In certain embodiments, 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.
In certain embodiments, 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).
In one embodiment, 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 another embodiment, 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.
In certain embodiments, the method includes acquiring a value for the expression of a plurality of genes. In certain embodiments, 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;
b) a plurality, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10, or more, MMF-differentially expressed genes;
c) a DMF-differentially expressed gene and an MMF-differentially expressed gene;
d) a DMF-differentially expressed gene and a gene that is both DMF-differentially expressed and MMF-differentially expressed; and
e) an MMF-differentially expressed gene and a gene that is both DMF-differentially expressed and MMF-differentially expressed.
In certain embodiments, 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).
In one embodiment, 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. In certain embodiments, the gene is selected from one or more of the genes in Table 1 or Table 9. In embodiments, the gene is a gene from Table 9 that shows differential expression as measured by mRNA levels. In one embodiment, 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). In one embodiment, the gene is chosen from one, two, three, four or all of: Granzyme A (Gzma), Natural cytotoxicity triggering receptor 1 (Ncr1), Killer cell lectin-like receptor subfamily C member 1 (Klrc1), Killer cell lectin-like receptor subfamily B member 1B (Klrb1b), or Killer cell lectin-like receptor family E member 1 (Klre1). In one embodiment, the gene is chosen from one, two, three or all of: Granzyme A (Gzma), Natural cytotoxicity triggering receptor 1 (Ncr1), Killer cell lectin-like receptor subfamily C member 1 (Klrc1), or Killer cell lectin-like receptor subfamily B member 1B (Klrb1b). In an embodiment, the gene is an NFkB activated gene, e.g., a gene chosen from one, two, three, or all of: Fc Fragment Of IgG, High Affinity Ia, Receptor (FCGR1A), Suppression Of Tumorigenicity 18 (ST18), Chemokine (C-C motif) ligand 3-like 1 (CCL3L1), or Vascular cell adhesion protein 1 (VCAM1). In an embodiment, the gene is an IL-2 activated gene, e.g., a gene chosen from one, two, three or all of: chemokine (C-C motif) receptor 3 (CCR3), Killer cell lectin-like receptor subfamily B member 1C (Klrb1c), Natural cytotoxicity triggering receptor 1 (Ncr1), or Chemokine (C-C motif) ligand 3-like 1 (CCL3L1). In an embodiment, the gene is decidual protein induced by progesterone (DEPP). In an embodiment, the gene is zinc finger and BTB domain containing 16 (Zbtb16), or an isoform thereof. In an embodiment, the gene is selected from 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In an embodiment, the method, e.g., method described herein, includes acquiring a value for the expression of FCGR1A. In an embodiment, the method, e.g., method described herein, includes acquiring a value for the expression of ST18. In an embodiment, the method, e.g., method described herein, includes acquiring a value for the expression of CCL3L1. In an embodiment, the method, e.g., method described herein, includes acquiring a value for the expression of VCAM1. In an embodiment, the method, e.g., method described herein, includes acquiring a value for the expression of, CCR3. In an embodiment, the method, e.g., method described herein, includes acquiring a value for the expression of Klrb1c. In an embodiment, the method, e.g., method described herein, includes acquiring a value for the expression of Ncr1. In an embodiment, the method, e.g., method described herein, includes acquiring a value for the expression of DEPP. In an embodiment, the method, e.g., method described herein, includes acquiring a value for the expression of Zbtb16.
In one embodiment, 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, Ncr1, Klrc1, Klrb1b, and Klre1. In one embodiment, 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, Ncr1, Klrc1, and Klrb1b. In an embodiment, 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, FCGR1A, ST18, CCL3L1, or VCAM1. In an embodiment, 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 CCR3, Klrb1c, Ncr1, or CCL3L1. In an embodiment, 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 DEPP. In an embodiment, 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 Zbtb16, or an isoform thereof. In an embodiment, In an embodiment, 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, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In other embodiments, 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. In certain embodiments, the gene is selected from one or more of the genes in Table 1 or Table 9. In embodiments, the gene is a gene from Table 9 that shows differential expression as measured by protein levels. In one embodiment, 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). In certain embodiments, the gene is chosen from one, two, three, or all of: Killer cell lectin-like receptor subfamily C member 1 (Klrc1), Killer cell lectin-like receptor subfamily B member 1B (Klrb1b), NKKG2d (Klrk1), or Natural killer cells (CD94) (Klrd1). In an embodiment, the gene is an NFkB activated gene, e.g., a gene chosen from one, two, three, or all of: Fc Fragment Of IgG, High Affinity Ia, Receptor (FCGR1A), Suppression Of Tumorigenicity 18 (ST18), Chemokine (C-C motif) ligand 3-like 1 (CCL3L1), or Vascular cell adhesion protein 1 (VCAM1). In an embodiment, the gene is an IL-2 activated gene, e.g., a gene chosen from one, two, three or all of: chemokine (C-C motif) receptor 3 (CCR3), Killer cell lectin-like receptor subfamily B member 1C (Klrb1c), Natural cytotoxicity triggering receptor 1 (Ncr1), or Chemokine (C-C motif) ligand 3-like 1 (CCL3L1). In an embodiment, the gene is decidual protein induced by progesterone (DEPP). In an embodiment, the gene is zinc finger and BTB domain containing 16 (Zbtb16), or an isoform thereof. In an embodiment, the gene is chosen from 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In one embodiment, 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, Klrc1, Klrb1b, Klrk1, and Klrd1. In an embodiment, 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 FCGR1A, ST18, CCL3L1, or VCAM1. In an embodiment, 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 CCR3, Klrb1c, Ncr1, or CCL3L1. In an embodiment, 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 DEPP. In an embodiment, 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 Zbtb16, or an isoform thereof. In an embodiment, 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, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In certain embodiments, 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.
In certain embodiments, the blood comprises, greater than background levels, e.g., therapeutic levels, of DMF, MMF, or both.
In certain embodiments, the value for expression of the gene is for a tissue, e.g., a tissue selected from cortical tissue, hippocampus, striatum, jejunum, kidney, liver, or spleen. In an embodiment, the value for expression of the gene is for spinal cord, brain, or combination thereof. In certain embodiments, the value for expression of the gene is for cerebrospinal fluid. In an embodiment, the value for expression of the gene is for lymph node, spleen, or combination thereof.
In certain embodiments, said gene is selected from the genes in Table 2, Table 3, Table 4, Table 5a, Table 5b, Table 6, Table 7, Table 8, Appendix A, Appendix B, Appendix C, Appendix D, or Appendix E.
In one embodiment, 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 tissue, e.g., whole tissue. In certain embodiments, the gene is selected from one or more of the genes in Table 1 or Table 9. In embodiments, the gene is a gene from Table 9 that shows differential expression as measured by mRNA levels. In one embodiment, 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). In one embodiment, the gene is chosen from one, two, three, four or all of: Granzyme A (Gzma), Natural cytotoxicity triggering receptor 1 (Ncr1), Killer cell lectin-like receptor subfamily C member 1 (Klrc1), Killer cell lectin-like receptor subfamily B member 1B (Klrb1b), or Killer cell lectin-like receptor family E member 1 (Klre1). In one embodiment, the gene is chosen from one, two, three or all of: Granzyme A (Gzma), Natural cytotoxicity triggering receptor 1 (Ncr1), Killer cell lectin-like receptor subfamily C member 1 (Klrc1), or Killer cell lectin-like receptor subfamily B member 1B (Klrb1b). In an embodiment, the gene is an NFkB activated gene, e.g., a gene chosen from one, two, three, or all of: Fc Fragment Of IgG, High Affinity Ia, Receptor (FCGR1A), Suppression Of Tumorigenicity 18 (ST18), Chemokine (C-C motif) ligand 3-like 1 (CCL3L1), or Vascular cell adhesion protein 1 (VCAM1). In an embodiment, the gene is an IL-2 activated gene, e.g., a gene chosen from one, two, three or all of: chemokine (C-C motif) receptor 3 (CCR3), Killer cell lectin-like receptor subfamily B member 1C (Klrb1c), Natural cytotoxicity triggering receptor 1 (Ncr1), or Chemokine (C-C motif) ligand 3-like 1 (CCL3L1). In an embodiment, the gene is decidual protein induced by progesterone (DEPP). In an embodiment, the gene is zinc finger and BTB domain containing 16 (Zbtb16), or an isoform thereof. In one embodiment, the gene is chosen from 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof
In one embodiment, 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 tissue, for 1, 2, 3, 4, or all of, Gzma, Ncr1, Klrc1, Klrb1b, and Klre1. In one embodiment, 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 tissue, for 1, 2, 3, or all of, Gzma, Ncr1, Klrc1, and Klrb1b. In an embodiment, 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 tissue, for 1, 2, 3, or all of, FCGR1A, ST18, CCL3L1, or VCAM1. In an embodiment, 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 tissue, for 1, 2, 3, or all of CCR3, Klrb1c, Ncr1, or CCL3L1. In an embodiment, 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 tissue, for DEPP. In an embodiment, 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 tissue, for Zbtb16, or an isoform thereof. In one embodiment, 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 tissue, for 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In other embodiments, 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 tissue, e.g., whole tissue. In certain embodiments, the gene is selected from one or more of the genes in Table 1 or Table 9. In embodiments, the gene is a gene from Table 9 that shows differential expression as measured by protein levels. In one embodiment, 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). In certain embodiments, the gene is chosen from one, two, three, or all of: Killer cell lectin-like receptor subfamily C member 1 (Klrc1), Killer cell lectin-like receptor subfamily B member 1B (Klrb1b), NKKG2d (Klrk1), or Natural killer cells (CD94) (Klrd1). In an embodiment, the gene is an NFkB activated gene, e.g., a gene chosen from one, two, three, or all of: Fc Fragment Of IgG, High Affinity Ia, Receptor (FCGR1A), Suppression Of Tumorigenicity 18 (ST18), Chemokine (C-C motif) ligand 3-like 1 (CCL3L1), or Vascular cell adhesion protein 1 (VCAM1). In an embodiment, the gene is an IL-2 activated gene, e.g., a gene chosen from one, two, three or all of: chemokine (C-C motif) receptor 3 (CCR3), Killer cell lectin-like receptor subfamily B member 1C (Klrb1c), Natural cytotoxicity triggering receptor 1 (Ncr1), or Chemokine (C-C motif) ligand 3-like 1 (CCL3L1). In an embodiment, the gene is decidual protein induced by progesterone (DEPP). In an embodiment, the gene is zinc finger and BTB domain containing 16 (Zbtb16), or an isoform thereof. In one embodiment, the gene is chosen from 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In one embodiment, 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 tissue, for 1, 2, 3, or all of, Klrc1, Klrb1b, Klrk1, and Klrd1. In an embodiment, 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 tissue, for 1, 2, 3 or all of FCGR1A, ST18, CCL3L1, or VCAM1. In an embodiment, 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 tissue, for 1, 2, 3, or all of CCR3, Klrb1c, Ncr1, or CCL3L1. In an embodiment, 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 tissue, for DEPP. In an embodiment, 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 tissue, for Zbtb16, or an isoform thereof. In an embodiment, 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 tissue, for 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In some embodiments, 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. In embodiments, the treatment is an MS treatment (e.g., a treatment that includes a DMF).
In certain embodiments, the subject has been administered the treatment, e.g., the DMF, e.g., prior to, at the time of, or after, acquiring the value. In one embodiment, 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).
In one embodiment, 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). For example, 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.
In certain embodiments, 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. In an embodiment, 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.
In certain embodiments, a tissue (e.g., cerebrospinal 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.
In certain embodiments, the sample is chosen from a non-cellular body fluid; or a cellular or tissue fraction. In one embodiment, the non-cellular fraction is chosen from blood, e.g., whole blood, plasma or serum. In other embodiments, the cellular fraction comprises one or more of: T cells, B cells or myeloid cells. For example, the cellular fraction can include one or more of: natural killer (NK) cells, peripheral blood mononuclear cells (PBMC), CD8+ T cells, or Regulatory T cells. In an embodiment, the sample is cerebrospinal fluid.
In certain embodiments, 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.
For any of the methods, devices or kits disclosed herein, 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. In certain embodiments, 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. In other embodiments, the subject has MS-like symptoms, such as those having clinically isolated syndrome (CIS) or clinically defined MS (CDMS). In one embodiment, 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). In another embodiment, the subject is an MS patient (e.g., a relapsing MS patient) after administration of an MS therapy described herein (e.g., a DMF). In other embodiments, 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.
In one embodiment, the subject has a relapsing form of MS, e.g., RRMS.
Alternatively, or in combination with the methods described herein, the invention features a method of treating a subject having one or more symptoms associated with MS. In one embodiment, the subject is identified as responding or not responding to a therapy, using the methods, devices, or kits described herein.
In an embodiment the method comprises treating the subject with DMF, MMF, or a combination thereof.
In certain embodiments, the treatment includes reducing, retarding or preventing, a relapse, or the worsening of a disability, in the MS patients.
In one embodiment, 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.
In embodiments where a first therapy (e.g., the DMF therapy) is not detectably effective, 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 1a molecule or an IFN-b 1b molecule, including analogues and derivatives thereof (e.g., pegylated variants thereof)). In one embodiment, the other MS therapy includes an IFN-b 1a agent (e.g., Avonex®, Rebif®). In another embodiment, the other MS therapy includes an INF-b 1b agent (e.g., Betaseron®, Betaferon®). In other embodiments, 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. In certain embodiments, the methods include the use of one or more symptom management therapies, such as antidepressants, analgesics, anti-tremor agents, among others.
In certain embodiments, 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.
In certain embodiments, a probe is a nucleic acid that specifically hybridizes with a transcription product of the gene or genes. In other embodiments, the detection includes amplification of a transcription product of the gene or genes. In other embodiments, the detection includes reverse transcription and amplification of a transcription product of the gene or genes.
In other embodiments, a translation product of the gene or genes, e.g., a polypeptide, is detected. The polypeptide can be detected, or the level determined, by any means of polypeptide detection, or detection of the expression level of the polypeptides. For example, the polypeptide can be detected using a probe or reagent which specifically binds with the polypeptides. In another embodiment, 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. In one embodiment, 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. Polypeptide detection methods can be performed in any other assay format, including but not limited to, ELISA, RIA, and mass spectrometry.
In certain embodiments, the probe is an antibody. In one embodiment, 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.
In certain embodiments, the methods described herein further include: performing a neurological examination, evaluating the subject's status on the Expanded Disability Status Scale (EDSS), or detecting the subject's lesion status as assessed using an MRI.
In another aspect, the invention features a device comprising:
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 gene selected independently from:
i) a dimethyl fumarate (DMF)-differentially expressed gene,
ii) a monomethyl fumarate (MMF)-differentially expressed gene, or
iii) a DMF/MMF-differentially expressed gene.
In one embodiment, 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.
In one embodiment, the probe or probes of the device are specific for a gene or genes selected from the genes in Table 9. In an embodiment, the probe or probes of the device are specific for a gene or genes in Appendix A, Appendix B, Appendix C, Appendix D or Appendix E.
In other embodiments, 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.
In yet other embodiments, the device includes a probe specific for a transcriptional product of 1, 2, 3, 4, or all of, Gzma, Ncr1, Klrc1, Klrb1b, and Klre1. In yet other embodiments, the device includes a probe specific for a transcriptional product of 1, 2, 3, or all of, Gzma, Ncr1, Klrc1, and Klrb1b. In yet other embodiments, the device includes a probe specific for a transcriptional product of 1, 2, 3, or all of, FCGR1A, ST18, CCL3L1, or VCAM1. In yet other embodiments, the device includes a probe specific for a transcriptional product of 1, 2, 3, or all of CCR3, Klrb1c, Ncr1, or CCL3L1. In yet other embodiments, the device includes a probe specific for a transcriptional product of DEPP. In yet other embodiments, the device includes a probe specific for a transcriptional product of Zbtb16, or an isoform thereof. In yet other embodiments, the device includes a probe specific for a transcriptional product of 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In other embodiments, 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.
In other embodiments, the device includes a probe specific for a translational product of 1, 2, 3, or all of, Klrc1, Klrb1b, Klrk1, and Klrd1. In other embodiments, the device includes a probe specific for a translational product of 1, 2, 3, or all of, FCGR1A, ST18, CCL3L1, or VCAM1. In yet other embodiments, the device includes a probe specific for a translational product of 1, 2, 3, or all of CCR3, Klrb1c, Ncr1, or CCL3L1. In yet other embodiments, the device includes a probe specific for a translational product of DEPP. In yet other embodiments, the device includes a probe specific for a translational product of Zbtb16, or an isoform thereof. In other embodiments, the device includes a probe specific for a translational product of 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof
In one embodiment, the device further comprises a sample, e.g., a sample as described herein. In one embodiment, 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 administered DMF. In yet other embodiments, 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.
In other embodiments, 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.
In yet other embodiments, 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 5a, Table 5b, Table 6, Table 7, Table 8, Appendix A, Appendix B, Appendix C, Appendix D, or Appendix E.
In other embodiments, the probe is a nucleic acid that specifically hybridizes with a transcription product of the gene or genes.
In embodiments, the device is configured to allow amplification of a transcription product of the gene or genes.
In other embodiments, the device is configured to allow reverse transcription and amplification of a transcription product of the gene or genes.
In other embodiments, 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.
In other embodiments, 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.
In yet other embodiments, 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,
ii) a monomethyl fumarate (MMF)-differentially expressed gene, or
iii) a DMF/MMF-differentially expressed gene.
In one embodiment, 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.
In yet other embodiments, 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.
In other embodiments, 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:
i) a dimethyl fumarate (DMF)-differentially expressed gene,
ii) a monomethyl fumarate (MMF)-differentially expressed gene, or
iii) a DMF/MMF-differentially expressed gene.
In other embodiments, 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.
In other embodiments, 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.
In certain embodiments, the probe or probes are disposed on a surface of the device.
In another aspect, the invention features a method of using a device described herein. The method includes:
providing a device described herein;
contacting the device with a sample described herein,
thereby using the device.
In one embodiment, the method includes a step of capturing a signal, e.g., an electronic, or visual signal, to evaluate the sample.
In an aspect, 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
one, or a plurality of, probes each probe being specific for a product, e.g., a translational product or transcriptional product, of a gene described herein,
wherein 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 the gene described herein.
In another aspect, the invention features, a reaction mixture comprising:
a sample; and
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 gene selected independently from:
i) a dimethyl fumarate (DMF)-differentially expressed gene,
ii) a monomethyl fumarate (MMF)-differentially expressed gene, or
iii) a DMF/MMF-differentially expressed gene.
In an embodiment 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.
In another embodiment, the probe or probes are specific for a gene or genes selected from the genes in Table 9.
In another embodiment, 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.
In one embodiment, the reaction mixture comprises probes specific for a transcriptional product of 1, 2, 3, 4, or all of, Gzma, Ncr1, Klrc1, Klrb1b, and Klre1. In one embodiment, the reaction mixture comprises probes specific for a transcriptional product of 1, 2, 3, or all of, Gzma, Ncr1, Klrc1, and Klrb1b. In one embodiment, the reaction mixture comprises probes specific for a transcriptional product of 1, 2, 3, or all of, FCGR1A, ST18, CCL3L1, or VCAM1. In one embodiment, the reaction mixture comprises probes specific for a transcriptional product of 1, 2, 3, or all of CCR3, Klrb1c, Ncr1, or CCL3L1. In one embodiment, the reaction mixture comprises probes specific for a transcriptional product of DEPP. In one embodiment, the reaction mixture comprises probes specific for a transcriptional product of Zbtb16, or an isoform thereof. In one embodiment, the reaction mixture comprises probes specific for a transcriptional product of 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In another embodiment, 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.
In other embodiments, the reaction mixture comprises probes specific for a translational product of 1, 2, 3, or all of, Klrc1, Klrb1b, Klrk1, and Klrd1. In one embodiment, the reaction mixture comprises probes specific for a translational product of 1, 2, 3, or all of, FCGR1A, ST18, CCL3L1, or VCAM1. In one embodiment, the reaction mixture comprises probes specific for a translational product of 1, 2, 3, or all of CCR3, Klrb1c, Ncr1, or CCL3L1. In one embodiment, the reaction mixture comprises probes specific for a translational product of DEPP. In one embodiment, the reaction mixture comprises probes specific for a translational product of Zbtb16, or an isoform thereof. In one embodiment, the reaction mixture comprises probes specific for a translational product of 1, 2, 3, 4, 5, 6, 7, 8 or all of FCGR1A, ST18, CCL3L1, VCAM1, CCR3, Klrb1c, Ncr1, DEPP, or Zbtb16, or an isoform thereof.
In an embodiment, said sample is from a subject having an autoimmune disorder, e.g., MS, e.g., relapsing MS.
In one embodiment, said sample is from a subject that has been administered DMF.
In an embodiment, 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.
In an embodiment, said sample comprises blood, or a substance derived from blood, e.g., serum, or an NK-cell containing fraction.
In other embodiments, 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, Appendix A, Appendix B, Appendix C, Appendix D, or Appendix E.
In an embodiment, a probe is a nucleic acid that specifically hybridizes with a transcription product of the gene or genes.
In an embodiment, the reaction mixture further comprises reagents to allow for amplification of a transcription product of the gene or genes.
In an embodiment, the reaction mixture further comprises reagents to allow for reverse transcription and amplification of a transcription product of the gene or genes.
In an embodiment, 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.
In an embodiment, 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.
In other embodiments, 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
i) a dimethyl fumarate (DMF)-differentially expressed gene,
ii) a monomethyl fumarate (MMF)-differentially expressed gene, or
iii) a DMF/MMF-differentially expressed gene.
In other embodiments, 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.
In other embodiments, 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.
In other embodiments, 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:
i) a dimethyl fumarate (DMF)-differentially expressed gene,
ii) a monomethyl fumarate (MMF)-differentially expressed gene, or
iii) a DMF/MMF-differentially expressed gene.
In other embodiments, 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.
In other embodiments of the reaction mixture 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.
In another aspect, the invention features a method of making a reaction mixture comprising:
providing the a sample described herein;
contacting the sample with one or a plurality of probes described herein, or with a device described herein,
thereby making a reaction mixture.
In embodiments, the method of making includes capturing a signal, e.g., an electronic, or visual signal, to evaluate the sample.
In another aspect, the invention features 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 kit includes a means for detection of (e.g., a reagent that specifically detects) one or more genes as described herein. In certain embodiments, the kit includes an MS therapy. In one another embodiment, the kit comprises an antibody, an antibody derivative, or an antibody fragment to a protein produce of the gene. In one embodiment, the kit includes an antibody-based detection technique, such as immunofluorescence cell sorting (FACS), immunohistochemistry, antigen retrieval and/or microarray detection reagents. In one embodiment, at least one of the reagents in the kit is an antibody that binds to a gene product (optionally) with a detectable label (e.g., a fluorescent or a radioactive label). In certain embodiments, 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. In one embodiment, 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.
In a related aspect, 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:
administering the prodrug to the subject;
acquiring from said subject 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:
In certain embodiments, the method further comprises comparing the value with a reference value.
In an embodiment the drug is DMF and the metabolite is MMF
In an embodiment the drug metabolite is MMF and the drug or prodrug is a compound of Formula I:
or a pharmaceutically acceptable salt thereof, wherein
In certain embodiments of a compound of Formula (I), each substituent group is independently chosen from halogen, —OH, —CN, —CF3, —R11a, —OR11a, and —NR11a2 wherein each R11a is independently chosen from hydrogen and C1-4 alkyl. In certain embodiments, each substituent group is independently chosen from —OH, and —COOH.
In certain embodiments of a compound of Formula (I), each substituent group is independently chosen from ═O, C1-4 alkyl, and —COOR11a wherein R11a is chosen from hydrogen and C1-4 alkyl.
In certain embodiments of a compound of Formula (I), each of R1a and R2a is hydrogen.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is C1-4 alkyl.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is methyl.
In certain embodiments of a compound of Formula (I), R3a and R4a are independently chosen from hydrogen and C1-6 alkyl.
In certain embodiments of a compound of Formula (I), R3a and R4a are independently chosen from hydrogen and C1-4 alkyl.
In certain embodiments of a compound of Formula (I), R3a and R4a are independently chosen from hydrogen, methyl, and ethyl.
In certain embodiments of a compound of Formula (I), each of R3a and R4a is hydrogen; in certain embodiments, each of R3a and R4a is methyl; and in certain embodiments, each of R3a and R4a is ethyl.
In certain embodiments of a compound of Formula (I), R3a is hydrogen; and R4a is chosen from C1-4 alkyl, substituted C1-4 alkyl wherein the substituent group is chosen from ═O, —OR11a, —COOR11a, and —NR11a2, wherein each R11a is independently chosen form hydrogen and C1-4 alkyl.
In certain embodiments of a compound of Formula (I), R3a is hydrogen; and R4a is chosen from C1-4 alkyl, benzyl, 2-methoxyethyl, carboxymethyl, carboxypropyl, 1,2,4-thiadoxolyl, methoxy, 2-methoxycarbonyl, 2-oxo(1,3-oxazolidinyl), 2-(methylethoxy)ethyl, 2-ethoxyethyl, (tert-butyloxycarbonyl)methyl, (ethoxycarbonyl)methyl, carboxymethyl, (methylethyl)oxycarbonylmethyl, and ethoxycarbonylmethyl.
In certain embodiments of a compound of Formula (I), R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from a C5-6 heterocycloalkyl, substituted C5-6 heterocycloalkyl, C5-6 heteroaryl, and substituted C5-6 heteroaryl ring. In certain embodiments of a compound of Formula (I), R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from a C5 heterocycloalkyl, substituted C5 heterocycloalkyl, C5 heteroaryl, and substituted C5 heteroaryl ring. In certain embodiments of a compound of Formula (I), R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from a C6 heterocycloalkyl, substituted C6 heterocycloalkyl, C6 heteroaryl, and substituted C6 heteroaryl ring. In certain embodiments of a compound of Formula (I), R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from piperazine, 1,3-oxazolidinyl, pyrrolidine, and morpholine ring.
In certain embodiments of a compound of Formula (I), R3a and R4a together with the nitrogen to which they are bonded form a C5-10 heterocycloalkyl ring.
In certain embodiments of a compound of Formula (I), R5a is methyl.
In certain embodiments of a compound of Formula (I), R5a is ethyl.
In certain embodiments of a compound of Formula (I), R5a is C3-6 alkyl.
In certain embodiments of a compound of Formula (I), R5a is chosen from methyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.
In certain embodiments of a compound of Formula (I), R5a is chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is C1-6 alkyl; R3a is hydrogen; R4a is chosen from hydrogen, C1-6 alkyl, and benzyl.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is C1-6 alkyl; R3a is hydrogen; R4a is chosen from hydrogen, C1-6 alkyl, and benzyl; and R5a is methyl.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is chosen from hydrogen and C1-6 alkyl; and each of R3a and R4a is C1-6 alkyl.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is chosen from hydrogen and C1-6 alkyl; each of R3a and R4a is C1-6 alkyl; and R5a is methyl. In certain embodiments of a compound of Formula (I), each of R1a and R2a is hydrogen; each of R3a and R4a is C1-6 alkyl; and R5a is methyl.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is chosen from hydrogen and C1-4 alkyl; R3a is hydrogen; R4a is chosen from C1-4 alkyl, substituted C1-4 alkyl wherein the substituent group is chosen from ═O, —OR11a, —COOR11a, and —NR11a2, wherein each R11a is independently chosen form hydrogen and C1-4 alkyl; and R5a is methyl. In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is methyl; R3a is hydrogen; R4a is chosen from C1-4 alkyl, substituted C1-4 alkyl wherein the substituent group is chosen from ═O, —OR11a, —COOR11a, and
—NR11a2, wherein each R11a is independently chosen form hydrogen and C1-4 alkyl; and R5a is methyl. In certain embodiments of a compound of Formula (I), each of R1a and R2a is hydrogen; R3a is hydrogen; R4a is chosen from C1-4 alkyl, substituted C1-4 alkyl wherein the substituent group is chosen from ═O, —OR11a, —COOR11a, and —NR11a2, wherein each R11a is independently chosen form hydrogen and C1-4 alkyl; and R5a is methyl.
In certain embodiments of a compound of Formula (I), R3a and R4a together with the nitrogen to which they are bonded form a C5-10 heterocycloalkyl ring.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is chosen from hydrogen and C1-6 alkyl; R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from a C5-6 heterocycloalkyl, substituted C5-6 heterocycloalkyl, C5-6 heteroaryl, and substituted C5-6 heteroaryl ring; and R5a is methyl. In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is methyl; R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from a C5-6 heterocycloalkyl, substituted C5-6 heterocycloalkyl, C5-6 heteroaryl, and substituted C5-6 heteroaryl ring; and R5a is methyl. In certain embodiments of a compound of Formula (I), each of R1a and R2a is hydrogen; R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from a C5-6 heterocycloalkyl, substituted C5-6 heterocycloalkyl, C5-6 heteroaryl, and substituted C5-6 heteroaryl ring; and R5a is methyl.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is chosen from hydrogen and C1-6 alkyl; and R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from morpholine, piperazine, and N-substituted piperazine.
In certain embodiments of a compound of Formula (I), one of R1a and R2a is hydrogen and the other of R1a and R2a is chosen from hydrogen and C1-6 alkyl; R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from morpholine, piperazine, and N-substituted piperazine; and R5a is methyl.
In certain embodiments of a compound of Formula (I), R5a is not methyl.
In certain embodiments of a compound of Formula (I), R1a is hydrogen, and in certain embodiments, R2a is hydrogen.
In certain embodiments of a compound of Formula (I), 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-1,4-dioate; (N-butylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-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-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic acid; methyl(N-(1,3,4-thiadiazol-2-yl)carbamoyl)methyl(2E)but-2ene-1,4-dioate; (N,N-dimethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate; (N-methoxy-N-methylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate; bis-(2-methoxyethylamino)carbamoyl]methyl methyl(2E)but-2-ene-1,4-dioate; [N-(methoxycarbonyl)carbamoyl]methyl methyl(2E)but-2ene-1,4-dioate; 4-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic acid, sodium salt; methyl 2-oxo-2-piperazinylethyl(2E)but-2-ene-1,4-dioate; methyl 2-oxo-2-(2-oxo(1,3-oxazolidin-3-yl)ethyl(2E)but-2ene-1,4-dioate; {N-[2-(dimethylamino)ethyl]carbamoy}methyl methyl(2E)but-2ene-1,4 dioate; methyl 2-(4-methylpiperazinyl)-2-oxoethyl(2E)but-2-ene-1,4-dioate; methyl {N-[(propylamino)carbonyl]carbamoyl}methyl(2E)but-2ene-1,4-dioate; 2-(4-acetylpiperazinyl)-2-oxoethyl methyl(2E)but-2ene-1,4-dioate; {N,N-bis[2-(methylethoxy)ethyl]carbamoy}methyl methyl(2E)but-2-ene-1,4-dioate; methyl 2-(4-benzylpiperazinyl)-2-oxoethyl(2E)but-2-ene-1,4-dioate; [N,N-bis(2-ethoxyethyl)carbamoyl]methyl methyl(2E)but-2-ene-1,4-dioate; 2-{(2S)-2-[(tert-butyl)oxycarbonyl]pyrrolidinyl}-2-oxoethyl methyl(2E)but-2ene-1,4-dioate; 1-{2-{(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetyl}(2S)pyrrolidine-2-carboxylic acid; (N-{[tert-butyl)oxycarbonyl]methyl}-N-methylcarbamoyl)methyl methyl(2E)but-2ene1,4-dioate; {N-(ethoxycarbonyl)methyl]-N-methylcarbamoyl}methyl methyl(2E)but-2-ene-1,4-dioate; methyl 1-methyl-2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-1,4-dioate; [N,N-bis(2-methoxyethyl)carbamoyl]ethyl methyl(2E)but-2-ene-1,4-dioate; (N,N-dimethylcarbamoyl)ethyl methyl(2E)but-2-ene-1,4-dioate; 2-{2-[(2E)-3-(methoxy carbonyl)prop-2-enoyloxyl]-N-methylacetylamin}acetic acid; (N-{[(tert-butyl)oxycarbonyl]methyl}carbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate; (2E)but-methyl-N-{[(methylethyl)oxycarbonyl]methyl}carbamoyl)methyl(2E)but-2-ene-1,4-dioate; {N-[(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}methyl methyl(2E)but-2-ene-1,4-dioate; {N-[(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}ethyl methyl(2E)but-2-ene-1,4-dioate; {N-[(ethoxycarbonyl)methyl]-N-methylcarbamoyl}ethyl methyl(2E)but-2-ene-1,4-dioate; (1S)-1-methyl-2-morpholin-4-yl-2-oxo ethyl methyl(2E)but-2-ene-1,4-dioate; (1S)-1-[N,N-bis(2-methoxyethyl)carbamoyl]ethyl methyl(2E)but-2-ene-1,4-dioate; (1R)-1-(N,N-diethylcarbamoyl)ethyl methyl(2E)but-2-ene-1,4-dioate; and a pharmaceutically acceptable salt of any of the foregoing.
In certain embodiments of a compound of Formula (I), 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-1,4-dioate; (N-butylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-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; {2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic acid; methyl(N-(1,3,4-thiadiazol-2-yl)carbamoyl)methyl(2E)but-2ene-1,4-dioate; (N,N-dimethylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate; (N-methoxy-N-methylcarbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate; bis-(2-methoxyethylamino)carbamoyl]methyl methyl(2E)but-2-ene-1,4-dioate; [N-(methoxycarbonyl)carbamoyl]methyl methyl(2E)but-2ene-1,4-dioate; methyl 2-oxo-2-piperazinylethyl(2E)but-2-ene-1,4-dioate; methyl 2-oxo-2-(2-oxo(1,3-oxazolidin-3-yl)ethyl(2E)but-2ene-1,4-dioate; {N-[2-(dimethylamino)ethyl]carbamoyl}methyl methyl(2E)but-2ene-1,4-dioate; (N-[(methoxycarbonyl)ethyl]carbamoyl)methyl methyl(2E)but-2-ene-1,4-dioate; 2-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}propanoic acid; and a pharmaceutically acceptable salt of any of the foregoing.
In certain embodiments of a compound of Formula (I), R3a and R4a are independently chosen from hydrogen, C1-6 alkyl, substituted C1-6 alkyl, C6-10 aryl, substituted C6-10 aryl, C4-12 cycloalkylalkyl, substituted C4-12 cycloalkylalkyl, C7-12 arylalkyl, substituted C7-12 arylalkyl, C1-6 heteroalkyl, substituted C1-6 heteroalkyl, C6-10 heteroaryl, substituted C6-10 heteroaryl, C4-12 heterocycloalkylalkyl, substituted C4-12 heterocycloalkylalkyl, C7-12 heteroarylalkyl, substituted C7-12 heteroarylalkyl; or R3a and R4a together with the nitrogen to which they are bonded form a ring chosen from a C5-10 heteroaryl, substituted C5-10 heteroaryl, C5-10 heterocycloalkyl, and substituted C5-10 heterocycloalkyl.
In some embodiments, the compound that metabolizes to MMF is a compound of Formula II:
or a pharmaceutically acceptable salt thereof, wherein
In certain embodiments of a compound of Formula (II), each substituent group is independently chosen from halogen, —OH, —CN, —CF3, —R11b, —OR11b, and —NR11b2 wherein each R11b is independently chosen from hydrogen and C1-4 alkyl.
In certain embodiments of a compound of Formula (I), each substituent group is independently chosen from ═O, C1-4 alkyl, and —COOR11b wherein R11b is chosen from hydrogen and C1-4 alkyl.
In certain embodiments of a compound of Formula (II), one of R7b and R8b is hydrogen and the other of R7b and R8b is C1-6 alkyl. In certain embodiments of a compound of Formula (II), one of R7b and R8b is hydrogen and the other of R7b and R8b is C1-4 alkyl.
In certain embodiments of a compound of Formula (II), one of R7b and R8b is hydrogen and the other of R7b and R8b is chosen from methyl, ethyl, n-propyl, and isopropyl. In certain embodiments of a compound of Formula (II), each of R7b and R8b is hydrogen.
In certain embodiments of a compound of Formula (II), R9b is chosen from substituted C1-6 alkyl and —OR11b wherein R11b is independently C1-4 alkyl.
In certain embodiments of a compound of Formula (II), R9b is C1-6 alkyl, in certain embodiments, R9b is C1-3 alkyl; and in certain embodiments, R9b is chosen from methyl and ethyl.
In certain embodiments of a compound of Formula (II), R9b is methyl.
In certain embodiments of a compound of Formula (II), R9b is chosen from ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.
In certain embodiments of a compound of Formula (II), R9b is chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.
In certain embodiments of a compound of Formula (II), R6b is C1-6 alkyl; one of R7b and R8b is hydrogen and the other of R7b and R8b is C1-6 alkyl; and R9b is chosen from C1-6 alkyl and substituted C1-6 alkyl.
In certain embodiments of a compound of Formula (II), R6b is —OR10b,
In certain embodiments of a compound of Formula (II), R10b is chosen from C1-4 alkyl, cyclohexyl, and phenyl.
In certain embodiments of a compound of Formula (II), R6b is chosen from methyl, ethyl, n-propyl, and isopropyl; one of R7b and R8b is hydrogen and the other of R7b and R8b is chosen from methyl, ethyl, n-propyl, and isopropyl.
In certain embodiments of a compound of Formula (II), R6b is substituted C1-2 alkyl, wherein each of the one or more substituent groups are chosen from —COOH,
—NHC(O)CH2NH2, and —NH2.
In certain embodiments of a compound of Formula (II), R6b is chosen from ethoxy, methylethoxy, isopropyl, phenyl, cyclohexyl, cyclohexyloxy,
—CH(NH2CH2COOH, —CH2CH(NH2)COOH, —CH(NHC(O)CH2NH2)—CH2COOH, and —CH2CH(NHC(O)CH2NH2)—COOH.
In certain embodiments of a compound of Formula (II), R9b is chosen from methyl and ethyl; one of R7b and R8b is hydrogen and the other of R7b and R8b is chosen from hydrogen, methyl, ethyl, n-propyl, and isopropyl; and R6b is chosen from C1-3 alkyl, substituted C1-2 alkyl wherein each of the one or more substituent groups are chosen —COOH, —NHC(O)CH2NH2, and —NH2, —OR10b wherein R10b is chosen from C1-3 alkyl and cyclohexyl, phenyl, and cyclohexyl.
In certain embodiments of a compound of Formula (II), the compound is chosen from: ethoxycarbonyloxyethyl methyl(2E)but-2-ene-1,4-dioate; methyl(methylethoxycarbonyloxy)ethyl(2E)but-2-ene-1,4-dioate; (cyclohexyloxycarbonyloxy)ethyl methyl(2E)but-2-ene-1,4-dioate; and a pharmaceutically acceptable salt of any of the foregoing.
In certain embodiments of a compound of Formula (II), the compound is chosen from: methyl(2-methylpropanoyloxy)ethyl(2E)but-2-ene-1,4-dioate; methyl phenylcarbonyloxyethyl(2E)but-2-ene-1,4-dioate; cyclohexylcarbonyloxybutyl methyl(2E)but-2-ene-1,4-dioate; [(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]ethyl methyl(2E)but-2-ene-1,4-dioate; methyl 2-methyl-1-phenylcarbonyloxypropyl(2E)but-2-ene-1,4-dioate; and a pharmaceutically acceptable salt of any of the foregoing.
In certain embodiments of a compound of Formula (II), the compound is chosen from: ethoxycarbonyloxyethyl methyl(2E)but-2-ene-1,4-dioate; methyl(methylethoxycarbonyloxy)ethyl(2E)but-2-ene-1,4-dioate; methyl(2-methylpropanoyloxy)ethyl(2E)but-2-ene-1,4-dioate; methyl phenylcarbonyloxyethyl(2E)but-2-ene-1,4-dioate; cyclohexylcarbonyloxybutyl methyl(2E)but-2-ene-1,4-dioate; [(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]ethyl methyl(2E)but-2-ene-1,4-dioate; (cyclohexyloxycarbonyloxy)ethyl methyl(2E)but-2-ene-1,4-dioate; methyl 2-methyl-1-phenylcarbonyloxypropyl(2E)but-2-ene-1,4-dioate; 3-({[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]methyl}oxycarbonyl)(3S)-3-aminopropanoic acid, 2,2,2-trifluoroacetic acid; 3-({[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]methyl}oxycarbonyl)(2S)-2-aminopropanoic acid, 2,2,2-trifluoroacetic acid; 3-({[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]methyl}oxycarbonyl)(3S)-3-(2-aminoacetylamino)propanoic acid, 2,2,2-trifluoroacetic acid; 3-({[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]methyl}oxycarbonyl)(2S)-2-aminopropanoic acid, 2,2,2-trifluoroacetic acid; 3-{[(2E)-3-(methoxycarbonyl)prop-2enoyloxy]ethoxycarbonyloxy}(2S)-2-aminopropanoic acid, chloride; and a pharmaceutically acceptable salt of any of the foregoing.
The compounds of Formulae (I)-(II) may be prepared using methods known to those skilled in the art, or the methods disclosed in U.S. Pat. No. 8,148,414 B2.
In another embodiment is provided silicon-containing compounds, which like DMF and the compounds of Formulae (I)-(II), can metabolize into MMF upon administration.
In some embodiments, the compound that metabolizes to MMF is a compound of Formula (III):
or a pharmaceutically acceptable salt thereof, wherein:
Another group of compounds of Formula III include compounds wherein R1c is optionally substituted C1-C24 alkyl. Another group of compounds of Formula III include compounds wherein R1c is optionally substituted C1-C6 alkyl. Another group of compounds of Formula III include compounds wherein R1c is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula III include compounds wherein R1c is optionally substituted C5-C50 aryl. Another group of compounds of Formula III include compounds wherein R1c is optionally substituted C5-C10 aryl. Another group of compounds of Formula III include compounds wherein R2c is C1-C10 alkyl. Another group of compounds of Formula III include compounds wherein R2c is optionally substituted C1-C6 alkyl. Another group of compounds of Formula III include compounds wherein R2c is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula III include compounds wherein R2c is optionally substituted C5-C15 aryl. Another group of compounds of Formula III include compounds wherein R2c is optionally substituted C5-C10 aryl.
In a further embodiment, the compound that metabolizes to MMF is a compound of Formula (III):
or a pharmaceutically acceptable salt thereof, wherein
In some embodiments, the compound that metabolizes to MMF is chosen from (dimethylsilanediyl)dimethyl difumarate; methyl ((trimethoxysilyl)methyl)fumarate; methyl ((trihydroxysilyl)methyl)fumarate; trimethyl (methylsilanetriyl)trifumarate; and a pharmaceutically acceptable salt of any of the foregoing.
In some embodiments, the compound that metabolizes to MMF is a compound of Formula (IV):
or a pharmaceutically acceptable salt thereof, wherein:
In another embodiment, compounds of Formula IV include compounds wherein each R1d is independently optionally substituted C1-C24 alkyl or C6-C10 aryl. In another embodiment, compounds of Formula IV include compounds wherein R1d is optionally substituted C1-C24 alkyl. Another group of compounds of Formula IV include compounds wherein R1d is optionally substituted C1-C6 alkyl. Another group of compounds of Formula IV include compounds wherein R1d is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula IV include compounds wherein R1d is optionally substituted C5-C50 aryl. Another group of compounds of Formula IV include compounds wherein R1d is optionally substituted C5-C10 aryl. Another group of compounds of Formula IV include compounds wherein each of R2d and R3d is, independently, optionally substituted C1-C10 alkyl. Another group of compounds of Formula IV include compounds wherein each of R2d and R3d is, independently, optionally substituted C1-C6 alkyl. Another group of compounds of Formula IV include compounds wherein each of R2d and R3d is, independently, optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula IV include compounds wherein each of R2d and R3d is, independently, optionally substituted C5-C15 aryl. Another group of compounds of Formula IV include compounds wherein each of R2d and R3d is, independently, optionally substituted C5-C10 aryl.
In a further embodiment, the compound that metabolizes to MMF is a compound of Formula (IV):
or a pharmaceutically acceptable salt thereof, wherein:
In some embodiments, the compound that metabolizes to MMF is a compound of Formula (V):
or a pharmaceutically acceptable salt thereof, wherein:
R1e is C1-C24 alkyl or C5-C50 aryl;
In another embodiment, compounds of Formula V include compounds wherein R1e is optionally substituted C1-C24 alkyl. Another group of compounds of Formula V include compounds wherein R1e is optionally substituted C1-C6 alkyl. Another group of compounds of Formula V include compounds wherein R1e is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula V include compounds wherein R1e is optionally substituted C5-C50 aryl. Another group of compounds of Formula V include compounds wherein R1e is optionally substituted C5-C10 aryl. Another group of compounds of Formula V include compounds wherein each of R2e, R3e, and R5e is, independently, hydroxyl. Another group of compounds of Formula V include compounds wherein each of R2e, R3e, and R5e is, independently, optionally substituted C1-C10 alkyl. Another group of compounds of Formula V include compounds wherein each of R2e, R3e, and R5e is, independently, optionally substituted C1-C6 alkyl. Another group of compounds of Formula V include compounds wherein each of R2e, R3e, and R5e is, independently, optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula V include compounds wherein each of R2e, R3e, and R5e is, independently, optionally substituted C5-C15 aryl. Another group of compounds of Formula V include compounds wherein each of R2e, R3e, and R5e is, independently, optionally substituted C5-C10 aryl.
In a further embodiment, the compound that metabolizes to MMF is a compound of Formula (V):
or a pharmaceutically acceptable salt thereof, wherein:
R1e is C1-C24 alkyl or C6-C10 aryl;
In some embodiments, the compound that metabolizes to MMF is a compound of Formula (VI):
or a pharmaceutically acceptable salt thereof, wherein:
R2f is C1-C10 alkyl.
In another embodiment, compounds of Formula VI include compounds wherein R1f is optionally substituted C1-C24 alkyl. Another group of compounds of Formula VI include compounds wherein R1f is optionally substituted C1-C6 alkyl. Another group of compounds of Formula VI include compounds wherein R1f is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula VI include compounds wherein R1f is optionally substituted C5-C50 aryl. Another group of compounds of Formula VI include compounds wherein R1f is optionally substituted C5-C10 aryl. Another group of compounds of Formula VI include compounds wherein R2f is optionally substituted C1-C6 alkyl. Another group of compounds of Formula VI include compounds wherein R2f is optionally substituted methyl, ethyl, or isopropyl.
In a further embodiment, the compound that metabolizes to MMF is a compound of Formula (VI):
or a pharmaceutically acceptable salt thereof, wherein:
R1f is C1-C24 alkyl or C6-C10 aryl; and
R2f is C1-C10 alkyl.
In another aspect, the invention features, a method of treating a subject having a natural killer (NK) function related disorder or condition comprising: administering to the subject in need of treatment a dialkyl fumarate in an amount sufficient to treat the disorder,
wherein the disorder or condition is selected from:
cancer, a viral infection, and inflammation.
In an embodiment, the dialkyl fumarate is:
wherein R1g and R2g, which may be the same or different, independently represent a linear, branched or cyclic, saturated or unsaturated C1-20 alkyl radical which may be optionally substituted with halogen (Cl, F, I, Br), hydroxy, C1-4 alkoxy, nitro or cyano.
In an embodiment, R1g and R2g, 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.
In an embodiment, R1g and R2g are identical and are methyl or ethyl.
In an embodiment, R1g and R2g are methyl.
In an embodiment, the compound is a monoalkyl fumarate. In an embodiment, the monoalkyl fumarate is:
wherein R1h represents a linear, branched or cyclic, saturated or unsaturated C1-20 alkyl radical which may be optionally substituted with halogen (Cl, F, I, Br), hydroxy, C1-4 alkoxy, nitro or cyano;
or a pharmaceutically acceptable salt thereof.
In an embodiment, R1h 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.
In an embodiment, R1h is methyl or ethyl.
In an embodiment, R1h is methyl.
In a further embodiment, the compound that metabolizes to MMF is a compound of Formula (VII):
wherein:
R1i is unsubstituted C1-C6 alkyl;
La is substituted or unsubstituted C1-C6 alkyl linker, substituted or unsubstituted C3-C10 carbocycle, substituted or unsubstituted C6-C10 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
R2i and R3i are each, independently, H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C3-C10 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, R2i and R3i, 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 unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S.
In some embodiments, the compound of Formula (VII) is selected from a compound of Formula (VIIa):
wherein:
R1i is unsubstituted C1-C6 alkyl;
La is substituted or unsubstituted C1-C6 alkyl linker, substituted or unsubstituted C3-C10 carbocycle, substituted or unsubstituted C6-C10 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
R2i is H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C3-C10 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.
In some embodiments, the compound of Formula (VII) is selected from a compound of Formula (VIIb):
A− is a pharmaceutically acceptable anion;
R1i is unsubstituted C1-C6 alkyl;
La is substituted or unsubstituted C1-C6 alkyl linker, substituted or unsubstituted C3-C10 carbocycle, substituted or unsubstituted C6-C10 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;
R3i′ is substituted or unsubstituted C1-C6 alkyl; and
R2i and R3i are each, independently, H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C3-C10 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, R2i and R3i, 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 unsubstituted heterocycle comprising one or two 5- or 6-member rings and 1-4 heteroatoms selected from N, O and S.
In some embodiments, the compound of Formula (VII) is selected from a compound of Formula (VIII):
wherein:
R1i is unsubstituted C1-C6 alkyl;
R4i and R5i are each, independently, H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C3-C10 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;
R6i, R7i, R8i and R9i are each, independently, H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl or C(O)ORa; and
Ra is H or substituted or unsubstituted C1-C6 alkyl.
In some embodiments, the compound of Formula (VII) is selected from a compound of Formula (IX):
wherein:
R1i is unsubstituted C1-C6 alkyl;
is selected from the group consisting of:
m is 0, 1, 2, or 3;
n is 1 or 2;
w is 0, 1, 2 or 3;
t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
R6i, R7i, R8i and R9i are each, independently, H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl or C(O)ORa; and
Ra is H or substituted or unsubstituted C1-C6 alkyl; and
each R10i is, independently, H, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C3-C10 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, two R10i's attached to the same carbon atom, together with the carbon atom to which they are attached, form a carbonyl, substituted or unsubstituted C3-C10 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, two R10i's attached to different atoms, together with the atoms to which they are attached, form a substituted or unsubstituted C3-C10 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.
In some embodiments, the compound is a compound listed in Table A herein.
Representative compounds of the present invention include compounds listed in Table A.
In some embodiments, the compound of Formula (VII) is the compound (X):
or a pharmaceutically acceptable salt thereof.
In an embodiment, the disorder or condition is cancer.
In an embodiment, the disorder or condition is a hematological malignancy.
In an embodiment, the hematological malignancy is selected from lymphocytic leukemia, chronic lymphocytic leukemia, and lymphoma.
In an embodiment, the disorder or condition is a solid tumor.
In an embodiment, the solid tumor is selected from gastrointestinal sarcoma, neuroblastoma, and kidney cancer.
In an embodiment, the disorder or condition is a viral infection.
In another aspect, 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.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
The invention is based, at least in part, on the discovery that the prodrug DMF makes a contribution to pharmacologic effect that is distinct from that of its metabolite, MMF.
As used herein, 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.
“Acquire” or “acquiring” as the terms are used herein, refer to obtaining possession of a physical entity, or a value, e.g., a numerical value, by “directly acquiring” or “indirectly acquiring” the physical entity or value. “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 analysis”), performing 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.
“Acquiring a sample” as the term is used herein, 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. 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, as the term is used herein, 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. By way of example, 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, as the term is used herein, 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. By way of example, 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. In an embodiment 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, as the term is used herein, 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. By way of example, 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. In an embodiment 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, as the term is used herein, is a gene that is differentially expressed for both DMF and MMF.
A drug-differentially expressed gene, as the term is used herein, 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. By way of example, 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, as the term is used herein, is a gene that is differentially expressed for both prodrug and drug.
As used herein, the “Expanded Disability Status Scale” or “EDSS” is intended to have its customary meaning in the medical practice. 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). Typically 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). More specifically, 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.
As used herein, the term “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.
As used herein, the term prodrug, or pro-drug, refers to a compound that is processed, in the body of a subject, into a drug. In an embodiment the processing comprises the breaking or formation of a bond, e.g., a covalent bond. Typically, breakage of a covalent bond releases the drug.
“Sample,” “tissue sample,” “subject or patient sample,” “subject or patient cell or tissue sample” or “specimen” 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. In other embodiments, the body fluid from which the sample is obtained from an individual comprises blood (e.g., whole blood). In certain embodiments, the blood can be further processed to obtain plasma or serum. In another embodiment, the sample contains a tissue, cells (e.g., peripheral blood mononuclear cells (PBMC)). In an embodiment the sample includes NK cells. For example, 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. The term 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.
The term “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 C1-C24 alkyl groups, e.g., C1-C10 alkyl groups. C1-C10 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.
The term “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.
The term “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 C5-15 aryl, e.g., phenyl, p-tolyl, 4-methoxyphenyl, 4-(tert-butoxy)phenyl, 3-methyl-4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 3-nitrophenyl, 3-aminophenyl, 3-acetamidophenyl, 4-acetamidophenyl, 2-methyl-3-acetamidophenyl, 2-methyl-3-aminophenyl, 3-methyl-4-aminophenyl, 2-amino-3-methylphenyl, 2,4-dimethyl-3-aminophenyl, 4-hydroxyphenyl, 3-methyl-4-hydroxyphenyl, 1-naphthyl, 3-amino-naphthyl, 2-methyl-3-amino-naphthyl, 6-amino-2-naphthyl, 4,6-dimethoxy-2-naphthyl, indanyl, biphenyl, phenanthryl, anthryl, and acenaphthyl. Unless otherwise indicated, all aryl groups described herein include both unsubstituted and substituted aryl groups.
The term “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.
The term “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[2.1.1]hexyl, bicyclo[2.2.1]heptyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, or bicyclo[2.2.2]octyl. Exemplary tricyclic carbocyclyl groups include adamantyl.
The term “cycloalkylalkyl” refers to a cycloalkyl group which is attached to another moiety through an alkyl group.
The term “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. Examples of heterocycloalkyl groups include [1,3]dioxolane, 1,4-dioxane, 1,4-dithiane, piperazinyl, 1,3-dioxolane, imidazolidinyl, imidazolinyl, pyrrolidine, dihydropyran, oxathiolane, dithiolane, I,3-dioxane, 1,3-dithianyl, oxathianyl, thiomorpholinyl, oxiranyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, and piperazinyl.
As used herein, the term “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. In one embodiment, the heteroaryl is monocyclic and has 5 or 6 ring members. Examples of monocyclic heteroaryl groups include pyridyl, thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl and tetrazolyl. In another embodiment, the heteroaryl is bicyclic and has from 8 to 10 ring members. Examples of 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.
The term “heteroarylalkyl” refers to an alkyl group which is attached to another moiety through an alkyl group.
Multiple sclerosis (MS) is a central nervous system disease that is characterized by inflammation and loss of myelin sheaths.
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. (McDonald et al., 2001, Recommended diagnostic criteria for Multiple sclerosis: guidelines from the International Panel on the Diagnosis of Multiple Sclerosis, Ann Neurol 50:121-127). 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. Follow-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. 36:573-79, 1994), while an increase of one full step will indicate the progression or worsening of disease (e.g., exacerbation). Typically 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).
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 Neurological Rating Scale (Sipe et al., Neurology 34:1368, 1984). An annual exacerbation rate and proportion of exacerbation-free patients are determined.
Therapy 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. In addition, 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, which can be treated with the methods described herein or managed using symptom management therapies, 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 pain, l'hermitte's, proprioceptive dysfunction, trigeminal neuralgia, ataxia, intention tremor, dysmetria, vestibular ataxia, vertigo, speech ataxia, dystonia, dysdiadochokinesia, frequent micturation, bladder spasticity, flaccid bladder, detrusor-sphincter dyssynergia, erectile dysfunction, anorgasmy, frigidity, constipation, fecal urgency, fecal incontinence, depression, cognitive dysfunction, dementia, mood swings, emotional lability, euphoria, bipolar syndrome, anxiety, aphasia, dysphasia, fatigue, Uhthoff's symptom, gastroesophageal reflux, and sleeping disorders.
Each case of MS displays one of several patterns of presentation and subsequent course. Most commonly, MS first manifests itself as a series of attacks followed by complete or partial remissions as symptoms mysteriously lessen, only to return later after a period of stability. This is called relapsing-remitting MS (RRMS). Primary-progressive MS (PPMS) is characterized by a gradual clinical decline with no distinct remissions, although there may be temporary plateaus or minor relief from symptoms. Secondary-progressive MS (SPMS) begins with a relapsing-remitting course followed by a later primary-progressive course. Rarely, patients may have a progressive-relapsing (PRMS) course in which the disease takes a progressive path punctuated by acute attacks. PPMS, SPMS, and PRMS are sometimes lumped together and called chronic progressive MS.
A few patients experience malignant MS, defined as a swift and relentless decline resulting in significant disability or even death shortly after disease onset. This decline may be arrested or decelerated by determining the likelihood of the patient to respond to a therapy early in the therapeutic regime and switching the patient to an agent that they have the highest likelihood of responding to.
As described in the Examples, transcriptional profiling of mouse genes was performed on 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, striatum and whole blood) were collected and analyzed by transcriptional profiling on mouse Affymetrix GeneChips. Differentially expressed genes were identified by comparing DMF or MMF treated mice to matched vehicle controls and exemplary genes that were identified are provided in TABLES 1-8 below. While the experiments were performed in mice, the human homolog of the identified murine gene transcript is included in the tables. Additional genes that were identified in the study are provided in APPENDIX A, filed herewith on even date, the contents of which are herein incorporated by reference in their entirety. Additional studies are described in the Examples, and genes identified in the studies are provided in APPENDIX B, APPENDIX C, APPENDIX D, and APPENDIX E filed herewith on even date, the contents of each of which are herein incorporated by reference in their entirety.
Probes and Methods for Detection of Translation Products
Probe-based methods, include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, liquid chromatography mass spectrometry (LC-MS), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), flow cytometry, laser scanning cytometry, hematology analyzer and assays based on a property of the protein including but not limited to DNA binding, ligand binding, or interaction with other protein partners.
The translation product or polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These can include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, immunohistochemistry and the like. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining the expression level of one or more biomarkers in a serum sample.
A useful probe for detecting a polypeptide is an antibody capable of binding to the polypeptide, e.g., an antibody with a detectable label. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
An antibody probe can be labeled, e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody. In another embodiment, an antibody derivative (e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair {e.g., biotin-streptavidin}), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically with a protein corresponding to the marker, such as the protein encoded by the open reading frame corresponding to the marker or such a protein which has undergone all or a portion of its normal post-translational modification, is used.
Immunohistochemistry or IHC refers to the process of localizing antigens (e.g. proteins), e.g., in cells of a tissue section or other sample, exploiting the principle of antibodies binding specifically to antigens in biological tissues. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyze a color-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein, rhodamine, DyLight Fluor or Alexa Fluor.
Proteins from cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
In one format, antibodies, or antibody fragments, can be used as probes in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, one can immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
One skilled in the art will know other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means. Means of detecting proteins using electrophoretic techniques are well known to those of skill in the art (see generally, R. Scopes (1982) Protein Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc., N.Y.).
In another embodiment, Western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide in the sample. This technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind a polypeptide. The anti-polypeptide antibodies specifically bind to the polypeptide on the solid support. These antibodies can be directly labeled or alternatively can be subsequently detected using labeled antibodies (e.g., labeled sheep anti-human antibodies) that specifically bind to the anti-polypeptide.
In another embodiment, the polypeptide is detected using an immunoassay. As used herein, an immunoassay is an assay that utilizes an antibody to specifically bind to the analyte. The immunoassay is thus characterized by detection of specific binding of a polypeptide to an anti-antibody as opposed to the use of other physical or chemical properties to isolate, target, and quantify the analyte.
The polypeptide is detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York; Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.
In another embodiment, the polypeptide is detected and/or quantified using Luminex™ assay technology. The Luminex™ assay separates tiny color-coded beads into e.g., distinct sets that are each coated with a reagent for a particular bioassay, allowing the capture and detection of specific analytes from a sample in a multiplex manner. The Luminex™ assay technology can be compared to a multiplex ELISA assay using bead-based fluorescence cytometry to detect analytes such as biomarkers.
Immunological binding assays (or immunoassays) typically utilize a “capture agent” to specifically bind to and often immobilize the analyte (polypeptide or subsequence). The capture agent is a moiety that specifically binds to the analyte. In another embodiment, the capture agent is an antibody that specifically binds a polypeptide. The antibody (anti-peptide) can be produced by any of a number of means well known to those of skill in the art.
Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent can itself be one of the moieties comprising the antibody/analyte complex. Thus, the labeling agent can be a labeled polypeptide or a labeled anti-antibody. Alternatively, the labeling agent can be a third moiety, such as another antibody, that specifically binds to the antibody/polypeptide complex.
In one embodiment, the labeling agent is a second human antibody bearing a label. Alternatively, the second antibody can lack a label, but it can, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, e.g., as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G can also be used as the label agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, and Akerstrom (1985) J. Immunol., 135: 2589-2542).
As indicated above, immunoassays for the detection and/or quantification of a polypeptide can take a wide variety of formats well known to those of skill in the art.
Exemplary immunoassays for detecting a polypeptide can be competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte is directly measured. In one “sandwich” assay, for example, the capture agent (anti-peptide antibodies) can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture polypeptide present in the test sample. The polypeptide thus immobilized is then bound by a labeling agent, such as a second human antibody bearing a label.
In competitive assays, the amount of analyte (polypeptide) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte (polypeptide) displaced (or competed away) from a capture agent (anti-peptide antibody) by the analyte present in the sample. In one competitive assay, a known amount of, in this case, a polypeptide is added to the sample and the sample is then contacted with a capture agent. The amount of polypeptide bound to the antibody is inversely proportional to the concentration of polypeptide present in the sample.
In another embodiment, the antibody is immobilized on a solid substrate. The amount of polypeptide bound to the antibody can be determined either by measuring the amount of polypeptide present in a polypeptide/antibody complex, or alternatively by measuring the amount of remaining uncomplexed polypeptide. The amount of polypeptide can be detected by providing a labeled polypeptide.
The assays described herein are scored (as positive or negative or quantity of polypeptide) according to standard methods well known to those of skill in the art. The particular method of scoring will depend on the assay format and choice of label. For example, a Western Blot assay can be scored by visualizing the colored product produced by the enzymatic label. A clearly visible colored band or spot at the correct molecular weight is scored as a positive result, while the absence of a clearly visible spot or band is scored as a negative. The intensity of the band or spot can provide a quantitative measure of polypeptide.
In another embodiment, level (activity) is assayed by measuring the enzymatic activity of the gene product. Methods of assaying the activity of an enzyme are well known to those of skill in the art.
In vivo techniques for detection of a marker protein include introducing into a subject a labeled antibody directed against the protein. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
Certain markers identified by the methods of the invention can be secreted proteins. It is a simple matter for the skilled artisan to determine whether any particular marker protein is a secreted protein. In order to make this determination, the marker protein is expressed in, for example, a mammalian cell, e.g., a human cell line, extracellular fluid is collected, and the presence or absence of the protein in the extracellular fluid is assessed (e.g., using a labeled antibody which binds specifically with the protein).
Antibodies can be used a probes for translation products. The terms “antibody” and “antibody substance” as used interchangeably herein refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention. A molecule which specifically binds to a given polypeptide is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. Probes can be polyclonal or monoclonal antibodies. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope.
An antibody directed against a polypeptide can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the marker (e.g., in a cellular lysate or cell supernatant) in order to evaluate the level and pattern of expression of the marker. The antibodies can also be used diagnostically to monitor protein levels in tissues or body fluids (e.g., in a tumor cell-containing body fluid) as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include, but are not limited to, streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes, but is not limited to, luminol; examples of bioluminescent materials include, but are not limited to, luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include, but are not limited to, 125I, 131I, 35S or 3H.
Probes and Methods for Detection of Transcription Products
Translational expression can be assessed by any of a wide variety of well known methods for detecting expression. Non-limiting examples of such methods include nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.
In certain embodiments, activity of a particular gene is characterized by a measure of gene transcript (e.g., mRNA). Detection can involve quantification of the level of gene expression (e.g., cDNA, mRNA), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.
Methods of detecting and/or quantifying the gene transcript (mRNA or cDNA made therefrom) using nucleic acid hybridization techniques are known to those of skill in the art (see e.g., Sambrook et al. supra). For example, one method for evaluating the presence, absence, or quantity of cDNA involves a Southern transfer as described above. Briefly, the mRNA is isolated (e.g., using an acid guanidinium-phenol-chloroform extraction method, Sambrook et al. supra.) and reverse transcribed to produce cDNA. The cDNA is then optionally digested and run on a gel in buffer and transferred to membranes. Hybridization is then carried out using the nucleic acid probes specific for the target cDNA.
A general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that can contain a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.
For example, one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.
There are many established methods for anchoring assay components to a solid phase. These include, without limitation, marker or probe molecules which are immobilized through conjugation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the surfaces with immobilized assay components can be prepared in advance and stored.
Other suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the marker or probe belongs. Well-known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
In order to conduct assays with the above-mentioned approaches, the non-immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components can be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of marker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.
In another embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.
It is also possible to directly detect marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule can simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label can be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).
In another embodiment, determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C., 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surface plasmon resonance” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.
Alternatively, in another embodiment, analogous diagnostic and prognostic assays can be conducted with marker and probe as solutes in a liquid phase. In such an assay, the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, marker/probe complexes can be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A. P., 1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographic techniques can also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex can be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the marker/probe complex as compared to the uncomplexed components can be exploited to differentiate the complex from uncomplexed components, for example, through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter 11(1-6):141-8; Hage, D. S., and Tweed, S. A. J Chromatogr B Biomed Sci Appl 1997 Oct. 10; 699(1-2):499-525). Gel electrophoresis can also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non-denaturing gel matrix materials and conditions in the absence of reducing agent are typical. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.
In a particular embodiment, the level of mRNA corresponding to the marker can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term “biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).
The isolated nucleic acid can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a marker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.
In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.
The probes can be full length or less than the full length of the nucleic acid sequence encoding the protein. Shorter probes are empirically tested for specificity. Exemplary nucleic acid probes are 20 bases or longer in length (See, e.g., Sambrook et al. for methods of selecting nucleic acid probe sequences for use in nucleic acid hybridization). Visualization of the hybridized portions allows the qualitative determination of the presence or absence of cDNA.
An alternative method for determining the level of a transcript involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. Fluorogenic rtPCR can also be used in the methods of the invention. In fluorogenic rtPCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
For in situ methods, mRNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker.
As an alternative to making determinations based on the absolute expression level of the marker, determinations can be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a subject sample, to another sample, e.g., a healthy subject, or between samples from different sources.
Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a marker, the level of expression of the marker is determined for 10 or more samples of normal versus MS isolates, or even 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker. The expression level of the marker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level.
In certain embodiments, the samples used in the baseline determination will be from samples derived from a subject having multiple sclerosis versus samples from a healthy subject of the same tissue type. The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the marker assayed is specific to the tissue from which the cell was derived (versus normal cells). In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from normal cells provides a means for grading the severity of the multiple sclerosis disease state.
In another embodiment, expression of a marker is assessed by preparing mRNA/cDNA (i.e., a transcribed polynucleotide) from cells in a subject sample, and by hybridizing the genomic DNA or mRNA/cDNA with a reference polynucleotide which is a complement of a polynucleotide comprising the marker, and fragments thereof. cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide. Expression of one or more markers can likewise be detected using quantitative PCR (QPCR) to assess the level of expression of the marker(s). Alternatively, any of the many known methods of detecting mutations or variants (e.g., single nucleotide polymorphisms, deletions, etc.) of a marker of the invention can be used to detect occurrence of a mutated marker in a subject.
In a related embodiment, a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g., at least 7, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 500, or more nucleotide residues) of a marker of the invention. If polynucleotides complementary to or homologous with a marker of the invention are differentially detectable on the substrate (e.g., detectable using different chromophores or fluorophores, or fixed to different selected positions), then the levels of expression of a plurality of markers can be assessed simultaneously using a single substrate (e.g., a “gene chip” microarray of polynucleotides fixed at selected positions). When a method of assessing marker expression is used which involves hybridization of one nucleic acid with another, the hybridization can be performed under stringent hybridization conditions.
In another embodiment, a combination of methods to assess the expression of a marker is utilized.
Because the compositions, kits, and methods of the invention rely on detection of a difference in expression levels of one or more markers of the invention, in certain embodiments the level of expression of the marker is significantly greater than the minimum detection limit of the method used to assess expression in at least one of a biological sample from a subject with MS or a healthy control.
A nucleic acid molecule of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecules so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts (e.g., mRNA) or genomic sequences corresponding to one or more markers of the invention. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
The methods described herein can also include molecular beacon nucleic acid molecules having at least one region which is complementary to a nucleic acid molecule of the invention, such that the molecular beacon is useful for quantitating the presence of the nucleic acid molecule of the invention in a sample. A “molecular beacon” nucleic acid is a nucleic acid molecule comprising a pair of complementary regions and having a fluorophore and a fluorescent quencher associated therewith. The fluorophore and quencher are associated with different portions of the nucleic acid in such an orientation that when the complementary regions are annealed with one another, fluorescence of the fluorophore is quenched by the quencher. When the complementary regions of the nucleic acid molecules are not annealed with one another, fluorescence of the fluorophore is quenched to a lesser degree. Molecular beacon nucleic acid molecules are described, for example, in U.S. Pat. No. 5,876,930.
A kit is any manufacture (e.g., a package or container) comprising at least one reagent, e.g., a probe, e.g., a nucleic acid probe or an antibody, for specifically detecting a translation or transcription product described herein.
The invention also encompasses kits having probes for detecting the presence of a polypeptide or nucleic acid in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow. For example, the kit can comprise a labeled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). Kits can also include instructions for interpreting the results obtained using the kit.
A kit can include a plurality of probes for detecting a plurality of translation or transcription products. If a plurality of expression products are to be analysed the kit can comprise a probe for each.
The kit can comprise one or more probes capable of identifying one or more of gene products described herein, e.g., gene products identified herein (e.g., the markers set forth in Table 9). Suitable probes for a polypeptide include antibodies, antibody derivatives, antibody fragments, and the like. Suitable probes for a transcription product include a nucleic acid, e.g., complementary nucleic acids. For example, a kit can include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like.
The kit of the invention can optionally comprise additional components useful for performing the methods of the invention. By way of example, the kit can comprise fluids (e.g., SSC buffer) suitable for annealing complementary nucleic acids or for binding an antibody with a protein with which it specifically binds, one or more sample compartments, an instructional material which describes performance of a method of the invention, a reference sample for comparison of expression levels of the biomarkers described herein, and the like.
A kit can include a device described herein.
For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable label.
For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker of the invention. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
regions.
There are several medications presently used to modify the course of multiple sclerosis. Such agents include, but are not limited to, dialkyl fumarates (e.g., DMF or others of Formula A herein), Beta interferons (e.g., Avonex®, Rebif®, Betaseron®, Betaferon®, among others)), glatiramer (Copaxone®), natalizumab (Tysabri®), and mitoxantrone (Novantrone®).
“Treat,” “treatment,” and other forms of this word refer to the administration of an agent, e.g., an agent described herein, alone or in combination with one or more symptom management agents, to a subject, e.g., an MS patient, to impede progression of multiple sclerosis, to induce remission, to extend the expected survival time of the subject and or reduce the need for medical interventions (e.g., hospitalizations). In those subjects, treatment can include, but is not limited to, inhibiting or reducing one or more symptoms such as numbness, tingling, muscle weakness; reducing relapse rate, reducing size or number of sclerotic lesions; inhibiting or retarding the development of new lesions; prolonging survival, or prolonging progression-free survival, and/or enhanced quality of life.
As used herein, unless otherwise specified, the terms “prevent,” “preventing” and “prevention” contemplate an action that occurs before a subject begins to suffer from the a multiple sclerosis relapse and/or which inhibits or reduces the severity of the disease.
As used herein, and unless otherwise specified, the terms “manage,” “managing” and “management” encompass preventing the progression of MS symptoms in a patient who has already suffered from the disease, and/or lengthening the time that a patient who has suffered from MS remains in remission. The terms encompass modulating the threshold, development and/or duration of MS, or changing the way that a patient responds to the disease.
As used herein, and unless otherwise specified, a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of multiple sclerosis, or to delay or minimize one or more symptoms associated with MS. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of MS. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease, or enhances the therapeutic efficacy of another therapeutic agent.
As used herein, and unless otherwise specified, a “prophylactically effective amount” of a compound is an amount sufficient to prevent relapse of MS, or one or more symptoms associated with the disease, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of the compound, alone or in combination with other therapeutic agents, which provides a prophylactic benefit in the prevention of MS relapse. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
As used herein, the term “patient” or “subject” refers to an animal, typically a human (i.e., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult) or other mammal, such as a primate (e.g., cynomolgus monkey, rhesus monkey); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys, that will be or has been the object of treatment, observation, and/or experiment. When the term is used in conjunction with administration of a compound or drug, then the patient has been the object of treatment, observation, and/or administration of the compound or drug.
The methods described herein permit one of skill in the art to identify a monotherapy that an MS patient is most likely to respond to, thus eliminating the need for administration of multiple therapies to the patient to ensure that a therapeutic effect is observed. However, in one embodiment, combination treatment of an individual with MS is contemplated.
It will be appreciated that the MS therapies, as described above and herein, can be administered in combination with one or more additional therapies to treat and/or reduce the symptoms of MS described herein, particularly to treat patients with moderate to severe disability (e.g., EDSS score of 5.5 or higher). The pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutic agent utilized in this combination can be administered together in a single composition or administered separately in different compositions. The particular combination to employ in a regimen will take into account compatibility of the pharmaceutical composition with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved. In general, it is expected that additional therapeutic agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
In other embodiments, alternative therapies to the DMF can be administered.
In one embodiment, the alternative therapy includes an interferon beta, 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®)). In other embodiments, the alternative therapy includes an antibody or fragment thereof against alpha-4 integrin (e.g., natalizumab (Tysabri®)). In yet other embodiments, the alternative therapy includes an anthracenedione molecule (e.g., mitoxantrone (Novantrone®)). In yet another embodiment, the alternative therapy includes a fingolimod (e.g., FTY720; Gilenya®). In other embodiments, the alternative therapy is an antibody to the alpha subunit of the IL-2 receptor of T cells (e.g., Daclizumab; described in, e.g., Rose, J. W. et al. (2007) Neurology 69 (8): 785-789). In yet other embodiments, the alternative therapy is an antibody against CD52 (e.g., alemtuzumab (Lemtrada®)). In yet another embodiment, the alternative therapy includes an anti-LINGO-1 antibody (described in, e.g., U.S. Pat. No. 8,058,406, entitled “Composition comprising antibodies to LINGO or fragments thereof.”).
Steroids, e.g., corticosteroid, and ACTH agents can be used to treat acute relapses in relapsing-remitting MS or secondary progressive MS. Such agents include, but are not limited to, Depo-Medrol®, Solu-Medrol®, Deltasone®, Delta-Cortef®, Medrol®, Decadron®, and Acthar®.
Dialkyl fumarates, e.g., those of Formula A, can be used to treat NK function related disorders and conditions. While not wishing to be bound by theory it is believed that these disorders are ameliorated by NK cells. Such disorders include: cancer, e.g., hematopoietic malignancies, e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, and lymphoma; solid tumors, e.g., gastrointestinal sarcoma, neuroblastoma, and kidney cancer; viral infection; autoimmune disorders; and inflammation. Such conditions also include transplantation, e.g., solid organ transplantation, and GVHD.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, figures, sequence listing, patents and published patent applications cited throughout this application are hereby incorporated by reference.
Tecfidera (BG-12, dimethyl fumarate, DMF) is an oral therapeutic approved in the U.S., Canada and Australia for the treatment of relapsing multiple sclerosis (MS). The mechanism by which Tecfidera exerts clinical effects is unknown, but preclinical studies indicate activation of the nuclear factor (erythroid-derived 2)-like 2(Nrf2) pathway is involved. Preclinical studies indicate that DMF promotes anti-inflammatory and neuroprotective responses, both of which may be useful in amelioration of MS pathophysioliogy. In vivo, DMF is rapidly metabolized to monomethyl fumarate (MMF), and both compounds are pharmacologically active.
In vitro, DMF and MMF share some common effects, but also have divergent pharmacological properties. To understand if in vitro differences translate into differential in vivo biology, DMF and MMF pharmacodynamic responses were characterized and compared in mice. This example describes the discovery and evaluation of differential transcriptional responses in multiple tissues and whole blood after oral dosing of DMF or MMF.
C57BL/6 mice were dosed with vehicle, DMF or MMF (100 mg/kg) and sacrificed at 2, 7, and 12 hours. Tissues (liver, spleen, kidney, jejunum, cortex, hippocampus, striatum and whole blood) were collected and analyzed by transcriptional profiling on mouse Affymetrix GeneChips. Differentially expressed genes were identified by comparing DMF or MMF treated mice to matched vehicle controls.
A separate cohort was sacrificed 30 minutes after dosing to evaluate MMF exposure. More specifically, 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. These analyses demonstrate that in mice receiving DMF or MMF, no significant differences in MMF exposure was observed, and this was consistent across tissues. As shown in
A specific transcriptional response to DMF and MMF treatment was observed in all tissues and whole blood. The overall number and identity of differentially regulated transcripts varied between tissues and treatment (
The incomplete overlap of transcriptional signatures induced by DMF and MMF indicates that not all DMF pharmacodynamic effects are conveyed through MMF, as may have been predicted due to the rapid in vivo metabolism of DMF to MMF. This suggests DMF may directly drive unique pharmacology not captured by MMF alone. Characterizing the potential biological consequences of these responses, such as effects on NK cells, and how they may contribute to the therapeutic benefit derived from oral administration of DMF, will be further investigated.
Transcriptional differences in NK cell markers were confirmed at the protein level by flow cytometry.
Among other things, these data confirm and expand the findings described in Example 1. For example, flow cytometry analysis confirmed transcriptional data identifying a number of DMF specific transcriptional changes related to NK cell function in blood. Furthermore, the data demonstrate that DMF exerts effects on NK cells in the spleen that were not observed with MMF.
In vivo, DMF is rapidly metabolized to monomethyl fumarate (MMF). The field has long sought to define the relative contributions of DMF and MMF to the therapeutic benefit derived from BG-12. Although only MMF can be detected in systemic circulation following an oral dose of DMF, clinically and preclinically, DMF conjugates have been detected in urine indicating that some DMF survives first pass metabolism. Additionally, in patients receiving BG-12, there is likely exposure to DMF in the intestines once the enteric coated microtablets dissolve and the active pharmaceutical ingredient is released. Understanding the direct effects of DMF and MMF in vitro and in vivo would provide important insights into the mechanisms of action of BG-12. This example demonstrates transcriptional profiling of pharmacodynamic effects of oral administration of DMF and MMF in single or multi-dose regiments in naïve mice.
The number of independent mice whose tissues were harvested for transcript profiling studies and whose data passed QC is shown in TABLES 10 and 11.
Animals were exposed to CO2 and whole blood collected via cardiac puncture. Two 100 μl aliquots of whole blood were collected in 1.5 mL microcentrifuge tubes and snap frozen in liquid nitrogen. Peripheral (liver, spleen, kidney and jejunum) and CNS brain (cerebellum, hippocampus, striatum and cortex) tissues were harvested and snap frozen in liquid nitrogen, with special care taken to collect tissues of similar size and from the same location. All samples were stored at −80° C. until RNA extraction was conducted.
For RNA preparation, frozen tissues were placed in 2 mL RNAse-free 96-well blocks with 1.5 mL QIAzol Lysis Reagent (QIAgen) and a 3.2 mm stainless steel bead (BioSpec Products, Bartlesville, Okla.). Tissues were disrupted for four cycles of 45 seconds in a Mini-Beadbeater (Biospec Products). RNA was extracted in chloroform and the aqueous phase was mixed with an equal volume of 70% ethanol. Extracted RNA was applied to RNeasy 96 plates and purified by the spin method according to the manufacturer's protocol (RNeasy 96 Universal Tissue Protocol, QIAgen, Hilden Germany).
Blood RNA Extraction and QC (Allaire N E, et al. BMC Res Notes. 2013 Jan. 5; 6:8)
50 ul of snap frozen mouse blood was re-suspended in lysis buffer with proteinase K and arrayed into deep-well plates for automated RNA extraction. RNA extractions were completed on Arrayplex (Beckman Coulter, Indianapolis, Ind.) using Agencourt RNAdvance Blood kit (Part number A35604) according to the manufacturer's specifications. RNA integrity was assessed using the HT RNA reagent kit (Part number 760410, Caliper Life Sciences, Hopkinton, Mass.) using a LabChip GX (PerkinElmer, Waltham, Mass.). RNA samples with a RQS score of >8.0 were considered high quality for downstream microarray processing.
Automated sample amplifications and biotin labelings were carried out using the NuGEN Ovation RNA Amplification system V2 (Cat #3100), Ovation WB reagent (Cat #1300) and Encore Biotin module (Cat #4200) (NuGEN Technologies, Inc, San Carlos, Calif.) according to manufacturer's recommendations using an Arrayplex automated liquid handler (Beckman Coulter, Indianapolis, Ind.). Two micrograms of biotin labeled sscDNA probe were hybridized to Affymetrix GeneChip Affymetrix HT_MG-430_PM plate arrays with modified conditions as described in Allaire et al. Washing and staining of the hybridized arrays were completed as described in the GeneChip Expression analysis technical manual for HT plate arrays using the Genechip® Array Station (Affymetrix, Santa Clara, Calif.) with modifications as described in Allaire et al. The processed GeneChip® plate arrays were scanned using GeneTitan scanner (Affymetrix, Santa Clara, Calif.).
Affymetrix scans were subject to quality control (QC) measures. These tests included a visual inspection of the distribution of raw signal intensities and an assessment of RNA degradation, relative log expression (RLE), and normalized unsealed standard error (NUSE). All sample scans that passed these QC metrics were included in the analysis.
All CEL files were subjected to GC-content-based Robust Multi-array Average (GCRMA) normalization (version 2.20.0) (Irizarry R A, et al. Nucleic Acids Res 2003; 31:e15; Li C, et al. Genome Biol 2001; 2:RESEARCH0032). Expression levels were log (base 2) transformed.
Analyses were applied to discover genes that were differentially expressed (DEGs) between different animal treatments. The contrasts carried out are shown in TABLES 12 and 13. To identify differentially expressed genes between groups of samples, we applied the linear modeling approach (ANOVA) to fit gene expression levels (log 2 transformed) according to the defined groups of samples and Bayesian posterior error analysis as implemented by Smyth (limma, version 3.4.5) (Smyth G K. Stat Appl Genet Mol Biol 2004; 3:Article3). Genes were considered significantly different if they met the following criteria: (i) average normalized signal intensity greater than four; (ii) logarithm [base 10] of odds [LOD] score greater than zero; and (iii) fold change greater than 1.5. All calculations and analyses were carried out using R (version 2.11.1) and Bioconductor computational tools (Gentleman R. Springer Science+Business Media 2005).
Changes in gene expression were studied by applying a linear modeling approach to fit gene expression levels according to the defined groups of samples (e.g. DMF-, MMF-, and Vehicle-treated animals) and Bayesian posterior error analysis as implemented by Smyth (Smyth G K. Stat Appl Genet Mol Biol 2004; 3:Article3). The comparisons considered included: DMF-vs-MMF, DMF-vs-Vehicle, and MMF-vs-Vehicle. Differentially expressed genes (DEGs) were defined in each comparison as those Affymetrix probesets (henceforth referred to as “genes”) that exhibited an average normalized signal intensity greater than 4, a LOD score >0, and absolute fold change value greater than 1.5. TABLES 12 and 13 show the number of DEGs identified in each contrast. In general, more DEGs are apparent with the multi-dosing than the single dosing regimen, and no clear trend is seen between the 2 h, 7 h, and 12 h time points after a single dose. Very few DEGs are observed in blood and tissues derived from the central nervous system (brain, cerebellum, cortex, striatum, and spinal cord). The jejunum and kidney exhibited the highest number of DEGs in the animals receiving a single dose of treatment, whereas in the multi-dosed animals, the largest number of DEGs for both MMF and DMF treatments was consistently observed in the spleen.
A large number of DEGs (280 DEGs) was observed in the brains of multi-dosed MMF animals as compared to the Vehicle treatment; DMF treatment did not induce this large effect. Interestingly, this same trend was seen in the brains of EAE animals chronically dosed with MMF (158 DEGs). These two sets of DEGs overlap by 61 genes, all of them down-regulated with MMF as compared to Vehicle treatment; this overlap is significant, with p-value 5.67×10−94 (
The gene expression results generally indicate that treatment of naive mice with DMF and MMF elicits unique pharmacodynamic responses in the tissues.
The DEPP gene is robustly induced with DMF but not with MMF in the brains and spinal cords of naïve mice that were administered a multi-dosing regimen of these compounds (TABLE 14 and
As shown in
As shown in Examples 1 and 3, a transcriptional comparison of single-dose MMF vs DMF in naïve mice revealed an NK cell “signature” in DMF-treated naïve mice. Example 2 describes FACS analysis which confirmed and extended the findings of an NK cell signature. The present example describes immunophenotyoing analysis of immune cell subsets in Experimental Autoimmune Encephalomyelitis (EAE) mice treated with a single dose or chronic administration of DMF or MMF.
EAE induction is generally performed by immunization with brain extracts, CNS proteins (such as myelin basic protein), or peptides from such protein emulsified in an adjuvant such as complete Freund's adjuvant, e.g., as described in Linker et al., Brain. 2011 March 134(Pt3): 678-92. Vehicle, MMF or DMF was administered to EAE mice by a chronic or single dose administration, as described below Immune cells were obtained from various mouse tissues and analyzed by flow cytometry.
In the first study (chronic dose study), EAE mice were chronically dosed with vehicle, MMF, or DMF beginning at day 4 post-immunization. Mice were sacrificed on day 17 post-immunization at 12-hours after receiving the last dose.
In the second study (single dose study), EAE mice were treated with a single dose of vehicle, MMF, or DMF on day 17 post-immunization. Animals were sacrificed 12 hours after receiving the dose.
Additional immune cell types were analyzed, including T cells, B cells, and myeloid cells. In particular, T cells from EAE mice treated with vehicle, MMF, or DMF were analyzed by flow cytometry.
B cells from EAE mice treated with vehicle, MMF, or DMF were also analyzed by flow cytometry.
Myeloid cells from EAE mice treated with vehicle, MMF, or DMF were also analyzed by flow cytometry.
This example demonstrates transcriptional profiling of pharmacodynamic effects of oral administration of DMF and MMF in single or multi-dose regiments in EAE mice. The number of independent mice whose tissues were harvested for transcript profiling studies and whose data passed QC is shown in TABLE 15.
Numbers in parentheses indicate the number of samples included in a sub-analysis for which only the animals with the highest cumulative EAE score were used. Naïve mice do not have EAE nor were they administered any treatment.
Animals were exposed to CO2 and whole blood collected via cardiac puncture. Two 100 μl aliquots of whole blood were collected in 1.5 mL microcentrifuge tubes and snap frozen in liquid nitrogen. Peripheral (liver, spleen, kidney and jejunum) and CNS brain (cerebellum, hippocampus, striatum and cortex) tissues were harvested and snap frozen in liquid nitrogen, with special care taken to collect tissues of similar size and from the same location. All samples were stored at −80° C. until RNA extraction was conducted.
For RNA preparation, frozen tissues were placed in 2 mL RNAse-free 96-well blocks with 1.5 mL QIAzol Lysis Reagent (QIAgen) and a 3.2 mm stainless steel bead (BioSpec Products, Bartlesville, Okla.). Tissues were disrupted for four cycles of 45 seconds in a Mini-Beadbeater (Biospec Products). RNA was extracted in chloroform and the aqueous phase was mixed with an equal volume of 70% ethanol. Extracted RNA was applied to RNeasy 96 plates and purified by the spin method according to the manufacturer's protocol (RNeasy 96 Universal Tissue Protocol, QIAgen, Hilden Germany).
Blood RNA Extraction and QC (Allaire N E, et al. BMC Res Notes. 2013 Jan. 5; 6:8)
50 ul of snap frozen mouse blood was re-suspended in lysis buffer with proteinase K and arrayed into deep-well plates for automated RNA extraction. RNA extractions were completed on Arrayplex (Beckman Coulter, Indianapolis, Ind.) using Agencourt RNAdvance Blood kit (Part number A35604) according to the manufacturer's specifications. RNA integrity was assessed using the HT RNA reagent kit (Part number 760410, Caliper Life Sciences, Hopkinton, Mass.) using a LabChip GX (PerkinElmer, Waltham, Mass.). RNA samples with a RQS score of >8.0 were considered high quality for downstream microarray processing.
Automated sample amplifications and biotin labelings were carried out using the NuGEN Ovation RNA Amplification system V2 (Cat #3100), Ovation WB reagent (Cat #1300) and Encore Biotin module (Cat #4200) (NuGEN Technologies, Inc, San Carlos, Calif.) according to manufacturer's recommendations using an Arrayplex automated liquid handler (Beckman Coulter, Indianapolis, Ind.). 2 ug of biotin labeled sscDNA probe were hybridized to Affymetrix HT_MG-430_PM plate arrays with modified conditions as described in Allaire et al. Washing and staining of the hybridized arrays were completed as described in the GeneChip Expression analysis technical manual for HT plate arrays using the Genechip® Array Station (Affymetrix, Santa Clara, Calif.) with modifications as described in Allaire et al. The processed GeneChip® plate arrays were scanned using GeneTitan scanner (Affymetrix, Santa Clara, Calif.).
Affymetrix scans were subject to quality control (QC) measures. These tests included a visual inspection of the distribution of raw signal intensities and an assessment of RNA degradation, relative log expression (RLE), and normalized unscaled standard error (NUSE). All sample scans that passed these QC metrics were included in the analysis.
All CEL files were subjected to GC-content-based Robust Multi-array Average (GCRMA) normalization (version 2.20.0) (Irizarry R A, et al. Nucleic Acids Res 2003; 31:e15; Li C, et al. Genome Biol 2001; 2:RESEARCH0032). Expression levels were log (base 2) transformed.
Analyses were applied to discover genes that were differentially expressed (DEGs) between different animal treatments. The contrasts carried out are shown in TABLE 16. To identify differentially expressed genes between groups of samples, we applied the linear modeling approach (ANOVA) to fit gene expression levels (log 2 transformed) according to the defined groups of samples and Bayesian posterior error analysis as implemented by Smyth (limma, version 3.4.5) (Smyth G K. Stat Appl Genet Mol Biol 2004; 3:Article3). Genes were considered significantly different if they met the following criteria: (i) average normalized signal intensity greater than four; (ii) logarithm [base 10] of odds [LOD] score greater than zero; and (iii) fold change greater than 1.5. All calculations and analyses were carried out using R (version 2.11.1) and Bioconductor computational tools (Gentleman R. Springer Science+Business Media 2005).
In order to understand if global gene expression patterns might arise in each tissue from experimental treatments, the set of Affymetrix probesets (henceforth referred to as “genes”) that exhibited an average normalized intensity greater than 4 within a treatment group and with coefficient of variation (CV) greater than 0.05 was subjected to unsupervised clustering. Generally, no distinct sample groupings were apparent from this analysis; however, in the following cohorts, differences in EAE severity across the mice were manifest in global gene expression patterns:
An example of this animal grouping is shown in
Changes in gene expression were studied by applying a linear modeling approach to fit gene expression levels according to the defined groups of samples (e.g. DMF-, MMF-, and Vehicle-treated animals) and Bayesian posterior error analysis as implemented by Smyth (Smyth G K. Stat Appl Genet Mol Biol 2004; 3:Article3). The comparisons considered included: DMF-vs-MMF, DMF-vs-Vehicle, MMF-vs-Vehicle, and Vehicle-vs-Naïve. Differentially expressed genes (DEGs) were defined in each comparison as those genes that exhibited an average normalized signal intensity greater than 4, a LOD score >0, and absolute fold change value greater than 1.5. TABLE 16 shows the number of DEGs identified in each contrast. In general, more DEGs are apparent with chronic dosing than an acute dosing regimen, and no clear trend was seen between the 7 h and 12 h time points in either dosing regimen. Very few DEGs were observed in blood and tissues derived from the central nervous system (brain, cerebellum, and spinal cord). The lymph node and spleen exhibited the highest number of DEGs.
The gene expression results indicate that treatment of EAE mice with DMF and MMF elicits unique pharmacodynamic responses in the tissues.
Finally, several Affymetrix probe sets that represent the Zbtb16 transcript exhibited increased signal in DMF-treated animals as compared to the MMF-treated animals in the lymph node and spleen (
The compounds of Formulae (III)-(VI) may be prepared using methods known to those skilled in the art, or the methods disclosed in the present invention.
Specifically, the compounds of this invention of Formula IV may be prepared by the exemplary reaction in Scheme 1.
wherein R1d, R2d, and R3d are each defined above for Formula IV.
Reaction of fumaric acid ester 1 with silane diacetate intermediate 2 in a refluxing organic solvent such as diethyl ether, toluene, or hexane to give the desired siloxane 3.
Some of the fumaric acid esters 1 are commercially available. Fumaric acid ester 1′ can be prepared, for example, using synthetic methods known by one of ordinary skill in the art. For example, fumaric acid can be converted by reacting alcohol (R1c—OH) with a catalytic amount of p-toluene sulfonic acid at room temperature for a few hours to overnight as shown in Scheme 2.
wherein R1c is defined above for Formula III.
Alternatively, fumaric acid ester 1′ can be prepared by reacting alcohol (R1c—OH) under the coupling conditions of hydroxybenzotriazole (HOBT), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), and diisopropyl amine (DIPEA) as shown in Scheme 3.
wherein R1c is defined above for Formula III.
Some of the silanes that can be used in the present invention are commercially available. Commercially available silyl halides include trimethylsilyl chloride, dichloro-methylphenylsilane, dimethyldichlorosilane, methyltrichlorosilane, (4-aminobutyl)diethoxymethylsilane, trichloro(chloromethyl)silane, trichloro(dichlorophenyl)silane, trichloroethylsilane, trichlorophenylsilane, and trimethylchlorosilane. Commercial sources for silyl halides include Sigma Aldrich and Acros Organics.
Silanes used in the present invention can be prepared, for example, using synthetic methods known by one of ordinary skill in the art. For example, trichlorosilane may be prepared by the exemplary reaction in Scheme 4.
The silylation of styrene derivatives catalyzed by palladium is described in Zhang, F. and Fan, Q.-H., Organic & Biomolecular Chemistry 7:4470-4474 (2009) and in Bell, J. R., et al., Tetrahedron 65:9368-9372 (2009).
Diacetate intermediate 2 may be prepared by treatment of dichlorosubstituted silicon compound 4 with sodium acetate in diethyl ether under reflux as shown in Scheme 5.
wherein R2d and R3d are each defined above for Formula IV.
Specifically, the compounds of this invention of Formula V may be prepared by the exemplary reaction in Scheme 6.
wherein R1e, R2e, R3e, and R5e are as defined above for Formula V.
Fumaric acid ester 1″ can be converted to the sodium salt 5 using, for example, sodium methoxide in methanol at room temperature. Removal of the solvent would afford sodium salt 5. Treatment of the sodium salt 5 with silane 6 in an organic solvent such as dimethylformamide under reflux would generate ester 7. The synthesis of structurally related (trimethoxysilyl)-methyl esters is described in Voronkov, M. G., et al., Zhurnal Obshchei Khimii 52:2052-2055 (1982).
Alternatively, the compounds of this invention of Formula V may be prepared by the exemplary reaction in Scheme 7.
wherein R1e, R4e, R5e, R6e, and n are as defined above for Formula V.
Treatment of the sodium salt 5 with silane 6 in an organic solvent such as dimethylformamide under heating with or without an acid scavenger would generate ester 7.
wherein R1e, R4e, R5e, R6e, and n are as defined above for Formula V.
Reaction of fumaric acid ester 1″ with tri-substituted silane alcohol 8 in methylene chloride with mild base such as triethyl amine and 4-N,N-dimethyl amino pyridine (DMAP) at room temperature generates fumarate 7. See Coelho, P. J., et al., Eur. J. Org. Chem. 3039-3046 (2000).
Specifically, the compounds of this invention of Formula VI can be prepared by the exemplary reaction in Scheme 9.
wherein R1f and R2f are as defined above for Formula VI.
Reaction of fumaric acid 1′″ with trichlorosilane 9 in a refluxing organic solvent such as hexane or toluene using a catalytic amount of a base such as triethylamine generates the trifumarate silane 10. The reaction of acetic and methacrylic acids with 1-silyladamantanes is described in Fedotov, N. S., et al., Zhurnal Obshchei Khimii 52:1837-1842 (1982).
To a slurry of sodium acetate (8.2 g, 100 mmol, 2.0 equiv.) in anhydrous diethyl ether (40 mL) was slowly added a solution of dimethyldichloro silane 11A (6.45 g, 50 mmol, 1.0 equiv.) in anhydrous diethyl ether (10 mL). After addition was completed, the mixture was heated at reflux for 2 hours, and then filtered under N2. The filtrate was concentrated under vacuum at 40° C. to give diacetate 11B as a colorless oil (6.1 g, 70%). 1H NMR (400 MHz, CDCl3) δ ppm: 2.08 (s, 6H), 0.48 (s, 6H).
A mixture of 11B (2.0 mL, 12 mmol, 1.5 equiv.) and 11C (1.04 g, 8.0 mmol, 1.0 equiv.) in a sealed tube was heated at 170° C. with stirring under microwave condition for 1 hour. After cooling to 50° C., the mixture was transferred to a round bottom flask and the excess silica reactant 11B was removed under vacuum at 100° C. to afford compound 11 as brown oil (1.47 g, 60%). 1H NMR (400 MHz, CDCl3) δ ppm: 6.82-6.80 (m, 4H), 3.79 (s, 6H), 0.57 (s, 6H).
To a stirred solution of monomethyl fumarate (3.5 g, 27 mmol, 1.0 equiv.) in anhydrous THF (35 mL) at room temperature was added sodium hydride (1.08 g, 27 mmol, 1.0 equiv.) in small portions. After addition, the mixture was heated to reflux for 3 hours, and then cooled to room temperature. The solid was collected by filtration and washed twice with diethyl ether, and further dried in vacuo to give 3.8 g of 12B (93%).
To a suspension of 12B (760 mg, 5.0 mmol, 1.0 equiv.) in dry DMA (5 mL) at 100° C. under nitrogen was added a solution of 12A (1.03 g, 6.0 mmol, 1.2 equiv.) in dry DMA (1 mL) dropwise. The resulting mixture was heated to 160° C. and stirred for 1 hour, and then cooled to room temperature. The solid was filtered, and the filtrate was evaporated under reduced pressure to give the titled compound 12, 513 mg (37%), as a red viscous liquid.
1H NMR (400 MHz, CDCl3) δ ppm: 6.90-6.86 (m, 2H), 3.97 (s, 2H), 3.82 (s, 3H), 3.62 (s, 9H).
To a solution of 12 (1.0 g, 3.8 mmol, 1.0 equiv., prepared in Example 2) in MeOH (10 mL) at room temperature was added water (341 mg, 19.0 mmol, 5.0 equiv.) dropwise. After addition, the mixture was stirred at room temperature for 30 minutes, with white solids precipitated out. The solids were collected through filtration, washed with methanol three times, and dried at 60° C. in vacuo, to provide the titled compound 13, 500 mg (59%), as a white solid.
1H NMR (400 MHz, DMSO-d6) δ ppm: 6.79-6.74 (m, 2H), 3.91-3.58 (m, 6H), 3.18-3.15 (m, 2H).
Following the procedure described in Scheme 9, monomethyl fumarate 14A would react with trichloromethane-silane 14B in refluxing toluene or hexanes with a catalytic amount of triethylamine to provide (2′E,2″E)-trimethyl O,O′,O″-(methylsilanetriyl)trifumarate 14C.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed.
This application claims the benefit of U.S. Provisional Application No. 61/825,938, filed May 21, 2013, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/38973 | 5/21/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61825938 | May 2013 | US |
