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WO2010005387A1 - Procédé et marqueurs inédits pour le diagnostic de la sclérose en plaques - Google Patents

Procédé et marqueurs inédits pour le diagnostic de la sclérose en plaques Download PDF

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Publication number
WO2010005387A1
WO2010005387A1 PCT/SE2009/050885 SE2009050885W WO2010005387A1 WO 2010005387 A1 WO2010005387 A1 WO 2010005387A1 SE 2009050885 W SE2009050885 W SE 2009050885W WO 2010005387 A1 WO2010005387 A1 WO 2010005387A1
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Prior art keywords
alpha
multiple sclerosis
subject
protein
msps
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Inventor
Lars I Andersson
Bo Franzén
Kerstin C Nilsson
Jan Ottervald
Hugh Salter
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AstraZeneca AB
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AstraZeneca AB
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the present invention relates to biological markers for multiple sclerosis. More specifically, the present invention relates to the use of such markers to diagnose multiple sclerosis, to monitor progression of the disease in clinical or preclinical trials, as well as for drug screening and drug development.
  • MS Multiple sclerosis
  • MS can be divided into four different forms; clinical isolated syndrome (CIS), relapsing remitting (RR), secondary progressive (SP) and primary progressive (PP) respectively.
  • CIS can be the first step in developing the disease from which 30-80% actually develops MS.
  • RR is characterized by a series of exacerbations that result in varying degrees of disability from which the patient recovers. The course of the disease in about 60-80% of RR patients steadily changes to SP in which the patient does not experience exacerbations, but instead reports a gradual decline.
  • PP does not include the typical exacerbations as in RR instead the disease progression gradually progress.
  • MS is a chronic demyelinating disease in which inflammation of the CNS is associated with lesions appearing typically in plaques within white matter. This inflammatory process involves activation and recruitment of T cells, macrophages and microglia to lesion sites. Symptoms are believed to occur from axonal demyelination that inhibits or blocks conduction throughout the nervous system. Plaques may be found throughout the brain and spinal cord. Recovery of symptoms has been attributed to partial remyelination and resolution of inflammation. Based on accumulating data from immunological studies of MS patients and a wealth of animal model data, autoimmune dysregulation has been viewed as the major contributor to tissue damage.
  • the current model of MS immunopathology suggests that autoreactive T cells within the periphery become activated.
  • Activated T cells express up-regulated levels of adhesion molecules and are able to migrate across the blood- brain barrier much more efficiently than naive, resting T cells.
  • Extravasation across the blood-brain barrier is thought to involve a sequence of overlapping molecular interactions between inducible ligand- receptor pairs on the surface of the migrating cell and the endothelial barrier.
  • Selective expression of adhesion molecules, chemokines and chemokine receptors and matrix metalloproteinases are likely to be important in mediating the transmigration of effector cells across the blood-brain barrier and into the central nervous system (CNS) perivascular tissue in demyelinating diseases.
  • CNS central nervous system
  • the pathogenic mechanisms of MS may not be limited to autoimmunity. Demyelination may occur through many proposed mechanisms: Fas/Fas ligand interactions, toxic cytokines, reactive oxygen species, antibody dependent cellular toxicity and metabolic instability of oligodendrocytes.
  • axonal damage is increasingly recognized as a prominent pathological feature in MS lesions as well as in normal appearing white matter in MS brains. Whereas these observations do not preclude the role of inflammatory demyelination in MS pathogenesis, axonal compromise may predate the inflammatory lesions, raising the possibility that an independent axonal pathology may contribute to the primary pathobiology of the disease.
  • Studies of the mechanisms of axonal damage and neurodegeneration in MS are in their infancy. However, axonal damage may determine clinical outcome to a large extent. CNS tissue destruction markers would be useful not only for inflammatory demyelination but for neurodegenerative processes in MS.
  • MS is a systemic disease in terms of its autoimmune pathogenesis and a compartmental disease in as much as the end-organ damage is in the CNS.
  • biomarkers of the disease could likely be found in the CSF that surrounds the brain, as well as in other more easily obtainable fluids, such as serum or urine, that are reflective of systemic disease.
  • MS The disease course of MS is highly variable within and between patients indicating that there is disease heterogeneity. Indeed, heterogeneity in MS lesions has been shown in MRI and pathologic studies. MRI affords the ability to identify atrophy and different types of lesions, however it lacks pathologic specificity. Because of its intimate association with the CNS, considerable efforts have been made to identify prognostic and diagnostic markers in the CSF from patients with MS.
  • Phosphorylation of proteins is also regarded as a post-translational modification that can act as on or off signal for protein action, (see Principles of interleukin (IL)-6-type cytokine signalling and its regulation by. Heinrich, P, et al. J374, 1-20. (2003) for discussion).
  • the area, phosphorylations and glycosylations, with a proteomics approach on CSF has not been well investigated although some studies shows examples (Yuko Ogata, M. et al., Journal of Proteome Research, 4, 837- 845 837 (2005)). Characterization of proteins in CSF with proteomic approaches has been sparse.
  • the inventors have used depletion of Albumin and IgG combined with fluorescent stain for total protein. This is a novel approach for quantification and identification of proteins in CSF from MS patients.
  • the present invention provides biological markers (“biomarkers”) indicative of Multiple Sclerosis (MS). These biomarkers can be used to diagnose the disease, monitor its progression, assess response to therapy and screen drugs for treating MS. Early diagnosis and knowledge of disease progression could allow early institution of treatment when it is most appropriate and would be of the greatest benefit to the patient. In addition, such information will allow prediction of exacerbations and classification of potential MS subtypes. The ability to evaluate response to therapy will allow the personalized treatment of the disease and provided the basis for clinical trials aimed at evaluating the effectiveness of candidate drugs.
  • biomarkers biological markers indicative of Multiple Sclerosis
  • Such neuroinflammatory or neurodegenerative disorders could be, but are not limited to, Parkinson's disease, Alzheimer's Disease, Mild Cognitive Impairment, Dementia, Age-Associated Memory Impairment, Age-Related Cognitive Decline, Disorder(s) associated with neurofibrillar tangle pathologies, Dementia due to Alzheimer's Disease, Dementia due to Schizophrenia, Dementia due to Parkinson's Disease, Dementia due to Creutzfeld- Jacob Disease, Dementia due to Huntington's Disease, Dementia due to Pick's Disease, Stroke, Head Trauma, Spinal Injury, Multiple Sclerosis, Migraine, Pain, Systemic Pain, Localized Pain, Nociceptive Pain, Neuropathic Pain, Urinary Incontinence, Sexual Dysfunction, Premature Ejaculation, Motor Disorder(s), Endocrine Disorder(s), Gastrointestinal Disorder(s), and Vasospasm.
  • the biomarkers of the present invention include the level of particular proteins whose measurement values in a biological sample are different (either higher or lower) in a subject with MS as compared to a standard level or reference range established by obtaining measurement values for the biomarker in subjects who do not have the disease ("normal controls") and such differences may be statistically significant.
  • CSF from individual patients may be analysed longitudinal, prior to and during treatment.
  • the invention provides a method for determining whether a subject has MS. Further, the invention provides a method for determining whether a subject is more likely than not to have MS, or is more likely to have MS than to have another disease.
  • the method is performed by analysing a biological sample, such as serum or CSF, from the subject; measuring the level of protein of at least one of the biomarkers in the biological sample; and comparing the measured level with a standard level or reference range.
  • the standard level or reference range is obtained by measuring the same marker or markers in a normal control or, more preferably, a set of normal controls.
  • the patient can be diagnosed as having MS, or as not having MS.
  • a standard level or reference range is specific to the biological sample at issue.
  • a standard level or reference range for the marker in serum that is indicative of MS would be expected to be different from the standard level or reference range (if one exists) for that same marker in CSF, urine or another tissue, fluid or compartment.
  • references herein to measuring biomarkers will be understood to refer to measuring the level of the biomarker.
  • references herein to comparisons between a marker measurement level and a standard level or reference range will be understood to refer to such levels or ranges for the same type of biological sample.
  • the invention provides a method for monitoring a MS patient over time to determine whether the disease is progressing.
  • the method is performed by analysing a biological sample, such as serum or CSF, from the subject at a certain time; measuring the level of at least one of the biomarkers in the biological sample; and comparing the measured level with the level measured with respect to a biological sample obtained from the subject at an earlier time. Depending upon the difference between the measured levels, it can be seen whether the marker level has increased, decreased, or remained constant over the interval. Subsequent sample acquisitions and measurements can be performed as many times as desired over a range of times. The same type of method also can be used to assess the efficacy of a therapeutic intervention in a subject where the therapy is instituted, or an ongoing therapy is changed.
  • a biological sample such as serum or CSF
  • the invention provides a method for conducting a clinical trial to determine whether a candidate drug is effective in treating MS.
  • the method is performed by analysing a biological sample from each subject in a population of subjects diagnosed with MS, and measuring the level of at least one of the biomarkers in the biological samples. Then, a dose of a candidate drug is administered to one portion or sub-population of the same subject population ("experimental group") while a placebo is administered to the other members of the subject population ("control group"). After drug or placebo administration, a biological sample is acquired from the experimental and control groups and the same assays are performed on the biological samples as were previously performed to obtain measurement values. Depending upon the difference between the measured levels between the experimental and control groups, it can be seen whether the candidate drug is effective.
  • the relative efficacy of two different drugs or other therapies for treating MS can be evaluated using this method by administering the drug or other therapy in place of the placebo.
  • the methods of the present invention may be used to evaluate an existing drug, being used to treat another indication, for its efficacy in treating MS (e.g., by comparing the efficacy of the drug relative to one currently used for treating MS in a clinical trial, as described above).
  • the present invention also provides molecules (for example antibodies) that specifically bind to protein and low molecular weight markers.
  • marker specific reagents have utility in isolating the markers and in detecting the presence of the markers, e.g., in immunoassays.
  • kits for diagnosing MS, monitoring progression of the disease and assessing response to therapy comprising a container for a sample collected from a subject and at least one marker specific reagent.
  • biomarkers include protein and low molecular weight molecules.
  • a biological marker (“biomarker”) is "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacological responses to therapeutic interventions.” NIH Biomarker Definitions Working Group (1998).
  • Biomarkers can also include patterns or ensembles of characteristics indicative of particular biological processes.
  • the biomarker measurement can increase or decrease to indicate a particular biological event or process.
  • a biomarker measurement typically changes in the absence of a particular biological process, a constant measurement can indicate occurrence of that process.
  • the biomarkers are primarily used for diagnostic purposes. However they may also be used for therapeutic, drug screening and patient stratification purposes (e.g., to group patients into a number of "subsets" for evaluation).
  • the present invention is based on the findings of a study designed to identify biological markers for MS. Samples of CSF and serum from patients with MS were analyzed using liquid chromatography, 2D gel electrophoresis and mass spectrometry. The markers of the present invention were identified by comparing the levels of markers measured in samples obtained from MS patients with the levels of markers measured in samples obtained from patients who did not have the disease. Peaks consistently higher or lower in patients with MS were further investigated by using liquid chromatography mass combined with tandem mass spectrometry techniques to identify the molecules at issue.
  • the present invention includes all methods relying on correlations between the biomarkers described herein and the presence of MS.
  • the invention provides methods for determining whether a candidate drug is effective at treating MS by evaluating the effect it has on the biomarker values.
  • the term "effective" is to be understood broadly to include reducing or alleviating the signs or symptoms of MS, improving the clinical course of the disease, decreasing the number or severity of exacerbations, reducing the number of plaques, reducing the amount or rate of axonal demyelination, reducing the number of inflammatory cells in existing plaque or reducing in any other objective or subjective indicia of the disease.
  • Different drugs, doses and delivery routes can be evaluated by performing the method using different drug administration conditions. The method may also be used to compare the efficacy of two different drugs or other treatments or therapies for MS.
  • the present invention provides a method for determining whether a subject has MS. Biomarker level measurements are taken of a biological sample from a patient suspected of having the disease and compared with a standard level or reference range. Typically, the standard biomarker level or reference range is obtained by measuring the same marker or markers in a set of normal controls.
  • Measurement of the standard biomarker level or reference range need not be made contemporaneously; it may be a historical measurement.
  • the normal control is matched to the patient with respect to some attribute(s) (e.g., age or sex).
  • the patient can be diagnosed as having MS or as not having MS.
  • a method for screening or diagnosis of multiple sclerosis in a subject for determining the stage or severity of multiple sclerosis in a subject, for identifying a subject at risk of developing multiple sclerosis, or for monitoring the effect of therapy administered to a subject having multiple sclerosis, said method comprising:
  • MSPs multiple sclerosis proteins
  • alpha- 1 -antitrypsin alpha- 1 -antitrypsin
  • leucine-rich alpha-2-glycoprotein alpha-2-macroglobulin
  • amyloid beta A4 protein alpha- 1-antichymotrypsin
  • agrin alpha- 1 -microglobulin
  • angiotensinogen Apolipoprotein E
  • ceruloplasmin cetinase-3-like protein 1
  • carnosinase 1 contactin 1; complement C3 fragment; complement C4-A; component C9; f ⁇ bulin-1; alpha-2-HS- glycoprotein; gelsolin 3408; haptoglobin; hemopexin; kallikrein 6; neural cell adhesion molecule 1; nidogen-2 precursor; neuronal cell
  • MSPs multiple sclerosis proteins
  • a method for screening or diagnosis of multiple sclerosis in a subject for determining the stage of multiple sclerosis in a subject or for monitoring the effect of therapy administered to a subject having multiple sclerosis, said method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following multiple sclerosis proteins (MSPs): alpha- 1 -antitrypsin; leucine-rich alpha-2-glycoprotein; alpha-2-macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1 -microglobulin; angiotensinogen; Apolipoprotein E; ceruloplasmin; chitinase-3-like protein 1; carnosinase 1; contactin 1; complement C3 fragment; complement C4-A; component C9; f ⁇ bulin-1; alpha-2-HS-glycoprotein; gelsolin 3408;
  • MSPs multiple sclerosis proteins
  • a method which is for determining the stage of multiple sclerosis in a subject, wherein said stage is clinical isolated syndrome multiple sclerosis.
  • a method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following MSPs: alpha- 1 -antitrypsin; leucine-rich alpha-2-glycoprotein; alpha-2- macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1- microglobulin; angiotensinogen; apolipoprotein E; ceruloplasmin; chitinase-3-like protein 1 ; carnosinase 1 ; contactin 1 ; complement C3 fragment; complement C4-A; component C9; f ⁇ bulin-1; alpha-2-HS-glycoprotein; gelsolin 3408; haptoglobin; hemopexin; kallikre
  • a method which is for determining the stage of multiple sclerosis in a subject, wherein said stage is relapsing remitting multiple sclerosis.
  • a method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following MSPs: alpha- 1 -antitrypsin; leucine-rich alpha-2-glycoprotein; alpha-2- macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1- microglobulin; angiotensinogen; apolipoprotein E; ceruloplasmin; chitinase-3-like protein 1 ; carnosinase 1 ; contactin 1 ; complement C3 fragment; complement C4-A; component C9; fibulin-1; alpha-2-HS-glycoprotein; gelsolin 3408; haptoglobin; hemopexin;
  • a method which is for determining the stage of multiple sclerosis in a subject, wherein said stage is secondary progressive multiple sclerosis.
  • a method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following MSPs: alpha- 1 -antitrypsin; leucine-rich alpha-2-glycoprotein; alpha-2- macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1- microglobulin; angiotensinogen; apolipoprotein E; ceruloplasmin; chitinase-3-like protein 1 ; carnosinase 1 ; contactin 1 ; complement C3 fragment; complement C4-A; component C9; fibulin-1; alpha-2-HS-glycoprotein; gelsolin 3408; haptoglobin; hemopexin; kallikrein 6;
  • a method which is for determining the stage of multiple sclerosis in a subject, wherein said stage is neurodegenerative stage of multiple sclerosis.
  • a method comprising quantitatively detecting, in a test sample of body fluid from the subject, one or more of the following MSPs: alpha- 1 -antitrypsin; leucine-rich alpha-2-glycoprotein; alpha- 2-macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1- microglobulin; angiotensinogen; apolipoprotein E; ceruloplasmin; chitinase-3-like protein 1 ; carnosinase 1 ; contactin 1 ; complement C3 fragment; complement C4-A; component C9; fibulin-1; alpha-2-HS-glycoprotein; gelsolin 3408; haptoglobin; hemopexin; kal
  • test sample of body fluid is selected from blood, serum, plasma, cerebrospinal fluid, urine and saliva.
  • an antibody capable of binding to the MSPs alpha- 1 -antitrypsin; leucine-rich alpha-2-glycoprotein; alpha-2- macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1- microglobulin; angiotensinogen; apolipoprotein E; ceruloplasmin; chitinase-3-like protein 1 ; carnosinase 1 ; contactin 1 ; complement C3 fragment; complement C4-A; component C9; f ⁇ bulin-1; alpha-2-HS-glycoprotein; gelsolin 3408; haptoglobin; hemopexin; kallikrein 6; neural cell adhesion molecule 1; nidogen-2 precursor; neuronal cell adhesion molecule; peptidylglycine alpha-amidating monooxygenase isoform b; pigment epithelium-derived
  • said antibody is a monoclonal antibody.
  • kits comprising one or more of said antibodies, other reagents and instructions for use.
  • Said kit can be used in the screening or diagnosis of multiple sclerosis in a subject, for determining the stage or severity of multiple sclerosis in a subject, for identifying a subject at risk of developing multiple sclerosis, or for monitoring the effect of therapy administered to a subject having multiple sclerosis.
  • Aid kit may comprise a plurality of said antibodies.
  • a pharmaceutical composition comprising a therapeutically effective amount of said antibody, or a fragment or derivative of an antibody, and a pharmaceutically acceptable carrier.
  • a method of treating or preventing multiple sclerosis comprising administering to a subject in need of such treatment a therapeutically effective amount of said antibody.
  • a method of screening for agents that interact with one or more MSPs alpha- 1 -antitrypsin; leucine -rich alpha-2- glycoprotein; alpha-2-macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1 -microglobulin; angiotensinogen; apo lipoprotein E; ceruloplasmin; chitinase- 3-like protein 1; carnosinase 1; contactin 1; complement C3 fragment; complement C4-A; component C9; fibulin-1; alpha-2-HS-glycoprotein; gelsolin 3408; haptoglobin; hemopexin; kallikrein 6; neural cell adhesion molecule 1;
  • said determination of interaction between the candidate agent and the MSP comprises quantitatively detecting binding of the candidate agent and the MSP.
  • a method of screening for or identifying agents that modulates the expression or activity of one or more more MSPs alpha- 1 -antitrypsin; leucine-rich alpha-2-glycoprotein; alpha-2-macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1 -microglobulin; angiotensinogen; apolipoprotein E; ceruloplasmin; chitinase-3-like protein 1; carnosinase 1; contactin 1; complement C3 fragment; complement C4-A; component C9; f ⁇ bulin-1; alpha-2-HS- glycoprotein; gelsolin 3408; haptoglobin; hemopexin; kallikrein 6; neural cell adhesion molecule 1; nidogen-2 precursor; neuronal cell adhesion molecule; peptidylglycine alpha- amidating monooxygenase
  • a method of screening for or identifying agents that modulate the expression or activity of one or more more MSPs alpha- 1 -antitrypsin; leucine-rich alpha-2-glycoprotein; alpha-2-macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1 -microglobulin; angiotensinogen; apolipoprotein E; ceruloplasmin; chitinase-3-like protein 1; carnosinase 1; contactin 1; complement C3 fragment; complement C4-A; component C9; f ⁇ bulin-1; alpha-2-HS- glycoprotein; gelsolin 3408; haptoglobin; hemopexin; kallikrein 6; neural cell adhesion molecule 1; nidogen-2 precursor; neuronal cell adhesion molecule; peptidylglycine alpha- amidating monooxygenase
  • said administration of a candidate agent results in an increase in the level of said MSPs, or mRNA encoding said MSPs, or said downstream effecter in the first population of cells or mammals compared to the second population of cells or mammals.
  • said administration of a candidate agent results in a decrease in the level of said MSPs, or mRNA encoding said MSPs , or said downstream effecter in the first population of cells or mammals compared to the second population of cells or mammals.
  • a method of screening for or identifying agents that modulate the activity of one or more of the MSPs alpha- 1- antitrypsin; leucine -rich alpha-2-glycoprotein; alpha-2-macroglobulin; amyloid beta A4 protein; alpha- 1-antichymotrypsin; agrin; alpha- 1 -microglobulin; angiotensinogen; apolipoprotein E; ceruloplasmin; chitinase-3-like protein 1; carnosinase 1; contactin 1; complement C3 fragment; complement C4-A; component C9; f ⁇ bulin-1; alpha-2-HS- glycoprotein; gelsolin 3408; haptoglobin; hemopexin; kallikrein 6; neural cell adhesion molecule 1; nidogen-2 precursor; neuronal cell adhesion molecule; peptidylglycine alpha- amidating monooxygenase iso
  • the above-mentioned methods may relates to MSP(s), wherein said MSP(s) is a recombinant protein.
  • MS may turn out to be a number of related, but distinguishable conditions. Indeed, four types of MS have already been recognized: (i) early MS e.g. clinical isolated syndrome (CIS), (ii) relapsing remitting MS (RR), (iii) secondary chronic progressive MS (SP), and (iv) primary progressive MS (PP). Additional classifications may be made, and these types may be further distinguished into subtypes. Any and all of the various forms of MS are intended to be within the scope of the present invention. Indeed, by providing a method for subsetting patients based on biomarker measurement level, the compositions and methods of the present invention may be used to uncover and define various forms of the disease.
  • CIS clinical isolated syndrome
  • RR relapsing remitting MS
  • SP secondary chronic progressive MS
  • PP primary progressive MS
  • the methods of the present invention may be used to make the diagnosis of MS, independently from other information such as the patient's symptoms or the results of other clinical or paraclinical tests. However, the methods of the present invention are preferably used in conjunction with such other data points.
  • the method may be used to determine whether a subject is more likely than not to have MS, or is more likely to have MS than to have another disease, based on the difference between the measured and standard level or reference range of the biomarker.
  • a patient with a putative diagnosis of MS may be diagnosed as being "more likely” or “less likely” to have MS in light of the information provided by a method of the present invention.
  • the biological sample may be of any tissue or fluid.
  • the sample is a CSF or serum sample, but other biological fluids or tissue may be used.
  • Possible biological fluids include, but are not limited to, plasma, urine and neural tissue.
  • CSF represents a preferred biological sample to analyze for MS markers as it bathes the brain and removes metabolites and molecular debris from its liquid environment.
  • biomolecules associated with the presence and/or progression of MS is expected to be present at highest concentrations in this body fluid.
  • a CSF biomarker in itself may be particularly useful for early diagnosis of disease.
  • molecules initially identified in CSF may also be present, presumably at lower concentrations, in more easily obtainable fluids such as serum and urine. Such biomarkers may be valuable for monitoring all stages of the disease and response to therapy.
  • Serum and urine also represent preferred biological samples as they are expected to be reflective of the systemic manifestations of the disease.
  • the level of a marker may be compared to the level of another marker or some other component in a different tissue, fluid or biological "compartment.”
  • a differential comparison may be made of a marker in CSF and serum. It is also within the scope of the invention to compare the level of a marker with the level of another marker or some other component within the same compartment.
  • biomarker levels are measured using conventional techniques.
  • a wide variety of techniques are available, such as mass spectrometry, chromatographic separations, 2 -D gel separations, binding assays (e.g., luminex immunoassays) and competitive inhibition assays.
  • Any effective method in the art for measuring the level of a protein or low molecular weight marker is included in the invention. It is within the ability of one of ordinary skill in the art to determine which method would be most appropriate for measuring a specific marker. Thus, for example, a robust ELISA assay may be best suited for use in a physician's office while a measurement requiring more sophisticated instrumentation may be best suited for use in a clinical laboratory. Regardless of the method selected, it is important that the measurements are reproducible.
  • the markers of the invention can be measured by mass spectrometry, which allows direct measurements of analytes with high sensitivity and reproducibility.
  • mass spectrometric methods are available and could be used to accomplish the measurement.
  • Electrospray ionization (ESI) allows quantification of differences in relative concentration of various species in one sample against another; absolute quantification is possible by normalization techniques (e.g., using an internal standard).
  • Matrix-assisted laser desorption ionization (MALDI) or the related SELDI® technology (Ciphergen, Inc.) also could be used to make a determination of whether a marker was present, and the relative or absolute level of the marker.
  • mass spectrometers that allow time-of- flight (TOF) measurements have high accuracy and resolution and are able to measure low abundant species, even in complex matrices like serum or CSF.
  • quantification can be based on derivatization in combination with isotopic labeling, referred to as isotope coded affinity tags ("ICAT").
  • ICAT isotope coded affinity tags
  • one- and two-dimensional gels have been used to separate proteins and quantify gels spots by silver staining, fluorescence or radioactive labeling. These differently stained spots have been detected using mass spectrometry, and identified by tandem mass spectrometry techniques.
  • the markers may also be measured using mass spectrometry in connection with a separation technology, such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry. It is preferable to couple reverse-phase liquid chromatography to high resolution, high mass accuracy ESI time-of-flight (TOF) mass spectroscopy. This allows spectral intensity measurement of a large number of biomolecules from a relatively small amount of any complex biological material without sacrificing sensitivity or throughput. Analyzing a sample will allow the marker (specified by a specific retention time and m/z) to be determined and quantified. As will be appreciated by one of skill in the art, many other separation technologies may be used in connection with mass spectrometry.
  • a separation technology such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry. It is preferable to couple reverse-phase liquid chromatography to high resolution, high mass accuracy ESI time-of-flight (TOF) mass spectroscopy. This allows
  • separations may be performed using custom chromatographic surfaces (e.g., a bead on which a marker specific reagent has been immobilized). Molecules retained on the media subsequently may be eluted for analysis by mass spectrometry.
  • Analysis by liquid chromatography-mass spectrometry produces a mass intensity spectrum, the peaks of which represent various components of the sample, each component having a characteristic mass- to-charge ratio (m/z) and retention time (r.t.).
  • the presence of a peak with the m/z and retention time of a biomarker indicates that the marker is present.
  • the peak representing a marker may be compared to a corresponding peak from another spectrum (e.g., from a control sample) to obtain a relative measurement.
  • Any normalization technique in the art e. g., an internal standard
  • deconvoluting software is available to separate overlapping peaks.
  • the retention time depends to some degree on the conditions employed in performing the liquid chromatography separation.
  • the mass spectrometer selected for this purpose preferably provides high mass accuracy and high mass resolution.
  • the mass accuracy of a well- calibrated Micromass TOF instrument, for example, is reported to be approximately 2 mDa, with resolution m/Am exceeding 5000.
  • a number of the assays discussed above employ a reagent that specifically binds to the marker ("marker specific reagent"). Any molecule that is capable of specifically binding to a marker is included within the invention.
  • the marker specific reagents are antibodies or antibody fragments. In other embodiments, the marker specific reagents are non-antibody species.
  • a marker specific reagent may be an enzyme for which the marker is a substrate. The marker specific reagents may recognize any epitope of the targeted markers.
  • a marker specific reagent may be identified and produced by any method accepted in the art. Methods for identifying and producing antibodies and antibody fragments specific for an analyte are well known. Examples of other methods used to identify marker specific reagents include binding assays with random peptide libraries (e.g., phage display) and design methods based on an analysis of the structure of the marker.
  • the markers of the invention may also be detected or measured using a number of chemical derivatization or reaction techniques known in the art. Reagents for use in such techniques are known in the art, and are commercially available for certain classes of target molecules.
  • the chromatographic separation techniques described above also may be coupled to an analytical technique other than mass spectrometry such as fluorescence detection of tagged molecules, NMR, capillary UV, evaporative light scattering or electrochemical detection.
  • an analytical technique other than mass spectrometry such as fluorescence detection of tagged molecules, NMR, capillary UV, evaporative light scattering or electrochemical detection.
  • a method for monitoring an MS patient over time to determine whether the disease is progressing The specific techniques used in implementing this embodiment are similar to those used in the embodiments described above. The method is performed by obtaining a biological sample, such as serum or CSF, from the subject at a certain time (t 1); measuring the level of at least one of the biomarkers in the biological sample; and comparing the measured level with the protein level measured with respect to a biological sample obtained from the subject at an earlier time.
  • the ability to monitor a patient by making serial marker level determinations would represent a valuable clinical tool. Rather than the limited "snapshot" provided by a single test, such monitoring would reveal trends in marker levels over time.
  • tracking the marker levels in a patient could be used to predict exacerbations or indicate the clinical course of the disease.
  • the biomarkers of the present invention could be further investigated to distinguish between any or all of the known forms of MS (early MS, relapsing remitting MS, secondary chronic progressive MS, and primary progressive MS) or any later described types or subtypes of the disease.
  • the sensitivity and specificity of any method of the present invention could be further investigated with respect to distinguishing MS from other diseases of autoimmunity, or other nervous system disorders, or to predict relapse and remission.
  • the markers of the present invention can be used to assess the efficacy of a therapeutic intervention in a subject.
  • the same approach described above would be used, except a suitable treatment would be started, or an ongoing treatment would be changed, before the second measurement.
  • the treatment can be any therapeutic intervention, such as drug administration, dietary restriction or surgery, and can follow any suitable schedule over any time period.
  • the measurements before and after could then be compared to determine whether or not the treatment had an effect effective.
  • the determination may be confounded by other superimposed processes (e.g., an exacerbation of the disease during the same period).
  • the markers may be used to screen candidate drugs in a clinical trial to determine whether a candidate drug is effective in treating MS.
  • a biological sample is obtained from each subject in population of subjects diagnosed with MS.
  • assays are performed on each subject's sample to measure levels of a biological marker. In some embodiments, only a single marker is monitored, while in other embodiments, several markers are monitored.
  • a predetermined dose of a candidate drug is administered to a portion or sub-population of the same subject population. Drug administration can follow any suitable schedule over any time period. In some cases, varying doses are administered to different subjects within the sub-population, or the drug is administered by different routes.
  • a biological sample is acquired from the sub-population and the same assays are performed on the biological samples as were previously performed to obtain measurement values. As before, subsequent sample acquisitions and measurements can be performed as many times as desired over a range of times.
  • a different subpopulation of the subject population serves as a control group, to which a placebo is administered. The same procedure is then followed for the control group: obtaining the biological sample, processing the sample, and measuring the level of the biological markers to obtain a measurement chart.
  • Specific doses and delivery routes can also be examined.
  • the method is performed by administering the candidate drug at specified dose or delivery routes to subjects with MS; obtaining biological samples, such as serum or CSF, from the subjects; measuring the level of at least one of the biomarkers in each of the biological samples; and, comparing the measured level for each sample with other samples and/or a standard level.
  • the standard level is obtained by measuring the same marker or markers in the subject before drug administration.
  • the drug can be considered to have an effect on MS. If multiple biomarkers are measured, at least one and up to all of the biomarkers must change, in the expected direction, for the drug to be considered effective. Preferably, multiple markers must change for the drug to be considered effective, and preferably, such change is statistically significant.
  • a subject population having MS is selected.
  • the population is typically selected using standard protocols for selecting clinical trial subjects.
  • the subjects are generally healthy, are not taking other medication, and are evenly distributed in age and sex.
  • the subject population can also be divided into multiple groups; for example, different sub-populations may be suffering from different types or different degrees of the disorder to which the candidate drug is addressed. Alternatively, subgroups may be defined by the level of biomarkers.
  • biomarker measurements can be detected following drug administration.
  • the amount of change in a biomarker depends upon a number of factors, including strength of the drug, dose of the drug, and treatment schedule. It will be apparent to one skilled in statistics how to determine appropriate subject population sizes. Preferably, the study is designed to detect relatively small effect sizes.
  • the subjects optionally may be "washed out” from any previous drug use for a suitable period of time. Washout removes effects of any previous medications so that an accurate baseline measurement can be taken.
  • a biological sample is obtained from each subject in the population.
  • the sample is blood or CSF, but other biological fluids may be used (e.g., urine).
  • an assay or variety of assays is performed on each subject's sample to measure levels of particular biomarkers of the invention.
  • the assays can use conventional methods and reagents, as described above. If the sample is blood, then the assays typically are performed on either serum or plasma. For other fluids, additional sample preparation steps are included as necessary before the assays are performed.
  • the assays measure values of at least one of the biological markers described herein.
  • markers may also be monitored in conjunction with other measurements and factors associated with MS (e.g., MRI imaging).
  • MS e.g., MRI imaging.
  • the number of biological markers whose values are measured depends upon, for example, the availability of assay reagents, biological fluid, and other resources.
  • a predetermined dose of a candidate drug is administered to a portion or sub- population of the same subject population.
  • Drug administration can follow any suitable schedule over any time period, and the sub-population can include some or all of the subjects in the population.
  • varying doses are administered to different subjects within the sub-population, or the drug is administered by different routes. Suitable doses and administration routes depend upon specific characteristics of the drug.
  • another biological sample is acquired from the sub-population.
  • the sample is the same type of sample and processed in the same manner (for example, CSF or blood) as the sample acquired from the subject population before drug administration (the "t ⁇ sample”).
  • the same assays are performed on the samples to obtain measurement values. Subsequent sample acquisitions and measurements can be performed as many times as desired over a range of times.
  • a different sub-population of the subject population is used as a control group, to which a placebo is administered.
  • the same procedure is then followed for the control group: obtaining the biological sample, processing the sample, and measuring the level of biological markers to obtain measurement values.
  • different drugs can be administered to any number of different sub-populations to compare the effects of the multiple drugs. Allocation of treatment to participating subjects should be done randomly, and the trial should preferably be double-blinded. As will be apparent to those of ordinary skill in the art, the above description is a highly simplified description of a method involving a clinical trial. Clinical trials have many more procedural requirements, and it is to be understood that the method is typically implemented following all such requirements.
  • post- treatment measurements should be used to compare the treated group versus the placebo group. Pre-treatment measurements may be used in the analysis to adjust for potential differences in baseline values between patients.
  • biomarker If only one biomarker is measured, then that value must be different between the placebo and drug-treated groups to indicate drug efficacy. If more than one biomarker is measured, then drug efficacy can be indicated by change in only one biomarker, all biomarkers, or any number in between. In some embodiments, multiple markers are measured, and drug efficacy is indicated by changes in multiple markers. Measurements can be of both biomarkers of the present invention and other measurements and factors associated with MS (e.g., measurement of biomarkers reported in the literature and/or MRI imaging). Furthermore, the amount of change in a biomarker level may be an indication of the relatively efficacy of the drug.
  • biomarkers of the invention can also be used to examine dose effects of a candidate drug.
  • dose effects of a candidate drug There are a number of different ways that varying doses can be examined. For example, different doses of a drug can be administered to different subject populations, and measurements corresponding to each dose analyzed and, optionally,compared to placebo to determine if the differences in the inventive biomarkers are significant. In this way, a minimal dose required to effect a change can be estimated.
  • results from different doses can be compared with each other to determine how each biomarker behaves as a function of dose.
  • Example The invention is hereby exemplified by the below non-limiting example.
  • Material Samples were collected at Karolinska Hospital Sweden (provided by Professor Tomas Olsson, CMM, Karolinska Institute, Sweden), during investigation of patients with possible Multiple Sclerosis, diagnosis criteria described in Recommended Diagnostic Criteria for Multiple Sclerosis: Guidelines from the International Panel on the Diagnosis of Multiple Sclerosis, W. Ian McDonald et al, Ann Neurol; 50, 121-127 (2001).
  • samples were wortexed 3(Tand centrifuged 15" at 13000Xg to erase insoluble molecules. Thereafter the estimated equal protein amounts in their respectively volume were transferred to 1.5 ml eppendorf tubes and rehydration solution (RH; Urea 8 M (Sigma, USA) DTT 19.5 mM, NP-40 (10%) 0.5 % (v/v) (USB Corporation, USA) IPG-buffer 4-7, 0.5 % (v/v) (GE healthcare, USA) Glycerol 7 % (v/v) CHAPS 1.5 % (Genomic solutions, USA), thiourea 2M, (Fluka, Germany)) was added to a final volume of 460 ⁇ l.
  • the IPG strips Prior to the 2-D run, the IPG strips were subjected to a two-step reduction and alkylation step by equilibrating the strips for 15 min first in 50 mM Tris-HCl, pH 6.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS (Bio-Rad, Hercules, CA, USA), and 65 mM DTT, and then for 15 min in 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS, and 259 mM iodoacetamide (IAA, Matrix Scientific, USA).
  • the autostainer program included the following step; 2X 15'MQ, 3X 30'fixation, 12h Sypro Ruby ® , 3X 15'MQ for and final step in fixation solution. Thereafter the gels were scanned with Molecular Imager ® FX at 100 ⁇ m resolution. After scanning the gels is stored in plastic bags in a 0.01% sodium azid solution. Image analysis
  • Image files (16 bit grey levels and lOO ⁇ m resolution) representing SyproRuby® stained gels were processed for background subtraction and protein spot detection using one defined set of parameters. These parameters were optimized using tools given by the software PDQuest (version 7.3, Bio-Rad, Hercules, CA, USA). Detected protein spots were then matched between gels and a synthetic master image was prepared to represent most of the protein spots present in all gels. The quantity of each protein spot was expressed as ppm (parts per million) of the total sum of the integrated spot volumes of the given gel image. This procedure allow for quantitative comparison of all protein spots detected in all gels. Protein spots of interest were excised from gels using a spot cutter robot (Bio-Rad, Hercules, CA, USA), transferred to 96-well plates.
  • PLS Partial Least Squares
  • VIP is a weighted sum of squares of the PLS weights, with the weights calculated from the amount of Y-variance of each PLS component in the model.
  • the predictor matrix is the 2DE data
  • the response is a binary vector denoting class membership, in this case zeros represents the OND/HC class and ones each of the MS classes (Tab. 1).
  • a cutoff needs to be set for the y, such that if the predicted y value is below the cutoff, the predicted class membership will be OND/HC in this case, and if the predicted y value is above the cutoff, the predicted class membership will be CIS, RR rem, RR rel or SP (Tab.l).
  • the PLS modeling was carried out in Matlab (The Mathworks, Inc) using the PLS toolbox (Eigenvector Research) Hierarchical clustering was performed in Spotfire in order to provide a visualisation of the data. Protein identification
  • Protein spots of interest were excised from gels using the EXQUEST spot cutter robot (Bio-Rad, Hercules, CA, USA), transferred to 96-well plates. Up to 6 protein spots of same spot number were pooled to each well in order to facilitate the identification of low abundant protein spots.
  • the excised gel plugs were subjected to distaining using a wash solution consisting of 70% ACN in 25 mM ammonium bicarbonate. The gel plugs were incubated this wash solution for 10 minutes. This washing procedure was repeated three times. Finally, the wash solution was removed followed by speed vaccing for 20 minutes.
  • High Sensitivity Micro-Tech Workstation for protein identification A microtechnology workstation was used for high sensitivity protein analysis and identification of targets and biomarkers within the study.
  • This microtech platform builds on chip integrated solid-phase microextraction array and a microdispenser for sample purification and trace enrichment of peptides as previously described (Wallman et al, Electrophoresis, 2004, 25, 3778-3787). Briefly, the capillary mircofluidic system is operated in an automated set-up. Chip- integrated sample clean-up of the protein sample, performed in a 96-array chip format.
  • the microextraction array was loaded with solid-phase media (Poros R2 50 ⁇ m beads) for purification and enrichment of proteomic samples.
  • Samples bound to the microchip were eluted in a volume of 100 nL.
  • the protein sample is eluted by utilizing a sequential capillary action that is docking to piezoelectric microdispencer.
  • the subsequent transfer is made to the MALSI TOF mass spectrometry target plate where typically a burst of 1000 droplets of the sample is spotted onto the MALDI plate.
  • MALSI TOF mass spectrometry target plate typically a burst of 1000 droplets of the sample is spotted onto the MALDI plate.
  • This micro-tech principle provide high quality data from samples in the picomolar range.
  • the built-in force feed-back control also further ensures precise and robust integration and interfacing of solid phase chip enrichment and piezodispencing technology.
  • the micro-proteomic platform was compared to corresponding commercial preparation protocols, showing higher MS signal intensities for peptides generated from the resulting 2D-gel spots.
  • LC-MALDI-MS/MS Reversed-phase chromatography was performed with an Agilent nano 1100 HPLC system (Agilent Technologies, Waldbronn, Germany). The HPLC effluent was directly fractionated onto a 144 position ABI MALDI target plate using an Agilent micro fraction collector and spots were deposited every 30 seconds during the gradient (90 spots/run). The spots were allowed to dry completely prior to addition of I ⁇ L of CHCA matrix. The MS/MS data from the MALDI-TOF/TOF instrument was acquired accordinging to a method previously described by Zhen et al. (J. Am. Soc. Mass Spectrom. 2004, 15, 803-822). A standard peptide mixture containing 6 peptides diluted to about 500 fmol/ ⁇ L were applied on the six calibration spot positions of the target plate and used as external calibration points.
  • MS/MS data obtained from the MALDI- TOF/TOF instrument were searched using Mascot as the search engine. All searches were performed against the human, rat, and mouse subset of in-house protein sequence databases (Genseq P, RefSeqP, PDB, PIR, SwissProt and TREMBL).
  • the Global Proteome Server (GPS) was used for submitting data acquired from the TOF/TOF for database searching.
  • the Mascot searching was performed using the default settings for the TOF/TOF instrument as supplied by Matrix Science (peptide mass tolerance of 50 ppm and a fragment mass tolerance of 0.2 Da). Oxidation (M) and carbamidomethyl were allowed as variable modifications.
  • VIP variable importance weights
  • Microsphere-based multiplex assays for ten proteins were developed using antigen-specific capture and detection antibodies in a sandwich format (the assay for ⁇ -2 macroglubulin is a competitive assay) and other optimized reagents. Due to different requirements on sample dilution a three-plex assay for alpha- 1-antichymotrypsin, alpha-2 macroglobulin and angiotensinogen, and a seven-plex assay for contactin 1, alpha-2-HS-glycoprotein (fetuin), fibulin-1, neural cell adhesion molecule 1 (NCAM 1), neuronal cell adhesion molecule(NrCAM), superoxide dismutase 1, vitamin D-binding protein were developed.
  • CSF samples Prior to assay CSF samples were pre-diluted 10-fold for the three-plex and 200-fold for the seven-plex. All incubations toke place at room temperature in the dark.
  • a diluted mixture of capture-antibody microspheres (5 ⁇ L) were mixed with 5 ⁇ L blocking buffer and 10 ⁇ L standard, pre-diluted sample or control. The plate was incubated for 1 hour.
  • 10 ⁇ L biotinylated detection antibody or for ⁇ -2 macroglubulin a biotinylated antigen
  • alpha- 1-antichymotrypsin alpha-2 macroglobulin, angiotensinogen, contactin 1, alpha-2-HS-glycoprotein (fetuin), fibulin-1, neural cell adhesion molecule 1 (NCAM 1), neuronal cell adhesion molecule(NrC AM), superoxide dismutase 1 , vitamin D-binding protein were measured using the multiplex assay.
  • Patient information and concentrations of each protein are provided in Table 7.
  • FIG. 1 A representative 2D-gel is shown in figure 1 and the locations of the 43 identified protein spots are zoomed in. Each spot is given a unique database SSP number by the PDQuest software.
  • Tables 2-5 show protein identities obtained by mass spectrometry analysis and estimated molecular weight/isoelectric point. Table 6 below exemplify a selection of proteins, which may be feasible for antibody-based validation.
  • Vitamin D-binding protein Mean 935 978 884 1240 872
  • Fibulin 1 Mean 5790 6930 6700 7090 6030
  • Vitamin D-binding protein Mean 908 916 1440 1210 (VDBP) SD 349 419 612 769
  • the sensitivity and specificity of the models was calculated using the average of LOO ("Leave-one-out”) cross-validation rounds at a range of cut-offs. These are presented as ROC (Receiver Operator Characteristic) plots in figure 2.
  • ROC Receiveiver Operator Characteristic
  • Protein concentrations were also compared based on pre vs. post Tysabri treatment.
  • patient data was modelled using a mixed effects model with patient as random effect and time as fixed effect. This model takes into account that observations from the same patients are correlated, but assumes that observations from different patients are independent. Six and 12 months duration data were modeled separately. Estimated differences and p-values are presented in Table 10. No adjustments for multiplicity were performed.
  • VDBP RR rem vs OND 1.09 0.91 1.30 0.3369
  • Alpha- 1 -antichymotrypsin 6 0.80 0.71 0.91 0.0029
  • Alpha-2-macroglobulin 6 0.82 0.66 1.02 0.0667
  • NCAM 1 6 0.70 0.54 0.90 0.0123
  • VDBP 6 0.69 0.48 1.01 0.0560

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Abstract

La présente invention concerne des marqueurs biologiques de la sclérose en plaques. Plus précisément, la présente invention concerne l'utilisation desdits marqueurs en vue du diagnostic de la sclérose en plaques, du suivi de la progression de la maladie lors d'essais cliniques ou précliniques, ainsi que du criblage et de la mise au point de médicaments.
PCT/SE2009/050885 2008-07-10 2009-07-09 Procédé et marqueurs inédits pour le diagnostic de la sclérose en plaques Ceased WO2010005387A1 (fr)

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WO2010136232A1 (fr) * 2009-05-28 2010-12-02 Fundació Institut De Recerca De L´Hospital Universitari Vall D'hebron Procédé in vitro approprié pour des patients souffrant d'un syndrome clinique isolé pour le diagnostic précoce ou le pronostic de la sclérose en plaques
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