JP2014517300A - Method for diagnosing multiple sclerosis - Google Patents

Abstract

  The present invention relates to the field of diagnostic methods. Specifically, the present invention provides a method for diagnosing multiple sclerosis in a subject, a method for identifying whether a subject requires treatment for multiple sclerosis, or determining whether multiple sclerosis treatment is successful. Intended how to do. Furthermore, it contributes to a method of diagnosing or predicting the risk of multiple sclerosis active state in a subject. The invention also relates to a tool for performing the above-described method, such as a diagnostic device.

Description

  The present invention relates to the field of diagnostic methods. Specifically, the present invention provides a method for diagnosing multiple sclerosis, a method for identifying whether a subject is in need of treatment for multiple sclerosis, or a method for determining whether multiple sclerosis treatment is successful. Intended. It further contributes to a method of diagnosing or predicting the active state or risk of progression of multiple sclerosis in a subject. The invention also relates to a tool for performing the above-described method, such as a diagnostic device.

  Multiple sclerosis (MS) is a very common disease of the central nervous system (CNS) that affects approximately 1,000,000 individuals worldwide and causes long-term severe disability in young adults. Although its etiology remains unclear, strong evidence supports the notion that a T cell-mediated inflammatory process for self-molecules in the white matter of the brain and spinal cord underlies its pathogenicity. Since myelin-reactive T cells are present in both MS patients and healthy individuals, primary immune abnormalities in MS are accompanied by dysfunctional regulatory mechanisms leading to enhanced T cell activation status and strict activation requirements Most likely. Thus, the pathogenesis is encephalitogenic, ie, activation of autoimmune myelin-specific T cells outside the CNS, followed by blood-brain barrier opening, T cell and macrophage infiltration, microglia activation, demyelination and Includes irreversible neuronal damage (Aktas 2005, Neuron 46, 421-432, Zamvil 2003, Neuron 38: 685-688 or Zipp 2006, Trends Neurosci. 29, 518-527). Much is known about the mechanisms responsible for the induction of T cell encephalitis, but little is known about the body's intrinsic regulatory mechanisms that regulate harmful lymphocyte responses to and in the CNS. . In addition, despite extensive research on T cell-mediated demyelination, the damage process within the CNS in vivo is not well understood.

  Currently, diagnostic tools such as neuroimaging, cerebrospinal fluid analysis and evoked potentials are used to diagnose MS. Demyelination (lesions or plaques) can be visualized by magnetic resonance images of the brain and spine. By administering gadolinium intravenously as a contrast agent to mark and remove active plaques, the presence of past lesions not associated with symptoms at the time of evaluation can be demonstrated. Analysis of cerebrospinal fluid taken from lumbar puncture provides evidence of chronic inflammation of the central nervous system. Cerebrospinal fluid can be analyzed for oligoclonal bands, which are inflammatory markers found in 75-85% of people with MS (Link 2006, J Neuroimmunol. 180 (1-2): 17 ~ 28 pages). However, none of the techniques described above are specific to MS alone. Therefore, in most cases, a reliable diagnosis can be obtained only from biopsy or autopsy.

  Multiple sclerosis is a chronic demyelinating disease of the central nervous system that shows various signs and symptoms. This diversity in clinical symptoms can lead to patient misdiagnosis, unnecessary specialist referrals and misleading information. Because MS is a clinically heterogeneous disease, diagnostic and prognostic markers are required to facilitate diagnosis and predict the course of the disease, the need for treatment, and the type of treatment in individual patients ( Bielekova and Martin 2004, Brain 127: 1463-1478). Responses to currently available treatments vary from patient to patient and no evidence is available from the course of the disease. Markers reduce the choice of drugs to use and will become more important in subsequent years if additional drugs are marketed. Furthermore, rapidly progressing patients should be treated more aggressively from the start than patients with a rather benign disease course. Tissue damage, specifically neuronal damage markers, may be expressed only in or with high expression in patients with rapid progression and subsequent injury. On the other hand, treatment of patients with aggressive treatment with potentially devastating side effects requires therapeutic response markers and risk management. Thus, biomarkers of disease activity and response to treatment can help determine patient prognosis and allow individual adjustment of treatment.

  Therefore, there is a great need for a means and method for reliably diagnosing MS and assessing the success of treatment, but it is not currently available.

Accordingly, the present invention is a method of diagnosing multiple sclerosis in a subject,
a) determining the amount of at least one ceramide or the total amount of ceramide in the sample of interest;
b) comparing the amount of said at least one ceramide, or the total amount of ceramide with a reference amount, whereby multiple sclerosis will be diagnosed.

In particular, the present invention is a method for diagnosing multiple sclerosis in a subject,
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 1 and / or Table 2 in the sample of interest;
b) comparing the amount of said at least one biomarker with a reference amount, thereby diagnosing multiple sclerosis.

  The method defined in accordance with the present invention includes a method consisting essentially of the steps described above or a method comprising further steps. However, it will be appreciated that in a preferred embodiment, the method is performed ex vivo, i.e. not performed on a human or animal body. The method can preferably be assisted by automation.

  As used herein, the term “diagnose” refers to determining whether a subject has the disease MS. As known to those skilled in the art, such a determination is preferably accurate for 100% of the subjects studied, but it is not usually the case. However, the term requires that a statistically significant portion of the subject can be correctly determined, i.e., diagnosed. Whether a part is statistically significant can be determined by using a variety of well-known statistical evaluation tools such as determination of confidence intervals, determination of p-value, Student's t test, Annova, Mann-Whitney test, etc. Can be determined immediately. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p value is preferably 0.2, 0.1, 0.05, 0.01, 0.005, 0.001.

  The term includes individual diagnosis of MS or its symptoms and continuous patient monitoring. Monitoring, ie, diagnosing the presence of MS or its associated symptoms at various time points, monitoring patients who are known to have MS and subjects known to be at risk of developing MS Monitoring is included. In addition, monitoring can be used to determine whether the patient's treatment is successful or whether the particular treatment is gradually improving at least the symptoms of MS.

  As used herein, the term “MS (multiple sclerosis)” relates to diseases of the central nervous system (CNS) that cause long-term severe disability in the subject. Methods for diagnosing MS are well known in the art. Specifically, MS can be diagnosed according to the criteria disclosed in Polman et al. (“Revised McDonald criteria”, Ann Neurol 2005; 58: 840-846) and is hereby incorporated by reference in its entirety for its entire disclosure. Incorporate into. The pathogenesis of MS is encephalitis-induced, ie activation of autoimmune myelin-specific T cells outside the CNS, followed by blood-brain barrier opening, T cell and macrophage infiltration, microglia activation, demyelination and irreversible Includes sexual neuron damage. MS has a unified definition of four subtypes. The following subtype definitions are also encompassed by the terminology used by the present invention: relapsing-remitting, secondary progressive, primary progressive, and progressive relapse. A relapsing-remitting subtype is characterized by an unpredictable relapse with months to years of remission with no new signs of disease activity. Defects experienced during seizures (active state) may recover or remain sequelae. This represents the initial course of 85-90% of subjects suffering from MS. In the case of so-called benign MS, the defect always recovers during the active state. Secondary progressive MS has an early relapsing-remitting MS, after which progressive neurodegeneration begins without a clear remission period during an acute attack. Occasional recurrence and mild remission may appear. The average period between disease onset and relapse-remitting to secondary progressive MS is about 19 years. Primary progressive subtype refers to about 10-15% of subjects who never remission after initial MS symptoms. This type is characterized by progression from onset, no remission and improvement, or a rare and minor disorder. The age of onset of the primary progressive subtype is later than the other subtypes. Progressive relapsing MS refers to a subject who has steadily reduced neuropathy since onset but also has an obvious mixed seizure. This type is the rarest of all subtypes. There are also some cases of atypical MS that cannot be assigned to the above subtype group. As used herein, the term “multiple sclerosis” also encompasses clinically isolated syndrome (often referred to as “CIS”).

  Symptoms associated with MS include sensory changes (decreased and abnormal sensations), muscle weakness, muscle cramps, movement difficulties, coordination and balance difficulties (ataxia), speech (articulation disorder) or swallowing problems (dysphagia), vision Problems include nystagmus, optic neuritis or diplopia, fatigue, headache, acute or chronic pain, bladder and bowel difficulties. Symptoms of variable amounts of cognitive impairment and emotional symptoms of depression or emotional instability may also occur. The primary clinical measure of disability progression and symptom severity is the Expanded Disability Status Scale (EDSS).

  Additional symptoms of MS are well known in the art and are described in standard medical textbooks such as Stedman or Pschyrembl.

  In studies conducted in connection with the present invention, the biomarkers shown in Tables 1 and 2 have been shown to be particularly useful for early diagnosis of MS. Therefore, the present invention particularly relates to a method for early diagnosis of MS, and thus diagnosis of MS at an early stage of MS. The initial stage of MS includes a period of preferably 3 years, more preferably 2 years, even more preferably 1 year, most preferably 6 months after the onset of MS. Also preferably, the early stage of MS includes the period from the first acute neurological event (seizure) of MS to the second acute neurological event (seizure) of MS. It can further encompass the period from the first acute neurological event (seizure) of MS to the third acute neurological event (seizure) of MS. A neurological event preferably refers to a demyelinating event, more preferably an inflammatory demyelinating event. Preferably, subjects in the early stages of MS have two lesions in the central nervous system. More preferably, the subject has a lesion in the central nervous system. Preferably, the lesion is a region of the central nervous system that is inflamed and / or demyelinated. Such a lesion can be detected, for example, by MRI. Preferably, the subject at an early stage also exhibits symptoms of MS. Preferred symptoms, particularly neurological symptoms, are disclosed elsewhere herein.

  As used herein, the term “biomarker” refers to a molecular species that functions as an indicator for a disease or effect as defined herein. The molecular species can be the metabolite itself found in the sample of interest. Furthermore, the biomarker may be a molecular species obtained from the metabolite. In such cases, the actual metabolite may be chemically modified in the sample or during the measurement process, and the modification results in a chemically different molecular species, i.e. the molecular species from which the analyte is determined. . In such a case, it will be appreciated that the analyte represents the actual metabolite and has the same potential as the indicator for each disease state.

  Furthermore, the biomarker according to the present invention does not necessarily correspond to one kind of molecular species. Rather, a biomarker may include a stereoisomer or an optical isomer of a compound. In addition, a biomarker can represent the sum of isomers of a biological classification of isomeric molecules. The isomers may in some cases exhibit the same analytical properties and are therefore indistinguishable by various analytical methods, including the analytical methods utilized in the accompanying examples described below. However, the isomers share at least the same total formula parameters and thus share the same chain length and the same number of double bonds in the fatty acid and / or sphingo base moiety, for example in the case of lipids.

  The biomarker used for the method of the invention is preferably ceramide. Ceramide is a lipid molecule composed of an amino alcohol and bound to a single fatty acid by an amide bond. Sphingosine (2-aminooctadeca-4-ene-1,3-diol). Preferably, the amino alcohol is sphingosine (2-aminooctadeca-4-ene-1,3-diol), a long chain aliphatic amine. Other amino alcohols are also referred to as “long-chain bases” (LCB) with different chain lengths, such as d16-, d17- or d20-sphingosine, and also give rise to their respective saturated amino alcohols (dihydrosphingosine or sphinganine). be able to. The nomenclature used to describe different ceramide species is `` Update of the LIPID MAPS comprehensive classification system for lipids ''; Fahy E., Subramaniam S., Murphy R., Nishijima M., Raetz C., Shimizu T. , Spener F., van Meer G., Wakelam M., and Dennis E., Journal of Lipid Research 50, S9-S14 (2009), followed the “Lipidmaps” classification scheme.

  Fatty acids attached to sphingosine can have various chain lengths and different degrees of saturation / unsaturation. Preferred ceramides are C21-, C22, C23 or C24 ceramide. Particularly preferred ceramides used as biomarkers in connection with the method of the present invention are listed herein below and / or in Table 1 and / or Table 2.

  Accordingly, the ceramide related to the method of the present invention is preferably ceramide (d16: 1, C22: 0), ceramide (d16: 1, C23: 0), ceramide (d16: 1, C24: 0), ceramide ( d17: 1, C22: 0), ceramide (d17: 1, C23: 0), ceramide (d17: 1, C24: 0), ceramide (d17: 1, C24: 1), ceramide (d18: 1, C21: 0), ceramide (d18: 1, C22: 0), ceramide (d18: 1, C22: 1), ceramide (d18: 1, C23: 0), ceramide (d18: 1, C23: 1), ceramide (d18 : 1, C24: 0), ceramide (d18: 1, C24: 1), ceramide (d18: 2, C22: 0), ceramide (d18: 2, C23: 0), ceramide (d18: 2, C24: 0) ) And ceramide (d18: 2, C24: 1).

  In a more preferred embodiment, the ceramide is ceramide (d16: 1, C22: 0), ceramide (d16: 1, C23: 0), ceramide (d16: 1, C24: 0), ceramide (d17: 1, C22: 0), ceramide (d17: 1, C23: 0), ceramide (d17: 1, C24: 0), ceramide (d17: 1, C24: 1), ceramide (d18: 1, C21: 0), ceramide (d18 : 1, C22: 0), ceramide (d18: 1, C22: 1), ceramide (d18: 1, C23: 0), ceramide (d18: 1, C23: 1), ceramide (d18: 2, C22: 0) ), Ceramide (d18: 2, C23: 0), ceramide (d18: 2, C24: 0) and ceramide (d18: 2, C24: 1). In one embodiment, the ceramide is not ceramide (d18: 1, C24: 0) or ceramide (d18: 1, C24: 1).

  Preferred methods for measuring ceramide are disclosed in the Examples or are known in the art (see, for example, Kasumov et al., Analytical Biochemistry 401 (2010) pages 154-161), see for full disclosure. Is incorporated herein by reference.

  In the method according to the invention, at least one metabolite of the above-mentioned group of biomarkers, ie the biomarkers shown in Table 1 and / or Table 2, is measured. More preferably, however, a group of biomarkers can be measured to enhance the specificity and / or sensitivity of the determination. Such groups preferably comprise at least two, at least three, at least four, at least five, at least ten or all of the biomarkers listed in the table. In addition to the specific biomarkers described herein, preferably other biomarkers may be further measured in the methods of the invention.

  In the context of the method of the invention, in step a) of the method of the invention, instead of the amount of at least one biomarker listed in Tables 1 to 3, specifically Tables 1 and 2, It is also envisaged to determine the total amount of ceramide. That is, the total ceramide content shall be determined. In step b), the total amount of ceramide shall be compared with a reference amount. Preferably, the total amount of C21 ceramide, C22 ceramide, C23 ceramide or C24 ceramide is determined. More preferably, the total amount of C21 to C24 ceramide is determined. Preferably, an increase in the total amount of ceramide compared to the reference is an indicator of the diagnosis of MS, specifically the early stage diagnosis of MS.

  In a preferred embodiment of the method of the invention, said at least one biomarker is selected from the group of biomarkers listed in Table 1a and / or Table 2a. Such an increase in biomarkers is an indicator of multiple sclerosis, specifically, an early stage of multiple sclerosis. Preferably, an increase of at least 10%, at least 20%, at least 30%, or at least 50% is an indicator of MS diagnosis (priority in order of preference). The same applies when the biomarker is ceramide. The same applies when the total amount of ceramide is determined in the sample.

  In another preferred embodiment of the method of the invention, said at least one biomarker is selected from the group of biomarkers listed in Table 1b and / or Table 2b. Such a decrease in biomarkers is an indicator of multiple sclerosis, specifically, the initial stage of multiple sclerosis. Preferably, a reduction of at least 10%, at least 20%, at least 30%, at least 50% is an indicator of MS diagnosis (priority in order of preference).

  In a preferred embodiment of the method of the invention, when the subject to be tested is suspected of being in the acute phase of MS, the amount of at least one biomarker shown in Table 2 is determined. Subjects suspected of having an acute phase of MS preferably exhibit symptoms of the acute phase of MS, while subjects not suspected of having an acute phase of MS preferably do not exhibit symptoms of the acute phase of MS.

  Where the subject to be tested is suspected of being in the acute phase of MS, preferably the at least one biomarker is selected from the group of biomarkers listed in Table 2a. Such an increase in biomarkers is an indicator of multiple sclerosis. Preferably, the increase is an increase of at least 10%, at least 20%, at least 30%, at least 50% (priority in descending order).

  Where the subject to be tested is suspected of being in the acute phase of MS, preferably the at least one biomarker is selected from the group of biomarkers listed in Table 2b. Such a decrease in biomarkers is indicative of multiple sclerosis.

  As used herein, a metabolite refers to a molecule of at least one specific metabolite up to a plurality of molecules of the specific metabolite. It will be further understood that the group of metabolites means a plurality of chemically different molecules, and for each metabolite, there can be at least one molecule up to a plurality of molecules. The metabolites according to the present invention include all classes of organic or inorganic compounds including those contained in biological materials such as living organisms. Preferably, the metabolite according to the present invention is a small molecule compound. More preferably, when multiple types of metabolites are envisioned, the multiple types of metabolites are metabolomics (i.e., metabolites contained in an organism, organ, tissue, body fluid or cell at a specific time and under specific conditions). Collection).

  Metabolites are small molecule compounds such as enzyme substrates of metabolic pathways, intermediate products of such pathways or products obtained by metabolic pathways. Metabolic pathways are well known in the art and may vary between species. Preferably, the pathway includes at least a citrate cycle, respiratory chain, glycolysis, gluconeogenesis, hexose monophosphate pathway, oxidative pentose phosphate pathway, fatty acid production and β-oxidation, urea cycle, amino acid biosynthesis pathway , Proteolytic pathways (such as proteasome degradation), amino acid degradation pathways, lipids, polyketides (including flavonoids and isoflavonoids), isoprenoids (including terpenes, sterols, steroids, carotenoids, xanthophylls), carbohydrates, phenylpropanoids and Derivatives, alkaloids, benzenoids, indoles, indole sulfur compounds, porphyrins, anthocyanins, hormones, vitamins, cofactors (such as prosthetic groups or electron carriers), lignins, glucosinolates, purines, pyrimidines, nucleosides, nucleotides Related molecules include biosynthesis or degradation pathway (tRNA, micro RNA (miRNA) or mRNA, etc.). Thus, small molecule compound metabolites are preferably of the following compound types: alcohols, alkanes, alkenes, alkynes, aromatics, ketones, aldehydes, carboxylic acids, esters, amines, imines, amides, cyanides, amino acids, It is composed of peptides, thiols, thioesters, phosphate esters, sulfate esters, thioethers, sulfoxides, ethers or combinations or derivatives of the aforementioned compounds. Small molecules of metabolites can be primary metabolites required for normal cellular function, organ function or animal growth, development or health. In addition, small molecule metabolites further include secondary metabolites having essential ecological functions, such as metabolites that allow an organism to adapt to its environment. Furthermore, metabolites are not limited to the primary and secondary metabolites, but further include artificial small molecule compounds. The artificial small molecule compound is derived from an exogenously provided small molecule that is administered or taken up by an organism, but is not a primary or secondary metabolite as defined above. For example, an artificial small molecule compound may be a metabolite obtained from a drug by the animal's metabolic pathway. In addition, metabolites further include peptides, oligopeptides, polypeptides, oligonucleotides and polynucleotides (such as RNA or DNA). More preferably, the metabolite is 50 Da (Dalton) to 30,000 Da, most preferably less than 30,000 Da, less than 20,000 Da, less than 15,000 Da, less than 10,000 Da, less than 8,000 Da, less than 7,000 Da, less than 6,000 Da, less than 5,000 Da Less than 4,000 Da, less than 3,000 Da, less than 2,000 Da, less than 1,000 Da, less than 500 Da, less than 300 Da, less than 200 Da, less than 100 Da. However, preferably the metabolite has a molecular weight of at least 50 Da. Most preferably, the metabolite according to the present invention has a molecular weight of 50 Da up to 1,500 Da.

  As used herein, the term “sample” refers to a sample derived from a bodily fluid (preferably blood, plasma, serum, saliva, urine or cerebrospinal fluid) or, for example, a cell, tissue or organ (specifically by biopsy). Refers to a sample obtained from the CNS including the brain and spine. More preferably, the sample is a blood, plasma or serum sample, most preferably a serum or plasma sample. The biological sample is obtained from a subject specified elsewhere herein. Techniques for obtaining the different types of biological samples described above are well known in the art. For example, a blood sample is obtained by collecting blood when collecting a tissue or organ sample (eg, by biopsy).

  Preferably, the sample described above is used in the method of the invention after pretreatment. As will be described in more detail later, the pretreatment may include treatments necessary to liberate or separate compounds or to remove excess material or waste. Suitable techniques include centrifugation, extraction, fractionation, ultrafiltration, protein precipitation, subsequent filtration and purification, and / or compound concentration. In addition, other pretreatments are performed to obtain the compound in the form or concentration suitable for compound analysis. For example, when gas chromatography coupled mass spectrometry is used in the method of the present invention, it is necessary to derivatize the compound prior to the gas chromatography. Appropriate and necessary pretreatment depends on the means used to carry out the method of the invention and is well known to those skilled in the art. As mentioned above, pretreated samples are also included in the term “sample” as used by the present invention.

  The term “subject” as used herein relates to animals and preferably mammals. More preferably, the subject is a primate, most preferably a human. Preferably, the subject may be suspected of having MS, ie already exhibiting some or all of the symptoms associated with the disease.

  The term “determining the amount” as used herein refers to measuring at least one property of the biomarker to be measured in a sample by the method of the invention. Properties according to the invention are features that characterize physical and / or chemical properties, including biochemical properties of biomarkers. Such properties include, for example, molecular weight, viscosity, density, charge, rotation, optical activity, color, fluorescence, chemiluminescence, elemental composition, chemical structure, ability to react with other compounds, biological readouts. out) system (for example, induction of reporter gene) and the like. The value of the property may function as a property and can be determined by techniques well known in the art. Further, the property may be any feature obtained from the physical and / or chemical property values of the biomarker by standard operations, for example, mathematical calculations such as multiplication, division or logarithmic calculation. Most preferably, at least one characteristic allows determination and / or chemical identification of the at least one biomarker and its amount. Therefore, preferably, the characteristic value also includes information regarding the abundance of the biomarker from which the characteristic value is obtained. For example, the characteristic value of the biomarker may be a mass spectrum peak. Such peaks include biomarker characteristic information, ie m / z information (mass / charge ratio or index) and intensity values related to the abundance of the biomarker in the sample (ie its amount).

  As mentioned above, preferably each biomarker contained in a sample can be measured quantitatively or semi-quantitatively according to the present invention. For quantitative measurements, either the absolute amount or the exact amount of the biomarker will be determined, and the relative amount of the biomarker was determined for the characteristic (s) described herein above. It will be determined based on the value. If the exact amount of biomarker cannot or cannot be determined, the relative amount can be determined. In such cases, it can be determined whether the abundance of the biomarker is increased or decreased relative to a second sample comprising the biomarker in a second amount. In a preferred embodiment, the second sample containing the biomarker is a calculated reference as specified elsewhere herein. Thus, quantitative analysis of biomarkers includes what is sometimes referred to as semi-quantitative analysis of biomarkers.

Furthermore, the measurement used in the method of the present invention preferably comprises using the analytical step defined above after the compound separation step. Preferably, the compound separation step provides time-resolved separation of metabolites contained in the sample. Thus, suitable separation techniques used by the present invention preferably include liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography, size exclusion or affinity. All chromatographic separation techniques such as chromatography are included. These techniques are well known in the art and are readily available to those skilled in the art. Most preferably, LC and / or GC are chromatographic techniques envisioned by the method of the present invention. Suitable devices for such determination of biomarkers are well known in the art. Preferably, mass spectrometry, specifically gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), direct injection mass spectrometry or Fourier transform ion cyclotron resonance mass spectrometry ( FT-ICR-MS), capillary electrophoresis mass spectrometry (CE-MS), high performance liquid chromatography coupled mass spectrometry (HPLC-MS), quadrupole mass spectrometry, arbitrary coupled mass spectrometry (MS-MS) Or MS-MS-MS), inductively coupled plasma mass spectrometry (ICP-MS), pyrolysis mass spectrometry (Py-MS), ion mobility mass spectrometry or time-of-flight mass spectrometry (TOF) Is done. Most preferably, LC-MS and / or GC-MS is used as detailed below. Such techniques are disclosed, for example, in Nissen 1995, Journal of Chromatography A, 703: 37-57, US Pat. No. 4,540,884 or US Pat. No. 5,397,894, the disclosure of which is incorporated herein by reference. As a variant or in addition to mass spectrometry techniques, the following techniques can be used for compound measurements: nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), Fourier transform infrared analysis (FT-IR) , Ultraviolet spectroscopy (UV), refractive index (RI), fluorescence detection, radiochemical detection, electrochemical detection, light scattering (LS), dispersive Raman spectroscopy or flame ionization detection (FID). These techniques are well known to those skilled in the art and are readily available. The method of the invention should preferably be assisted by automation. For example, sample processing or pretreatment can be automated by robotics. Preferably, data processing and comparison is supported by a suitable computer program and database. As previously described herein, automation allows the method of the present invention to be used in a high throughput manner.

Furthermore, at least one biomarker can be measured by a specific chemical or biological assay. Said assay shall comprise means enabling specific detection of at least one biomarker in the sample. Preferably, the means is capable of specifically recognizing the chemical structure of the biomarker and is capable of reacting with other compounds or inducing a response in a biological readout system (e.g., induction of a reporter gene). Based on this, it is possible to specifically identify the biomarker. The means capable of specifically recognizing the chemical structure of a biomarker is preferably an antibody or other protein that interacts specifically with a chemical structure such as a receptor or enzyme. Specific antibodies can be obtained, for example, using biomarkers as antigens by methods well known in the art. Antibodies as defined herein include both polyclonal and monoclonal antibodies, as well as fragments thereof (such as Fv, Fab and F (ab) ₂ fragments capable of binding to antigen or hapten). The invention also includes humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody that exhibits the desired antigen specificity is combined with the sequence of a human acceptor antibody. In addition, single chain antibodies are included. The donor sequence will usually include at least the antigen-binding amino acid residues of the donor, but may also include other structurally and / or functionally important donor antibody amino acid residues. Such hybrids can be prepared by several methods well known in the art. A suitable protein capable of specifically recognizing a biomarker is preferably an enzyme involved in metabolic conversion of the biomarker. The enzyme may use a biomarker as a substrate or convert the substrate into a biomarker. Furthermore, the antibodies can be used as a basis for generating oligopeptides that specifically recognize biomarkers. These oligopeptides include, for example, an enzyme binding domain or pocket for the biomarker. Suitable antibody and / or enzyme based assays include RIA (radioimmunoassay), ELISA (enzyme linked antibody immunoassay), sandwich enzyme immunoassay, electrochemiluminescence sandwich immunoassay (ECLIA), dissociation- It can be an enhanced lanthanide fluorescent immunoassay (DELFIA) or a solid phase immunoassay. Furthermore, biomarkers can also be determined based on their ability to react with other compounds (ie by specific chemical reactions). Furthermore, biomarkers can be measured in samples by their ability to induce responses in biological readout systems. The biological response shall be detected as a readout indicating the presence and / or amount of biomarker contained in the sample. A biological response can be, for example, the induction of gene expression or a phenotypic response of a cell or organism. In a preferred embodiment, determination of at least one biomarker is a quantitative process, for example, allowing determination of the amount of at least one biomarker present in a sample.

  As described above, the measurement of at least one biomarker may preferably include mass spectrometry (MS). Mass spectrometry as used herein encompasses all techniques that allow the determination of the molecular weight (ie, mass) or mass variation corresponding to a compound, ie, a biomarker to be measured according to the present invention. Preferably, the mass spectrometry used herein is GC-MS, LC-MS, direct injection mass spectrometry, FT-ICR-MS, CE-MS, HPLC-MS, quadrupole mass spectrometry, It relates to any coupled mass spectrometry (such as MS-MS or MS-MS-MS), ICP-MS, Py-MS, TOF or any combination technique using the techniques described above. Methods utilizing these techniques are well known to those skilled in the art. In addition, suitable equipment is commercially available. More preferably, mass spectrometry as used herein relates to LC-MS and / or GC-MS, ie mass spectrometry operatively linked to a previous chromatographic separation step. More preferably, mass spectrometry as used herein includes quadrupole MS. Most preferably, the quadrupole MS is: a) selection of the mass / charge index (m / z) of ions produced by ionization in the first analytical quadrupole of the mass spectrometer, and b) the collision gas is Fragmentation of the ions selected in step a) by applying an acceleration voltage in the further next quadrupole that is filled and functioning as a collision chamber, and c) in the further next quadrupole, the step b) by the selection of the mass / charge index of the ions produced by the fragmentation process, wherein steps a) to c) of the method are carried out at least once, in the mixture of substances as a result of the ionization process An analysis of the mass / charge index of all ions present is performed, in which the quadrupole is filled with a collision gas, but no accelerating voltage is applied during the analysis. Details regarding the most preferred mass spectrometry used according to the invention can be found in WO03 / 073464.

  More preferably, the mass spectrometry is liquid chromatography (LC) MS and / or gas chromatography (GC) MS. Liquid chromatography as used herein refers to any technique that allows for the separation of compounds (ie, metabolites) in a liquid or supercritical phase. Liquid chromatography is characterized in that the compound in the mobile phase passes through the stationary phase. Since each individual compound has a unique retention time (ie, the time required for the compound to pass through the system), it is separated in time as the compound passes through the stationary phase at different rates. Liquid chromatography as used herein also includes HPLC. Liquid chromatography equipment is commercially available from, for example, Agilent Technologies (USA). The gas chromatography utilized by the present invention works in principle as liquid chromatography. However, rather than having the compound (ie, metabolite) in the liquid mobile phase that passes through the stationary phase, the compound will be present in the gas volume. The compound passes through a column that can contain a solid support material as the stationary phase or a wall that can function as or is coated with the stationary phase. Again, each compound has a unique time required to pass through the column. Further, in the case of gas chromatography, it is preferably assumed that gas chromatography is performed after derivatization of the compound. Suitable techniques for derivatization are well known in the art. Preferably, derivatization according to the invention preferably relates to methoxymation and trimethylsilylation of polar compounds, and preferably to methyl group transfer, methoxylation and trimethylsilylation of nonpolar (ie lipophilic) compounds.

  The term “reference” refers to the characteristic value of each of the biomarkers associated with a disease state (ie, the presence or absence of a disease), a disease state, or an effect as defined herein. Where the biomarker is selected from the biomarkers shown in Table 1a, Table 2a, or Table 3a, preferably the reference is a threshold amount for the biomarker that is higher or essentially higher than the threshold in the sample to be investigated The same amount is an indicator of the presence of a medical condition, while a lower amount is an indicator of the absence of a medical condition. When the biomarker is selected from the biomarkers shown in Table 1b, Table 2b, or Table 3b, preferably the reference is a threshold amount for the biomarker and is below or identical to the threshold in the sample to be investigated. An amount is an indicator of the presence of a medical condition, while a higher amount is an indicator of the absence of a medical condition.

  According to the above method of the invention, the reference is preferably a reference amount obtained from a sample derived from a subject known to suffer from MS. Specifically, the reference amount is obtained from a sample derived from a subject known to be in the early stages of MS. Preferably, the amount of at least one biomarker in essentially the same test sample is indicative of the presence of the disease.

  Furthermore, the reference is preferably also derived from a subject known not to suffer from MS, preferably an apparently healthy subject. More preferably, the reference is obtained from a subject (or from the group of subjects) known to be free of MS but exhibiting MS symptoms. Thus, the subject may exhibit MS-like symptoms. The subject's MS or MS-like symptoms will be caused by a neurological disorder other than MS. Preferred symptoms are disclosed elsewhere herein. Furthermore, the symptoms are preferentially similar or identical to MS symptoms, sensory changes (decreased sensation and sensory abnormalities), muscle weakness, muscle spasms, difficulty in movement, coordination and balance difficulties (ataxia), conversation ( Articulation disorders) or swallowing problems (dysphagia), visual problems (nystagmus, optic neuritis or double vision), fatigue, headache. Where the reference amount is obtained from the above-mentioned subject or group thereof, the amount of at least one biomarker found in the test sample that is altered relative to the reference is an indication of the presence of the disease. Preferably, the amount varies by at least 10%, at least 20%, at least 30%, at least 50%. Preferably, if the biomarker is selected from the group of biomarkers shown in Table 1a or Table 2a (and if the biomarker is ceramide or if the total amount of ceramide is determined), compare to the reference. The amount of biomarker that is increasing in the test sample is an indicator of the presence of MS. Preferably, when the biomarker is selected from the group of biomarkers shown in Table 1b or Table 2b, the amount of biomarker that is decreased in the test sample relative to the reference is an indication of the presence of MS Become.

  The same applies mutatis mutandis to the calculated reference (most preferably the mean or median) and to the relative or absolute amount of at least one biomarker of the population of individuals comprising the study subject. As specified elsewhere herein, the absolute or relative amount of at least one biomarker of the individual in a population can be determined. Methods for calculating an appropriate reference value, preferably an average or median value are well known in the art. The previously defined population of subjects should include multiple subjects, preferably at least 5, 10, 50, 100, 1,000 or 10,000 subjects. It will be understood that the subject to be diagnosed by the method of the present invention and the subject of the plurality of subjects are the same species.

  If the characteristic value and (in the case of quantitative measurement) the intensity value are essentially the same, the amount of the test sample and the reference amount are essentially the same. Essentially identical means that the difference between the two quantities is preferably not significant, and the intensity value is at least the 1-99 percentile, 5-95 percentile, 10-90 percentile, 20-80 of the reference value. It will be characterized by a percentile, a range of 30-70 percentile, a range of 40-60 percentile, preferably a reference value of 50, 60, 70, 80, 90 or 95 percentile. Statistical tests for determining whether two quantities are essentially identical are well known in the art and are described elsewhere herein.

  On the other hand, the difference observed for the two quantities is statistically significant. The difference in relative or absolute amount is preferably in the range of 45-55 percentile, 40-60 percentile, 30-70 percentile, 20-80 percentile, 10-90 percentile, 5-95 percentile, 1-99 percentile of the reference value Is significantly different. Preferred changes and magnification adjustments are described in the accompanying tables and examples.

  Preferably, the reference, ie the value of at least one characteristic of the at least one biomarker, is stored in a suitable data storage medium such as a database and can therefore be used for further determination.

  The term “comparison” refers to determining whether the amount of biomarker determined is essentially the same or different from the reference. Preferably, a biomarker is considered different from a reference if the observed difference that can be determined by statistical techniques defined elsewhere in this description is statistically significant. If the difference is not statistically significant, the amount of the biomarker and the reference amount are essentially the same. Based on the above comparison, it can be determined whether the subject is suffering from a disease.

  For certain biomarkers described herein, a preferred value (i.e., modulation `` magnification '') or change type (i.e., `` up '' or `` down '') as a result of a change in relative amount, relative to (Or absolute amounts are obtained) are shown in the following tables and examples. If the table indicates that a given biomarker is “up-regulated” in a subject, the relative and / or absolute amount will have increased, and if it is “down-regulated” The relative and / or absolute amount of marker will be reduced. For “upward” adjustments, the median ratio will be greater than 1.0, and for “downward” adjustments, it will be less than 1.0. Furthermore, the “magnification” change indicates the degree of increase or decrease, for example, a two-fold increase means that the median of one group (eg, MS group) is the median of biomarkers of another group (eg, reference group) Means twice as much. Therefore, the direction of adjustment is obtained from the table as well.

  Preferably, the comparison is assisted by automation. For example, a suitable computer program that includes an algorithm for comparing two different data sets (eg, a data set that includes the characteristic value (s)) can be used. Such computer programs and algorithms are well known in the art. Despite the above, the comparison can also be performed manually.

  Conveniently, it was found in the basic study of the present invention that the amount of the specific biomarker is indicative of MS. Thus, it is possible in principle to determine whether the subject is MS using at least one biomarker specified above in the sample. This is particularly useful for effective diagnosis of disease and improved preclinical and clinical management of MS, as well as effective patient monitoring. Furthermore, as described in detail below, the basic discovery of the present invention also facilitates the development of effective drug therapies for MS.

  The definitions and explanations of the terms made above apply mutatis mutandis to the following embodiments of the present invention, except as otherwise specified herein below.

Further, the present invention provides i) a subject who exhibits symptoms of multiple sclerosis and suffers from multiple sclerosis and ii) a subject who exhibits symptoms of multiple sclerosis but does not suffer from multiple sclerosis. A method of discrimination, wherein the method
a) determining the amount of at least one ceramide, or the total amount of ceramide, in a sample of a subject exhibiting multiple sclerosis symptoms;
b) a method of comparing the amount of said at least one ceramide or the total amount of ceramide with at least one reference amount, whereby i) and ii) are differentiated.

Specifically, the present invention relates to i) subjects with multiple sclerosis symptoms and suffering from multiple sclerosis and ii) multiple sclerosis symptoms but not suffering from multiple sclerosis. A method for differentiating an object, wherein the method
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 1 and / or Table 2 in a sample of a subject exhibiting multiple sclerosis symptoms;
b) comparing the amount of said at least one biomarker with at least one reference amount, whereby i) and ii) are differentiated.

  As used herein, the term “differentiate” preferably refers to a subject afflicted with multiple sclerosis that exhibits symptoms of multiple sclerosis and symptoms of multiple sclerosis, but multiple sclerosis Means to distinguish from subjects not suffering from. Therefore, according to the above-described method, it is possible to determine whether or not a subject exhibiting multiple sclerosis symptoms suffers from MS. As is known to those skilled in the art, such a determination is usually not intended to be accurate for 100% of the subjects to be differentially diagnosed. However, the term requires that a statistically significant portion of the subject can be correctly diagnosed. The accuracy of the diagnosis can be confirmed by methods well known in the art. Furthermore, whether a part is statistically significant can be determined using various well-known statistical evaluation tools (for example, determination of confidence interval, determination of p-value, student's t test, Mann-Whitney test, ANOVA, etc.). Can be readily determined by one skilled in the art. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p value is preferably 0.2, 0.1, 0.05, 0.01, 0.005, or 0.0001.

  In a preferred embodiment of the above method, i) a subject who exhibits symptoms of multiple sclerosis and suffers from an early stage of multiple sclerosis; and ii) who exhibits symptoms of multiple sclerosis but suffers from multiple sclerosis. Differentiated objects are identified.

  The term “subject” is defined elsewhere in this specification. According to the method described above, preferably the subject will exhibit symptoms of MS, in particular neurological symptoms of MS. A subject who exhibits symptoms of MS but is not suffering from MS is preferably a neurological disorder other than MS. Preferred diseases are disclosed in the Examples section.

  MS symptoms were described elsewhere in this specification. Preferred symptoms include sensory changes (sensory decline and sensory abnormalities), muscle weakness, muscle spasms, difficulty in movement, difficulty in coordination and balance (ataxia), speech (articulation disorder) or swallowing problems (dysphagia), visual problems (eye problems) Tremor, visual neuritis or double vision), fatigue, headache. Preferably, a subject exhibiting symptoms of MS may have a headache, specifically migraine, decreased perception, abnormal sensation or meningitis or a combination thereof.

  In a preferred embodiment of the method of the invention, the at least one biomarker is selected from the group of biomarkers listed in Table 1a and / or Table 2a. An increase in such a biomarker compared to a reference preferably indicates that the subject exhibiting symptoms of MS suffers from MS, but there is no change or amount of such biomarker compared to the reference Preferably indicates that the subject exhibiting MS symptoms is not suffering from MS. The same applies when the biomarker is ceramide. The same applies when the total amount of ceramide is determined in the sample. Preferred references have been disclosed elsewhere herein.

  Preferably, said increase or decrease is an increase / decrease of at least 10%, at least 20%, at least 30%, or at least 50% (priority in descending order).

  In a further preferred embodiment of the method of the invention, the at least one biomarker is selected from the group of biomarkers listed in Table 1b and / or Table 2b. A decrease in such a biomarker compared to a reference preferably indicates that a subject exhibiting symptoms of MS suffers from MS, but there is no change or amount of such biomarker compared to the reference Preferably indicates that the subject exhibiting symptoms of MS is not suffering from MS. Preferred references are disclosed above.

  If the subject to be tested is suspected of being in the acute phase of MS, preferably the at least one biomarker is selected from the group of biomarkers listed in Table 2, Table 2a and Table 2b, respectively.

  The present invention is a method for identifying whether a subject is in need of treatment for multiple sclerosis, comprising the steps of the method described above for diagnosing MS, and multiple sclerosis when diagnosed with multiple sclerosis. And a further step of identifying a subject in need of treatment.

  As used herein, the phrase “in need of treatment for multiple sclerosis” refers to the subject's disease requiring or beneficial therapeutic intervention to ameliorate or treat MS or a related condition. It means being in a situation. Thus, the discovery of basic research of the present invention not only allows for the diagnosis of MS in a subject, but also allows the identification of subjects to be treated with MS therapy. Once the subject has been identified, the method can further comprise recommending treatment for MS.

  The treatment of multiple sclerosis used by the present invention is preferably interferon β1a, interferon β1b, azathioprine, cyclophosphamide, galatiramel acetate, immunoglobulin methotrexate, mitoxantrone, leustatin, IVIg, natalizumab, teriflunomide, statin , Daclizumab, alemtuzumab, rituximab, sphingosine monophosphate antagonist (fingolimod (FTY720)), cladribine, fumarate, laquinimod, drugs that act on B cells, and antisense drugs and drugs against CD49d It relates to a treatment comprising or consisting of the administration of at least one drug.

Further, the present invention is a method for determining whether multiple sclerosis treatment is successful,
a) determining the total amount of at least one ceramide or ceramide in the first and second samples of interest, wherein the first sample is taken before or at the start of multiple sclerosis treatment And wherein the second sample is taken after initiation of the treatment;
b) comparing the amount of the at least one ceramide in the first sample or the total amount of ceramide with the amount or total amount in the second sample.

  In the context of the above-described method, a decrease in the amount measured in the second sample compared to the first sample is preferably an indication that multiple sclerosis treatment has been successful. An increase in the amount measured in the second sample compared to the first sample preferably indicates that the multiple sclerosis treatment has not been successful. Preferably, the increase is an increase of at least 10%, at least 20%, at least 30%, or at least 50% (priority in descending order).

  The term “ceramide” is described elsewhere in this specification. Preferably, the ceramide is selected from the ceramides listed in Table 1 and / or Table 2.

  Accordingly, the ceramide related to the method of the present invention is preferably ceramide (d16: 1, C22: 0), ceramide (d16: 1, C23: 0), ceramide (d16: 1, C24: 0), ceramide ( d17: 1, C22: 0), ceramide (d17: 1, C23: 0), ceramide (d17: 1, C24: 0), ceramide (d17: 1, C24: 1), ceramide (d18: 1, C21: 0), ceramide (d18: 1, C22: 0), ceramide (d18: 1, C22: 1), ceramide (d18: 1, C23: 0), ceramide (d18: 1, C23: 1), ceramide (d18 : 1, C24: 0), ceramide (d18: 1, C24: 1), ceramide (d18: 2, C22: 0), ceramide (d18: 2, C23: 0), ceramide (d18: 2, C24: 0) ) And ceramide (d18: 2, C24: 1).

  In a more preferred embodiment, the ceramide is ceramide (d16: 1, C22: 0), ceramide (d16: 1, C23: 0), ceramide (d16: 1, C24: 0), ceramide (d17: 1, C22: 0), ceramide (d17: 1, C23: 0), ceramide (d17: 1, C24: 0), ceramide (d17: 1, C24: 1), ceramide (d18: 1, C21: 0), ceramide (d18 : 1, C22: 0), ceramide (d18: 1, C22: 1), ceramide (d18: 1, C23: 0), ceramide (d18: 1, C23: 1), ceramide (d18: 2, C22: 0) ), Ceramide (d18: 2, C23: 0), ceramide (d18: 2, C24: 0) and ceramide (d18: 2, C24: 1). In one embodiment, the ceramide is not ceramide (d18: 1, C24: 0) or ceramide (d18: 1, C24: 1).

Furthermore, the present invention is a method for determining whether multiple sclerosis treatment is successful,
a) measuring at least one biomarker selected from the biomarkers listed in Table 1 and / or Table 2 in the first and second samples of the subject, wherein the first sample is a multiple sclerosis treatment The second sample is taken before or at the start of the treatment, and the second sample is taken after the treatment starts;
b) comparing the amount of the at least one biomarker in the first sample with the amount in the second sample, whereby the change in the amount determined in the second sample compared to the first sample And a step that is an indicator of successful treatment of multiple sclerosis.

  It will be appreciated that MS treatment will be successful if MS or at least some of its symptoms can be treated or ameliorated compared to an untreated subject. Furthermore, treatment can also be successful in the sense herein if disease progression can be prevented or at least slowed compared to an untreated subject.

  In a preferred embodiment of the above method, the at least one biomarker is selected from the biomarkers listed in Table 1a and / or Table 2a. In such a case, the change in step b) is a decrease. Thus, a decrease preferably indicates that the multiple sclerosis treatment is successful. Preferably, said reduction is a reduction of at least 10%, at least 20%, at least 30%, at least 50% (highest priority). The same applies when the biomarker is ceramide. The same applies when the total amount of ceramide is determined in the sample.

  In another more preferred embodiment of the above method, the at least one biomarker is selected from the biomarkers listed in Table 1b and / or Table 2b. In such a case, the change in step b) is an increase. Thus, an increase preferably indicates that the multiple sclerosis treatment is successful.

The present invention is further a method of diagnosing the active state of multiple sclerosis in a subject comprising:
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 3 in the sample of interest;
b) comparing the amount of said at least one biomarker with a reference amount, whereby the active state of multiple sclerosis will be diagnosed.

  In a preferred embodiment of the above method, the at least one biomarker is selected from the group of biomarkers listed in Table 3a, and an increase in the at least one biomarker is an indicator of the active state of MS. Preferably, the increase is an increase of at least 10%, at least 20%, at least 30%, at least 50% (highest priority).

  In another preferred embodiment of the above method, the at least one biomarker is selected from the group of biomarkers listed in Table 3b, and a decrease in the at least one biomarker is indicative of the activity state of MS. Preferably, said reduction is a reduction of at least 10%, at least 20%, at least 30%, at least 50% (highest priority).

  Preferably, the method of diagnosing the active state of MS in a subject can also be used to monitor disease progression. Specifically, when determining the presence of an active state in all samples taken at a particular time window with samples that do not exhibit a remission state, the method allows the progression of disease, for example, from relapsing-remitting subtypes to An indicator of progress to the next progressive subtype is also obtained. Thus, the frequency and number of active states that can be diagnosed by the methods of the present invention can be used to further diagnose the malignancy and progression of MS.

The present invention is a method for predicting whether a subject is at risk of developing multiple sclerosis, comprising:
a) determining the amount of at least one ceramide or the total amount of ceramide in the sample of interest;
b) comparing the amount of said at least one ceramide, or the total amount of ceramide with a reference amount, thereby predicting whether the subject is at risk of developing multiple sclerosis. Also related.

  Preferred ceramides are described elsewhere in this specification. Preferably, the ceramide is selected from the ceramides listed in Table 1 and / or Table 2.

  Accordingly, the ceramide related to the method of the present invention is preferably ceramide (d16: 1, C22: 0), ceramide (d16: 1, C23: 0), ceramide (d16: 1, C24: 0), ceramide ( d17: 1, C22: 0), ceramide (d17: 1, C23: 0), ceramide (d17: 1, C24: 0), ceramide (d17: 1, C24: 1), ceramide (d18: 1, C21: 0), ceramide (d18: 1, C22: 0), ceramide (d18: 1, C22: 1), ceramide (d18: 1, C23: 0), ceramide (d18: 1, C23: 1), ceramide (d18 : 1, C24: 0), ceramide (d18: 1, C24: 1), ceramide (d18: 2, C22: 0), ceramide (d18: 2, C23: 0), ceramide (d18: 2, C24: 0) ) And ceramide (d18: 2, C24: 1).

  In a more preferred embodiment, the ceramide is ceramide (d16: 1, C22: 0), ceramide (d16: 1, C23: 0), ceramide (d16: 1, C24: 0), ceramide (d17: 1, C22: 0), ceramide (d17: 1, C23: 0), ceramide (d17: 1, C24: 0), ceramide (d17: 1, C24: 1), ceramide (d18: 1, C21: 0), ceramide (d18 : 1, C22: 0), ceramide (d18: 1, C22: 1), ceramide (d18: 1, C23: 0), ceramide (d18: 1, C23: 1), ceramide (d18: 2, C22: 0) ), Ceramide (d18: 2, C23: 0), ceramide (d18: 2, C24: 0) and ceramide (d18: 2, C24: 1). In one embodiment, the ceramide is not ceramide (d18: 1, C24: 0) or ceramide (d18: 1, C24: 1).

Furthermore, the present invention is a method for predicting whether a subject is at risk of developing multiple sclerosis, comprising:
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 1 and / or Table 2 in the sample of interest;
b) comparing the amount of said at least one biomarker with a reference amount, thereby predicting whether the subject is at risk of developing multiple sclerosis.

  As used herein, the term “predict” generally determines the probability that a subject will develop a medical condition or its associated symptoms within a specific time window (ie, the prediction window) after taking a sample. Refers to that. It will be appreciated that such predictions may not be accurate for all people surveyed (100%). However, this prediction is assumed to be accurate for a statistically significant portion of subjects in a population of subjects (eg, subjects of a cohort study). Whether a portion is statistically significant can be determined by statistical techniques described elsewhere herein.

  In a preferred embodiment of the above method for predicting whether a subject is at risk of developing multiple sclerosis, for one or more further samples of the subject taken after taking the (first) sample mentioned above Repeat this method. Therefore, the prediction power of the present method can be further enhanced by repeating the prediction several times after the initial prediction is performed.

The present invention further relates to a method for predicting whether a subject is at risk of developing an active state of multiple sclerosis. The method
a) determining the amount of at least one ceramide or the total amount of ceramide in the sample of interest;
b) comparing the amount of the at least one ceramide, or the total amount of ceramide, with a reference amount, thereby predicting whether the subject is at risk of developing an active state of multiple sclerosis; including.

  The term “ceramide” is described elsewhere in this specification. In connection with the method described above, it is particularly preferred that the ceramide is selected from the ceramides listed in Table 3. Preferably, an increase in the amount of at least one ceramide compared to the reference amount (or an increase in the total amount of ceramide) is an indicator of a subject at risk of developing an active state of multiple sclerosis.

A method for predicting whether a subject is at risk for developing an active state of multiple sclerosis is also envisaged by the present invention. The method
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 3 in the sample of interest;
b) comparing the amount of the at least one biomarker with a reference amount, thereby predicting whether the subject is at risk of developing an active state of multiple sclerosis.

  Furthermore, the present invention is a method for identifying whether a subject is in need of treatment for an active state of multiple sclerosis, for predicting whether the subject is at risk of developing an active state of multiple sclerosis. And a further step of identifying the subject in need of the treatment if the subject is predicted to be at risk of developing an active state of multiple sclerosis. .

The present invention assists in diagnosing whether a subject is afflicted with MS, is afflicted with an active state of MS, or is at risk of developing MS or an active state thereof. A method of establishing, wherein the method comprises:
a) determining the amount of the at least one biomarker in the sample of interest, wherein the determination comprises: (i) a detection agent that specifically binds to the at least one biomarker and the sample; Contacting with a sample-derived biomarker for a time sufficient to form a complex; (ii) measuring the amount of complex formed, wherein said amount of complex formed is Converting the amount of complex formed to an amount of biomarker that reflects the amount of biomarker present in the sample, proportional to the amount of biomarker present therein When,
b) comparing said amount to a reference;
c) Based on the comparison result generated in step b), whether the subject is affected by MS, whether it is affected by the active state of MS, or the risk of developing MS or its active state And a method comprising the step of establishing assistance for diagnosing whether or not there is.

  Preferably, a suitable detection agent may be an antibody that specifically binds to at least one biomarker in the sample of interest to be investigated by the method of the invention. Preferably, another available detection agent may be an aptamer that specifically binds to at least one biomarker in the sample. In a further preferred embodiment, after removing the sample from the complex formed between the detection agent and at least one biomarker, the amount of complex formed is measured. Thus, in a preferred embodiment, the detection agent may be immobilized on a solid support. In a further preferred embodiment, the sample can be removed from the complex formed on the solid support by utilizing a wash solution. The complex formed is proportional to the amount of at least one biomarker present in the sample. It will be appreciated that the specificity and / or sensitivity of the detection agent utilized defines the degree of proportion of at least one biomarker that can be specifically bound and contained in the sample. Further details regarding how the measurements can be performed are also elsewhere in this specification. The amount of complex formed will be converted to an amount of at least one biomarker that reflects the amount actually present in the sample. Preferably, such an amount can be essentially the amount present in the sample, and preferably from a fixed percentage of the relationship between the complex formed and the amount present in the original sample. Amount.

  In a further preferred embodiment of the method described above, step a) can be performed by an analysis unit, in one aspect, an analysis unit as defined elsewhere herein.

  In a preferred embodiment of the method of the invention, the amount determined in step a) is compared with a reference. Preferably, the reference is a reference as defined elsewhere herein. In yet another preferred embodiment, the reference considers a proportional relationship between the amount of complex measured and the amount present in the original sample. Thus, the reference utilized in a preferred embodiment of the method of the present invention is an artificial reference employed to reflect the limitations of the detection agent used. In another preferred embodiment, the comparison is performed, for example, by including a normalization and / or correction calculation step on the determined quantity before actually comparing the value of the determined quantity with the reference value. In doing so, the relationship can also be taken into account. Furthermore, the normalization and / or correction calculation step for the determined amount employs a comparison step that appropriately reflects the limitations of the detection agent used. Preferably, the comparison is performed automatically with the aid of, for example, a computer system.

  To diagnose with a certain degree of accuracy, based on the comparison performed in step b) by assigning the subject to either a group of subjects suffering from MS or a group of subjects not suffering from MS Assistance is established. As already mentioned elsewhere in this specification, the distribution of the subject being investigated is not accurate for 100% of the cases investigated. Furthermore, the subject group to which the subject to be investigated is assigned is an artificial group established on the basis of statistical considerations (i.e. a certain preselected accuracy on which the method of the invention is performed). Thus, the method can, in one aspect, establish diagnostic assistance that may require further enhancement of the diagnosis by other techniques. Preferably, the assistance for diagnosis is established automatically with the aid of eg a computer system.

  In a preferred embodiment of the method of the present invention, the measurement of at least one biomarker is accomplished by mass spectrometry techniques (preferably GCMS and / or LCMS), NMR or other techniques as described herein above. In such cases, preferably the sample to be analyzed is pretreated. Preferably, the pretreatment comprises obtaining at least one biomarker from the sample material (e.g. plasma or serum can be obtained from whole blood, or at least one biomarker is further specifically from the sample material. Can be extracted). Furthermore, in the case of GCMS, further sample pretreatment such as derivatization of at least one biomarker is preferably required. Furthermore, preferably the pretreatment also includes dilution of the sample material and adjustment or normalization of the concentrations of the components contained therein. For this purpose, preferably a normalization standard is added to the sample in a predetermined amount that allows a comparison of the amount of at least one biomarker and the reference amount and / or the amount between different samples to be analyzed. Can do.

  In a preferred embodiment of the method of the present invention, the method further comprises the step of recommending and / or managing the subject according to the result of the diagnostic assistance established in step c). Such recommendations may include, in one aspect, lifestyle and nutrition aimed at improving the living environment, therapeutic measures described in detail elsewhere herein, and / or periodic disease monitoring, etc. Can be adapted.

  In another preferred embodiment of the above-described method, steps b) and / or c) are performed by an evaluation unit as described elsewhere herein.

  In another preferred embodiment, the method also includes the step of managing or treating the subject according to recommendations or diagnostic results. Preferably, the treatment comprises interferon β1a, interferon β1b, azathioprine, cyclophosphamide, galatiramel acetate, immunoglobulin methotrexate, mitoxantrone, leustatin, IVIg, natalizumab, teriflunomide, statin, daclizumab, alemtuzumab, rituximablin, sphingosine 1 At least one drug selected from the group consisting of acid antagonists (fingolimod (FTY720)), cladribine, fumarate, laquinimod, drugs acting on B cells and antisense drugs and drugs against CD49d at a therapeutically effective dose To include.

  Accordingly, the present invention provides a method for treating it in a subject suffering from MS, wherein (a) by utilizing the methods described herein above, the activity status of MS or MS in the subject is determined. (B) interferon β1a, interferon β1b, azathioprine, cyclophosphamide, galatiramel acetate, immunoglobulin methotrexate, mitoxantrone, leustatin, IVIg, natalizumab, teriflunomide, statin, daclizumab, alemtuzumab, rituximab A therapeutically effective amount of at least one drug selected from the group consisting of sphingosine monophosphate antagonist (fingolimod (FTY720)), cladribine, fumarate, laquinimod, drugs acting on B cells and antisense drugs and drugs against CD49d , MS or MS Method comprising the step of administering the active state of the identified are or MS or MS as suffering from active state to a subject at risk of developing also contemplated.

  The above-described method for measuring at least one biomarker can be performed in an apparatus. The device used herein comprises at least the means described above. Furthermore, the apparatus preferably further comprises means for comparing and evaluating the detected characteristic (s) of the at least one biomarker and preferably also the determined signal strength. The means of the device are preferably operatively connected to each other. The method of linking the means in an operating manner depends on the type of means included in the device. For example, if a means for automatically qualitatively or quantitatively determining a biomarker is utilized, the data obtained by the means that operates automatically can be processed, for example, by a computer program to facilitate determination. In such a case, preferably the means is included in a single device. Thus, the apparatus can include an analysis unit for the biomarker and a computer unit for processing and determining result data. Preferred devices are devices that can be used without special knowledge of a specialist clinician (eg, electronic devices that only require sample loading).

  Alternatively, the method of measuring at least one biomarker can be performed by a system comprising several devices, preferably operatively linked to each other. In particular, the means must be linked in a manner that allows the method of the invention detailed above to be carried out. Thus, as used herein, operably linked preferably means functionally linked. Depending on the means used in the system of the invention, each means is connected to the other by means that allow data transmission between said means (for example glass fiber cables and other cables for high throughput data transmission). Thus, the means can be functionally connected. Moreover, wireless data transfer between means is also assumed by the present invention, for example via a LAN (wireless LAN, W-LAN). A preferred system includes a means for measuring a biomarker. As used herein, means for measuring biomarkers includes means for separating biomarkers (such as chromatography devices) and means for determining metabolites (such as mass spectrometers). Suitable devices have been described in detail above. Preferred means for separating the compounds used in the system of the present invention include chromatography devices, more preferably liquid chromatography, HPLC and / or gas chromatography devices. A preferred instrument for measuring compounds is a mass spectrometer, more preferably GC-MS, LC-MS, direct injection mass spectrometry, FT-ICR-MS, CE-MS, HPLC-MS, quadrupole mass Includes analytical methods, coupled mass spectrometry (including MS-MS or MS-MS-MS), ICP-MS, Py-MS or TOF. The separating and measuring means are preferably connected to one another. Most preferably, LC-MS and / or GC-MS are used in the system of the present invention, as detailed elsewhere herein. Further included are means for comparing and / or analyzing the results obtained from the means for measuring the biomarkers. The means for comparing and / or analyzing the results may comprise at least one database and a computer program executed to compare the results. Preferred embodiments of the systems and devices described above are also described in detail below.

Accordingly, the present invention is a diagnostic device comprising:
a) An analysis unit comprising a detector for at least one biomarker listed in any one of Table 1, Table 1a, Table 1b, Table 2, Table 2a, Table 2b, Table 3, Table 3a, and Table 3b. Operatively coupled to the analysis unit adapted to determine the amount of the biomarker detected by the detector,
b) Evaluation comprising a computer comprising computer program code specifically incorporated to perform a comparison between the determined amount of at least one biomarker and a reference amount, and a database containing the reference amount for the biomarker A comparison result of at least one metabolite comprising at least one of Table 1, Table 1a, Table 1b, Table 2, Table 2a, Table 2b, Table 3, Table 3a or Table 3b Multiple sclerosis in a subject, whether the subject requires treatment for multiple sclerosis, or multiple, if essentially the same as the type of modulation and / or the modulation factor indicated for each of the biomarkers Successful sclerosis identified, i) subjects with multiple sclerosis and suffering from multiple sclerosis and ii) multiple sclerosis symptoms but multiple sclerosis It relates to a diagnostic device and a subject not is discrimination.

In a preferred embodiment, the present invention is a diagnostic device comprising:
a) At least one ceramide detector listed in any one of Table 1, Table 1a, Table 2, Table 2a, Table 3 or Table 3a, or an analysis unit comprising a ceramide detector, Operatively coupled to the analysis unit adapted to determine the amount of the at least one ceramide detected by
b) Evaluation comprising a computer comprising computer program code specifically incorporated to perform a comparison between the determined amount of at least one biomarker and a reference amount, and a database containing the reference amount for the biomarker The at least one ceramide comparison result for each of the at least one ceramide in any one of Table 1, Table 1a, Table 2, Table 2a, Table 3 or Table 3a, and / or Or multiple sclerosis in the subject, whether the subject is in need of treatment for multiple sclerosis, or a response to multiple sclerosis, if essentially the same as the rate of modulation, i) A subject who exhibits symptoms of multiple sclerosis and suffers from multiple sclerosis and ii) a subject who exhibits symptoms of multiple sclerosis but does not suffer from multiple sclerosis Bets are differential, a diagnostic device comprising said evaluation unit.

  In a preferred embodiment, the device comprises at least one biomarker in any one of Table 1, Table 1a, Table 1b, Table 2, Table 2a, Table 2b, Table 3, Table 3a, Table 3b. In order to carry out a comparison between a further database containing the type of adjustment and / or the magnification of the adjustment value indicated for each and the adjustment type and / or the magnification of the adjustment value and those contained in the database Additional computer program code specifically incorporated.

  In addition, the present invention provides for the pathology or effects described above (i.e., diagnosis of multiple sclerosis in a subject, identification of whether a subject requires treatment for multiple sclerosis, or whether multiple sclerosis treatment is successful). The present invention relates to a data collection including characteristic values of at least one kind of biomarker that serves as an index of determination.

  The term “data collection” refers to a collection of data that can be grouped together physically and / or logically. Thus, data collection may be implemented on a single data storage medium or physically separate data storage media that are operatively coupled to each other. Preferably, the data collection is implemented using a database. Thus, the database used herein includes data collection on a suitable storage medium. Further, the database preferably further includes a database management system. Preferably, the database management system is a network-based hierarchical or object-oriented database management system. Further, the database may be a federated or integrated database. More preferably, the database can be implemented as a distributed (federated) system, such as a client server system. More preferably, the database is constructed such that a search algorithm can compare the test data set with the data set contained in the data collection. Specifically, by using such an algorithm, a database can be searched (for example, query search) for similar or identical data sets that serve as indicators of the above-mentioned medical condition or effect. Thus, if the same or similar data set can be identified in the data collection, the test data set will be associated with the disease state or effect. As a result, the information obtained from the data collection can be used, for example, as a reference for the inventive method described above. More preferably, the data collection includes characteristic values for all metabolites included in any one of the groups described above.

  In view of the above, the present invention encompasses a data storage medium that includes the data collection described above.

  The term “data storage medium” as used herein encompasses data storage media based on a single physical entity (such as a CD, CD-ROM, hard disk, optical storage medium or diskette, etc.). The term further includes a data storage medium consisting of physically separated entities that are operatively linked to each other in a manner that provides the data collection described above, preferably in a manner suitable for query retrieval.

The present invention
(a) a means for comparing characteristic values of at least one biomarker of the sample and operatively linked thereto;
(b) the data storage medium described above;
It also relates to systems that include

  As used herein, the term “system” relates to different means operatively connected to each other. Said means may be implemented in a single device or may be physically separated devices operatively connected to each other. The means for comparing the biomarker characteristic values is preferably based on the aforementioned comparison algorithm. The data storage medium preferably includes a data collection or database as described above, and each stored data set is indicative of a medical condition or effect referred to above. Thus, the system of the present invention makes it possible to identify whether a test data set is included in a data collection stored on a data storage medium. As a result, the method of the present invention can be executed by the system of the present invention.

  In a preferred embodiment of the system, means for determining the characteristic value of the sample biomarker is included. Preferably, the term “means for determining a characteristic value of a biomarker” is used to measure a metabolite, such as a mass spectrometer, an NMR device or a device for performing a biomarker chemical or biological assay. Relates to the above-mentioned apparatus.

  Furthermore, the present invention relates to a diagnostic means comprising means for measuring at least one biomarker selected from any one of the above groups.

  As specified in detail elsewhere in the description, the term “diagnostic means” preferably relates to a diagnostic device, system or biological or chemical assay.

  The expression “means for measuring at least one biomarker” refers to a device or agent capable of specifically recognizing a biomarker. A suitable device may be a spectroscopic device, such as a mass spectrometer, an NMR device or a device for performing a biomarker chemical or biological assay. A suitable agent may be a compound that specifically detects a biomarker. Detection as used herein may be a two-step process, i.e., the compound first specifically binds to the biomarker to be detected and then detects a detectable signal (e.g., fluorescent signal, chemical Luminescent signals, radioactive signals, etc.) can be generated. Additional compounds may be required to generate a detectable signal, which compounds are all included in the term “means for measuring at least one biomarker”. Compounds that specifically bind to biomarkers are described in detail elsewhere herein, and preferably are enzymes, antibodies, ligands, receptors or other organisms that specifically bind to biomarkers. Includes molecules or chemicals.

  Furthermore, the present invention relates to a diagnostic composition comprising at least one biomarker selected from any one of the above groups.

  At least one biomarker selected from any of the above groups can function as a biomarker, ie, an indicator molecule for a disease state or effect in a subject described elsewhere herein. Thus, by means described herein, the metabolite molecule itself can preferably function as a diagnostic composition in visualization or detection. Therefore, the diagnostic composition showing the presence of the biomarker according to the present invention can physically contain the biomarker. For example, a complex of an antibody and a metabolite to be detected is used as a diagnostic composition. Can function. Thus, as specified elsewhere in this description, the diagnostic composition can further comprise a means for detecting metabolites. Alternatively, when detection means (such as MS or NMR based techniques) are used, the molecular species that serve as an indicator for the risk state can be at least one biomarker included in the test sample to be investigated. Therefore, at least one biomarker defined by the present invention is identified as a biomarker and thus functions as a diagnostic composition.

  Overall, the present invention provides at least one biomarker selected from the biomarkers listed in any one of Table 1, Table 2, Table 1a, Table 2a, Table 1b, or Table 2b in a sample of interest. Use in the diagnosis of multiple sclerosis, and in the target sample, at least one biomarker selected from the biomarkers listed in Table 3, Table 3a or Table 3b is the activity of multiple sclerosis Using for diagnosing a condition, or in a sample of a subject, using at least one biomarker selected from the biomarkers of Table 1 and / or Table 2 to predict multiple sclerosis, And at least one biomarker selected from the biomarkers in Table 3 in the subject sample to predict the active state of multiple sclerosis It intended to be.

  All references cited herein are hereby incorporated by reference with respect to their entire disclosure content or the specific disclosure content mentioned above.

  The invention is illustrated herein by the following examples which are not intended to limit or limit the scope of the invention.

[Example 1]
Study design sample specifications for differential diagnosis of multiple sclerosis (MS) from other neurological symptoms or disorders:
Cases: 41 newly diagnosed untreated patients with MS (n = 15) or clinical isolated syndrome (CIS) (n = 26).

  Controls: patients with different neurological symptoms or diseases that are not CIS or MS (e.g. headache (n = 11), hypoperception (n = 10), sensory abnormalities (n = 6)), meningitis patients (n = 4) And 42 patients (n = 11) with other symptoms.

  Among patients with other symptoms, one patient was diagnosed with abductal nerve palsy, one patient had leg pain, one patient had dysarthria, and one patient Elsberg syndrome, 1 patient with idiopathic facial nerve palsy, 1 patient with headache and hyposensory, 1 patient with neuropathy, 1 patient with pseudobrain tumor Yes, two patients were dizzy.

  Of 41 CIS and MS patients, 18 had changes in signs or symptoms (acute seizures), while 23 had no clinical changes (stable).

  As described in detail in Example 3, additional information from all subjects (eg gender, age, BMI, date of sample collection, disease status) was used for the analysis.

  Serum was prepared from all samples by centrifugation and samples were stored at -80 ° C. until measurements were performed.

[Example 2]
Metabolite measurements Human serum samples were prepared and subjected to LC-MS / MS and GC-MS or SPE-LC-MS / MS (hormone) analysis as described below:
Proteins were separated by precipitation from serum. After addition of water and a mixture of ethanol and dichloromethane, the remaining sample was fractionated into an aqueous, polar and organic, lipophilic phase.

  For trans-methanol solvation of the lipid extract, a mixture of 140 μl chloroform, 37 μl hydrochloric acid (37% by weight HCl in water), 320 μl methanol and 20 μl toluene was added to the evaporated extract. The vessel was sealed tightly and heated at 100 ° C. with shaking for 2 hours. The solution was then evaporated to dryness. The residue was completely dried.

  Methoximation of the carbonyl group was carried out by reaction with methoxyamine hydrochloride (20 mg / ml in pyridine; 100 μl, 1.5 hours at 60 ° C.) in a tightly sealed container. Solution of odd linear fatty acids (0.3 mg / mL of each fatty acid of 7 to 25 carbon atoms and 0.6 mg / mL of each fatty acid of 27, 29 and 31 carbon atoms, 3/7 (v / v) pyridine / toluene Solution) 20 μl was added as a time standard. Finally, derivatization with 100 μl of N-methyl-N- (trimethylsilyl) -2,2,2-trifluoroacetamide (MSTFA) was performed again at 60 ° C. for 30 minutes in a tightly sealed container. The final volume before injection into the GC was 220 μl.

  For the polar phase, derivatization was carried out in the following manner: by reacting with methoxyamine hydrochloride (20 mg / ml in pyridine; 50 μl, 1.5 hours at 60 ° C. in pyridine) in a tightly sealed container. The methoxylation of was performed. Odd linear fatty acid solution (3/7 (v / v) pyridine / toluene solution of 0.3 mg / mL of each fatty acid of 7 to 25 carbon atoms and 0.6 mg / mL of each fatty acid of 27, 29 and 31 carbon atoms ) 10 μl was added as a time standard. Finally, derivatization with 50 μl of N-methyl-N- (trimethylsilyl) -2,2,2-trifluoroacetamide (MSTFA) was performed again at 60 ° C. for 30 minutes in a tightly sealed vessel. The final volume before injection into the GC was 110 μl.

  The GC-MS system consists of an Agilent 6890 GC connected to an Agilent 5973 MSD. The autosampler is CompiPal or GCPal made by CTC.

  For analysis, depending on the sample material analyzed and the fraction from the phase separation step, a conventional commercial capillary separation column with different polymethylsiloxane stationary phases containing from 0% to 35% aromatic moieties ( 30 m × 0.25 mm × 0.25 μm) were used (eg: DB-1ms, HP-5ms, DB-XLB, DB-35ms, Agilent Technologies). Splitless injection of a final volume of 1 μL or less, and oven temperature program, starting at 70 ° C. and ending at 340 ° C. with different heating rates depending on the sample material and fraction from the phase separation step, sufficient chromatographic separation and each The number of scans within the analyte peak range was achieved. In addition, RTL (Retention Time Locking, Agilent Technologies) is used for the analysis, using normal GC-MS standard conditions, e.g. nominally 1-1.7 ml / min constant flow, helium as mobile phase gas, 70 eV Ionization was performed by electron impact and scanned at m / z range 15-600, scan speed 2.5-3 scans / second and standard pacing conditions.

  The HPLC-MS system consists of an Agilent 1100 LC system (Agilent Technologies, Waldbronn, Germany) connected to an API 4000 mass spectrometer (Applied Biosystem / MDS SCIEX, Toronto, Canada). HPLC analysis was performed on a commercial reverse phase separation column with a C18 stationary phase (eg GROM ODS 7pH, Thermo Betasil C18). Up to 10 μL of the final sample volume of evaporated and reconstituted polar and lipophilic phase was injected. Separations were performed with gradient elution using a methanol / water / formic acid or acetonitrile / water / formic acid gradient at a flow rate of 200 μL / min.

  Using multi-reaction monitoring (MRM) mode, mass spectrometry was performed with electrospray ionization in positive mode for nonpolar fractions, negative mode for polar fractions, and full scans from 100 to 1000 amu. .

  Steroids and their metabolites were measured by online SPE-LC-MS (solid phase extraction-LC-MS). Catecholamine and its metabolites were measured by online SPE-LC-MS as described by Yamada et al. (J. Anal. Toxicol. (26), 2002, pp. 17-22). For both catecholamines and related metabolites and steroids and related metabolites, quantification was achieved using stable isotope labeled standards and absolute concentrations were calculated.

Analysis of complex lipids in serum samples:
Total lipids were extracted from serum by liquid / liquid extraction using chloroform / methanol.

  The lipid extracts were then fractionated into 11 different lipid groups by normal phase liquid chromatography (NPLC) according to Christie (Journal of Lipid Research (26), 1985, 507-512).

  Lipid classification monoacylglyceride (MAG), triacylglyceride (TAG), phosphatidylcholine (PC), and phosphatidylserine (PS) were measured by GC.

  Fractions were derivatized with TMSH (trimethylsulfonium hydroxide) and then analyzed by GC-MS to obtain fatty acid methyl esters (FAME) corresponding to the acyl portion of the classified lipid. The concentration of C14-C24 FAME is determined in each fraction.

  Use electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) while detecting unique multiple reaction monitoring (MRM) transitions for cholesterol ester (CE), sphingomyelin (SM) and ceramide (CER), respectively Fractions were analyzed by LC-MS / MS.

  LC-MS using electrospray ionization (ESI) while detecting unique multiple reaction monitoring (MRM) transitions as described in Schmidt H et al., Prostaglandins & other Lipid Mediators 81 (2006), pages 162-170 Sphingosine and sphingosine-1-phosphate (SP) were analyzed by / MS.

  The metabolites in Tables 1 to 3 and / or Tables 1a to 3a and / or Tables 1b to 3b obtained from one of these fractions are preceded by their respective abbreviations before their names separated by underscores. including.

  Eicosanoids and related metabolites were measured from serum by offline and online SPE LC-MS / MS (solid phase extraction-LC-MS / MS) (Masoodi M and Nicolaou A: Rapid Commun Mass Spectrom. 2006 20 (20) : 3023-3029). Absolute quantification was performed using stable isotope labeled standards.

[Example 3]
Data analysis and statistical evaluation Serum samples were analyzed in a randomized analysis sequence design using pooled samples (so-called “pools”) made from aliquots of each sample. After the comprehensive analysis verification step, the raw peak data for each analyte was normalized to the pool median for each analysis order that caused process variation (so-called “pool normalization ratio”). Where available, absolute concentrations of metabolites were used for statistical analysis. In all other cases, a pool normalization ratio was used. All the data were log10 transformed and distributed normally.

  In the case of the study described in Example 1, age (numerical value), BMI (numerical value), gender (classification), sample preparation time (numerical value), and diagnosis (all CASES (cases) (with or without acute attacks), A fixed-effect ANOVA model was designed to include CASES with acute seizures, CASES without acute seizures, and control-reference: control) factors. CASES includes MS patients and CIS patients. Because of the large number of metabolites affected by these factors, pairwise interaction of factors such as age, BMI and gender was considered. Statistical significance was read out from the p-value of t statistic. By inversely transforming the contrast estimate by calculating the median ratio for the groups to be compared ("ratio = 10 ^ contrast_estimate"), the direction and intensity of adjustment were obtained on a ratio scale. . The control type is `` upper '' when increased (ratio> 1) in each group (e.g. CASES with or without acute seizures) vs. reference (control), and decreased (ratio <1) relative to reference Was determined for each metabolite as "down". All metabolite tables were sorted by significant difference (p value <0.05).

  To identify biomarkers for multiple sclerosis, we examined a readout that diagnosed all CASES (with or without acute seizures) versus control reference comparison (Table 1). Compare CASES with acute seizures to the reference control (Table 2), and also CASES with acute seizures and acute seizures, to read out the duration of acute seizures associated with diagnostic effects during multiple sclerosis disease (Table 2) A CASES comparison with no analysis was analyzed (Table 3).

The analysis results are summarized in the following table. The biomarkers determined by the method of the present invention are listed in the table below. Biomarkers that are not precisely defined by name are further characterized in Table 4.

Claims (15)

A method of diagnosing multiple sclerosis (MS) in a subject comprising:
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 1 and / or Table 2 in the sample of interest;
b) comparing the amount of said at least one biomarker with a reference amount, whereby multiple sclerosis will be diagnosed.   The at least one biomarker is selected from the group of biomarkers listed in Table 1a and / or Table 2a, and an increase in the at least one biomarker compared to a reference amount is an indicator of multiple sclerosis. Item 2. The method according to Item 1.   The at least one biomarker is selected from the group of biomarkers listed in Table 1b and / or Table 2b, and a decrease in the at least one biomarker compared to a reference amount is an indicator of multiple sclerosis. Item 2. The method according to Item 1.   4. The method according to any one of claims 1 to 3, wherein multiple sclerosis is diagnosed at an early stage of multiple sclerosis.   5. The reference amount according to any one of claims 1 to 4, wherein the reference amount is obtained from an apparently healthy subject or group thereof, or from a subject or group thereof that is known not to have MS but exhibits MS symptoms. the method of.   A method for identifying whether a subject is in need of treatment for multiple sclerosis, comprising the steps of the method of any one of claims 1 to 5 and a subject in need of treatment for multiple sclerosis. And further comprising steps. i) a method for differentiating a subject who exhibits symptoms of multiple sclerosis and suffers from multiple sclerosis and ii) a subject who exhibits MS symptoms but does not suffer from multiple sclerosis,
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 1 and / or Table 2 in a sample of a subject exhibiting multiple sclerosis symptoms;
b) comparing the amount of said at least one biomarker with at least one reference amount, whereby i) and ii) are differentiated. A method of determining whether multiple sclerosis treatment is successful,
a) measuring at least one biomarker selected from the biomarkers listed in Table 1 and / or Table 2 in the first and second samples of the subject, wherein the first sample is a multiple sclerosis treatment The second sample is taken before or at the start of the treatment, and the second sample is taken after the treatment starts, and
b) comparing the amount of the at least one biomarker in the first sample with the amount in the second sample, whereby the change in the amount determined in the second sample compared to the first sample is frequent. And a step that serves as an indicator of successful treatment of systemic sclerosis. i) the change is reduced and the at least one biomarker is selected from the biomarkers listed in Table 1a and / or Table 2a, or
9. The method of claim 8, wherein the change is an increase and the at least one biomarker is selected from the biomarkers listed in Table 1b and / or Table 2b.   The treatment is interferon β1a, interferon β1b, azathioprine, cyclophosphamide, galatiramel acetate, immunoglobulin methotrexate, mitoxantrone, leustatin, IVIg, natalizumab, teriflunomide, statin, daclizumab, alemtuzumab, rituximab, sphingosine monophosphate antagonist The administration of at least one drug selected from the group consisting of golimod (FTY720), cladribine, fumarate, laquinimod, a drug that acts on B cells and an antisense drug and a drug against CD49d. Method. A method for diagnosing an active state of multiple sclerosis in a subject comprising:
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 3 in the sample of interest;
b) comparing the amount of said at least one biomarker with a reference amount, whereby multiple sclerosis will be diagnosed. i) the at least one biomarker is selected from the group of biomarkers listed in Table 3a, and an increase in the at least one biomarker is indicative of the active state of multiple sclerosis, or
ii) The at least one biomarker is selected from the group of biomarkers listed in Table 3b, and a decrease in the at least one biomarker is an indicator of an active state of multiple sclerosis. Method.   13. The method of claim 11 or 12, wherein the reference amount is obtained from a subject or subject group that exhibits a stable state of multiple sclerosis. A method of predicting whether a subject is at risk of developing multiple sclerosis,
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 1 and / or Table 2 in the sample of interest;
b) comparing the amount of said at least one biomarker with a reference amount, whereby the subject is predicted to be at risk of developing multiple sclerosis. A method of predicting whether a subject is at risk of developing an active state of multiple sclerosis, comprising:
a) determining the amount of at least one biomarker selected from the biomarkers listed in Table 3 in the sample of interest;
b) comparing the amount of said at least one biomarker with a reference amount, thereby predicting whether the subject is at risk of developing an active state of multiple sclerosis.