The present invention relates to methods for monitoring the progression of multiple sclerosis in a patient, and corresponding methods for identifying a therapy to suppress progression.

Multiple sclerosis (MS) is the most common cause of progressive disability in the Western world. Being a progressive disease, MS patients are at risk of suffering deteriorating disability over time, with the speed and aggression of deterioration differing amongst individuals. A decreased quality of life (relative to the healthy population) follows not only because of increasing symptoms of disability, but also because of the uncertainty of how a patient’s prognosis will progress.

However, evaluation of progression in multiple sclerosis (MS) patients remains a clinical challenge. Previous efforts to evaluate and monitor progression have employed PET imaging, notably in combination with innate immune cell 18kDa translocator protein (TSPO) radioligand(s) that can reveal inflammation in the brain, which is associated with disease progression. However, radioligands/ isotopes are costly and regular serial PET scanning is likely to raise safety concerns, rendering this strategy impractical for routine use (in addition to being resource intensive). Additionally or alternatively, the “Expanded Disability Status Scale” (EDSS) has been used as a method of quantifying disability in multiple sclerosis. This scale is principally investigated through a neurological examination by a clinician, quantifying disability in eight Functional Systems (FS) by assigning a Functional System Score (FSS) in each of these functional systems. However, this method is time consuming and diverts yet further time and effort toward monitoring progression as opposed to suppressing it.

To overcome the above-mentioned problems, the present invention is predicated on a metabolomics-based approach to monitoring progression, and on the utilization of an advantageous panel of metabolite biomarkers that has been demonstrated to correlate with disease progression. More particularly (as explained in more detail in the Examples section) the present inventors have demonstrated that concentrations of this panel of key metabolites correlates with the magnitude of inflammation in the brain (a known indicator of MS progression). Indeed, the metabolite’s concentrations have been shown to be significantly increased in patients that suffer disease progression when compared with corresponding concentrations in patients that have not progressed (else not substantially progressed). Thus, overcoming prior reliance on PET imaging and EDSS analysis, the present invention provides readily detectable and quantifiable metabolite panel as a surrogate marker of brain inflammation and that is predictive of increased disability. This provides for a metabolomics test (e.g. blood-based test) toward aiding clinicians to monitor disease progression and the impact of therapy in MS patients in a minimally invasive and cost-effective manner.

The invention will now be described in more detail below.

A broad aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: identifying a concentration of one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from the patient; comparing the concentrations of said one or more metabolite(s) with a reference value; and determining there has been progression of MS in the patient or determining there has been no progression of MS in the patient (e.g. based on the comparison).

Anther broad aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: a. identifying an intensity of a chemical shift region(s) of a 1 H-NMR spectrum for one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from the patient; b. comparing the intensity of said chemical shift region(s) for said one or more metabolite(s) with a reference value(s); and c. determining there has been progression of MS in the patient or determining there has been no progression of MS in the patient (e.g. based on the comparison).

Another broad aspect of the invention provides a method for monitoring a multiple sclerosis (MS) patient’s response to a therapy, the method comprising: a. identifying a concentration of one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from the patient (e.g. that has been administered the therapy); b. comparing the concentrations of said one or more metabolite(s) with a reference value; and c. determining that the patient is responsive to the therapy or determining that the patient is not responsive to the therapy (e.g. based on the comparison). Another broad aspect of the invention provides a method for monitoring a multiple sclerosis (MS) patient’s response to a therapy, the method comprising: a. identifying an intensity of a chemical shift region(s) of a 1 H-NMR spectrum for one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from the patient (e.g. that has been administered the therapy); b. comparing the intensity of said chemical shift region(s) for said one or more metabolite(s) with a reference value(s); and c. determining that the patient is responsive to the therapy or determining that the patient is not responsive to the therapy (e.g. based on the comparison).

The one or more metabolite(s) may be selected from glucose, p-hydroxybutyrate, myoinositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a - (CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 (e.g. present in a sample obtained from the patient).

For example, the one or more metabolite(s) may be selected from glucose, p- hydroxybutyrate, and myo-inositol. Methods of the invention preferably comprise identifying a concentration of two or more metabolite(s) (or all three metabolites) selected from glucose, P-hydroxybutyrate, myo-inositol.

The reference value may represent the concentration of said same one or more metabolite(s) present in a sample obtained from the patient at an earlier timepoint. Additionally or alternatively, the reference value may represent the concentration of said same one or more metabolite(s) present in a sample obtained from a patient (e.g. corresponding control patient) suffering from MS that has not progressed. Additionally or alternatively, the reference value may represent the concentration of said same one or more metabolite(s) present in a sample obtained from a patient (e.g. corresponding control patient) that has progressed.

Methods of the invention may comprise determining there has been progression of MS in the patient when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from a patient (e.g. corresponding control patient) suffering from MS that has not progressed. Said patient suffering from MS that has not progressed may be referred to as a corresponding control patient that is representative of a non- progressor.

Additionally or alternatively, methods of the invention may comprise determining there has been progression of MS in the patient when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myoinositol is lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from the patient at an earlier timepoint.

Additionally or alternatively, methods of the invention may comprise determining there has been progression of MS in the patient when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myoinositol is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from a (e.g. control) patient that has progressed. Additionally or alternatively, methods of the invention may comprise determining there has been progression of MS in the patient when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from a (e.g. control) patient suffering from MS that has progressed. Said patient (e.g. suffering from MS) that has progressed may be referred to as a corresponding control patient that is representative of progressor (also known as “an MS progressor”).

Methods of the invention may comprise determining there has been progression of MS in the patient when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not progressed. Said patient suffering from MS that has not progressed may be referred to as a corresponding control patient that is representative of a non-progressor. Additionally or alternatively, methods of the invention may comprise determining there has been progression of MS in the patient when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient at an earlier timepoint.

Additionally or alternatively, methods of the invention may comprise determining there has been progression of MS in the patient when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from a (e.g. control) patient suffering MS that has progressed. Said patient (e.g. suffering from MS) that has progressed may be referred to as a corresponding control patient that is representative of progressor (also known as “an MS progressor”).

Methods of the invention may comprise determining there has been no progression of MS in the patient when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from a (e.g. control) patient suffering from MS that has not progressed. Said patient suffering from MS that has not progressed may be referred to as a corresponding control patient that is representative of a non-progressor.

Additionally or alternatively, methods of the invention may comprise determining there has been no progression of MS in the patient when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from the patient at an earlier timepoint. Additionally or alternatively, methods of the invention may comprise determining there has been no progression of MS in the patient when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) (preferably two or more metabolites) present in a sample obtained from a (e.g. control) patient suffering MS that has progressed. Said patient (e.g. suffering from MS) that has progressed may be referred to as a corresponding control patient that is representative of progressor (also known as “an MS progressor”).

Methods of the invention may comprise determining there has been no progression of MS in the patient when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not progressed. Said patient suffering from MS that has not progressed may be referred to as a corresponding control patient that is representative of a non-progressor.

Additionally or alternatively, methods of the invention may comprise determining there has been no progression of MS in the patient when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CHS)3 group is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient at an earlier timepoint.

Additionally or alternatively, methods of the invention may comprise determining there has been no progression of MS in the patient when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CHS)3 group is lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from a (e.g. control) patient suffering MS that has progressed. Said patient (e.g. suffering from MS) that has progressed may be referred to as a corresponding control patient that is representative of progressor (also known as “an MS progressor”).

The reference value may represent the intensity of said same chemical shift region(s) present in a sample obtained from the patient at an earlier timepoint. Additionally or alternatively, the reference value may represent the intensity of said same chemical shift region(s) present in a sample obtained from a patient suffering from MS that has not progressed (said patient suffering from MS that has not progressed may be referred to as a corresponding control patient that is representative of a non-progressor). Additionally or alternatively, the reference value may represent the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient that suffering MS has progressed (said patient (e.g. suffering from MS) that has progressed may be referred to as a corresponding control patient that is representative of progressor (also known as “an MS progressor”)).

Methods of the invention may comprise determining there has been progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same (e.g. at least one) chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not progressed.

Additionally or alternatively, methods of the invention may comprise determining there has been progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient at an earlier timepoint.

Additionally or alternatively, methods of the invention may comprise determining there has been progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s), when the reference value(s) represents the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering MS that has progressed.

Methods of the invention may comprise determining there has been progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CHS)3 group is higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not progressed.

Additionally or alternatively, methods of the invention may comprise determining there has been progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient at an earlier timepoint.

Additionally or alternatively, methods of the invention may comprise determining there has been progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering MS that has progressed.

Methods of the invention may comprise determining there has been no progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not progressed. Additionally or alternatively, methods of the invention may comprise determining there has been no progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient at an earlier timepoint.

Additionally or alternatively, methods of the invention may comprise determining there has been no progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering MS that has progressed.

Methods of the invention may comprise determining there has been no progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CHS)3 group is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not progressed.

Additionally or alternatively, methods of the invention may comprise determining there has been no progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained the patient at an earlier timepoint.

Additionally or alternatively, methods of the invention may comprise determining there has been no progression of MS in the patient when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering MS that has progressed.

The 1 H-NMR chemical shift range for glucose may be one or more selected from 3.17-3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm. The 1 H-NMR chemical shift range for myo-inositol may be one or more selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25- 3.29 ppm. The 1 H-NMR chemical shift range for p-hydroxybutyrate may be one or more selected from 1.19-1.21 ppm and 2.27-2.45 ppm. The 1 H-NMR chemical shift range for a lipoprotein having a -CH3 group of an HDL and/or LDL may be 0.80-0.86 ppm. The 1 H-NMR chemical shift range for a lipoprotein having a -(CH2)n group of an HDL and/or LDL may be 1.15-1.30 ppm. The 1 H-NMR chemical shift range for a lipoprotein having an -N(CHs)3 group may be 3.17-3.31 ppm. Said 1 H-NMR chemical shift range may be determinable by a method as described herein. The metabolites described herein may be referred to (e.g. individually or collectively) as follows: glucose may be referred to as a molecule that demonstrates one or more 1 H- NMR chemical shift range selected from 3.17-3.95 ppm, 4.63-4.66 ppm, and 5.22- 5.25 ppm; myo-inositol may be referred to as a molecule that demonstrates one or more 1 H- NMR chemical shift range selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25-3.29 ppm;

P-hydroxybutyrate may be referred to as a molecule that demonstrates one or more 1 H-NMR chemical shift range selected from 1.19-1.21 ppm and 2.27-2.45 ppm; a lipoprotein having a -CH3 group of an HDL and/or LDL may be referred to as a molecule that demonstrates a 1 H-NMR chemical shift range from 0.80-0.86 ppm. a lipoprotein having a -(CH2)n group of an HDL and/or LDL may be referred to as a molecule that demonstrates a 1 H-NMR chemical shift range selected from 1.15- 1.30 ppm; a lipoprotein having an -N(CHs)3 group may be referred to as a molecule that demonstrates a 1 H-NMR chemical shift range from 3.17-3.31 ppm.

One aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: a. identifying a concentration of one or more metabolite(s) selected from glucose, p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient; b. comparing the concentration of said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the concentration of said same one or more metabolite(s) present in a sample obtained from: the patient at an earlier timepoint; and/or a patient suffering from MS that has not progressed; and c. determining there has been progression of MS in the patient when: i. the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is lower in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is higher in the patient sample relative to the reference value(s); or d. determining there has been no progression of MS in the patient when: i. the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or higher in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is the same or lower in the patient sample relative to the reference value(s).

One aspect of the invention provides a method, the method comprising: a. obtaining a biofluid sample derived from a patient, optionally a multiple sclerosis patient; b. assaying the biofluid sample for a concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, myo-inositol, a lipoprotein having a - CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in the sample; optionally c. comparing the assayed concentration of said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the concentration of said same one or more metabolite(s) present in a biofluid sample obtained from: the patient at an earlier timepoint; and/or a patient suffering from MS that has not progressed; and d. confirming there has been progression of MS in the patient when: i. the assayed concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is lower in the patient sample relative to the reference value(s); and/or ii. the assayed concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is higher in the patient sample relative to the reference value(s); or e. confirming there has been no progression of MS in the patient when: i. the assayed concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or higher in the patient sample relative to the reference value(s); and/or ii. the assayed concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is the same or lower in the patient sample relative to the reference value(s).

Another aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: a. identifying an intensity of a (e.g. at least one) chemical shift region(s) of a 1 H- NMR spectrum for one or more metabolites selected from glucose, p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient, wherein the 1 H-NMR chemical shift range for glucose is selected from 3.17- 3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm; the 1 H-NMR chemical shift range for myo-inositol is selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25-3.29 ppm; the 1 H-NMR chemical shift range for p-hydroxybutyrate is selected from 1.19-1.21 ppm and 2.27-2.45 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -CH3 group of an HDL and/or LDL is 0.80-0.86 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -(CH2)n group of an HDL and/or LDL is 1.15-1.30 ppm; the 1 H-NMR chemical shift range for a lipoprotein having an -N(CHs)3group is 3.17-3.31 ppm; b. comparing the intensity of said chemical shift region(s) for said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the intensity of said same chemical shift region(s) present in a sample obtained from: the patient at an earlier timepoint; and/or a patient suffering from MS that has not progressed; and c. determining there has been progression of MS in the patient when: i. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is lower in the patient sample relative to the reference value(s); and/or ii. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is higher in the patient sample relative to the reference value(s); or d. determining there has been no progression of MS in the patient when: i. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or higher in the patient sample relative to the reference value(s); and/or ii. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or lower in the patient sample relative to the reference value(s).

One aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: a. identifying a concentration of one or more metabolite(s) selected from glucose, p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient; b. comparing the concentration of said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the concentration of said same one or more metabolite(s) present in a sample obtained from: a patient suffering from MS that has progressed; and c. determining there has been progression of MS in the patient when: i. the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is the same or higher in the patient sample relative to the reference value(s); or d. determining there has been no progression of MS in the patient when: i. the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is lower in the patient sample relative to the reference value(s).

One aspect of the invention provides a method, the method comprising: a. obtaining a biofluid sample derived from a patient, optionally a multiple sclerosis patient; b. assaying the biofluid sample for a concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, myo-inositol, a lipoprotein having a - CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in the sample; optionally c. comparing the assayed concentration of said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the concentration of said same one or more metabolite(s) present in a biofluid sample obtained from: a patient suffering from MS that has progressed; and d. confirming there has been progression of MS in the patient when: i. the assayed concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s); and/or ii. the assayed concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is the same or higher in the patient sample relative to the reference value(s); or e. confirming there has been no progression of MS in the patient when: i. the assayed concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s); and/or ii. the assayed concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is lower in the patient sample relative to the reference value(s).

Another aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: a. identifying an intensity of a (e.g. at least one) chemical shift region(s) of a 1 H- NMR spectrum for one or more metabolites selected from glucose, p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient, wherein the 1 H-NMR chemical shift range for glucose is selected from 3.17- 3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm; the 1 H-NMR chemical shift range for myo-inositol is selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25-3.29 ppm; the 1 H-NMR chemical shift range for p-hydroxybutyrate is selected from 1.19-1.21 ppm and 2.27-2.45 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -CH3 group of an HDL and/or LDL is 0.80-0.86 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -(CH2)n group of an HDL and/or LDL is 1.15-1.30 ppm; the 1 H-NMR chemical shift range for a lipoprotein having an -N(CHs)3 group is 3.17-3.31 ppm; b. comparing the intensity of said chemical shift region(s) for said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the intensity of said same chemical shift region(s) present in a sample obtained from: a patient suffering from MS that has progressed; and c. determining there has been progression of MS in the patient when: i. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s); and/or ii. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s); or d. determining there has been no progression of MS in the patient when: i. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s); and/or ii. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s).

The MS patient may have been administered a therapy for MS.

The reference value may represent the concentration of said same one or more metabolite(s) present in a sample obtained from the patient pre-administration of the therapy. Additionally or alternatively, the reference value may represent the concentration of said same one or more metabolite(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not been administered the therapy.

Methods of the invention may comprise determining that the patient is responsive to the therapy when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient at an earlier timepoint (e.g. pre-administration of the therapy). For example, methods of the invention may comprise determining that the patient is responsive to the therapy when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient pre-administration of the therapy.

Additionally or alternatively, methods of the invention may comprise determining that the patient is responsive to the therapy when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myoinositol is higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not been administered the therapy.

Methods of the invention may comprise determining that the patient is responsive to the therapy when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient at an earlier timepoint (e.g. pre-administration of the therapy). For example, methods of the invention may comprise determining that the patient is responsive to the therapy when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient pre-administration of the therapy.

Additionally or alternatively, methods of the invention may comprise determining that the patient is responsive to the therapy when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s), when the reference value(s) represents the concentration of said same one or more metabolite(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not been administered the therapy.

Methods of the invention may comprise determining that the patient is not responsive to the therapy when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient at an earlier timepoint (e.g. pre-administration of the therapy). For example, methods of the invention may comprise determining that the patient is responsive to the therapy when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s), when the reference value(s) represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient pre-administration of the therapy.

Additionally or alternatively, methods of the invention may comprise determining that the patient is not responsive to the therapy when: the concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myoinositol is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not been administered the therapy.

Methods of the invention may comprise determining that the patient is not responsive to the therapy when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient at an earlier timepoint (e.g. pre-administration of the therapy). For example, methods of the invention may comprise determining that the patient is not responsive to the therapy when: the concentration of one or more metabolite(s) selected from a lipoprotein having a - CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from the patient preadministration of the therapy.

Additionally or alternatively, methods of the invention may comprise determining that the patient is not responsive to the therapy when: the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same one or more metabolite(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not been administered the therapy.

Methods of the invention may comprise determining that the patient is responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient at an earlier timepoint (e.g. pre-administration of the therapy). For example, methods of the invention may comprise determining that the patient is responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p- hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient pre-administration of the therapy.

Additionally or alternatively, methods of the invention may comprise determining that the patient is responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not been administered the therapy. Methods of the invention may comprise determining that the patient is responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CHS)3 group is lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient at an earlier timepoint (e.g. pre-administration of the therapy). For example, methods of the invention may comprise determining that the patient is responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient pre-administration of the therapy.

Additionally or alternatively, methods of the invention may comprise determining that the patient is responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not been administered the therapy.

Methods of the invention may comprise determining that the patient is not responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient at an earlier timepoint (e.g. pre-administration of the therapy). For example, methods of the invention may comprise determining that the patient is not responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient preadministration of the therapy.

Additionally or alternatively, methods of the invention may comprise determining that the patient is not responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s), when the reference value represents the intensity of said chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not been administered the therapy.

Methods of the invention may comprise determining that the patient is not responsive to the therapy when: the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the concentration of said same chemical shift region(s) present in a sample obtained from the patient at an earlier timepoint (e.g. pre-administration of the therapy). For example, methods of the invention may comprise determining that the patient is not responsive to the therapy when: the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CHS)3 group is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from the patient pre-administration of the therapy.

Additionally or alternatively, methods of the invention may comprise determining that the patient is not responsive to the therapy when: the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 is the same or higher in the patient sample relative to the reference value(s), when the reference value represents the intensity of said same chemical shift region(s) present in a sample obtained from a (e.g. control) patient suffering from MS that has not been administered the therapy. One aspect of the invention provides a method for monitoring a multiple sclerosis (MS) patient’s response to a therapy, the method comprising: a. identifying a concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p-hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient that has been administered the therapy; b. comparing the concentration of said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the concentration of said same one or more metabolite(s) present in a sample obtained from: the patient at an earlier timepoint (e.g. pre-administration of the therapy); and/or a (e.g. control) patient suffering from MS that has not been administered the therapy; and c. determining that the patient is responsive to the therapy when: i. the concentration of one or more metabolite(s) selected from glucose, p- hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s); or d. determining that the patient is not responsive to the therapy when: i. the concentration of one or more metabolite(s) selected from glucose, p- hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s).

Preferably, a method as described herein comprises identifying a concentration of one or more metabolite(s) (preferably two or more metabolites) selected from glucose, p- hydroxybutyrate, and myo-inositol. One aspect of the invention provides a method, the method comprising: a. obtaining a biofluid sample derived from a patient that has been administered an MS therapy, optionally a multiple sclerosis patient; b. assaying the biofluid sample for a concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, myo-inositol, a lipoprotein having a - CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient; optionally c. comparing the assayed concentration of said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the concentration of said same one or more metabolite(s) present in a sample obtained from: the patient at an earlier timepoint (e.g. pre-administration of the therapy); and/or a (e.g. control) patient suffering from MS that has not been administered the therapy; and d. confirming that the patient is responsive to the therapy when: i. the assayed concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s); and/or ii. the assayed concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is lower in the patient sample relative to the reference value(s); or e. confirming that the patient is not responsive to the therapy when: i. the assayed concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s); and/or ii. the assayed concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is the same or higher in the patient sample relative to the reference value(s).

Another aspect of the invention provides a method for monitoring a multiple sclerosis (MS) patient’s response to a therapy, the method comprising: a. identifying an intensity of a (e.g. at least one) chemical shift region(s) of a 1 H- NMR spectrum for one or more metabolites selected from glucose, p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient that has been administered the therapy, wherein the 1 H-NMR chemical shift range for glucose is selected from 3.17- 3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm; the 1 H-NMR chemical shift range for myo-inositol is selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25-3.29 ppm; the 1 H-NMR chemical shift range for p-hydroxybutyrate is selected from 1.19-1.21 ppm and 2.27-2.45 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -CH3 group of an HDL and/or LDL is 0.80-0.86 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -(CH2)n group of an HDL and/or LDL is 1.15-1.30 ppm; the 1 H-NMR chemical shift range for a lipoprotein having an -N(CH3)3 group is 3.17-3.31 ppm; b. comparing the intensity of said chemical shift region(s) for said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the intensity of said same chemical shift regions present in a sample obtained from: the patient at an earlier timepoint (e.g. pre-administration of the therapy); and/or a (e.g. control) patient suffering from MS that has not been administered the therapy; and c. determining that the patient is responsive to the therapy when: i. the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s); and/or ii. the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s); or d. determining that the patient is not responsive to the therapy when: i. the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s); and/or ii. the intensity of a (e.g. at least one) chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s).

In a preferable embodiment, methods of the invention (whether for monitoring MS progress, or monitoring response to therapy) utilise one or more metabolite(s) (preferably two or more metabolites; more preferably three or more metabolites) selected from glucose, p- hydroxybutyrate, and myo-inositol. In another preferable embodiment, methods of the invention utilise one or more metabolite(s) (preferably two or more metabolites; more preferably three or more metabolites) selected from glucose, p-hydroxybutyrate, and myoinositol; and one or more metabolites (preferably two or more metabolites; more preferably three or more metabolites) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CH ₃ )3 group.

An aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: a. identifying a concentration for each of two or more metabolites (preferably three or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol present in a sample obtained from the patient; b. comparing the concentrations of said two or more metabolites (preferably three or more metabolites) with reference values, wherein said reference values represent the concentrations of said same two or more metabolites (preferably three or more metabolites) present in a sample obtained from: i. the patient at an earlier timepoint; and/or ii. a patient suffering from MS that has not progressed

(preferably i., sample from the patient at an earlier timepoint); and c. determining there has been progression of MS in the patient when the concentration for each of said two or more metabolites (preferably three or more metabolites) is the lower in the patient sample relative to the reference values; or d. determining there has been no progression of MS in the patient when the concentration for each of said two or more metabolites (preferably three or more metabolites) is the same or higher in the patient sample relative to the reference values.

Another aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: a. identifying an intensity of a (e.g. at least one) chemical shift region(s) of a 1 H- NMR spectrum for each of two or more metabolites (preferably three or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol present in a sample obtained from the patient, wherein the 1 H-NMR chemical shift range for glucose is selected from 3.17- 3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm; the 1 H-NMR chemical shift range for myo-inositol is selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25-3.29 ppm; and the 1 H-NMR chemical shift range for p-hydroxybutyrate is selected from 1.19-1.21 ppm and 2.27-2.45 ppm; b. comparing the intensity of said chemical shift regions with reference values, wherein said reference values represent the intensity of said same chemical shift regions present in a sample obtained from: i. the patient at an earlier timepoint; and/or ii. a patient suffering from MS that has not progressed

(preferably i., sample from the patient at an earlier timepoint); and c. determining there has been progression of MS in the patient when the intensity of each of said chemical shift regions is lower in the patient sample relative to the reference values; or d. determining there has been no progression of MS in the patient when the intensity of each of said chemical shift regions is the same or higher in the patient sample relative to the reference values.

An aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: a. identifying a concentration for each of two or more metabolites (preferably three or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol present in a sample obtained from the patient; b. comparing the concentrations of said two or more metabolites (preferably three or more metabolites) with reference values, wherein said reference values represent the concentrations of said same two or more metabolites (preferably three or more metabolites) present in a sample obtained from: a patient suffering from MS that has progressed; and c. determining there has been progression of MS in the patient when the concentration for each of said two or more metabolites (preferably three or more metabolites) is the same or lower in the patient sample relative to the reference values; or d. determining there has been no progression of MS in the patient when the concentration for each of said two or more metabolites (preferably three or more metabolites) is higher in the patient sample relative to the reference values.

Another aspect of the invention provides a method for monitoring progression of multiple sclerosis (MS) in a patient, the method comprising: a. identifying an intensity of a (e.g. at least one) chemical shift region(s) of a 1 H- NMR spectrum for each of two or more metabolites (preferably three or more metabolites) selected from glucose, p-hydroxybutyrate, and myo-inositol present in a sample obtained from the patient, wherein the 1 H-NMR chemical shift range for glucose is selected from 3.17- 3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm; the 1 H-NMR chemical shift range for myo-inositol is selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25-3.29 ppm; and the 1 H-NMR chemical shift range for p-hydroxybutyrate is selected from 1.19-1.21 ppm and 2.27-2.45 ppm; b. comparing the intensity of said chemical shift regions with reference values, wherein said reference values represent the intensity of said same chemical shift regions present in a sample obtained from: a patient suffering from MS that has progressed; and c. determining there has been progression of MS in the patient when the intensity of each of said chemical shift regions is the same or lower in the patient sample relative to the reference values; or d. determining there has been no progression of MS in the patient when the intensity of each of said chemical shift regions higher in the patient sample relative to the reference values.

Furthermore, demonstrating a particularly advantageous technical effect of the invention, the inventors have demonstrated that the presently described metabolite concentration change(s), correlating with disease progression, can be abrogated (or reversed) by a series of known disease-modifying therapies. Thus, the present metabolite panel can be used to reliably inform response to therapy, allowing for identification of responder patients and also for screening of drugs.

Thus, the present invention finds utility in clinical trial assessment of MS therapy, or personalised therapy for a given MS patient, and overcomes previous reliance on techniques such as PET imaging and EDSS scoring in the assessment of treatment response.

Thus, a another aspect provides a method for monitoring a multiple sclerosis (MS) patient’s response to a therapy, the method comprising: a. identifying a concentration for each of two or more (preferably three or more) metabolites selected from glucose, p-hydroxybutyrate, and myo-inositol present in a sample obtained from the patient that has been administered the therapy; b. comparing the concentration of said two or more metabolites (preferably three or more metabolites) with reference values, wherein said reference values represent the concentrations of said same two or more metabolites (preferably three or more metabolites) present in a sample obtained from: i. the patient at an earlier timepoint (e.g. pre-administration of the therapy); and/or ii. a patient suffering from MS that has not been administered the therapy (preferably i., sample from the patient at an earlier timepoint, such as pre-administration of the therapy); and c. determining that the patient is responsive to the therapy when the concentration for each of said two or more metabolites (preferably three or more metabolites) is higher in the patient sample relative to the reference values; or d. determining that the patient is not responsive to the therapy when the concentration for any one of (preferably each of) said two or more metabolites (preferably three or more metabolites) is the same or lower in the patient sample relative to the reference values.

In a yet further aspect, the invention provides a method for monitoring a multiple sclerosis (MS) patient’s response to a therapy, the method comprising: a. identifying an intensity of a (e.g. at least one) chemical shift region(s) of a 1 H- NMR spectrum for each of two or more (preferably three or more) metabolites selected from glucose, p-hydroxybutyrate, and myo-inositol present in a sample obtained from the patient, wherein the 1 H-NMR chemical shift range for glucose is selected from 3.17- 3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm; the 1 H-NMR chemical shift range for myo-inositol is selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25-3.29 ppm; and the 1 H-NMR chemical shift range for p-hydroxybutyrate is selected from 1.19-1.21 ppm and 2.27-2.45 ppm; b. comparing the intensity of said chemical shift regions with reference values, wherein said reference values represent the intensity of said same chemical shift regions present in a sample obtained from: i. the patient at an earlier timepoint (e.g. pre-administration of the therapy); ii. a patient suffering from MS that has not been administered the therapy; and/or iii. a patient suffering from MS that has not progressed

(preferably i., sample from the patient at an earlier timepoint, such as pre-administration of the therapy); and c. determining that the patient is responsive to the therapy when the intensity of each of said chemical shift regions is higher in the patient sample relative to the reference value(s); or d. determining that the patient is not responsive to the therapy when the intensity for any one of (preferably each of) said chemical shift regions is the same or lower in the patient sample relative to the reference value(s).

As described in the Examples section in more detail, the trio of metabolites “glucose”, “P- hydroxybutyrate”, and “myo-inositol” are particularly advantageous for discriminating between patients suffering high or low levels of brain inflammation (a particular outcome of MS) as measurable by PET imaging (11C-PK-11195 DVR). Indeed, the panel is indicative of the magnitude of brain inflammation, and actually outperformed PK-11195 DVR at predicting progression.

In addition to the utility of glucose, p-hydroxybutyrate, and myo-inositol (the concentrations of which become lower as an indication of MS progression), it has been demonstrated that the panel may be supplemented with one or more of mobile -CH3 HDL/LDL, mobile (-CH2-)n LDL, -N(CHS)3/ free choline (the concentrations of which become higher as an indication of MS progression). Thus, robustness of the method may be yet further enhanced by the introduction of a further metabolite shown to drive separation of the metabolome of progressor vs non-progressor patients. Such embodiments allow focus around a minimal panel of biomarkers represented by a trio of metabolites, any two of which can be assayed/ monitored to reliably inform progression of MS. Advantageously, the methods of the invention are particularly accurate and/or sensitive and/or specific.

In one embodiment, a method for monitoring progression of multiple sclerosis (MS) in a patient further comprises: a. identifying a concentration for one or more (preferably two or more, more preferably three or more) further metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group in a sample obtained from the patient; b. comparing the concentration of said one or more (preferably two or more, more preferably three or more) further metabolite(s) with reference value(s), wherein said reference value(s) represent the concentrations of said one or more (preferably two or more, more preferably three or more) further metabolite(s) present in a sample obtained from: i. the patient at an earlier timepoint; and/or ii. a patient suffering from MS that has not progressed

(preferably i., sample form the patient at an earlier timepoint); and c. determining there has been progression of MS in the patient when the concentration for said one or more (preferably two or more, more preferably three or more) further metabolite(s) is higher in the patient sample relative to the reference values; or d. determining there has been no progression of MS in the patient when the concentration for said one or more (preferably two or more, more preferably three or more) further metabolite(s) is the same or lower in the patient sample relative to the reference values.

In one embodiment, a method for monitoring progression of multiple sclerosis (MS) in a patient further comprises: a. identifying a concentration for one or more (preferably two or more, more preferably three or more) further metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group in a sample obtained from the patient; b. comparing the concentration of said one or more (preferably two or more, more preferably three or more) further metabolite(s) with reference value(s), wherein said reference value(s) represent the concentrations of said one or more (preferably two or more, more preferably three or more) further metabolite(s) present in a sample obtained from: a patient suffering from MS that has progressed; and c. determining there has been progression of MS in the patient when the concentration for said one or more (preferably two or more, more preferably three or more) further metabolite(s) is the same or higher in the patient sample relative to the reference values; or d. determining there has been no progression of MS in the patient when the concentration for said one or more (preferably two or more, more preferably three or more) further metabolite(s) is lower in the patient sample relative to the reference values.

Said further metabolites likewise find utility in monitoring patient response to therapy.

In one embodiment, a method for monitoring a MS patient’s response to therapy further comprises: a. identifying a concentration for one or more (preferably two or more, more preferably three or more) further metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group in a sample obtained from the patient; b. comparing the concentration of said one or more (preferably two or more, more preferably three or more) further metabolite(s) with reference value(s), wherein said reference value(s) represent the concentrations of said one or more further metabolite(s) present in a sample obtained from: i. the patient at an earlier timepoint (e.g. pre-administration of the therapy); and/or ii. a patient suffering from MS that has not been administered the therapy (preferably i., sample from the patient at an earlier timepoint, such as preadministration of the therapy); and c. determining that the patient is responsive to the therapy when the concentration for said one or more (preferably two or more, more preferably three or more) further metabolite(s) is lower in the patient sample relative to the reference values; or d. determining that the patient is not responsive to the therapy when the concentration for said one or more (preferably two or more, more preferably three or more) further metabolite(s) is the same or higher in the patient sample relative to the reference values.

In all aspects and embodiments described herein e.g. as related to a “a method for monitoring a multiple sclerosis (MS) patient’s response to a therapy” described herein, the “therapy” is preferably a drug; more preferably a candidate drug. Said drug (for example, candidate drug) may be comprised within a pharmaceutical composition, optionally together with one or more pharmaceutically acceptable excipient(s).

A suitable therapy is preferably a disease modifying therapy/ therapies for MS, which may suppress/ delay progression of disability. For example, therapy may include an injectable medication, such as Avonex (interferon beta-1 a); Betaseron (interferon beta-1 b); Copaxone (glatiramer acetate), Extavia (interferon beta-1 b), Glatiramer Acetate Injection (glatiramer acetate, generic equivalent of Copaxone 20 mg and 40 mg doses), Glatopa (glatiramer acetate, generic equivalent of Copaxone 20mg and 40mg doses), Kesimpta® (ofatumumab), Plegridy (peginterferon beta-1 a), and/or Rebif (interferon beta-1 a); an oral medication, such as Aubagio (teriflunomide), Bafiertam (monomethyl fumarate), Gilenya (fingolimod), Mavenclad (cladribine), Mayzent (siponimod), Tecfidera (dimethyl fumarate), Vumerity (diroximel fumarate), oral methylprednisolone, and/or Zeposia (ozanimod); and/or an infused medication, such as Lemtrada (alemtuzumab), Novantrone (mitoxantrone), Ocrevus (ocrelizumab), intravenous methylprednisolone, and/or Tysabri (natalizumab). The treatment may additionally or alternatively comprise/ include autologous hematopoietic stem cell transplantation (AHSCT).

The therapy may be a Nuclear factor-erythroid factor 2-related factor 2 (Nrf2) activator (such as dimethyl fumarate), a sphingosine-1 -phosphate receptor modulator (such as fingolimod), a myelin basis protein or polypeptide that simulates myelin basic protein (such as glatiramer acetate), and/or an interferon (such as interferon beta 1a and/or interferon beta 1b).

Such sphingosine-1 -phosphate receptor modulator preferably functions to promote sequestration of lymphocytes in lymph nodes, suppressing lymphocyte contribution to an autoimmune reaction. In a preferable embodiment, the therapy is selected from the list comprising dimethyl fumarate, fingolimod, glatiramer acetate, interferon beta 1a and interferon beta 1b. For example, the therapy may be glatiramer acetate.

In a preferable embodiment, a reference value may represent the concentration of said same one or more metabolite(s) (preferably two or more metabolites, more preferably three or more metabolites) present in a sample obtained from the patient at an earlier timepoint. The term “a sample obtained from the patient at an earlier timepoint” as used throughout this disclosure may refer to a sample obtained 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months (e.g. 10, 12, or 14 months) prior to the test sample (i.e. the patient sample of step a.). The term “a sample obtained from the patient at an earlier timepoint” may refer to a sample obtained 2-24 months, 4-22 months, 6-20 months, 8-18 months, 10-16 months, or 12-14 months (preferably 11-13 months) prior to the test sample (i.e. the patient sample of step a.).

In the context of a method for monitoring a multiple sclerosis (MS) patient’s response to a therapy, a reference value may represent the concentration of said same one or more metabolite(s) (e.g. two or more metabolites) present in a sample obtained from the patient pre-administration of the therapy. The term “sample obtained from the patient preadministration of the therapy” may refer to a sample obtained 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months (e.g. 10, 12, or 14 months) prior to the test sample (i.e. the patient sample of step a.). The term “sample obtained from the patient pre-administration of the therapy” may refer to a sample obtained 2-24 months, 4-22 months, 6-20 months, 8-18 months, 10-16 months, or 12-14 months (preferably 11-13 months) prior to test sample (e.g. the patient sample of step a.).

A reference value may represent the concentration of said same one or more metabolite(s) (e.g. two or more metabolites) present in a sample obtained from a patient suffering from MS that has not progressed. A “patient suffering from MS that has not progressed” may refer to a control MS patient demonstrating no increase in a level of MS symptoms over time. The term “over time” throughout this disclosure may mean over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months (e.g. 10, 12, or 14 months). The term “over time” may mean over 2-24 months, 4-22 months, 6-20 months, 8-18 months, 10-16 months, or 12-14 months (preferably over 11-13 months). Throughout this disclosure, a suitable MS symptom may be brain inflammation (e.g. in the NAWN and/or perilesion, preferably NAWN). Brain inflammation may be referred to as “encephalitis”. In one embodiment, determining there has been progression of MS in the patient means determining there has been progression of brain inflammation (e.g. encephalitis) in the patient. In other words, determining there has been progression of MS in the patient may mean determining there has been an increase in brain inflammation (e.g. encephalitis) in the patient. Determining there has been no progression of MS in the patient may mean determining there has been no progression of brain inflammation (e.g. encephalitis) in the patient. In other words, determining there has been progression of MS in the patient may mean determining there has been no increase in brain inflammation (e.g. encephalitis) in the patient.

In one embodiment, determining that the patient is responsive to the therapy means determining that brain inflammation (e.g. encephalitis) has been suppressed. Determining that the patient is not responsive to the therapy may mean determining that brain inflammation (e.g. encephalitis) has not been suppressed.

The skilled person will understand that all references to a “patient” as part of a reference value described herein means an otherwise comparable patient (e.g. to allow for appropriately ‘controlled’ comparison between the patient sample and reference value). Any patient of a reference value described herein is thus a suitable ‘control’ patient. For example, “a patient suffering from MS that has not progressed”, as described herein as part of a reference value, is understood by the skilled person to be an otherwise comparable patient (e.g. in terms of disease stage/ prognosis) to a test patient (whose MS progression is being monitored by the invention) that “has” demonstrated progression, but for the fact the reference/ control patient’s MS did not progress (from an equivalent baseline level in both the reference/ control patient) over a controlled time period.

A “patient suffering from MS that has not progressed” may refer to a control MS patient that was stage-matched to the test patient (i.e. the patient being monitored for progression/ response to therapy in methods of the invention) at an earlier timepoint (prior to isolation of the patient sample for use in the invention), wherein the control MS patient did not demonstrate progression of MS at the time of isolation of the patient sample for use in the invention relative to said earlier timepoint.

Stage-matched means the control patient and test patient had the same (e.g. substantially the same) MS prognosis (e.g. level of MS symptoms), for example as measured by EDSS and/or by determining and comparing a level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) in the control patient and test patient (said level being the same, e.g. substantially the same); preferably by determining and comparing a level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN).

A “patient suffering from MS that has progressed” may refer to a control MS patient that was stage-matched to the test patient (i.e. the patient being monitored for progression/ response to therapy in methods of the invention) at an earlier timepoint (prior to isolation of the patient sample for use in the invention), wherein the control MS patient did demonstrate progression of MS at the time of isolation of the patient sample for use in the invention relative to said earlier timepoint.

Progression may be defined as a higher level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) in the control patient at the time of isolation of the patient sample for use in the invention relative to said earlier timepoint. Additionally or alternatively, progression may be defined as a change in EDSS score (AEDSS) of >0.5 in the control patient at the time of isolation of the patient sample for use in the invention relative to said earlier timepoint.

No progression may be defined as the same or higher level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) in the control patient at the time of isolation of the patient sample for use in the invention relative to said earlier timepoint. Additionally or alternatively, no progression may be defined as a change in EDSS score (AEDSS) of <0.0 in the control patient at the time of isolation of the patient sample for use in the invention relative to said earlier timepoint.

The “earlier timepoint” may refer to 2-24 months, 4-22 months, 6-20 months, 8-18 months, 10-16 months, or 12-14 months (preferably 11-13 months) prior to the time of isolation of the patient sample for use in the invention. The “earlier timepoint” may refer to about at least 2, 4, 8, 10, 12, 14, 16, 18, or 24 months prior to the time of isolation of the patient sample for use in the invention. For example, the “earlier timepoint” may refer to about at least 10, 12, or 14 months prior to the time of isolation of the patient sample for use in the invention.

The “earlier timepoint” may refer to about 2, 4, 8, 10, 12, 14, 16, 18, or 24 months prior to the time of isolation of the patient sample for use in the invention. For example, the “earlier timepoint” may refer to about 10, 12, or 14 months prior to the time of isolation of the patient sample for use in the invention. A “patient suffering from MS that has not progressed” may refer to a control MS patient having a lower level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) relative to the patient being monitored (in methods of the invention).

A “patient suffering from MS that has not progressed” may refer to a control MS patient having a lower level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) relative to the patient being monitored (in methods of the invention), the (non-progressor) patient demonstrating no increase in a level of MS symptoms (e.g. brain inflammation, such as in the NAWN) over time.

A “patient suffering from MS that has not progressed” may refer to a control patient that had an equivalent (e.g. the same) MS prognosis as the patient being monitored (in methods of the invention) at a first (earlier) timepoint, wherein said control patient’s MS prognosis is unchanged (relative to the first timepoint) at a second timepoint when a sample for use in a method of the invention has been obtained.

The term “no increase” preferably means substantially no increase. The term “substantially no increase” may refer to an increase of <5%, <2% or <1% in the level of MS symptoms over time. The term “substantially no increase” may suitably refer to an increase of 0% in the level of MS symptoms over time (e.g. may refer to no increase at all of MS symptoms over time).

The term “unchanged” preferably means substantially no change. The term “substantially no increase” may refer to a worsening of <5%, <2% or <1% in the patient’s prognosis over time. The term “substantially no increase” may suitably refer a worsening of 0% in the patient’s prognosis over time (e.g. may refer to no increase at all of MS symptoms over time).

A reference value may represent the concentration of said same one or more metabolite(s) (e.g. two or more metabolites) present in a sample obtained from a patient suffering from MS that has progressed. A “patient suffering from MS that has progressed” may refer to a control MS patient demonstrating an increase in a level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) over time. The term “over time” throughout this disclosure may mean over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months (e.g. 10, 12, or 14 months). The term “over time” may mean over 2-24 months, 4-22 months, 6-20 months, 8-18 months, 10-16 months, or 12-14 months (preferably over 11-13 months). A “patient suffering from MS that has progressed” may refer to a control MS patient having an equivalent or higher level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) relative to the patient being monitored (in methods of the invention).

A “patient suffering from MS that has progressed” may refer to a control MS patient having an equivalent or higher level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) relative to the patient being monitored (in methods of the invention), the (progressor) patient demonstrating an increase in a level of MS symptom(s) over time.

A “patient suffering from MS that has progressed” may refer to a control patient that had an equivalent (e.g. the same) MS prognosis as the patient being monitored (in methods of the invention) at a first (earlier) timepoint, wherein said control patient’s MS prognosis has worsened (relative to the first timepoint) at a second timepoint when a sample for use in a method of the invention has been obtained.

The term “increase” may refer to an increase of at least 5%, 8%, 10%, 12%, 14%, 16%, 18% or 20% in the level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) over time. The term “increase” may refer to an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in the level of MS symptom(s) (e.g. brain inflammation, such as in the NAWN) over time.

MS is a progressive neurological disorder that is untreatably, and thus any given patient’s disease state may progress over time albeit without consistency amongst MS patients and at an unpredictable rate. For example, a patient suffering relapsing-remitting MS (RRMS) may suffer disease progression even while still classed as an RRMS patient. When progression passes a threshold, such (previously) RRMS patient can be considered to have “transitioned” to secondary-progressive MS (SPMS), a more aggressive of the disease. Thus, “progression” may be defined as worsening of the patient’s prognosis. In other words, “progression” may refer to deterioration of patient health caused by MS.

The monitoring or progression (or no progression) may be performed within a given stage of MS. For example, the patient may have RRMS, and methods of the invention may involve monitoring the progression (e.g. worsening prognosis) of said RRMS. In one embodiment, the patient may have the patient may have SPMS, and methods of the invention may involve monitoring the progression (e.g. worsening prognosis) of said SPMS. In one embodiment, the patient may have primary-progressive MS (PPMS), and methods of the invention may involve monitoring the progression (e.g. worsening prognosis) of said PPMS.

For example, it may be said that methods of the invention do no comprise determining conversion of a subject from one clinical stage (e.g. one stage of MS) to another stages of MS. For example, it may be said that methods of the invention do not comprise determining conversion of a subject from clinically isolated syndrome to clinically definite multiple sclerosis.

The term “monitoring progression of multiple sclerosis (MS) in a patient” may be used synonymously with the term “monitoring an MS patient’s prognosis”. The term “determining there has been progression of MS” may be used synonymously with the term “determining that the patient’s prognosis has worsened, or has become poorer, or has deteriorated”. Similarly, the term “determining there has been no progression of MS” may be used synonymously with the term “determining that the patient’s prognosis has not worsened, or has not become poorer, or has not deteriorated”.

A reference value for an MS patient that “has progressed” may alternatively be referred to as a reference value for an MS patient “whose prognosis has worsened, or become poorer, or deteriorated”. A reference value for an MS patient that “has not progressed” may alternatively be referred to as a reference value for a patient “whose prognosis has not worsened, or not become poorer, or not deteriorated”.

In terms of clinical scales, progression may be defined as a worsening score measured by the Expanded Disability Status Scale (EDSS), a method of quantifying disability in MS, widely use in clinical assessment. Indeed, in the study described here (see the Examples section), the EDSS was used to assess patient disability at baseline and approximately 1 year after sampling. Patients were considered “progressors” (e.g. progression of MS was determined) when a change in EDSS score (AEDSS) was >0.5, and patients were considered non-progressors (e.g. progression of MS was not determined) when an AEDSS score was <0.

A “patient suffering from MS that has not progressed” may refer to a control MS patient demonstrating an increase in EDSS score (AEDSS) of <0.5 over time. The term “over time” throughout this disclosure may mean over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months (e.g. 10, 12, or 14 months). The term “over time” may mean over 2-24 months, 4-22 months, 6-20 months, 8-18 months, 10-16 months, or 12-14 months (preferably over 11-13 months).

A “patient suffering from MS that has not progressed” may refer to a control MS patient having a lower EDSS score relative to the patient being monitored (in methods of the invention).

A “patient suffering from MS that has not progressed” may refer to a control MS patient having a lower EDSS score relative to the patient being monitored (in methods of the invention), the (non-progressor) patient demonstrating an increase in EDSS score (AEDSS) of <0.5 over time.

A “patient suffering from MS that has progressed” may refer to a control MS patient demonstrating an increase in EDSS score (AEDSS) of >0.5 over time. The term “over time” throughout this disclosure may mean over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months (e.g. 10, 12, or 14 months). The term “over time” may mean over 2-24 months, 4-22 months, 6-20 months, 8-18 months, 10-16 months, or 12-14 months (preferably over 11-13 months).

A “patient suffering from MS that progressed” may refer to a control MS patient having an equivalent or higher EDSS score relative to the patient being monitored (in methods of the invention).

A “patient suffering from MS that has progressed” may refer to a control MS patient having an equivalent or higher EDSS score relative to the patient being monitored (in methods of the invention), the (progressor) patient demonstrating an increase in EDSS score (AEDSS) of >0.5 over time.

The Expanded Disability Status Scale (EDSS) is a method of quantifying disability in multiple sclerosis and monitoring changes in the level of disability over time. It is widely used in clinical trials and in the assessment of people with MS. The scale was developed by a neurologist called John Kurtzke in 1983 as an advance from his previous 10 step Disability Status Scale (DSS).

The EDSS scale ranges from 0 to 10 in 0.5 unit increments that represent higher levels of disability. Scoring is based on an examination by a neurologist. EDSS steps 1.0 to 4.5 refer to people with MS who are able to walk without any aid and is based on measures of impairment in eight functional systems (FS): pyramidal - muscle weakness or difficulty moving limbs cerebellar - ataxia, loss of balance, coordination or tremor brainstem - problems with speech, swallowing and nystagmus sensory - numbness or loss of sensations bowel and bladder function visual function - problems with sight cerebral functions - problems with thinking and memory other

A functional system (FS) represents a network of neurons in the brain with responsibility for particular tasks. Each FS is scored on a scale of 0 (no disability) to 5 or 6 (more severe disability).

EDSS steps 5.0 to 9.5 are defined by the impairment to walking. EDSS steps 1.0 to 4.5 refer to people with MS who are fully ambulatory. EDSS steps 5.0 to 9.5 are defined by the impairment to ambulation.

The clinical meaning of each possible result (e.g. score) is the following:

0.0: Normal Neurological Exam

1.0: No disability, minimal signs in 1 FS

1.5: No disability, minimal signs in more than 1 FS

2.0: Minimal disability in 1 FS

2.5: Mild disability in 1 or Minimal disability in 2 FS

3.0: Moderate disability in 1 FS or mild disability in 3 - 4 FS, though fully ambulatory

3.5: Fully ambulatory but with moderate disability in 1 FS and mild disability in 1 or 2 FS; or moderate disability in 2 FS; or mild disability in 5 FS

4.0: Fully ambulatory without aid, up and about 12hrs a day despite relatively severe disability. Able to walk without aid 500 meters

4.5: Fully ambulatory without aid, up and about much of day, able to work a full day, may otherwise have some limitations of full activity or require minimal assistance. Relatively severe disability. Able to walk without aid 300 meters

5.0: Ambulatory without aid for about 200 meters. Disability impairs full daily activities

5.5: Ambulatory for 100 meters, disability precludes full daily activities 6.0: Intermittent or unilateral constant assistance (cane, crutch or brace) required to walk 100 meters with or without resting

6.5: Constant bilateral support (cane, crutch or braces) required to walk 20 meters without resting

7.0: Unable to walk beyond 5 meters even with aid, essentially restricted to wheelchair, wheels self, transfers alone; active in wheelchair about 12 hours a day

7.5: Unable to take more than a few steps, restricted to wheelchair, may need aid to transfer; wheels self, but may require motorized chair for full day's activities

8.0: Essentially restricted to bed, chair, or wheelchair, but may be out of bed much of day; retains self care functions, generally effective use of arms

8.5: Essentially restricted to bed much of day, some effective use of arms, retains some self care functions

9.0: Helpless bed patient, can communicate and eat

9.5: Unable to communicate effectively or eat/swallow

10.0: Death due to MS

Additionally or alternatively, progression may refer to a change in brain inflammation in the patient (e.g. increasing brain inflammation over time).

Thus, progression may be defined with reference to brain inflammation, such as inflammation in the normal appearing white matter (NAWM) and/or in the perilesional area (e.g. perilesion) of the brain (preferably in the NAWM). MS patients can suffer brain inflammation that typically results from the immune system attacking the myelin sheath around nerves.

The term “a method for monitoring progression of multiple sclerosis (MS) in a patient” may be used synonymously with the term “a method for monitoring brain inflammation (e.g. of the NAWM and/or perilesion, preferably NAWM) in a multiple sclerosis (MS) patient”. The term “determining there has been progression of MS” may be used synonymously with the term “determining there has been progression of brain inflammation”, and the term “determining there has been no progression of MS” may be used synonymously with the term “determining there has been no progression of brain inflammation”.

Thus, aspects of the invention may be worded alternatively, as outlined below (all embodiments of the invention, described elsewhere herein, apply equally to the following aspects wherein “progression” is defined in terms of “brain inflammation”. An aspect of the invention provides a method for monitoring brain inflammation (e.g. of the NAWM and/or perilesion, preferably NAWM) in a multiple sclerosis (MS) patient, the method comprising: a. identifying a concentration of one or more metabolite(s) selected from glucose, p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient; b. comparing the concentration of said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the concentration of said same one or more metabolite(s) present in a sample obtained from: the patient at an earlier timepoint; and/or a patient suffering from MS whose brain inflammation has not progressed; c. determining there has been progression of brain inflammation in the patient when: i. the concentration of one or more metabolite(s) selected from glucose, p- hydroxybutyrate, and myo-inositol is lower in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is higher in the patient sample relative to the reference value(s); or d. determining there has been no progression of brain inflammation in the patient when: i. the concentration of one or more metabolite(s) selected from glucose, p- hydroxybutyrate, and myo-inositol is the same or higher in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is the same or lower in the patient sample relative to the reference value(s).

Throughout this disclosure, reference to brain inflammation may preferably mean encephalitis. An aspect of the invention provides a method for monitoring brain inflammation (e.g. of the NAWM and/or perilesion, preferably NAWM) in a multiple sclerosis (MS) patient, the method comprising: a. identifying a concentration for each of two or more (preferably three or more) metabolites selected from glucose, p-hydroxybutyrate, and myo-inositol present in a sample obtained from the patient; b. comparing the concentrations of said two or more metabolites (preferably three or more) with reference values, wherein said reference values represent the concentrations of said same two or more metabolites present in a sample obtained from: i. the patient at an earlier timepoint; and/or ii. a patient suffering from MS whose brain inflammation has not progressed; and c. determining there has been progression of brain inflammation in the patient when the concentration for each of said two or more (preferably three or more) metabolites is lower in the patient sample relative to the reference value(s); or d. determining there has been no progression of brain inflammation in the patient when the concentration for each of said two or more (preferably three or more) metabolites is the same or higher in the patient sample relative to the reference value(s).

An aspect of the invention provides a method for monitoring brain inflammation (e.g. of the NAWM and/or perilesion, preferably NAWM) in a multiple sclerosis (MS) patient, the method comprising: a. identifying an intensity of a (e.g. at least one) chemical shift region(s) of a 1 H- NMR spectrum for one or more metabolites selected from glucose, p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient, wherein the 1 H-NMR chemical shift range for glucose is selected from 3.17- 3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm; the 1 H-NMR chemical shift range for myo-inositol is selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25-3.29 ppm; the 1 H-NMR chemical shift range for p-hydroxybutyrate is selected from 1.19-1.21 ppm and 2.27-2.45 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -CH3 group of an HDL and/or LDL is 0.80-0.86 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -(CH2)n group of an HDL and/or LDL is 1.15-1.30 ppm; the 1 H-NMR chemical shift range for a lipoprotein having an -N(CHs)3 group is 3.17-3.31 ppm; b. comparing the intensity of said chemical shift region(s) for said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the intensity of said same chemical shift region(s) present in a sample obtained from: the patient at an earlier timepoint; and/or a patient suffering from MS whose brain inflammation has not progressed; c. determining there has been progression of brain inflammation in the patient when: i. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is lower in the patient sample relative to the reference value(s); and/or ii. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is higher in the patient sample relative to the reference value(s); or d. determining there has been no progression of brain inflammation in the patient when: i. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or higher in the patient sample relative to the reference value(s); and/or ii. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or lower in the patient sample relative to the reference value(s).

An aspect of the invention provides a method for monitoring brain inflammation (e.g. of the NAWM and/or perilesion, preferably NAWM) in a multiple sclerosis (MS) patient, the method comprising: a. identifying a concentration of one or more metabolite(s) selected from glucose, p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient; b. comparing the concentration of said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the concentration of said same one or more metabolite(s) present in a sample obtained from: a patient suffering from MS whose brain inflammation has progressed; c. determining there has been progression of brain inflammation in the patient when: i. the concentration of one or more metabolite(s) selected from glucose, - hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s); or d. determining there has been no progression of brain inflammation in the patient when: i. the concentration of one or more metabolite(s) selected from glucose, p- hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s); and/or ii. the concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is lower in the patient sample relative to the reference value(s).

Another aspect of the invention provides a method for monitoring brain inflammation (e.g. of the NAWM and/or perilesion, preferably NAWM) in a multiple sclerosis (MS) patient, the method comprising: a. identifying an intensity of a (e.g. at least one) chemical shift region(s) of a 1 H- NMR spectrum for one or more metabolites selected from glucose, p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in a sample obtained from the patient, wherein the 1 H-NMR chemical shift range for glucose is selected from 3.17- 3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm; the 1 H-NMR chemical shift range for myo-inositol is selected from 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25-3.29 ppm; the 1 H-NMR chemical shift range for p-hydroxybutyrate is selected from 1.19-1.21 ppm and 2.27-2.45 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -CH3 group of an HDL and/or LDL is 0.80-0.86 ppm; the 1 H-NMR chemical shift range for a lipoprotein having a -(CH2)n group of an HDL and/or LDL is 1.15-1.30 ppm; the 1 H-NMR chemical shift range for a lipoprotein having an -N(CHs)3group is 3.17-3.31 ppm; b. comparing the intensity of said chemical shift region(s) for said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the intensity of said same chemical shift region(s) present in a sample obtained from: a patient suffering from MS whose brain inflammation has progressed; and c. determining there has been progression of brain inflammation in the patient when: i. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s); and/or ii. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is the same or higher in the patient sample relative to the reference value(s); or d. determining there has been no progression of brain inflammation in the patient when: i. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s); and/or ii. the intensity of a chemical shift region(s) for one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group is lower in the patient sample relative to the reference value(s).

An aspect of the invention provides a method for monitoring brain inflammation (e.g. of the NAWM and/or perilesion, preferably NAWM) in a multiple sclerosis (MS) patient, the method comprising: a. identifying a concentration for each of two or more (preferably three or more) metabolites selected from glucose, p-hydroxybutyrate, and myo-inositol present in a sample obtained from the patient; b. comparing the concentrations of said two or more metabolites (preferably three or more) with reference values, wherein said reference values represent the concentrations of said same two or more metabolites present in a sample obtained from: a patient suffering from MS whose brain inflammation has progressed; and c. determining there has been progression of brain inflammation in the patient when the concentration for each of said two or more (preferably three or more) metabolites is the same or lower in the patient sample relative to the reference value(s); or d. determining there has been no progression of brain inflammation in the patient when the concentration for each of said two or more (preferably three or more) metabolites is higher in the patient sample relative to the reference value(s).

One aspect of the invention provides a method, the method comprising: a. obtaining a biofluid sample derived from a patient, optionally a multiple sclerosis patient; b. assaying the biofluid sample for a concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, myo-inositol, a lipoprotein having a - CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group present in the sample; optionally c. comparing the assayed concentration of said one or more metabolite(s) with a reference value(s), wherein said reference value(s) represents the concentration of said same one or more metabolite(s) present in a biofluid sample obtained from: a patient (e.g. suffering from MS) having brain inflammation that has progressed; and d. confirming there has been progression of brain inflammation (e.g. in the MS patient) when: i. the assayed concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is the same or lower in the patient sample relative to the reference value(s); and/or ii. the assayed concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is the same or higher in the patient sample relative to the reference value(s); or e. confirming there has been no progression of brain inflammation (e.g. in the MS patient) when: i. the assayed concentration of one or more metabolite(s) selected from glucose, p-hydroxybutyrate, and myo-inositol is higher in the patient sample relative to the reference value(s); and/or ii. the assayed concentration of one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group is lower in the patient sample relative to the reference value(s).

The term “a patient suffering from MS whose brain inflammation has not progressed” may be used synonymously with the term “a patient suffering from MS having brain inflammation that has not progressed”.

The term “a patient suffering from MS whose brain inflammation has progressed” may be used synonymously with the term “a patient suffering from MS having brain inflammation that has progressed”.

The term “response to therapy” may mean “suppression of brain inflammation (e.g. of the NAWM and/or perilesion, preferably NAWM)”.

Brain inflammation may be detectable (e.g. and measurable) by (e.g. by PET imaging) with a TSPO-binding ligand (e.g. radioligand), such as 11C-PK-11195.

MS lesions develop in the central nervous system (e.g. brain) of MS patients. The term lesion refers to an area of damage or scarring (sclerosis) in the central nervous system caused by MS. Lesions may also be called plaques, and are typically caused by inflammation that results from the immune system attacking the myelin sheath around nerves. The area in the vicinity of a lesion may be referred to as a “perilesion”. A perilesion may be within 0.2mm, 0.4mm, 0.8mm, 1mm, 1.2mm, 1.4mm, 1.6mm, 1.8mm or 2mm of an MS lesion. A perilesion may be within 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, or 10mm or an MS lesion.

A “patient suffering from MS that has not progressed” may refer to a control MS patient demonstrating no increase in a level of brain inflammation (e.g. in the NAWN and/or perilesion) over time. The term “over time” throughout this disclosure may mean over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months (e.g. 10, 12, or 14 months). The term “over time” may mean over 2-24 months, 4-22 months, 6-20 months, 8-18 months, 10-16 months, or 12- 14 months (preferably over 11-13 months).

A “patient suffering from MS that has not progressed” may refer to a control MS patient having a lower level of brain inflammation (e.g. in the NAWN and/or perilesion) relative to the patient being monitored (in methods of the invention).

A “patient suffering from MS that has not progressed” may refer to a control MS patient having a lower level of brain inflammation (e.g. in the NAWN and/or perilesion) relative to the patient being monitored (in methods of the invention), the (non-progressor) patient demonstrating no increase in a level of brain inflammation (e.g. in the NAWN and/or perilesion) over time.

The term “no increase” preferably means substantially no increase. The term “substantially no increase” may refer to an increase of <5%, <2% or <1% in the level of brain inflammation (e.g. in the NAWN and/or perilesion) over time. The term “substantially no increase” may suitably refer an increase of 0% in the level of brain inflammation (e.g. in the NAWN and/or perilesion) over time (e.g. may refer to no increase at all of brain inflammation (e.g. in the NAWN and/or perilesion) over time).

A “patient suffering from MS that has progressed” may refer to a control MS patient demonstrating an increase in a level of brain inflammation (e.g. in the NAWN and/or perilesion) over time. The term “over time” throughout this disclosure may mean over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months (e.g. 10, 12, or 14 months). The term “over time” may mean over 2-24 months, 4-22 months, 6-20 months, 8-18 months, 10-16 months, or 12- 14 months (preferably over 11-13 months). A “patient suffering from MS that has progressed” may refer to a control MS patient having an equivalent or higher level of brain inflammation (e.g. in the NAWN and/or perilesion) relative to the patient being monitored (in methods of the invention).

A “patient suffering from MS that has progressed” may refer to a control MS patient having an equivalent or higher level of brain inflammation (e.g. in the NAWN and/or perilesion) relative to the patient being monitored (in methods of the invention), the (progressor) patient demonstrating an increase in a level of brain inflammation (e.g. in the NAWN and/or perilesion) over time.

The term “increase” may refer to an increase of at least 5%, 8%, 10%, 12%, 14%, 16%, 18% or 20% in the level of brain inflammation (e.g. in the NAWN and/or perilesion) over time. The term “increase” may refer to an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in the level of brain inflammation (e.g. in the NAWN and/or perilesion) over time.

A further advantage of the invention includes the ability to classify the clinical course of MS in the patient via relapse monitoring. For example, relapsing-remitting MS (RRMS) and primary progressive MS (PPMS) may be distinguished during initial diagnosis, and RRMS vs. secondary progressive MS (SPMS) may be distinguished in the transitional phase.

That being said, reference to a “multiple sclerosis patient” may embrace a patient having any one of the four basic MS disease types (also called types or courses or phenotypes), for example as have been defined by the International Advisory Committee on Clinical Trials of MS in 2013: clinically isolated syndrome (CIS), relapsing remitting (RRMS), secondary progressive (SPMS) and primary progressive (PPMS). For example, multiple sclerosis patient may have CIS (in such cases, the patient may have been diagnosed to have CIS, but not yet diagnosed to have CDMS). The multiple sclerosis patient may have SPMS. The multiple sclerosis patient may have PPMS.

In a preferable embodiment, the multiple sclerosis patient has relapsing remitting multiple sclerosis (RRMS). In other words, the patient may be an RRMS patient.

Preferably, metabolite concentrations described herein may be determined during a remission (e.g. as opposed to during a relapse). In a preferable embodiment, the MS patient (e.g. RRMS patient) is in remission. Toward discussion of the term “remission”, a “relapse’ will first be described in more detail below.

A “relapse” of MS (also known as an exacerbation, attack or flare-up) means an episode of the occurrence of new symptoms or the worsening of old symptoms. A relapse can be (very) mild, or severe enough to interfere with a person’s ability to function. Symptoms vary from person to person and from one exacerbation to another. For example, the exacerbation might include an episode of optic neuritis (e.g. caused by inflammation of the optic nerve that impairs vision), or problems with balance or severe fatigue. Some relapses produce only one symptom (e.g. related to inflammation in a single area of the central nervous system). Other relapses may cause two or more symptoms at the same time (e.g. related to inflammation in more than one area of the central nervous system). The “relapse” may be referred to as an “acute relapse”.

Suitably, to be a true exacerbation, the attack lasts at least 24 hours and can be separated from a (the) previous attack by at least 30 days. Suitably, the relapse occurs in the absence of infection, or other cause of symptoms. Most exacerbations last from a few days to several weeks or even months. Relapses often occur without warning, but are sometimes associated with a period of illness or stress. The symptoms of a relapse may disappear altogether, with or without treatment, although some symptoms often persist, with repeated attacks happening over several years.

Periods between attacks are known as periods of “remission”. These can last for months or even years at a time. An MS patient that is in remission is in a disease phase defined by mild or no symptoms of MS, and the absence of an (e.g. acute) relapse. It is during this remission phase where concentrations of metabolites of the invention may preferably be determined as an accurate surrogate of MS progression in the patient.

When progression is determined, it may be determined that the patient’s prognosis for developing symptoms has worsened (e.g. the risk of suffering a MS symptom has become higher due to progression of the disease). Examples of such symptoms include: fatigue, difficulty walking, vision problems (such as blurred vision), problems controlling the bladder, numbness (or tingling) in different parts of the body, muscle stiffness and spasms, problems with balance and co-ordination, and/or problems with thinking, learning and planning. Symptoms may also include optic neuritis (e.g. caused by inflammation of the optic nerve that impairs vision), or problems with balance or severe fatigue, and/or inflammation in one or more area of the central nervous system.

When progression is determined, it may be determined that the patient’s prognosis for suffering a relapse has worsened (e.g. the risk of suffering a relapse has become higher due to progression of the disease).

A method of the invention may comprise a step of measuring a concentration of two or more metabolite(s) present in a sample obtained from a subject.

A method of the invention may comprise a step of obtaining a ¹ H-NMR spectrum of a sample obtained from a subject.

The concentrations of the metabolites in a sample can be measured using any suitable technique known in the art. By way of example, the following techniques may be used alone or in combination to detect and quantify molecules in solution, and are thus suitable for determining metabolite concentrations: Nuclear Magnetic Resonance (NMR) spectroscopy, mass spectrometry, gas chromatography, ultraviolet (UV) spectrometry (for example in combination with high-performance liquid chromatography [HPLC] as HPLC-UV), infrared spectroscopy, and a biochemical assay. A metabolite is preferably identified using NMR, more preferably ¹ H-NMR. The biochemical assay may be an enzymatic assay.

In one embodiment, the concentration of two or more metabolites is determined using NMR spectroscopy. In one embodiment, the concentration of two or more metabolites is determined using mass spectrometry. In one embodiment, the concentration of two or more metabolites is determined using HPLC-UV. In one embodiment, the concentration of two or more metabolites is determined using infrared spectroscopy.

The concentration of a metabolite in a sample can be expressed in a number of different ways, for example as a molar concentration (number of moles of metabolite per unit volume of sample) or a mass concentration (mass of metabolite per unit volume of sample). Alternatively, the concentration of a metabolite can be expressed as parts per million (ppm) or parts per billion (ppb). Such ways of expressing the concentration of a molecule in solution are known in the art. In some embodiments, a concentration of a metabolite may be expressed relative to a standard or to another metabolite within the sample. For example, when techniques such as NMR are employed a concentration may be expressed as a relative spectral intensity.

Thus, in one embodiment, the concentration of a metabolite in a sample is the molar concentration of said metabolite. In one embodiment, the concentration of a metabolite in a sample is the mass concentration of said metabolite.

The concentration of a metabolite in a sample may be expressed in absolute terms, for example as an absolute molar concentration or absolute mass concentration. Alternatively, the concentration of a metabolite in a sample can be expressed by comparison to the concentration of a different metabolite in the same sample (i.e. in relative terms). By way of example, the concentration of a metabolite in the sample can be normalised by comparison to the concentration of a different reference metabolite within the same sample.

The methods described herein are particularly sensitive and allow for accurate and/or sensitive and/or specific determination and/or diagnosis when using only one metabolite. Notably, even where the concentration of a metabolite has not been found to be statistically- significantly changed when compared to a reference standard, said metabolite has utility in a method of the invention, especially where used in combination with a further metabolite and/or when compared to multiple reference standards.

In one embodiment, a metabolite for use in the invention is a lipoprotein. A lipoprotein may be a very low density lipoprotein (VLDL), a low density lipoprotein (LDL) or a high density lipoprotein (HDL). In some embodiments, the methods employ the use of at least two of: a VLDL, a LDL, and an HDL.

A “lipoprotein having a -CH3 group” means “a lipoprotein having a -CH3 group of an HDL and/or LDL”. These two terms may be used synonymously herein.

A “lipoprotein having a -(CH2)n group” means “a lipoprotein having a -(CH2)n group of an HDL and/or LDL”. These two terms may be used synonymously herein.

A lipoprotein may be detected, and/or its concentration measured, by detecting a chemical group of the lipoprotein, for example a -CH3 group of a lipoprotein. When using NMR, certain chemical shift ranges are characteristic of such groups of the various density lipoproteins, as described below. In one embodiment, a method of the invention utilises a -(CH2)n group of an HDL and/or LDL. A ¹ H-NMR chemical shift range of 1.15-1.30 ppm may be characteristic of a -(CH2)n group of an HDL and/or LDL (preferably an LDL). Thus, methods of the invention may comprise comparison of the concentration of a lipoprotein having a -(CH2)n group of an HDL and/or LDL (preferably an LDL) with the concentration of said metabolite in a reference value as described herein. Said lipoprotein may be referred to as “lipoprotein (CH2)n (HDL/LDL dominated)”. Said lipoprotein may be referred to as “lipoprotein (CH2)n (LDL dominated)”.

In one embodiment, a method of the invention utilises a -CH3 group of an HDL and/or LDL. A ¹ H-NMR chemical shift range of 0.80-0.86 ppm may be characteristic of a -CH3 group of an HDL and/or LDL. Thus, methods of the invention may comprise comparison of the concentration of a lipoprotein having -CH3 group of an HDL and/or LDL with the concentration of said metabolite in a reference value as described herein. Said lipoprotein may be referred to as “lipoprotein CH3 (HDL/LDL dominated)”.

In one embodiment, a method utilises an -N(CHs)3 group of a lipoprotein. A ¹ H-NMR chemical shift range of 3.17-3.31 ppm may be characteristic of an -N(CHs)3 group of a lipoprotein. Thus, methods of the invention may comprise comparison of the concentration of a lipoprotein having an -N(CHs)3 group with the concentration of said metabolite in a reference value as described herein. Said lipoprotein may be referred to as “a lipoprotein having an -N(CH3)3”.

In one embodiment, a metabolite for use in the invention is glucose. Said metabolite may be defined via one or more ¹ H-NMR chemical shift range(s) of 3.17-3.95 ppm, 4.63-4.66 ppm, and/or 5.22-5.25 ppm. Preferably, said metabolite may be defined via ¹ H-NMR chemical shift ranges of 3.17-3.95 ppm, 4.63-4.66 ppm, and 5.22-5.25 ppm.

In one embodiment, a metabolite for use in the invention is p-hydroxybutyrate and/or p- hydroxybutyric acid (also known as 3-hydroxybutyrate). Said metabolites may be defined via one or more ¹ H-NMR chemical shift range(s) of 1.19-1.21 ppm and/or 2.27-2.45 ppm. Preferably, said metabolites may be defined via ¹ H-NMR chemical shift ranges of 1.19-1.21 ppm and 2.27-2.45 ppm.

In one embodiment, a metabolite for use in the invention is p-hydroxybutyrate (also known as 3-hydroxybutyrate). Said metabolite may be defined via one or more ¹ H-NMR chemical shift range(s) of 1.19-1.21 ppm and/or 2.27-2.45 ppm. Preferably, said metabolite may be defined via ¹ H-NMR chemical shift ranges of 1.19-1.21 ppm and 2.27-2.45 ppm.

In one embodiment, a metabolite for use in the invention is myo-inositol. Said metabolite may be defined via one or more ¹ H-NMR chemical shift range(s) of 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and/or 3.25-3.29 ppm. Preferably, said metabolite may be defined via ¹ H-NMR chemical shift ranges of 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and 3.25- 3.29 ppm.

In one aspect, the metabolites herein may instead be referred to by their ¹ H-NMR chemical shift range(s), as described above.

Methods of the present invention are also based on the identification of chemical shift regions of ¹ H-NMR spectra that allow for accurate and/or sensitive and/or specific diagnosis of cancer, a primary cancer, and/or a secondary cancer. By way of background, carrying out ¹ H-NMR produces a spectrum, as is known in the art. The spectrum can be characterised according to the chemical shift positions (in ppm), which define peak positions, and the intensity of the peaks. Intensity (i.e. peak/spectral intensity) corresponds to the concentration of a chemical (e.g. a metabolite) present in a sample. Intensity may be determined by any method known in the art, such as determining an area under the peak. The Examples herein define a particularly preferred method for carrying out ¹ H-NMR to produce a spectrum for use in the present invention.

The chemical shifts quoted herein may be considered to encompass a value that deviates from the quoted value by ± 0.01 ppm, preferably a value that deviates from the quoted value by less than ± 0.01 ppm, more preferably by 0 ppm.

In one embodiment, a suitable volume of a sample is diluted with an appropriate buffer (preferably having a pH meter reading of 7.4) and solvent. Preferably, the sample comprises D2O. Preferably, a suitable volume (e.g. 150 pl) of a sample is diluted with (e.g. 450 pl) sodium phosphate buffer prepared in D2O (pH meter reading of 7.4).

Said samples may be processed to remove any precipitate prior to carrying out NMR.

Preferably, the chemical shift regions quoted herein are reported relative to lactate -CH3 referenced at 1.33 ppm. In one embodiment, the ¹ H-NMR is carried out on samples at 298K. Preferably, the ¹ H-NMR is carried out on samples at 31 OK.

Most preferably, the chemical shift regions herein have been defined by carrying out “the ¹ H- NMR assay” described herein. “The ¹ H-NMR assay” comprises the following steps:

(a) diluting 150 pL of sample with 450 pL of 75 mM sodium phosphate buffer prepared in D2O (pH meter reading of 7.4);

(b) centrifuging said diluted sample at 16,000 x g for 3 minutes to remove any precipitate;

(c) transferring the supernatant to a 5mm NMR tube;

(d) obtaining an NMR spectrum of the sample, wherein the spectrum is obtained using a 1 D NOESY presaturation scheme for attenuation of the water resonance with a 2 s presaturation using a 700-MHz NMR spectrometer equipped with a helium-cooled cryogenic probe (e.g. a 700-MHz Bruker AVIII spectrometer operating at 16.4T equipped with a ¹ H ( ¹³ C/ ¹⁵ N) TCI cryoprobe) at a sample temperature of 298K or 31 OK (preferably 31 OK);

(e) zero-filling resulting free induction decays (Fl Ds) by a factor of 2 and multiplying by an exponential function corresponding to 0.30Hz line broadening prior to Fourier transformation;

(f) phasing and baseline correcting (using a 3 ^rd degree polynomial) the spectrum; and

(g) referencing chemical shifts to the lactate-CHs doublet resonance at 5 = 1.33 ppm; and

(h) optionally, when the sample is a serum or plasma sample, in addition to the 1D NOESY presaturation scheme, a spin-echo Carr-Purcell-Meiboom-Gill (CPMG) sequence with a T interval of 400ps, 80 loops, 32 data collections, an acquisition time of 1 ,5s, and a relaxation delay of 2s may be used to supress broad signals arising from large molecular weight blood components; and

(i) optionally, for blood plasma and blood serum spectra, the regions between 0.20 - 4.70 ppm and 5.00 - 9.60 ppm may be divided in to 0.01 ppm width ‘buckets’. For urine spectra, the regions between 0.20 - 4.70 ppm and 5.00 - 5.70 and 5.96 - 9.60 ppm may be divided in to 0.01 ppm width ‘buckets’; and

(j) optionally the absolute value of the integral of each spectral bucket may be Pareto scaled; and (k) optionally, resonances may be assigned by reference to literature values [Anal Biochem 325:260-272, . J Pharm Biomed Anal 33:1103-1115] and the Human Metabolome Database [Nucleic Acids Res 41:D801-807. doi:10.1093/nar/gks1065, Nucleic Acids Res 37:D603-610. doi:10.1093/nar/gkn810, Nucleic Acids Res 35:D521-526. doi:10.1093/nar/gkl923] and further confirmed by inspection of the 2D spectra, spiking of known compounds, and 1 D-TOCSY spectra.

In one embodiment, the invention encompasses ¹ H-NMR techniques carried out under conditions other than those defined herein, for example via the AXINON® lipoFIT® system (numares).

In the unlikely event that said techniques lead to one or more different chemical shift region(s), said different chemical shift region(s) are encompassed by the present invention so long as the different chemical shift region(s) correspond to the chemical shift region(s) presented herein when carried out using “the ¹ H-NMR assay” described herein.

The methods comprising the use of chemical shift regions of ¹ H-NMR spectra are accurate and/or sensitive and/or specific when using only one chemical shift region. Notably, even where the intensity of a chemical shift region has not been found to be statistically- significantly changed when compared to a reference standard, said chemical shift region has utility in a method of the invention, especially where used in combination with a further chemical shift region and/or when compared to multiple reference standards.

In some embodiments, more than one metabolite may be employed, i.e. a plurality of metabolites may be employed. In a preferred embodiment, at least 2 metabolites are employed in a method described herein.

The term “one or more” when used in the context of a metabolite described herein may mean at least 2, 3, 4, 5, or 6 metabolites. For example, the term “one or more” when used in the context of a metabolite described herein may mean at least 2, 3, 4, or 5 metabolites. Suitably, the term “one or more” when used in the context of a metabolite described herein may mean at least 3 metabolites. It is preferred that at least two metabolites selected from glucose, myo-inositol and p-hydroxybutyrate are employed. For example, each of the metabolites glucose, myo-inositol and p-hydroxybutyrate may be employed. In one embodiment, the invention utilises glucose; and one or more metabolite(s) selected from p-hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHS)3 group. In one embodiment, the invention utilises glucose; and two or more metabolite(s) selected from p-hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises glucose; and three or more metabolite(s) selected from p-hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises glucose; and four or more metabolite(s) selected from p-hydroxybutyrate, myoinositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a - (CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises glucose; and five or more metabolite(s) selected from p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CH ₃ )3 group.

In one embodiment, the invention utilises glucose; and one or more metabolite(s) selected from p-hydroxybutyrate, and myo-inositol. In one embodiment, the invention utilises glucose; and one or more metabolite(s) selected from p-hydroxybutyrate, and myo-inositol; and one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CH ₃ )3 group.

In one embodiment, the invention utilises p-hydroxybutyrate; and one or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CH ₃ )3 group.

In one embodiment, the invention utilises p-hydroxybutyrate; and two or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CHS)3 group. In one embodiment, the invention utilises p-hydroxybutyrate; and three or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises p- hydroxybutyrate; and four or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises p-hydroxybutyrate; and five or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group.

In one embodiment, the invention utilises p-hydroxybutyrate; and one or more metabolite(s) selected from glucose, and myo-inositol. In one embodiment, the invention utilises p- hydroxybutyrate; and one or more metabolite(s) selected from glucose, and myo-inositol; and one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CH ₃ )3 group.

In one embodiment, the invention utilises myo-inositol; and one or more metabolite(s) selected from glucose, p-hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises myo-inositol; and two or more metabolite(s) selected from glucose, p-hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises myoinositol; and three or more metabolite(s) selected from glucose, p-hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises myo-inositol; and four or more metabolite(s) selected from glucose, p- hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises myo-inositol; and five or more metabolite(s) selected from glucose, p-hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CH ₃ ) ₃ .

In one embodiment, the invention utilises myo-inositol; and one or more metabolite(s) selected from glucose and p-hydroxybutyrate. In one embodiment, the invention utilises myoinositol; and one or more metabolite(s) selected from glucose and p-hydroxybutyrate; and one or more metabolite(s) selected from a lipoprotein having a -CH ₃ group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CH ₃ )3 group.

In a preferable embodiment, the invention utilises myo-inositol, glucose and p- hydroxy butyrate; and one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In another preferable embodiment, the invention utilises myo-inositol, glucose and p-hydroxybutyrate; and two or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In another preferable embodiment, the invention utilises myo-inositol, glucose and p-hydroxybutyrate; and three or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group.

In one embodiment, the invention utilises one or more metabolite(s) selected from myoinositol, glucose and p-hydroxybutyrate; and one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises one or more metabolite(s) selected from myo-inositol, glucose and p- hydroxybutyrate; and two or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises one or more metabolite(s) selected from myo-inositol, glucose and p-hydroxybutyrate; and three or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CH ₃ )3 group.

In one embodiment, the invention utilises two or more metabolite(s) selected from myoinositol, glucose and p-hydroxybutyrate; and one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises two or more metabolite(s) selected from myo-inositol, glucose and p- hydroxybutyrate; and two or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises two or more metabolite(s) selected from myo-inositol, glucose and p-hydroxybutyrate; and three or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CH ₃ )3 group.

In one embodiment, the invention utilises three or more metabolite(s) selected from myoinositol, glucose and p-hydroxybutyrate; and one or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises three or more metabolite(s) selected from myo-inositol, glucose and p- hydroxybutyrate; and two or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises three or more metabolite(s) selected from myo-inositol, glucose and p-hydroxybutyrate; and three or more metabolite(s) selected from a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CH ₃ )3 group.

In some embodiments, more than one chemical shift region may be employed, i.e. a plurality of chemical shift regions may be employed. In a preferred embodiment, at least 2 chemical shift regions are employed in a method described herein.

In one embodiment, the invention utilises a chemical shift region for glucose; and a chemical shift region for one or more metabolite(s) selected from p-hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for glucose; and a chemical shift region for two or more metabolite(s) selected from p-hydroxybutyrate, myo-inositol, a lipoprotein having a - CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for glucose; and a chemical shift region for three or more metabolite(s) selected from p-hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for glucose; and a chemical shift region for four or more metabolite(s) selected from p- hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for glucose; and a chemical shift region for five or more metabolite(s) selected from p-hydroxybutyrate, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group.

In one embodiment, the invention utilises a chemical shift region for glucose; and a chemical shift region for one or more metabolite(s) selected from p-hydroxybutyrate, and myo-inositol.

In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for one or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for one or more metabolite(s) selected from glucose, and myo-inositol. In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for two or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for one or more metabolite(s) selected from glucose, and myo-inositol. In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for three or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for one or more metabolite(s) selected from glucose, and myo-inositol. In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for four or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for five or more metabolite(s) selected from glucose, and myo-inositol. In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for one or more metabolite(s) selected from glucose, myo-inositol, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group.

In one embodiment, the invention utilises a chemical shift region for p-hydroxybutyrate; and a chemical shift region for one or more metabolite(s) selected from glucose, and myo-inositol.

In one embodiment, the invention utilises a chemical shift region for myo-inositol; and a chemical shift region for one or more metabolite(s) selected from glucose, p-hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for myo-inositol; and a chemical shift region for two or more metabolite(s) selected from glucose, p-hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for myo-inositol; and a chemical shift region for three or more metabolite(s) selected from glucose, p-hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group. In one embodiment, the invention utilises a chemical shift region for myo-inositol; and a chemical shift region for four or more metabolite(s) selected from glucose, p-hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an - N(CHS)3 group. In one embodiment, the invention utilises a chemical shift region for myoinositol; and a chemical shift region for five or more metabolite(s) selected from glucose, p- hydroxybutyrate, a lipoprotein having a -CH3 group of an HDL and/or LDL, a lipoprotein having a -(CH2)n group of an HDL and/or LDL, and a lipoprotein having an -N(CHs)3 group.

In one embodiment, the invention utilises a chemical shift region for myo-inositol; and a chemical shift region for one or more metabolite(s) selected from glucose and p- hydroxybutyrate.

In a preferable embodiment, the invention utilises a chemical shift region for myo-inositol, a chemical shift region for glucose and a chemical shift region for p-hydroxybutyrate.

The terms “subject” and “patient” are used synonymously herein. The “subject” (aka. patient) may be a mammal, and preferably the subject is a human subject. A subject may be a subject that has or, is suspected of having, multiple sclerosis. The patient may be a MS patient that is at risk of suffering from a relapse.

Preferably, a patient is a patient that has presented with non-specific symptom(s)/sign(s) of MS progression. A method of the invention may further comprise detecting the presence or the absence of such non-specific symptom(s)/sign(s), and confirming or not confirming (respectively) that the patient is suffering progression. Such non-specific symptoms/signs may include optic neuritis (e.g. caused by inflammation of the optic nerve that impairs vision), problems with balance or severe fatigue, unexplained inflammation in the central nervous system (CNS), fatigue, difficulty walking, vision problems (such as blurred vision), problems controlling the bladder, numbness (or tingling) in different parts of the body, muscle stiffness and spasms, problems with balance and co-ordination, and/or problems with thinking, learning and planning, and/or GP clinical suspicion of a relapse (GP ‘gut feeling’).

Confirmation of the presence or absence of progression may also be corroborated by reference to PET imaging or magnetic resonance imaging (MRI) data obtained upon analysis of the patient. For example, PET imaging may be performed using an 11C-PK-11195 radioligand to monitor brain inflammation as a further confirmation of progression, noting brain inflammation is higher in progressors vs. non-progressors. For example, such MRI data may confirm the presence or absence of GAD-enhancing or new/enlarging T2 lesion/s within a CNS locale (e.g. consistent with the neurological deficits), offering further support for the presence or absence of a clinical relapse.

The sample that is to be tested using the method of the invention may be derived from any suitable biofluid. Thus, the sample is preferably a biofluid sample. In one embodiment, the biofluid is selected from blood, cerebrospinal fluid (CSF), or urine that has been obtained from a subject. Preferably the sample is a blood sample.

The term “blood” comprises whole blood, blood serum (henceforth “serum”) and blood plasma (henceforth “plasma”), preferably serum. Serum and plasma are derived from blood and thus may be considered as specific subtypes within the broader genus “blood”. Processes for obtaining serum or plasma from blood are known in the art. For example, it is known in the art that blood can be subjected to centrifugation in order to separate red blood cells, white blood cells, and plasma. Serum is defined as plasma that lacks clotting factors. Serum can be obtained by centrifugation of blood in which the clotting process has been triggered. Optionally, this can be carried out in specialised centrifuge tubes designed for this purpose.

A sample for use in a method of the present invention can be derived from a biofluid that has undergone processing after being obtained from a test subject. Alternatively, a sample can be derived from a biofluid that has not undergone any processing after being obtained from a test subject.

The methods of the invention may use samples that have undergone minimal or zero processing before testing. This provides a significant advantage over prior art methods in terms of time, cost and practicality. By way of example, a blood sample obtained from a test subject may be tested directly using the method of the present invention, without further processing. Serum and plasma samples can be readily obtained from blood samples using simple and readily available techniques that are well known in the art, as described above.

A sample for use in a method of the invention may be a cell-free sample. In other words, the sample of the invention may be processed to remove cells. The term “cell-free samples” are samples that contain substantially no cells. The term “substantially no” when used in the context of cells herein may mean less than 10,000, 5,000, 1,000, 100 or 10 cells/ml. The term “substantially no” when used in the context of cells herein preferably means less than 1 ,000 cells/ml, more preferably no cells. In some embodiments, the term “substantially no” when used in the context of cells herein may be expressed in absolute amounts. For example, the term “substantially no” when used in the context of cells herein may mean less than 10,000, 5,000, 1 ,000, 100 or 10 cells. Preferably less than 1 ,000 cells, more preferably no cells.

The methods of the invention comprise comparing a concentration of a metabolite to a reference value. Similarly, the methods of the invention may comprise comparing an intensity of one or more chemical shift regions of a ¹ H-NMR spectrum of a sample with a reference value.

The reference value may be obtained from a reference standard. In one embodiment, a reference standard comprises (or consists of) a sample (e.g. a biofluid sample described herein) obtained from a reference subject or subjects (e.g. patient or patients), wherein the reference subject is a subject other than the subject being tested in a method of the invention. In one embodiment, a “reference value” comprises (or consists of) a set of data relating to the concentration of one or more metabolites, and/or the intensity of one or more chemical shift regions of a ¹ H-NMR spectrum, obtained from a reference subject or subjects, wherein the reference subject is a subject other than the subject being tested in a method of the invention. The set of data may be derived by measuring the concentration of said one or more metabolites and/or measuring the intensity of one or more chemical shift regions of a ¹ H-NMR spectrum. Said measuring may be carried out using any suitable technique known in the art or described herein. It is particularly preferred that the set of data corresponding to the reference sample are obtained (or have been obtained) using the same or a similar technique used to obtain the concentration of the one or more metabolites or one or more chemical shift regions (respectively) in the sample being tested. As part of his common general knowledge, the skilled person knows which variables in an experimental protocol can be varied without affecting comparability of data and those that cannot be varied, and will thus select an appropriate experimental protocol to ensure comparability between a sample from a subject and a reference standard. Most preferably, the same technique and protocol will be used to obtain the concentration of the one or more metabolites or one or more chemical shift regions (respectively) in the sample and in the reference standard.

The skilled person understands that a “control” patient is a stage-matched patient.

In some embodiments, a reference value may be a dataset constructed based on a knowledge of metabolite concentrations, and/or chemical shift intensities, that are indicative of the presence of a progression, or the absence of progression. In some embodiments, a reference standard may be constructed based on metabolite concentrations and/or chemical shift intensities for a known population of MS patients suffering from progression and/or a known population of MS patients not suffering from progression (e.g. non-progressor population). In other words, in some embodiments, a reference value does not correspond to an actual sample obtained from a reference subject. However, it is preferred that a reference value comprises (or consists of) a set of data relating to the concentration of one or more metabolites, and/or the intensity of one or more chemical shift regions of a ¹ H-NMR spectrum, obtained from a reference subject or subjects, wherein the reference subject is a subject other than the subject being tested in a method of the invention.

In one embodiment, the reference value comprises (or consists of) a set of data relating to the concentration of said one or more metabolites, and/or the intensity of one or more chemical shift regions of a ¹ H-NMR spectrum, in a sample or samples derived from a single reference subject. In other embodiments, the reference value comprises (or consists of) a set of data relating to the concentration of said one or more metabolites, and/or the intensity of one or more chemical shift regions of a ¹ H-NMR spectrum, in a sample or samples derived from a plurality of reference subjects (e.g. two or more reference subjects). Thus, in one embodiment, the reference value is derived by pooling data obtained from two or more (e.g. three, four, five, 10, 15, 20 or 25) reference subjects and calculating an average (for example, mean or median) concentration for each metabolite, and/or an average intensity of one or more chemical shift regions of a ¹ H-NMR spectrum. Thus, the reference value may reflect average concentrations of said one or more metabolites, and/or average intensities of chemical shift regions of a ¹ H-NMR spectrum, in a given population of reference subjects. Said concentrations and/or intensities may be expressed in absolute or relative terms, in the same manner as described above in relation to the sample that is to be tested using the method of the invention.

In one embodiment, a method of the invention comprises the use of a plurality of reference values. In such embodiments, a method may comprise the use of multiple reference values. The use of multiple reference standards is particularly preferred when it is necessary to confirm whether or not a patient is suffering progression, but also whether a patient is predicted to suffer progression.

A metabolite concentration in a reference value may have been obtained (e.g. quantified) prior to carrying out a method of the invention.

When comparing concentrations between the sample and the reference value, the way in which the concentrations are expressed is matched between the sample and the reference value. Thus, an absolute concentration can be compared with an absolute concentration, and a relative concentration can be compared with a relative concentration.

An intensity of a chemical shift region of a ¹ H-NMR spectrum in a reference value may have been obtained (e.g. quantified) prior to carrying out a method of the invention.

When comparing intensities between the sample and the reference value, the way in which the intensities are expressed is matched between the sample and the reference value. Thus, an absolute intensity can be compared with an absolute intensity, and a relative intensity can be compared with a relative concentration. Moreover, the ¹ H-NMR protocol used for obtaining a spectrum for the sample and reference value should preferably be the same.

The reference value is preferably derived from the same sample type (e.g. biofluid) as the sample that is being tested, thus allowing for an appropriate comparison between the metabolites and/or chemical shifts.

The methods of the present invention are in vitro methods. Thus, the methods can be carried out in vitro on an isolated sample that has been obtained from a subject.

The methods of the invention may comprise comparing the (measured) concentrations of one or more metabolites to make a diagnosis. Thus, said (measured) concentrations may correlate with the presence of MS progression. Said diagnosis may be based on measuring/identifying a concentration difference. The term “concentration difference” embraces both positive and negative differences. Thus, a concentration difference can mean that the concentration of a metabolite is lower in the sample being tested than in the reference value. Alternatively, a concentration difference can mean that the concentration of a metabolite is higher in the sample than in the reference value.

Similarly, methods of the invention may comprise comparing the (measured) intensities of one or more chemical shift regions of a ¹ H-NMR spectrum to make a diagnosis. Thus, said (measured) intensities may correlate with the presence of MS progression. Said diagnosis may be based on measuring/identifying a difference in intensity. The term “difference in intensity” embraces both positive and negative differences. Thus, a difference in intensity can mean that the intensity of a chemical shift region is lower in the sample being tested than in the reference value. Alternatively, a difference in intensity can mean that the intensity of a chemical shift region is higher in the sample than in the reference value.

The comparison and/or identification of the presence or absence of a concentration difference (as described above) can be achieved using methods of statistical analysis. The comparison and/or identification of the presence or absence of a difference in intensity of a chemical shift region (as described above) can be achieved using methods of statistical analysis. In one embodiment, a method of statistical analysis suitable for use in the present invention includes orthogonal partial least squares discriminate analysis (OPLS-DA). Identifying a higher or lower concentration of a metabolite or intensity of a chemical shift region relative to the same metabolite or chemical shift region (respectively) in/of a reference standard preferably means identifying a statistically significant higher or lower concentration or intensity. Identifying the same concentration of a metabolite or intensity of a chemical shift region relative to the same metabolite or chemical shift region (respectively) in/of a reference standard preferably means identifying no statistically significant concentration difference or difference in intensity (respectively). More preferably, identifying the same concentration of a metabolite or intensity of a chemical shift region relative to the same metabolite or chemical shift region (respectively) in/of a reference standard preferably means identifying no concentration difference or difference in intensity (respectively).

In one embodiment, the method of the invention further comprises recording the output of at least one step on a data-storage medium. By way of example, the methods of the present invention can generate data relating to the subject, such data being recordable on a datastorage medium (for example, a form of computer memory such as a hard disk, compact disc, floppy disk, or solid state drive). Such data can comprise (or consist of) data relating to the concentration in a sample (from said subject) of any of two or more metabolites (as described herein) and/or data relating to the intensity in a sample (from said subject) of any of two or more chemical shift regions (as described) herein.

In one aspect the invention provides a data-storage medium, comprising data obtained by a method according to the present invention.

In one aspect, the invention provides a computer program product comprising program instructions to cause a processor to perform a method according to the invention.

In another aspect, the invention provides a device for use in a method of the invention, wherein said device is capable of performing the step of identifying: a concentration (e.g. a concentration difference) of two or more metabolites in the sample when compared to the reference standard and/or an intensity (e.g. a difference in intensity) of two or more chemical shift regions of a ¹ H-NMR spectrum of a sample obtained from a subject when compared to the reference value.

In one embodiment, following determining there has been progression of MS in the patient, the method further comprises determining that the patient is suitable for receiving an MS therapy. In one embodiment, following determining there has been no progression of MS in the patient, the method further comprises determining that the patient is not suitable for receiving an MS therapy.

In one aspect, the invention provides a method of suppressing MS in a patient, the method comprising:

(a) obtaining the results of a method according to the invention; and

(b) administering a therapy for MS when progression is confirmed.

Suppression (also referred to herein as “treatment”) may be carried out using any suitable therapeutic for a MS known in the art. A suitable therapy is preferably a disease modifying therapy/ therapies for MS, which may suppress progression (of disability) and/or the number of relapses. For example, therapy may include an injectable medication, such as Avonex (interferon beta-1 a); Betaseron (interferon beta-1 b); Copaxone (glatiramer acetate), Extavia (interferon beta-1 b), Glatiramer Acetate Injection (glatiramer acetate, generic equivalent of Copaxone 20 mg and 40 mg doses), Glatopa (glatiramer acetate, generic equivalent of Copaxone 20mg and 40mg doses), Kesimpta® (ofatumumab), Plegridy (peginterferon betala), and/or Rebif (interferon beta-1 a); an oral medication, such as Aubagio (teriflunomide), Bafiertam (monomethyl fumarate), Gilenya (fingolimod), Mavenclad (cladribine), Mayzent (siponimod), Tecfidera (dimethyl fumarate), Vumerity (diroximel fumarate), oral methylprednisolone, and/or Zeposia (ozanimod); and/or an infused medication, such as Lemtrada (alemtuzumab), Novantrone (mitoxantrone), Ocrevus (ocrelizumab), intravenous methylprednisolone, and/or Tysabri (natalizumab). The treatment may additionally or alternatively comprise include autologous hematopoietic stem cell transplantation (AHSCT).

The therapy may be a Nuclear factor-erythroid factor 2-related factor 2 (Nrf2) activator (such as dimethyl fumarate), a sphingosine-1 -phosphate receptor modulator (such as fingolimod), a myelin basis protein or polypeptide that simulates myelin basic protein (such as glatiramer acetate), and/or an interferon (such as interferon beta 1a and/or interferon beta 1b).

Such sphingosine-1 -phosphate receptor modulator preferably functions to promote sequestration of lymphocytes in lymph nodes, suppressing lymphocyte contribution to an autoimmune reaction.

In a preferable embodiment, the therapy is selected from the list comprising dimethyl fumarate, fingolimod, glatiramer acetate, interferon beta 1a and interferon beta 1b. For example, the therapy may be glatiramer acetate. The term “disorder” as used herein also encompasses a “disease”. In one embodiment, the disorder is a disease. The disorder treated in accordance with the invention is suitably MS, e.g. wherein the MS patient is suffering or is at risk of suffering progression.

The terms “suppress”, “suppressing”, “treat” or “treating” as used herein encompasses prophylactic treatment (e.g. to prevent onset of a disorder) as well as corrective treatment (treatment of a subject already suffering from a disorder). Preferably “treat” or “treating” as used herein means corrective treatment.

The term “treat” or “treating” as used herein refers to the disorder and/or a symptom thereof.

Therefore, a therapeutic may be administered to a subject in a therapeutically effective amount or a prophylactically effective amount.

A “therapeutically effective amount” is any amount of a therapeutic formulation, which when administered alone or in combination to a subject for treating said disorder (or a symptom thereof) is sufficient to effect such treatment of the disorder, or symptom thereof.

A “prophylactically effective amount” may be any amount of a therapeutic formulation that, when administered alone or in combination to a subject inhibits or delays the onset or reoccurrence of a disorder (or a symptom thereof). In some embodiments, the prophylactically effective amount prevents the onset or reoccurrence of a disorder entirely. “Inhibiting” the onset means either lessening the likelihood of a disorder’s onset (or symptom thereof), or preventing the onset entirely. Preferably, a “prophylactically effective amount” is any amount of a therapeutic formulation that, when administered alone or in combination to a subject inhibits or delays the onset or reoccurrence of a relapse (or a symptom thereof). In some embodiments, the prophylactically effective amount prevents the onset or reoccurrence of a relapse entirely. “Inhibiting” the onset means either lessening the likelihood of a relapse’s onset (or symptom thereof), or preventing the onset entirely.

Administration may be by any route known in the art and will typically be dependent on the nature of the therapeutic to be administered. For example, a therapeutic may be administered orally or parenterally. Methods of parenteral delivery include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intra-ventricular, intravenous, intraperitoneal, or intranasal administration. Embodiments related to the various methods of the invention are intended to be applied equally to other methods, therapeutic uses or methods, the data storage medium or device, the computer program product, and vice versa.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term “protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3- letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a chemical shift region” includes a plurality of such chemical shift regions and reference to “the chemical shift region” includes reference to one or more chemical shift regions and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

Figure 1 shows bar charts comparing extralesional 11C-PK-11195 DVR in the brains of MS patients that progress vs non-progressor, which is higher in the former. Data are presented as mean ±SEM and analysed by student’s unpaired t-test. *p<0.05, **p<0.01.

Figure 2 shows comparative metabolomics data that demonstrates that the serum metabolome can discriminate between patients exhibiting high or low 11C-PK-11195 DVR. Data are presented as mean ±SEM and analysed by student’s unpaired t-test. *p<0.05, **p<0.01, ****p<0.0001.

Figure 3 shows metabolomics data demonstrating that concentrations of key metabolites 3- hydroxybutyrate, myo-inositol and glucose are indicative of the magnitude of brain inflammation in MS patients.

Figure 4 shows metabolomics data demonstrating that concentrations of key metabolites 3- hydroxybutyrate, myo-inositol and glucose predict MS patient progression. Data presented as mean ±SEM, and analysed by student’s unpaired t-test. **p<0.01.

Figure 5 shows bar charts demonstrating that the metabolome discriminates between RRMS and SPMS patients in correlation with discrimination detected via extralesional 11C-PK- 11195 DVR. Data are presented as mean ±SEM and analysed by student’s unpaired t-test. *p<0.05, **p<0.01 , ***p<0.001.

Figure 6 shows metabolomics date demonstrating that the panel of key metabolites provides a marker of patient response to therapy. Data presented as mean ±SEM, and analysed by one-way ANOVA with Sidak’s post-hoc test. *p<0.05, **p<0.01 , ****p<0.0001.

EXAMPLES

Summary

Background and objectives: Evaluation of progression in multiple sclerosis (MS) patients remains a clinical challenge. PET imaging with innate immune cell 18kDa translocator protein (TSPO) radioligand can reveal inflammation in the brain, which is associated with disease progression. However, isotopes are costly and regular serial PET scanning is likely to raise safety concerns, and so this strategy is impractical for routine use. In this study, it is examined whether inflammation in the brain could be measured indirectly using the serum metabolome. Methods: The magnitude of brain inflammation was evaluated in MS patients by PET imaging with TSPO-binding radioligand ¹¹ C-PK-11195. Blood was collected, and serum metabolomics was performed using NMR and supervised multivariate analysis (OPLS-DA). The Expanded Disability Status Scale (EDSS) was used to assess patient disability at baseline and approximately 1 year after sampling. Results: n=69 patients were included in this study (n=56 RRMS, n=13 SPMS). TSPO binding in the brain was greater in patients who progressed within 1 year, compared with those who remained stable. Serum metabolomics was able to discriminate between patients with high and low TSPO binding with 80±4% accuracy (92±5% sensitivity and 70±6% specificity). Investigation of key metabolites driving separation identified 3-hydroxybutyrate, myo-inositol and glucose as being critical to the model, all of which correlated with the magnitude of inflammation in the brain. This result was confirmed when comparing RRMS and SPMS patients; TSPO binding was increased and key serum metabolites were decreased in SPMS patients, relative to RRMS patients. MultiROC analysis revealed that in combination, these 3 metabolites are predictive of worsening disability (AUC = 0.76) with the same accuracy as TSPO-PET imaging. Our findings are independent of disease modifying therapy. Discussion: Our results show that the serum metabolome can be used as a surrogate marker of brain inflammation and is predictive of increased disability. A blood-based metabolomics test could aid clinicians to monitor disease progression and the impact of therapy in MS patients in a minimally invasive and cost-effective manner.

Materials and Methods

In summary, the magnitude of brain inflammation was evaluated in MS patients by PET imaging with TSPO-binding radioligand ¹¹ C-PK-11195 (note: PET imaging with innate immune cell 18kDa translocator protein (TSPO) radioligand can reveal inflammation in the brain, which is associated with disease progression). Blood was collected, and serum metabolomics was performed using NMR and supervised multivariate analysis (OPLS-DA). The Expanded Disability Status Scale (EDSS) was used to assess patient disability at baseline and approximately 1 year after sampling.

Further details are outlined below.

Study subjects and procedures

The study cohort consisted of n=69 patients with either RRMS or SPMS (RRMS n=56, SPMS n=13). Expanded disability status scale (EDSS) score was evaluated by experienced clinicians at the time of, and 1 year after sampling. Patients were considered progressors when change in EDSS score (AEDSS) was >0.5, and patients were considered non- progressors when AEDSS score was <0.

PET imaging and analysis

PET imaging was performed using ¹¹ C-PK-11195 radioligand, as previously described (Nyland et al., 2022 [PMID: 34993478], Sucksdorff et al., 2020 [PMID: 33006604]; Rissanen et al., 2014 [PMID: 24711650]). Innate immune cell activation was evaluated as specific binding of ¹¹ C-PK-11195 using distribution volume ration (DVR), in pre-specified regions of interest: NAWM, perilesion, T1 and T2 lesion, and the thalamus. Biofluid sample collection

Serum was collected into BD additive-free tubes (e.g. by venepuncture into (BD™ Vacutainer™ 367837) and stored undisturbed at room temperature for 30 minutes minutes (to give sufficient time for clot formation in the case of serum), until centrifugation at 2,200g for 10 minutes to separate the clot. Serum was aliquoted and stored at -80°C, e.g. in polypropylene cryotubes (Crystal clear™, STARLAB UK Ltd).

NMR sample preparation

For NMR preparation, serum was thawed at room temperature and ultracentrifuged at 100,000g for 30 minutes at 4°C. 100pL of supernatant was combined with 450pL NMR buffer (75mM sodium phosphate buffer prepared in D2O, pH 7.4) and stored at -80°C until analysis. The sodium phosphate buffer was prepared by dissolving 62.5 mM of anhydrous sodium phosphate dibasic powder (CAS number 7558794) and 12.5 mM of anhydrous sodium phosphate monobasic powder (CAS number 7558807) in deuterium oxide (D2O) (CAS number 7789200), giving a final pH of 7.4 (all three reagents were obtained from Sigma- Aldrich, Dorset, UK). D2O was used as the NMR solvent for all NMR experiments.

On the day of NMR analysis, samples were thawed at room temperature and transferred to a 5mm NMR tube.

1H NMR metabolomics data acquisition and processing

All samples were measured using the 700-MHz Bruker AVII spectrometer operating at 16.4T, equipped with a ¹ H ( ¹³ C/ ¹⁵ N) TCI cryoprobe. Samples were held at 31 OK, and spectra were acquired using a water suppression with a transverse relaxation filter that eliminates distortions (WASTED) sequence, as previously described (Yeo et al., 2020 [PMID: 32709911], Yeo et al., 2021 [PMID: 34755110]).

All spectra were phased, baseline corrected and chemical shifts referenced to the lactate- CH3 doublet resonance at b=1.33ppm in Topspin 4.0.7 (Bruker). Spectra were then uploaded to ACD/NMR processor academic edition 12.01 (Advanced Chemistry development, Inc.) and regions were divided into 0.02ppm width ‘buckets’ and the absolute value of integral of each spectral bucket was unit variance scaled. Metabolite assignment was performed with references to literature reviews, the HMDB metabolite database, and 2D total correlation spectroscopy (TOCSY) experiments. Glucose was detected and quantified by measuring the intensity of one of more of the following 1 H-NMR chemical shift range(s): 3.17-3.95 ppm, 4.63-4.66 ppm, and/or 5.22-5.25 ppm.

Myo-inositol was detected and quantified by measuring the intensity of one of more of the following 1 H-NMR chemical shift range(s): 3.63-3.65 ppm, 3.53-3.58 ppm, 3.93-3.98 ppm, and/or 3.25-3.29 ppm.

3-hydroxybutyrate (aka beta-hydroxybutyrate) was detected and quantified by measuring the intensity of one of more of the following 1 H-NMR chemical shift range(s): 1.19-1.21 ppm and 2.27-2.45 ppm.

Mobile -CH3 HDL/LDL relates to the concentration of lipoprotein. This utilises a -CH3 group of an HDL and/or LDL. Said lipoprotein may be referred to as “lipoprotein CH3 (HDL/LDL dominated)” or “lipoprotein having -CH3 group of an HDL and/or LDL”. This metabolite was detected and quantified by measuring the intensity of the 1 H-NMR chemical shift range of 0.80-0.86 ppm.

Mobile (-CH2-)n LDL relates to the concentration of lipoprotein. This utilises a -(CH2)n group of an HDL and/or LDL. Said lipoprotein may be referred to as “lipoprotein (CH2)n (LDL dominated)” or “a lipoprotein having a -(CH2)n group of an HDL and/or LDL”. This metabolite was detected and quantified by measuring the intensity of the 1 H-NMR chemical shift range of 1.15-1.30 ppm.

N(CHS)3/ free choline relates to the concentration of lipoprotein. This utilises an -N(CHs)3 group of a lipoprotein. Said lipoprotein may be referred to as “a lipoprotein having an - N(CH3)3” or “a lipoprotein having an -N(CHs)3 group”. This metabolite was detected and quantified by measuring the intensity of the 1 H-NMR chemical shift range of 3.17-3.31 ppm.

Statistical analysis

For analysis of metabolomics data, the integrals of the scaled spectral buckets were imported into R software (R foundation for statistical computing). Multivariate analysis was carried out using in house R scripts and the ropls package. Differences between groups were determined using orthogonal partial least square discriminant analysis (OPLS-DA): unequal class sizes were corrected and were separated into a training set (90%) and a test set (10%). The training set was used to build a model on which the test set was applied, and the predictive accuracy of the model was determined. Ten-fold cross-validation with 100 iterations was performed, resulting in 1,000 model outcomes. The mean accuracy, sensitivity and specificity of the model was compared to that of a separate model set created by permuting the class assignments (random chance). Discriminatory variables driving the class separation were identified by a high average variable importance (VIP) score, meaning that their exclusion from the model resulted in a significant decrease in the model accuracy. These variables were investigated further by univariate analysis. To evaluate the diagnostic potential of metabolites, receiver operator curves (ROC), area under the curve (AUC) and optimal thresholds were calculated using the pROC package.

All other analyses were completed with GraphPad Prism 7 software. The D’Agostino- Pearson omnibus normality test was applied to all data, and the suitable parametric or nonparametric statistical analysis was used subsequently. Unpaired student’s t-test, Mann- Whitney test, and one-way analysis of variance (ANOVA) were employed, with Sidak’s post- hoc testing as appropriate. Results were considered significant at p<0.05 with 95% confidence intervals. All quantitative data are expressed as mean ± standard error of the mean (SEM).

EXAMPLES 1-6

In summary, n=69 patients were included in this study (n=56 RRMS, n=13 SPMS). TSPO binding in the brain was greater in patients who progressed within 1 year, compared with those who remained stable. Serum metabolomics was able to discriminate between patients with high and low TSPO binding with 80±4% accuracy (92±5% sensitivity and 70±6% specificity). Investigation of key metabolites driving separation identified 3-hydroxybutyrate, myo-inositol and glucose, all of which correlated with the magnitude of inflammation in the brain. This result was also confirmed when comparing RRMS and SPMS patients; TSPO binding was increased and key serum metabolites were decreased in SPMS patients, relative to RRMS patients. MultiROC analysis revealed that in combination, these 3 metabolites are predictive of worsening disability (AUC = 0.76) with the same accuracy as TSPO-PET imaging. Thus, the results described herein demonstrate that the metabolome can be used as a surrogate marker of brain inflammation and is predictive of increased disability.

Further details of the results are outlined below. 1 - Demonstrating that extralesional 11C-PK-11195 DVR is higher in the brains of MS patients that

Both the RRMS and SPMS patient cohorts underwent TSPO-PET imaging with ¹¹ C- PK- 11195 ligand. Results are illustrated in Figure 1.

Comparison of the distribution volume ratio (DVR) values for the normal appearing white matter (NAWM; A in Figure 1) and perilesion (B in Figure 1) revealed that DVR values were greater in patients that were shown to have progressed (via AEDSS) within 1 year of sampling (n=13), compared to those who did not progress (n=56). No differences were observed in other brain regions of interest, such as T1 lesion (C Figure 1), T2 lesion (D in Figure 1) and thalamus (E). that the serum metabolome can discriminate between w ¹¹ C-PK-11195 DVR

Both the RRMS and SPMS patient cohorts underwent TSPO-PET imaging with ¹¹ C- PK- 11195 ligand as a readout of brain inflammation. Concomitantly, a sample (blood) was isolated from each patient for metabolite analysis. Results are illustrated in Figure 2.

Patients demonstrating high (n=30) and low (n=39) brain inflammation were segregated/ classified by plotting NAWM DVR vs. perilesion DVR. In Figure 2A, the dotted line corresponds to the cut off of high/low inflammation in this cohort, as determined by DVR differences between progressors and non-progressors. In the parallel metabolite analysis, serum samples were measured by NMR (providing metabolome concentrations) and analysed by multivariate analysis. OPLS-DA was able to discriminate between patients classified as having high or low brain inflammation with 80±4% accuracy (see Figure 2B). Investigation of the metabolites driving separation identified 3-hydroxybutyrate (Figure 2C), myo-inositol (Figure 2D), and glucose (Figure 2F) as particularly key metabolites, each being reduced in the high inflammation cohort (vs low inflammation cohort) in a statistically significant as support by a p value of ****p<0.0001. Additional metabolites of note include - CH3 HDL/LDL (Figure 1 E), mobile -N(CHs)3/free choline (Figure 1G), and lactate and (-CH2- ) _n (Figure 2H). Example 3 - Demonstrating that the serum metabolome is indicative of the magnitude of brain inflammation

The magnitude of brain inflammation in the NAWM and perilesion, as determined by PK- 11195 DVR (e.g. as outlined in Example 1 above), was correlated with the key discriminatory metabolites detected in Example 2 above. Results are outlined in Figure 3.

Correlations with a coefficient that is significantly not 0 (Pearson’s correlation, p<0.05) are designated with a circle. The shading and size of the circle corresponds to the direction of correlation and the value of the coefficient, respectively. Arrows have been retrospectively inserted to roughly indicate where the shading of the circles lies against ‘direction of correlation’ scale in the right. Significant inverse correlations were identified between both brain regions and 3-hydroxybutyrate, myo-inositol and glucose (Figure 3A). Furthermore, a positive correlation between mobile -N(CHs)3/free choline and NAWM DVR was also identified. MultiROC analysis using 3-hydroxybutyrate, myo-inositol and glucose values (concentrations) was able to predict (and discriminate) patients previously demonstrated (e.g. as per Example 1) to have high or low brain inflammation, with an area under the curve (AUG) of 0.891 (Figure 3B). Correlations with the greatest significant coefficients were between NAWM PK-11195 DVR and serum 3-hydroxybutyrate (Figure 3C), myo-inositol (Figure 3D) and glucose (Figure 3E), and between perilesion PK-11195 DVR and 3- hydroxybutyrate (Figure 3F), myo-inositol (Figure 3G) and glucose (Figure 3H). nstrating that serum metabolites provide biomarkers to predict MS and outperform PK-11195 DVR

With the high inflammation and low inflammation cohorts, progression was assessed by EDSS and patients were considered progressors when change in EDSS score (AEDSS) was >0.5, and patients were considered non-progressors when AEDSS score was <0. It was then demonstrated that serum metabolites (e.g. as detected in Examples 2 and 3 above) provide biomarkers to predict MS patient progression. Results are illustrated in Figure 4.

It was found that 11/30 of patients with high brain inflammation progressed within 1 year of sampling, compared with 2/39 patients with low brain inflammation (Figure 4A). The serum levels of the three key metabolites that correlated with the magnitude of PK-11195 DVR (see Example 3) were evaluated in progressors and non-progressors. The serum levels of 3- hydroxybutyrate (Figure 4B), myo-inositol (Figure 4C) and glucose (Figure 4D) were all decreased in MS patients that progressed (n=13), compared with those that did not (n=56). Furthermore, MultiROC using these three key metabolites was able to predict between progressors and non-progressors, with an AUG of 0.762 (Figure 4E). Indeed, the serum metabolites actually outperformed PK-11195 DVR at predicting progression, as multiROC using NAWM and perilesion PK-11195 DVR values produced an AUG of 0.761 (Figure 4F).

Example 5 - Demonstrating that the serum metabolome discriminates between RRMS and SPMS patients and correlates with extralesional ¹¹ C-PK-11195 DVR differences

RRMS (n=56) and SPMS (n=13) patients underwent TSPO-PET imaging with ¹¹ C- PK-11195 ligand, and blood was taken for metabolite analysis. Complementing the results outlined in the examples above, the key metabolites described herein were demonstrated to discriminate between a cohort of RRMS and SPMS patients, correlating with extralesional 11C-PK-11195 DVR differences between said patient cohorts. Results are outlined in Figure 5.

NAWM DVR was significantly increased in SPMS patients, compared to RRMS patients (Figure 5A). A trending increase was also observed for the perilesion DVR (Figure 5B). Investigation of key metabolites as identified herein demonstrated significant differences between RRMS and SPMS patients - the relative serum levels of 3-hydroxybutyrate (Figure 5C), myo-inositol (Figure 5D), and glucose (Figure 5F) were decreased, and the relative serum levels of -CH3 HDL/LDL (Figure 5E), mobile (-CH2-)n LDL (Figure 5G), and mobile - N(CHs)3/free choline (H) were increased.

Example 6 - Demonstrating that disease modifying therapies can alter the concentration change of the key metabolites described herein such that said metabolites provide a marker of patient response to therapy

Demonstrating a particularly advantageous technical effect of the invention, the inventors have demonstrated that the concentration decrease, described above for 3-hydroxybutyrate, myo-inositol and glucose, can abrogated (or reversed) by a series of known diseasemodifying therapies (DMT). Thus, the present metabolite panel can be used to reliably inform response to treatment with such DMTs, allowing for identification of responder patients (and also for screening of drugs). Results are outlined in Figure 6. Within the study cohort described above, patients were administered a DMT selected from one of dimethyl fumarate (n=4), fingolimod (n=13), glatiramer acetate (n=6) or interferon (n=6), with control patients receiving no DMT (n=15). These drugs are known DMTs for MS, known to suppress disease progression. Patients administered with one of said DMTs had increased serum levels of 3-hydroxybutyrate (E), myo-inositol (F) and glucose (G), compared to patients on no DMT (n=15), demonstrating the utility this metabolite panel in the identification of responders to therapy.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.