Chondroitin sulfate and heparan sulfate as biomarkers for distinguishing between osteoarthritis and rheumatoid arthritis

Abstract

The present invention relates to a method for screening for osteoarthritis (OA) and rheumatoid arthritis (RA) in a subject with or suspected of having arthritis, and a method for distinguishing between OA and RA, which comprises determining the level of one or more glycosaminoglycans (GAGs) characteristic of the present invention in a body fluid sample. The present invention also provides a method for monitoring the progression of OA or RA in a subject with arthritis.

Description

[Technical Field]

[0001] The present invention relates to biomarkers associated with osteoarthritis (OA) and rheumatoid arthritis (RA), and to methods of screening for osteoarthritis or rheumatoid arthritis, which involve determining the levels and / or composition of certain biomarkers indicative of OA or RA.

[0002] Arthritis is a common and often debilitating group of conditions that affect the health of the body's joints. Arthritis can affect people of all ages, including children. Arthritis includes osteoarthritis (OA) and rheumatoid arthritis (RA), i.e., OA and RA are different types of arthritis.

[0003] OA is characterized by the deterioration of cartilage that cushions the ends of bones in joints. OA is sometimes referred to as "wear and tear" arthritis. OA is a condition that typically results in painful stiffness and swelling of joints. In the UK, OA is the most common type of arthritis. OA is a commonly misdiagnosed condition, and is often only diagnosed very late, after the disease has progressed and cartilage damage has become irreversible.

[0004] RA is an autoimmune disease in which the immune system attacks the cells that line the joints. Like OA, RA is also a condition that typically results in painful stiffness and swelling of the joints.

[0005] Treatments or other clinical management strategies for RA and OA are typically different (i.e., different for RA versus OA). For example, a subject with RA may be prescribed a disease-modifying anti-rheumatic drug (DMARD). DMARDs are not traditionally prescribed for OA because they do not provide clinically significant pain relief in OA.

[0006] As is clear from the above discussion, although OA and RA are both types of arthritis and both are typically characterized by common symptoms (e.g., joint pain, stiffness, and swelling), OA and RA are different conditions that have different underlying causes and are treated differently in the clinic (e.g., using different treatments or other clinical management strategies).

[0007] Because OA and RA share symptoms, it can be difficult for clinicians to determine whether a patient with arthritis or suspected arthritis has OA or RA using existing screening or diagnostic methods (e.g., radiological imaging), i.e., it can be difficult to distinguish between OA and RA.

[0008] There is an urgent need to improve the current screening and diagnostic landscape for arthritis. Specifically, affordable and practical tools for arthritis diagnosis are needed to assist healthcare professionals, for example, in determining whether a subject has osteoarthritis (OA) or rheumatoid arthritis (RA). This would help guide the clinical management of arthritis patients.

[0009] Circulating biomarkers are molecules that can be measured in an individual's available bodily fluids, e.g., urine, and whose levels are useful, for example, to aid in disease screening, disease diagnosis, and monitoring disease progression.

[0010] What is needed in the art is a new method for screening OA and RA (for example, distinguishing between OA and RA). The identification of new biomarkers for OA and RA could potentially have clinical implications for a large number of patients and would be an important clinical advance. Here, the inventors advantageously provide a method that uses certain markers that are useful in the screening of OA and RA (for example, to distinguish between OA and RA). Advantageously, such a method is non-invasive and can be performed on easily available samples.

[0011] Surprisingly and advantageously, the present inventors have found that the level of certain glycosaminoglycans (GAGs) and / or the chemical composition of these GAGs differ in body fluid samples from OA patients compared with those from RA patients.These differential levels of GAG CSs or HSs or the differential chemical composition of GAG CSs or HSs (GAG profile), characterized by the differential level of certain GAG characteristics according to the present invention, can act as useful biomarkers in screening for OA and RA (for example, to distinguish between OA and RA).Obviously, the finding that OA and RA screening (for example, distinguish between OA and RA) can be performed using available body fluid samples, such as urine, from subjects is extremely advantageous.

[0012] Thus, in one aspect, the present invention provides a method of screening for osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject having or suspected of having arthritis, the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; Determining the level and / or chemical composition of one or both of the GAGs may include: (i) 4S CS, (ii) NS HS; (iii) 0S HS, (iv) total concentration of CS, and (v) determining the level of one or more GAG ​​properties selected from the group consisting of: total concentration of HS; The sample is obtained from the subject.

[0013] In some embodiments, the methods of the invention comprise determining the level of 4S CS.

[0014] In some embodiments, the methods of the invention comprise determining the level of NS HS.

[0015] In some embodiments, the methods of the invention comprise determining the level of OS HS.

[0016] In some embodiments, the methods of the invention comprise determining the total concentration of CS.

[0017] In some embodiments, the methods of the present invention include determining the total concentration of HS.

[0018] In some embodiments, the methods of the invention involve determining the level of 4S CS or NS HS.

[0019] In some embodiments, the methods of the invention involve determining the levels of 4S CS and NS HS.

[0020] In some embodiments, the method of the present invention comprises: (i) absolute concentration of 4S CS, (ii) absolute concentrations of NS HS and / or relative concentrations of NS HS; (iii) absolute concentration of 0S HS; (iv) total concentration of CS, and (v) determining the level of one or more GAG ​​properties selected from the group consisting of: total concentration of HS;

[0021] The terms "absolute concentration," "relative concentration," and "total concentration" are discussed elsewhere herein.

[0022] Unless otherwise clear from the context, the term "one or more" includes "all."

[0023] In some embodiments, the methods of the invention comprise determining the absolute concentration of 4S CS.

[0024] In some embodiments, the methods of the present invention comprise determining the absolute concentration of NS HS.

[0025] In some embodiments, the methods of the present invention comprise determining the relative concentration of NS HS.

[0026] In some embodiments, the methods of the present invention comprise determining the absolute concentration of OS HS.

[0027] In some embodiments, the methods of the invention comprise determining the total concentration of CS.

[0028] In some embodiments, the methods of the present invention include determining the total concentration of HS.

[0029] In some embodiments, the methods of the invention involve determining the absolute concentration of 4S CS or the absolute concentration of NS HS.

[0030] In some embodiments, the methods of the invention involve determining the absolute concentrations of 4S CS and NS HS.

[0031] Therefore, in a preferred embodiment of the present invention, the level of 4S CS is the absolute concentration of 4S CS, the level of NS HS is the absolute concentration of NS HS or the relative concentration of NS HS (more preferably, the absolute concentration of NS HS), and the level of 0S HS is the absolute concentration of 0S HS.

[0032] Unless otherwise clear from the context, reference to one or more GAG ​​properties (or GAG forms or GAG characteristics or GAGome characteristics or GAGome properties) "according to the invention" or "according to the invention" (or equivalent phrases) is a reference to 4S CS (preferably the absolute concentration of 4S CS), NS HS (preferably the absolute concentration of NS HS and / or the relative concentration of NS HS, more preferably the absolute concentration of NS HS), OS HS (preferably the absolute concentration of 0S HS), total concentration of CS and / or total concentration of HS. Particularly preferred GAG properties "according to the invention" are 4S CS (preferably the absolute concentration of 4S CS) and / or NS HS (preferably the absolute concentration of NS HS).

[0033] In a preferred method of the present invention, an altered (possibly increased or decreased) level of one or more GAG ​​characteristics according to the present invention (4S CS (e.g., absolute concentration of 4S CS), NS HS (e.g., absolute concentration of NS HS and / or relative concentration of NS HS), OS HS (e.g., absolute concentration of OS HS), total CS concentration and / or total HS concentration) compared to a control level is indicative of either OA or RA in the subject (indicating that the subject has either OA or RA).

[0034] In certain methods of the present invention, an altered (possibly increased or higher) level of one or more GAG ​​characteristics of the present invention (4S CS (e.g., absolute concentration of 4S CS), NS HS (e.g., absolute concentration of NS HS and / or relative concentration of NS HS), OS HS (e.g., absolute concentration of OS HS), total concentration of CS and / or total concentration of HS) compared to a control level is indicative of OA in the subject (indicative of whether the subject has OA).

[0035] In certain methods of the present invention, an altered (possibly decreased or lower) level of one or more GAG ​​characteristics of the present invention (4S CS (e.g., absolute concentration of 4S CS), NS HS (e.g., absolute concentration of NS HS and / or relative concentration of NS HS), OS HS (e.g., absolute concentration of OS HS), total concentration of CS and / or total concentration of HS) compared to a control level is indicative of RA in the subject (indicates whether the subject has RA).

[0036] Those skilled in the art will be readily able to establish appropriate control levels for use in accordance with the present invention, and how certain appropriate control levels and indicators of OA or RA (discriminating between possible OA and possible RA) may be arrived at are discussed elsewhere herein.

[0037] In some embodiments of the methods of the present invention, a level of one or more of the GAG ​​characteristics of the present invention (4S CS (e.g., absolute concentration of 4S CS), NS HS (e.g., absolute concentration of NS HS and / or relative concentration of NS HS), OS HS (e.g., absolute concentration of OS HS), total CS concentration and / or total HS concentration) within an established (or given or appropriate) reference range (or reference interval), or an altered (possibly increased or decreased) level relative to the established (or given or appropriate) reference range (or reference interval) is indicative of either OA or RA in the subject (indicative of whether the subject has either OA or RA).

[0038] Those skilled in the art will be able to readily establish appropriate reference ranges (or reference intervals) for use in accordance with the present invention. Certain suitable reference ranges (or reference intervals) and how such reference ranges can be used to arrive at indicators of OA or RA are discussed elsewhere herein.

[0039] In preferred methods of the invention, both the level and chemical composition are determined, in other preferred methods of the invention, only the chemical composition is determined, or in other preferred methods, the level (total level or concentration) of only CS and / or HS is determined.

[0040] The methods of the invention include determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample. In some embodiments, the level and / or chemical composition of one of the GAGs is determined. In some embodiments, the level and / or chemical composition of chondroitin sulfate (CS) is determined. In some embodiments, the level and / or chemical composition of heparan sulfate (HS) is determined. In some embodiments, the level and / or chemical composition of chondroitin sulfate (CS) and heparan sulfate (HS) is determined.

[0041] As described elsewhere herein, in some preferred embodiments of the methods of the present invention, the methods comprise determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample. Thus, in some embodiments, the level and / or chemical composition of the protein-free fraction of chondroitin sulfate (CS) is determined. In some embodiments, the level and / or chemical composition of the protein-free fraction of heparan sulfate (HS) is determined. In some embodiments, the level and / or chemical composition of the protein-free fraction of chondroitin sulfate (CS) and heparan sulfate (HS) is determined.

[0042] In some embodiments of the methods of the invention, the methods involve determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS), although it is not essential in all aspects of the invention that the level and / or chemical composition of the "protein-free fraction" be specifically determined.

[0043] Glycosaminoglycans (GAGs) are sugar-containing molecules that can be attached to proteins on serine residues, i.e., form part of proteoglycans. They are formed from linear or unbranched chains of monosaccharides (i.e., polysaccharides) that may be sulfated. Heparan sulfate (HS), chondroitin sulfate (CS), keratan sulfate (KS), hyaluronic acid (HA), and heparin are common types of GAGs, of which HS and CS are examples of sulfated GAGs. Different types of GAGs are distinguished by different repeating disaccharide units.

[0044] When linked or attached to proteins, CS and HS are GAGs that share a common biosynthetic pathway in linking to the core protein, but then they differ in their polymerization in that the CS repeating disaccharide is composed of repeating N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residues, whereas the repeating disaccharide in HS is typically composed of repeating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) residues. Each monosaccharide is attached by a specific enzyme, allowing multiple levels of regulation over GAG synthesis.

[0045] Although GAGs can be bound to proteins, i.e., in the protein-bound or proteoglycan form, GAGs can also exist in a "free" form, i.e., in a non-protein-bound or non-proteoglycan form. Such "free" forms of GAGs are referred to herein as "protein-free GAGs."

[0046] Thus, in a body fluid (or body sample), there is typically a protein-free fraction of GAGs (or a protein-free pool of GAGs) and a protein-bound fraction of GAGs (or a protein-bound pool of GAGs). The two fractions combined (i.e., the protein-free fraction + the protein-bound fraction) can be referred to as the total GAG fraction or total GAG pool.

[0047] In a preferred method of the invention, the level or composition of the protein-free fraction of one or both of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample is determined.

[0048] Protein-free fraction GAGs (or disaccharide units derived therefrom as discussed elsewhere herein) for analysis can be obtained by any suitable means.

[0049] As discussed elsewhere herein, bodily fluid samples are typically processed prior to analysis. Such processing typically involves subjecting the GAGs to a processing step to obtain disaccharide units for analysis. Such processing steps typically involve contacting the sample (or the GAGs in the sample) with an enzyme (e.g., a GAG lyase such as chondroitinase or heparinase) that digests (or fragments) the GAGs into disaccharide units. Without wishing to be bound by theory, such enzymes are unable to access protein-bound GAGs, but rather act only (or essentially only) on (or use as a substrate for) protein-free GAGs. When proteoglycans (proteins with GAGs bound or attached thereto) are contacted with a proteolytic agent (e.g., a protease such as proteinase K), the protein component is digested by the proteolytic agent, liberating or releasing the protein-bound GAGs, meaning that the protein-bound GAGs are rendered (or converted) into a "free" or "released" form, which will then be available for digestion (or fragmentation) by an enzyme such as a GAG lyase. In certain preferred methods of the invention, what is determined is specifically the level and / or composition of the protein-free fraction (or native protein-free fraction) of one or both of the GAG ​​CS and HS, and therefore preferred methods of the invention do not involve contacting the sample with a proteolytic agent (e.g., a protease such as proteinase K). Thus, omitting the proteolytic agent during processing of a sample for analysis is a way to obtain (or obtain only, or obtain essentially only) the protein-free fraction of GAGs (or disaccharides thereafter derived therefrom) for analysis. Protein-bound GAGs (proteoglycan GAGs) that have been (or are) "liberated" or "released" by the action of a proteolytic agent (eg, a protease) are not protein-free GAGs according to the present invention.

[0050] Thus, protein-free GAGs (or protein-free fractions of GAGs) are GAGs (or fractions of GAGs) that are already (or naturally) free (i.e., not bound to proteins) in the absence (or absence) of a sample that has been treated with a proteolytic agent (e.g., a protease). In other words, protein-free GAGs (or protein-free fractions of GAGs) are GAGs (or fractions of GAGs) that are free (i.e., not bound to proteins) in the original, initial, or untreated sample. For example, protein-free GAGs (or protein-free fractions of GAGs) can be GAGs (or fractions of GAGs) that are present in a sample, e.g., an original or untreated sample, and that are susceptible to, accessible to, or available for (e.g., are substrates for) digestion (or fragmentation) into disaccharide units as described elsewhere herein, e.g., using an enzyme such as a lyase enzyme.

[0051] As noted above, protein-free GAGs (or protein-free fractions of GAGs) may be considered non-protein-bound (or non-protein-bound fractions) or non-proteoglycan (or non-proteoglycan fractions) forms of GAGs. In other words, protein-free GAGs (or protein-free fractions of GAGs) are GAGs that do not modify proteoglycans in the original, initial, or untreated sample.

[0052] Thus, in some preferred embodiments, the method comprises determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) according to the present invention in a body fluid sample, wherein the sample is subjected to a treatment prior to determining the level and / or composition, and the treatment does not include contacting the sample with a proteolytic agent (e.g., a protease such as proteinase K) or other agent capable of releasing protein-free GAGs from protein-bound GAGs.

[0053] Alternatively, in some preferred embodiments, the method comprises determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) according to the present invention in a body fluid sample, wherein the sample is subjected to a treatment prior to determining the level and / or composition, and the treatment does not include contacting the sample with a proteolytic agent (e.g., a protease such as proteinase K) or other agent capable of releasing GAGs (or GAG chains) from proteoglycans.

[0054] Thus, in some preferred embodiments, the method comprises determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) according to the present invention in a body fluid sample, the sample having been obtained from the subject and subjected to a treatment prior to determining the level and / or chemical composition, the treatment comprising: (a) fragmenting the one or both GAGs into disaccharide units (e.g., as described elsewhere herein); and (b) does not include contacting the sample with an agent capable of releasing GAGs (or GAG chains) from proteoglycans prior to (a).

[0055] Thus, in some preferred embodiments, the method comprises determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) according to the present invention in a body fluid sample, the sample having been obtained from the subject and subjected to a treatment prior to determining the level and / or chemical composition, the treatment comprising: (a) fragmenting the one or both GAGs into disaccharide units (e.g., as described elsewhere herein); and (b) does not involve contacting the sample with a proteolytic agent prior to (a).

[0056] As noted above, protein-bound GAGs (or protein-bound fractions of GAGs) may be considered proteoglycan GAGs (or proteoglycan fractions of GAGs). Alternatively, protein-bound GAGs (or protein-bound fractions of GAGs) may be considered GAGs that typically require a protein to which they are bound so that they can be liberated or released (or become liberated or released) upon contact with a proteolytic agent (e.g., a protease, such as a nonspecific protease).

[0057] In some other aspects of the methods of the invention, the level and / or chemical composition of the total fraction (or pool) of one or both of the GAGs CS and HS in a body fluid sample can be determined according to the invention (i.e., protein-free GAGs + protein-bound GAGs). In such embodiments, the sample is typically contacted with a proteolytic agent during sample processing.

[0058] The "level" of HS or CS referred to herein generally refers to the total level or amount (e.g., concentration) of HS or CS present in a sample. The level of CS and / or HS in a sample can be measured or determined by any suitable method known and described in the art. Some methods involve electrophoresis, particularly capillary electrophoresis, such as capillary electrophoresis with fluorescence detection, e.g., laser-induced fluorescence detection. Other suitable methods include gel electrophoresis, e.g., agarose gel electrophoresis (e.g., FACE, fluorophore-assisted carbohydrate electrophoresis), or mass spectrometry or liquid chromatography, e.g., HPLC, optionally combined with mass spectrometry (HPLC-MS). A preferred method involves high-performance liquid chromatography (HPLC), preferably ultra-HPLC (UHPLC), in combination with mass spectrometry, e.g., MS / MS or triple-quadrupole mass spectrometry. A preferred method includes ultra-high-performance liquid chromatography (UHPLC) combined with an electrospray ionization triple-quadrupole mass spectrometry system.

[0059] Conveniently, these levels may be measured as concentrations (e.g., actual or absolute levels or concentrations), for example, as micrograms per mL (μg / mL), although again, any suitable measure of level may be used.

[0060] In typical methods of the invention, the levels of HS and / or CS (e.g., the levels of HS and / or CS in the protein-free fraction) are determined separately or individually. In other words, the method does not involve measuring the total GAG level in the sample, or the total level of all GAGs present in combination (e.g., in the protein-free GAG ​​fraction), but rather involves measuring the levels of one or more of the individual GAGs, HS, or CS.

[0061] In certain embodiments, the level (e.g., total level or concentration) of CS and / or HS (e.g., the level of the protein-free fraction of CS and / or HS) can be determined in a bodily fluid sample, for example, a urine sample.

[0062] The individual monosaccharide units that make up CS and HS can have different sulfation patterns with respect to the position and amount / number of sulfate molecules. In the case of CS, sulfation can occur most commonly at one or more of position 2 of GlcA and positions 4 and 6 of GalNAc. In the case of HS, sulfation can occur at one or more of position 2 of GlcA after epimerization to IdoA (iduronic acid), positions 3 and 6 of GlcNAc, and N-sulfation of GlcNAc. Thus, each individual disaccharide in a GAG chain can have 0 (i.e., unsulfated), 1, 2, 3, or 4 (only in HS) sulfated forms, which in turn results in different overall chemical compositions of the GAG ​​chain with respect to sulfation level and specific disaccharide sulfation patterns.

[0063] As described elsewhere herein, preferred embodiments of the present invention involve determining the chemical composition of one or both of CS and HS. The term "chemical composition" as used herein can refer to both the level of GAGs and the disaccharide sulfation composition of GAGs. Specifically, this term includes determining one or more specific forms of the disaccharides that make up CS or HS GAGs, e.g., sulfated forms. In other words, the term "chemical composition," as described elsewhere herein, refers to the amount or level of one or more of the various sulfated and / or non-sulfated forms of CS or HS disaccharides, as well as some other characteristic of the individual GAGs present (e.g., total HS or CS GAG levels), or other characteristics related to GAG sulfation (e.g., HS charge or CS charge). Such chemical compositions analyzed or determined in the present invention may also be referred to herein as GAG profiles, GAG forms, GAG signatures, GAG characteristics, GAG-omes, or GAG-ome characteristics.

[0064] Thus, for example, the term "chemical composition" as used herein can refer to the determination or analysis of the sulfation pattern (e.g., one or more of the sulfated forms) of the disaccharides that make up the CS and / or HS.

[0065] As is clear from the above discussion, according to the present invention, it is essential to determine the level of one or more GAG ​​characteristics selected from the group consisting of 4S CS (preferably the absolute concentration of 4S CS), NS HS (preferably the absolute concentration of NS HS), 0S HS (preferably the absolute concentration of 0S HS), the total concentration of CS, and the total concentration of HS. Therefore, according to the present invention and disclosure, determining the "chemical composition" must include determining the level of one or more GAG ​​characteristics selected from the group consisting of 4S CS, NS HS, 0S HS, the total concentration of CS, and the total concentration of HS. Although the level of one or more other (additional) GAG characteristics (e.g., as described herein) can be additionally determined (or measured), according to the present invention and disclosure, it is the level of one or more GAG ​​characteristics selected from the group consisting of 4S CS, NS HS, 0S HS, the total concentration of CS, and the total concentration of HS that enables an indication (e.g., a diagnosis) of OA or RA to be made (or reached).

[0066] There are eight major sulfated and non-sulfated forms (sulfation patterns, disaccharide sulfate forms) of CS: 0S CS (also called non-sulfated CS or CS O unit), 2S CS (also called chondroitin-2-sulfate), 4S CS (also called chondroitin-4-sulfate or CS A unit), 6S CS (also called chondroitin-6-sulfate or CS C unit), 2S4S CS (also called chondroitin-2-4-sulfate), 2S6S CS (also called chondroitin-2-6-sulfate or CS D unit), 4S6S CS (also called chondroitin-4-6-sulfate or CS E unit), and Tris CS (also called chondroitin-2-4-6-sulfate or trisulfated CS).

[0067] Each of the above is a form of CS GAG (CS GAG form or property or characteristic or GAGome feature) that can be measured in the methods of the invention. One or more of these forms can be measured, for example, up to eight of these sulfated forms, for example, 1, 2, 3, 4, 5, 6, 7, or all eight. In some embodiments, measurement of all eight of these sulfated forms is preferred. In some embodiments, measurement of CS sulfated forms, or at least the CS sulfated forms 0S CS, 6S CS, 4S CS, 2S6S CS, 2S4S CS, 4S6S CS, is preferred.

[0068] Another GAG property of CS that can be measured in the methods of the invention is the total CS concentration (also referred to as CS tot or Tot CS or total CS) or total CS level. This is typically measured as a concentration, e.g., absolute concentration in μg / mL, as described elsewhere herein. In some embodiments, total CS is measured by summing the levels of all measured CS disaccharide forms (the eight major sulfated forms and the non-sulfated form) listed above. Thus, total CS can be the sum of the levels of all measured CS disaccharide forms (the eight major sulfated forms and the non-sulfated form) listed above. In some embodiments, total CS is measured by summing the levels of all CS disaccharide forms measured at (or above) an analytically detectable level or at (or above) a level selected as a minimum threshold level (the minimum threshold level considered analytically detectable or analytically meaningful, e.g., the detection limit or lower limit of quantification for such CS disaccharide forms). For example, total CS can be the sum of all CS disaccharide forms measured at levels equal to or greater than a given analytically detectable level or equal to or greater than a selected minimum threshold level (e.g., at concentrations greater than 0.1 μg / mL). Levels of CS disaccharide forms below a given analytically detectable level or below a minimum threshold level typically provide negligible (or insignificant) contributions to the total CS concentration, and therefore such CS disaccharide forms can, if desired, be excluded from the sum of CS disaccharide forms performed to determine or measure the total CS concentration.

[0069] In embodiments of the invention in which the total concentration of CS is measured as one of the GAG ​​profiles, it may be preferable to measure at least one other GAG or CS profile according to the invention, e.g., a profile that is not based on the total level of other individual GAGs present (e.g., not total HS). In some embodiments, the total concentration of CS is not measured. In some embodiments, measurement of one or more CS GAG profiles according to the invention is preferred.

[0070] "Charged CS" is another GAG form or property that can be measured in the present invention, for example, as part of a GAG profile. "Charged CS" refers to the total fraction of sulfated disaccharides of CS, i.e., the fraction of sulfated disaccharides of CS present, measured, or detected in a sample out of all CS disaccharides present, measured, or detected in the sample (i.e., sulfated CS disaccharides / sulfated + non-sulfated CS disaccharides). In some embodiments, "charged CS" refers to the weighted sum of the concentrations of all CS disaccharides divided by total CS, where the weight is the number of sulfo groups in the disaccharide, i.e., 0 for 0S CS, 1 for 4S CS, 6S CS, and 2S CS, 2 for 2S6S CS, 4S6S CS, and 2S4S CS, and 3 for Tris CS (this is the definition of "charged CS" used in connection with the term "charged CS" in the Examples section herein).

[0071] Because the measurement of "charged CS" depends on the measurement of other properties, namely the levels of sulfated and non-sulfated CS disaccharides, this property is not referred to herein as an independent GAG or CS property.

[0072] In some embodiments, it is preferred to measure up to eight (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) or all eight sulfated forms of CS (i.e., sulfated and non-sulfated forms) along with total CS. Charged CS may additionally be measured in some embodiments. In preferred embodiments, at least one (or at least 2, 3, 4, 5, 6, 7, or 8) sulfated forms of CS are measured.

[0073] In some embodiments, one or more (or all) of the following GAG properties may be measured or determined: the relative level of 4S CS to 6S CS (e.g., the 4S CS / 6S CS ratio or the inverse 6S CS / 4S CS ratio), the relative level of 6S CS to 0S CS (e.g., the 6S CS / 0S CS ratio or the inverse 0S CS / 6S CS ratio), or the relative level of 4S CS to 0S CS (e.g., the 4S CS / 0S CS ratio or the inverse 0S CS / 4S CS ratio). In some embodiments, the relative level of 4S CS to 6S CS (e.g., the 4S CS / 6S CS ratio or the inverse 6S CS / 4S CS ratio) is not measured or determined.

[0074] There are eight major sulfated and non-sulfated forms of HS (sulfation patterns, disaccharide sulfate forms): 0S HS (also called non-sulfated HS), 2S HS (sulfated at position 2 of GlcA), NS HS (sulfated at position N of GlcNAc), 6S HS (sulfated at position 6 of GlcNAc), 2S6S HS (sulfated at positions 2 of GlcA and 6 of GlcNAc), NS6S HS (sulfated at positions 6 and N of GlcNAc), NS2S HS (sulfated at positions 2 of GlcA and 6 and N of GlcNAc), and Tris HS (sulfated at positions 2 of GlcA and 6 and N of GlcNAc, also called trisulfated HS). Note that sulfation at position 3 of GlcNAc is also possible but is rarely observed.

[0075] Each of the above is a form of HS GAG (HS GAG form or property or characteristic or GAGome feature) that can be measured or determined in the methods of the present invention. However, due to its rarity, in preferred embodiments of the present invention, the sulfated form having sulfate at position 3 of GlcNAc is not measured. Thus, the methods of the present invention can measure one or more (or all) of these nine (or preferably eight) forms, for example, up to nine (or preferably up to eight) of these sulfated forms, for example, 1, 2, 3, 4, 5, 6, 7, 8, or all nine. In some embodiments, measurement of all eight of these sulfated forms (excluding the sulfated form having sulfate at position 3 of GlcNAc) is preferred.

[0076] Another GAG property of HS that can be measured in the methods of the invention is the total HS concentration (which may also be referred to as HS tot, Tot HS, or total HS) or total level of HS. This is typically measured as a concentration, e.g., an absolute concentration in μg / mL, as described elsewhere herein. In some embodiments, total HS is measured by summing the levels of all measured HS disaccharide forms listed above (preferably the eight major sulfated forms and the non-sulfated form, i.e., excluding the rare sulfated form having sulfate at position 3 of GlcNAc). Thus, total HS can be the sum of the levels of all measured HS disaccharide forms listed above (preferably the sum of the eight major sulfated forms and the non-sulfated form, i.e., excluding the rare sulfated form having sulfate at position 3 of GlcNAc). In some embodiments, total HS is measured by summing the levels of all of the HS disaccharide forms measured at (or above) an analytically detectable level or at (or above) a level selected as a minimum threshold level (the minimum threshold level considered analytically detectable or analytically meaningful, e.g., the limit of detection or lower limit of quantification for such HS disaccharide forms). For example, total HS can be the sum of all of the HS disaccharide forms measured at or above a given analytically detectable level or at a level above a selected minimum threshold level (e.g., at a concentration greater than 0.1 μg / mL). Because levels of HS disaccharide forms below a given analytically detectable level or below a minimum threshold level typically provide a negligible (or insignificant) contribution to the total HS concentration, such HS disaccharide forms can, if desired, be excluded from the sum of HS disaccharide forms performed to determine or measure the total HS concentration.

[0077] In embodiments of the invention in which total HS concentration is measured as one of the GAG ​​profiles, it may be preferable to measure at least one other GAG or HS profile according to the invention, e.g., a profile that is not based on the total level of other individual GAGs present (e.g., not total CS). In some embodiments, total HS concentration is not measured. In some embodiments, measurement of one or more HS GAG profiles according to the invention is preferred.

[0078] "Charged HS" is another GAG form or property that can be measured in the present invention, for example, as part of a GAG profile. "Charged HS" refers to the total fraction of sulfated HS disaccharides, i.e., the fraction of sulfated HS disaccharides present or measured in a sample out of all HS disaccharides present or measured in the sample (i.e., sulfated HS disaccharides / sulfated + non-sulfated HS disaccharides). In some embodiments, "charged HS" refers to the weighted sum of the concentrations of all HS disaccharides divided by total HS, where the weight is the number of sulfo groups in that disaccharide, i.e., 0 for 0S HS, 1 for NS HS, 6S HS, and 2S HS, 2 for 2S6S HS, NS6S HS, and NS2S HS, and 3 for Tris HS (this is the definition of "charged HS" used in connection with the term "charged HS" in the Examples section herein).

[0079] Because the measurement of "charged HS" depends on the measurement of other properties, namely sulfated and non-sulfated HS disaccharides, this property is not referred to herein as an independent GAG or HS property.

[0080] In some embodiments, it is preferred to measure up to eight (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) or all eight of the major sulfated forms of HS (i.e., the sulfated and non-sulfated forms listed above, excluding sulfated forms with sulfate at position 3 of GlcNAc), together with total HS. Charged HS may be additionally measured in some embodiments. In preferred embodiments, at least one (or at least 2, 3, 4, 5, 6, 7, or 8) sulfated forms of HS are measured.

[0081] In some embodiments, 18 GAG signatures are measured: 8 major sulfated and non-sulfated HS forms, total HS, 8 major sulfated and non-sulfated CS forms, and total CS.

[0082] In some embodiments, eight major sulfated and non-sulfated HS forms and eight major sulfated and non-sulfated CS forms, ie, 16 GAG signatures, are measured.

[0083] In some embodiments, the level and / or chemical composition of hyaluronic acid (HA) can be additionally determined in a body fluid sample. Hyaluronic acid (HA) is typically non-sulfated. Therefore, when HA is measured according to the present invention, it is typically and preferably the level (total level or concentration) of HA that is measured (which may also be referred to as total HA). This is typically measured as a concentration, e.g., in μg / mL, as described elsewhere herein. In some embodiments, HA is not measured.

[0084] In some embodiments, 19 GAG signatures are measured: the eight major sulfated and non-sulfated HS forms, total HS, the eight major sulfated and non-sulfated CS forms, total CS, and total HA.

[0085] CS (total CS) and HS (total HS) are typically measured in absolute concentrations, e.g., μg / mL, as described elsewhere herein.

[0086] The various CS sulfated forms and HS sulfated forms can also be measured in terms of absolute concentrations, e.g., in μg / mL. Thus, in some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form is the absolute level or concentration of the given CS sulfated form or a given HS sulfated form. In some embodiments, absolute levels or concentrations are preferred.

[0087] However, various CS and HS sulfated forms may alternatively or additionally be measured in terms of relative concentrations (or levels). Thus, in some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form is a relative level or concentration.

[0088] Thus, the "level" or "concentration" of a sulfated form of GAG can be its absolute concentration or its relative concentration. In some embodiments, both the absolute and relative concentrations of one or more sulfated GAG forms are measured (or determined).

[0089] A "relative concentration" can be considered as the mass fraction (e.g., %) of a given CS sulfated form or a given HS sulfated form obtained (or calculated or determined) by normalizing its absolute concentration to the total concentration of the relevant GAG class, i.e., by normalizing its absolute concentration to the total CS concentration or total HS concentration (as appropriate).

[0090] Therefore, the relative concentration of a given sulfated form of CS can be considered as the mass fraction (e.g., %) of that given sulfated form of CS obtained (or calculated or determined) by normalizing its absolute concentration by the total CS concentration.

[0091] The relative concentration of a given sulfated form of HS can be considered as the mass fraction (e.g., in %) of that given sulfated form of HS obtained (or calculated or determined) by normalizing its absolute concentration by the total HS concentration.

[0092] Relative concentrations can be expressed as a percentage (%).

[0093] Thus, in some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form can be the absolute concentration and / or the relative concentration of the given CS sulfated form or the given HS sulfated form. In some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form is the absolute concentration of the given CS sulfated form or the given HS sulfated form. In some embodiments, the level (or concentration) of a given CS sulfated form or a given HS sulfated form is the relative concentration of the given CS sulfated form or the given HS sulfated form.

[0094] Alternatively, the relative concentration can be considered as a "fraction" or "mass fraction" or "proportion" or "relative measure," as discussed below.

[0095] As described above, GAG characteristics or GAG forms, such as disaccharide sulfated forms (excluding total CS or total HS), may be measured not as absolute levels or concentrations, but as fraction sizes or fractions or mass fractions (e.g., μg / μg) or percentages or relative measurements, e.g., given a value less than 1 or normalized to 1 (or expressed as a %) depending on the levels of all sulfated forms of the relevant GAG class (or all major sulfated forms of the relevant GAG class) measured in the sample. In other words, the level of each desired sulfated form is measured independently and then normalized to 1. In other words, the level of each desired sulfated form is measured independently and then its mass fraction, volume fraction, or mole fraction is calculated. These fractions may be expressed as percentages. In other words, these fractions may be normalized to 100. For example, in some embodiments, the fraction size of a given sulfated or non-sulfated CS form can be determined by measuring the level of the given sulfated or non-sulfated CS form and dividing it by the sum of the levels of all CS sulfated forms (or all of the major sulfated forms) and non-sulfated CS forms measured (or present) in the sample. In some embodiments, the fraction size of a given sulfated or non-sulfated HS form can be determined by measuring the level of the given sulfated or non-sulfated HS form and dividing it by the sum of the levels of all HS sulfated forms (or the major sulfated forms) and non-sulfated HS forms measured (or present) in the sample. When calculating such fractions, it is preferred that at least the major sulfated form of CS or HS is measured so that the fraction of a particular individual sulfated form can be normalized to 1.

[0096] Relative measurements may be easier to interpret; for example, an 0s HS measurement of 0.6 indicates that 60% of the measured HS disaccharides are unsulfated. However, absolute levels can also be measured. Indeed, in some embodiments, it is preferable to measure the absolute concentration of one or more sulfated forms.

[0097] In some preferred embodiments of the present invention, the disaccharide composition (e.g., a particular sulfation pattern (e.g., sulfated form)) of one or more (or all) of the disaccharides constituting the CS and / or HS is measured or determined. In some embodiments, the sulfation characteristics or forms (or GAG features or properties or GAGome features or properties) of one or more (or all) of the CS and / or HS, such as those outlined above (e.g., 0S CS, 2S CS, etc.), are measured or determined. Suitable methods for doing this are well known to those skilled in the art, and any of these can be used. However, a convenient way to achieve such quantification of the disaccharide composition or suitable characteristics or forms of CS or HS (and separation of disaccharide forms) is to use electrophoresis, in particular capillary electrophoresis, for example capillary electrophoresis with fluorescence detection, for example capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). An alternative method is liquid chromatography, preferably HPLC (high performance liquid chromatography), for example SAX HPLC. Preferably, mass spectrometry (e.g., HPLC-MS), such as electrospray ionization mass spectrometry (ESI-MS), is also used. Alternatively, mass spectrometry can be used without chromatography (e.g., liquid chromatography). One example is capillary electrophoresis with laser-induced fluorescence detection. Another example is HPLC ESI-MS. A preferred method involves high performance liquid chromatography (HPLC), preferably ultra HPLC (UHPLC), in combination with mass spectrometry, such as MS / MS or triple quadrupole mass spectrometry. A preferred method includes ultra high performance liquid chromatography (UHPLC) coupled with electrospray ionization triple quadrupole mass spectrometry. Particularly preferred methods are outlined in the Examples.

[0098] In some methods of the invention in which the level of one or more individual disaccharide forms is measured, the GAG ​​is subjected to a processing step (e.g., a step of fragmentation or cleavage or digestion (e.g., by chemical digestion or enzymatic treatment)) to obtain the disaccharide units, which are then analyzed. The enzyme can be a GAG lyase, such as a chondroitinase or a heparinase, or a combination of chondroitinases, or a combination of heparinases, or a combination of one or more chondroitinases and one or more heparinases. Preferably, the chondroitinase is chondroitinase ABC or chondroitinase AC or chondroitinase A or chondroitinase B or chondroitinase C. Preferably, the heparinase is heparinase I-II-III. In a preferred embodiment, one or more chondroitinases and one or more heparinases, preferably chondroitinase ABC and heparinase I-II-III, are used.

[0099] In some methods of the invention, GAGs in a sample are subjected to extraction (e.g., using a proteolytic agent, e.g., a protease, e.g., a non-specific protease, e.g., proteinase K) and / or purification, e.g., using an anion exchange resin (or other means for purifying GAGs based on their negative charge).

[0100] However, in preferred methods of the invention, one or both of these steps are not performed (i.e., there is no such extraction and / or no such purification). For example, in preferred embodiments of the invention, such protein digestion (extraction) steps are not performed, e.g., when the level and / or composition of the protein-free fraction of one or more GAGs is determined. In other words, the method does not involve a processing step in which the sample is contacted with a proteolytic agent, e.g., a protease, e.g., proteinase K. As discussed elsewhere herein, omitting a processing step in which the sample is contacted with a proteolytic agent means that the protein-free fraction of GAGs can be specifically analyzed.

[0101] In other preferred embodiments of the present invention, the method does not involve a processing step in which GAGs (e.g., GAGs, one or both of CS and HS) are purified from the sample based on the negative charge of the GAGs, for example, using an anion exchange resin. Without wishing to be bound by theory, omitting a processing step in which GAGs (e.g., GAGs, one or both of CS and HS) are purified from the sample based on the negative charge of the GAGs (e.g., using an anion exchange resin) can simplify the method and provide efficiencies in terms of the yield of GAGs obtained during processing of a body fluid sample.

[0102] In preferred methods of the present invention, the method does not involve a processing step in which the sample is contacted with a proteolytic agent, e.g., a protease, e.g., proteinase K, and does not involve a processing step in which GAGs (e.g., one or both of GAG, CS and HS) are purified from the sample based on the negative charge of the GAGs, e.g., using an anion exchange resin (or other means of purifying GAGs based on the negative charge of the GAGs).

[0103] In some methods of the invention, GAGs in a sample (e.g., various different GAG forms in a sample) are subjected to a separation and / or quantification step, as described elsewhere herein. For example, HPLC in combination with mass spectrometry may be used in preferred embodiments, as discussed elsewhere herein. A particularly preferred method involves ultra-high performance liquid chromatography (UHPLC) coupled with electrospray ionization triple quadrupole mass spectrometry.

[0104] Other methods that can be used are known in the art, however, examples include analytical techniques that involve the use of antibodies against various GAG forms, such as Western blot, ELISA, or FACS, or methods that involve agarose gel electrophoresis (e.g., fluorophore-assisted carbohydrate electrophoresis (FACE)) or polyacrylamide gel electrophoresis (PAGE).

[0105] In any embodiment referring to the determination of one or more levels from a particular list of GAG forms (or GAG properties or GAG features or GAGome features) according to the present invention, in some such embodiments, the levels of all listed GAG forms (or GAG properties or GAG features or GAGome features) according to the present invention may be determined.

[0106] In some embodiments, the level of a single GAG ​​form (GAG profile or GAG signature or GAGome signature) is determined.

[0107] In other embodiments of the invention, the level of more than one GAG ​​form (GAG profile) is determined (e.g., the levels of two or more GAG ​​forms, or three or more GAG ​​forms, or four or more GAG ​​forms, or five or more GAG ​​forms are determined). "More than one" means 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, etc. For any list of markers or GAG profiles provided herein, in some embodiments, all are measured (or determined). Also, determining the level of each and every possible combination of GAG forms can be performed.

[0108] Thus, in some embodiments, a multi-marker approach is implemented. Determining the levels of multiple GAG ​​forms (biomarker multiplexing) according to the present invention can improve screening (e.g., diagnostic) accuracy.

[0109] Thus, although the markers according to the invention (GAG forms or GAG properties or characteristics or GAGome characteristics) can be used individually in the methods of the invention, they can also be used in combination (eg in the form of a multi-marker assay).

[0110] In some embodiments, the level of a single GAG ​​form (GAG profile) according to the present invention is used as the basis for screening for OA or RA, for example, discrimination (or differentiation) between OA and RA can be made in some embodiments based on the level of a single GAG ​​form according to the present invention (i.e., based on the level of 4S CS, NS HS, 0S HS, the total concentration of CS, or the total concentration of HS). In other embodiments, more than one (e.g., 2, 3, 4, or 5) GAG forms according to the present invention are used as the basis for screening for OA or RA, for example, discrimination (or differentiation) between OA and RA can be made based on the level of more than one GAG ​​form according to the present invention (i.e., based on more than one (e.g., all) levels of 4S CS, NS HS, 0S HS, the total concentration of CS, or the total concentration of HS, for example, based on any subgroup of these GAG ​​forms as discussed elsewhere herein).

[0111] In some embodiments, when one GAG ​​form or group of GAG forms (or a subgroup or subset) is used as the basis for screening for OA or RA (e.g., to distinguish or differentiate between OA and RA), the levels of one or more (or all) of the other GAG forms (or GAG characteristics) described herein may additionally be determined or measured.

[0112] Based on the observed changes in the levels of various GAG properties according to the present invention, if desired, one can design a scoring method, scoring system, marker or formula that uses such levels of various GAG forms to arrive at an index in the form of, for example, a value or score, which can then be used for screening (e.g., diagnosis, etc.). Suitable scoring systems and parameters (e.g., GAG forms) to be measured can be readily designed based on (or including) one or more of the individual GAG characteristics according to the present invention.

[0113] In some embodiments according to the present invention, the chemical composition may be expressed in terms of a score (or GAG score), which is based on the measured levels of one or more (preferably more than one) of the GAG ​​properties according to the present invention.

[0114] Thus, the score may be based on (or may be derived or calculated or computed using) one or more (or all) measured (or determined) GAG characteristics selected from the group consisting of 4S CS, NS HS, 0S HS, total CS and total HS.

[0115] In some embodiments, the score may be based on (or may be derived or calculated or computed using) measured levels of one or more GAG ​​signatures in more than one type of bodily fluid sample. In some such embodiments, the GAG ​​signatures measured in each type of bodily fluid may be the same or different.

[0116] In some embodiments, if necessary, an appropriate threshold or cutoff value for use with the score (e.g., used to declare a sample as OA or RA (or indicate probable OA or probable RA)) can be designed by one skilled in the art. As an example, the cutoff (or threshold) value can be calculated based on an ROC curve. In some cases, the most highly selected rank statistic can be used to identify the cutoff value (or cutoff score).

[0117] In some embodiments, screening (e.g., diagnosis, etc.) can be performed by comparing a given score for the sample (or subject) being screened to a threshold or cutoff value and assigning a result (e.g., an indication of OA or RA) based on whether the determined score is above or below (e.g., significantly above or significantly below) the cutoff value.

[0118] For example, any scoring method, scoring system, marker, or formula that includes any suitable combination of GAG characteristics according to the present invention can be used to arrive at an index in the form of a value or score, which can then be used to screen for (e.g., diagnose) OA or RA. For example, the method or the like can be an algorithm that includes any suitable combination of GAG characteristics according to the present invention as input, for example, to perform pattern recognition of samples, to arrive at an index in the form of a value or score, which can then be used to screen for (e.g., diagnose) OA or RA. Non-limiting examples of such algorithms include machine learning or deep learning algorithms that implement classification (algorithmic classifiers), such as linear classifiers (e.g., Fisher's linear discriminant, logistic regression, naive Bayes classifier, perceptron), support vector machines (e.g., least squares support vector machines), quadratic classifiers, kernel estimation (e.g., k-nearest neighbors), boosting, decision trees (e.g., random forests), neural networks, and learning vector quantization.

[0119] The use of such classifiers, for example, machine learning classifiers, such as random forest classifiers, would be within the skill of one of ordinary skill in the art.

[0120] In some embodiments, a multivariate logistic regression model can be used to calculate the GAG ​​score.

[0121] As described elsewhere herein, screening for OA or RA according to the present invention can involve using a score, or using a score to represent levels and / or chemical compositions according to the present invention. As described elsewhere herein, in some such embodiments, an altered (e.g., increased or decreased, as the case may be) score compared to a control score (or cutoff or threshold level) is indicative of OA or RA (as appropriate) in the subject.

[0122] As described above, the present invention provides a method for screening for OA or RA in a subject. The present invention also provides a method for diagnosing OA or RA in a subject. Therefore, the method for screening for OA or RA according to the present invention can be used, for example, to diagnose OA or RA.

[0123] Thus, in one aspect, the present invention provides methods for diagnosing OA or RA in a subject. In some embodiments, a positive diagnosis (i.e., the presence of OA or RA, as the case may be) is made when the level of one or more of the GAG ​​signatures according to the present invention in a sample is altered (as the case may be, increased or decreased) compared to an appropriate control level (e.g., as discussed elsewhere herein).

[0124] Thus, in a further aspect, the present invention provides a method of diagnosing osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject having or suspected of having arthritis, the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample is obtained from the subject.

[0125] Embodiments of the screening methods of the invention described elsewhere in this specification can be applied mutatis mutandis to this aspect of the invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0126] Screening (eg, diagnostic) methods according to the present invention can be used to determine (or distinguish or differentiate) OA or RA in subjects with or suspected of having arthritis.

[0127] Thus, in another aspect, the present invention provides a method of distinguishing (or distinguishing or differentiating) between OA and RA in a subject with or suspected of having arthritis, the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample is obtained from the subject.

[0128] Embodiments of the screening methods of the invention described elsewhere in this specification can be applied mutatis mutandis to this aspect of the invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0129] In a preferred aspect, the present invention provides a method for distinguishing (or differentiating or differentiating between) osteoarthritis (OA) and rheumatoid arthritis (RA) in a subject having or suspected of having arthritis, the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a urine sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample has been obtained from the subject. Embodiments of the screening methods of the invention described elsewhere herein may be applied to this aspect of the invention (e.g., preferred GAG form (or groups of GAG forms), preferred processing steps, preferred control levels, etc.) mutatis mutandis.

[0130] The level of one or more GAG ​​signatures according to the present invention may be used in (or for) the differential diagnosis of OA and RA (OA vs. RA) or as part of the process of differential diagnosis of OA and RA (OA vs. RA).

[0131] The concept of "differential diagnosis" is well understood in the art. Briefly, differential diagnosis can be considered as the process of distinguishing between two or more diseases (or conditions) that may cause the symptoms of a subject.Therefore, when the symptoms of a subject match the symptoms that can be caused by multiple conditions, differential diagnosis process can be used.

[0132] Thus, in another aspect, the present invention provides a method for the differential diagnosis of OA or RA (OA vs. RA) in a subject with or suspected of having arthritis, the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample is obtained from the subject.

[0133] Embodiments of the screening methods of the invention described elsewhere in this specification can be applied mutatis mutandis to this aspect of the invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0134] As described elsewhere herein, screening (eg, diagnostic) methods according to the present invention may be used in combination with other diagnostic tests or methods.

[0135] Thus, in another aspect, the present invention provides a method of confirming (or verifying) a previous diagnosis of OA or RA in a subject, the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample is obtained from the subject.

[0136] Embodiments of the screening methods of the invention described elsewhere in this specification can be applied mutatis mutandis to this aspect of the invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0137] In another aspect, the present invention provides a method of screening for osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject having or suspected of having arthritis, the method comprising: determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS in a body fluid (e.g., urine) sample; The sample is obtained from the subject.

[0138] Embodiments of the screening methods of the invention described elsewhere in this specification can be applied mutatis mutandis to this aspect of the invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0139] In another aspect, the present invention provides a method for distinguishing between osteoarthritis (OA) and rheumatoid arthritis (RA) in a subject having or suspected of having arthritis, the method comprising: determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS in a body fluid (e.g., urine) sample; The sample has been obtained from the subject. Embodiments of the screening methods of the invention described elsewhere herein may be applied to this aspect of the invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.) mutatis mutandis.

[0140] In another aspect, the present invention provides a method of diagnosing osteoarthritis (OA) in a subject having or suspected of having arthritis, the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample is obtained from the subject.

[0141] Embodiments of the screening methods of the invention described elsewhere in this specification can be applied mutatis mutandis to this aspect of the invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0142] In a further aspect, the invention provides a method for providing information useful in (or for) screening for (e.g., diagnosing, differentially diagnosing, distinguishing between, or confirming a diagnosis of) osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject having or suspected of having arthritis, the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample is obtained from the subject, and the information, of course, is typically the determined (or measured) level of one or more of the GAG ​​properties.

[0143] Embodiments of the screening methods of the invention described elsewhere in this specification can be applied mutatis mutandis to this aspect of the invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0144] A still further aspect of the invention provides a method of screening for (e.g., diagnosing, differentially diagnosing, distinguishing between, or confirming the diagnosis of) OA or RA in a subject, the method comprising analyzing disaccharide units derived from one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, the method comprising the level of one or more GAGs characteristic of the invention. Embodiments of the screening methods of the invention described elsewhere herein may be applied to this aspect of the invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.), mutatis mutandis.

[0145] A still further aspect of the present invention provides a method of screening for (e.g., diagnosing, differentially diagnosing, distinguishing between, or confirming the diagnosis of) OA or RA in a subject, the method comprising analyzing a population of disaccharide units consisting essentially of disaccharide units derived from the non-proteoglycan fraction (or protein-free fraction) of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, the method comprising the level of one or more GAGs characteristic of the present invention. Embodiments of the screening methods of the present invention described elsewhere herein can be applied to this aspect of the invention (e.g., preferred GAG form (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.), mutatis mutandis.

[0146] Unless otherwise clear from the context, the features and discussion herein relating to OA or RA screening methods (e.g., regarding preferred GAG characteristics or combinations thereof, or measurement scores, preferred treatment steps, preferred body fluids, preferred control levels, etc.) apply mutatis mutandis to other related methods of the invention (such as methods for diagnosing, differentially diagnosing, distinguishing between, or confirming the diagnosis of OA or RA, or methods for providing information useful for OA or RA).

[0147] In some embodiments, the present invention provides for the use of the methods of the present invention (e.g., methods of screening, diagnosing, or determining progression as described herein) in combination with other known screening, diagnostic, or progression monitoring methods for OA or RA (as appropriate), such as radiological imaging (e.g., computed tomography, CT, or positron emission tomography, PET, scan, or X-ray) or magnetic resonance imaging (MRI scan) or ultrasound imaging), or histological evaluation (e.g., using biopsy), or a set of criteria established to indicate (e.g., diagnose) OA or RA. For example, in the case of OA or RA, radiological imaging can be used in combination with the methods of the present invention. As another example, for OA or RA, other screening or diagnostic criteria for OA or RA (e.g., in the case of RA, the American College of Rheumatology criteria for RA) can be used in combination with the methods of the present invention. In the case of OA, the Kellgren-Lawrence (KL) system (or KL scoring system) can be used with the methods of the present invention. The KL system is a well-known radiological classification system for OA.

[0148] Thus, for example, the methods of the invention can be used to confirm a diagnosis (confirm a previous diagnosis) of OA or RA in a subject. In some embodiments, the methods of the invention are used alone.

[0149] The level of the GAG ​​form in question can be determined or measured by analyzing a sample obtained or removed from the subject by appropriate means. Measurements are typically carried out in vitro.

[0150] The level of one or more GAG ​​forms in a sample can be measured (determined) by any suitable assay or technique or method, several of which are well known and documented in the art. Electrophoresis, such as agarose gel electrophoresis or capillary electrophoresis (particularly capillary electrophoresis with fluorescence detection such as CE-LIF), is a technique that can be used to measure (determine) the level of one or more GAG ​​forms according to the present invention. Liquid chromatography, particularly HPLC (high performance liquid chromatography) combined with mass spectrometry (MS), is a preferred technique for measuring (determining) the level of one or more GAG ​​forms according to the present invention.

[0151] Suitable electrophoretic (e.g., capillary electrophoresis) and liquid chromatographic (e.g., HPLC) techniques for GAG conformation analysis, as well as suitable mass spectrometry methods (and associated data processing techniques), are well known and documented in the art.

[0152] One method that can be used in the present invention is capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) (e.g., as described in Galeotti et al., 2014, Electrophoresis 35:811-818, and Kottler et al., 2013, Electrophoresis 34:2323-2336). HPLC combined with post-column derivatization and fluorimetric detection can also be used, as described, for example, in Volpi 2006, Curr Pharm Des 12:639-658, HPLC combined with ESI-MS (electrospray ionization-mass spectrometry) can also be used, as described, for example, in Volpi and Linhardt, 2010, Nature protocols 5:993-1004, or HPLC combined with fluorimetric detection can also be used, as described, for example, in Galeotti and Volpi, 2011, Anal Chem 83:6770-6777 or Volpi et al., 2014, Nature Protocols 9:541-558. Agarose gel electrophoresis, e.g., FACE (fluorophore-assisted carbohydrate electrophoresis) as described in Volpi and Maccari, 2006, Analyt Technol Biomed Life Sci, 834:1-13, and Volpi and Maccari, 2002, Electrophoresis 23:4060-4066, can also be used.

[0153] Particularly preferred methods for determining the level of one or more GAG ​​forms in a sample are described in the Examples herein. Thus, preferred methods may involve high performance liquid chromatography (HPLC), preferably ultra-high performance liquid chromatography (UHPLC), in combination with mass spectrometry, such as MS / MS or triple quadrupole mass spectrometry. Particularly preferred methods include ultra-high performance liquid chromatography (UHPLC) coupled with an electrospray ionization triple quadrupole mass spectrometry system. An example of such a method is described in Tamburro et al. (Journal of Chromatography B, 1177 (2021) 122761).

[0154] Certain methods of sample preparation (or processing), such as GAG extraction and purification, are also known and described in the art, e.g., Volpi and Maccari, 2005, Biomacromolecules 6:3174-3180 and Clin Chim Acta 356:125-133, and Coppa et al., 2011 Glycobiology 21:295-303. Sample preparation (or processing) methods based on such reported techniques involve protease treatment (protease extraction) and a purification step based on the use of an anion exchange resin. In some methods of the present invention, such a protease treatment step and / or a purification step using an anion exchange resin may be performed. However, as discussed elsewhere herein, in preferred methods, a protease treatment step and / or a purification step using an anion exchange resin is not performed. Specifically, in preferred methods in which the protein-free fraction of GAGs is analyzed, a protease treatment step is not performed.

[0155] In some embodiments, HPLC and mass spectrometry (and associated data processing techniques) are used to obtain the fraction of the level of one or more specific GAG forms (e.g., sulfated or non-sulfated disaccharide forms) in a sample compared to the total amount. For example, after sample preparation, GAGs can be digested using enzymes, separated on an HPLC column, and identified using MS. As described elsewhere herein, the amount of one or more individual GAG forms (e.g., specific sulfated or non-sulfated disaccharide forms) can be conveniently normalized (i.e., divided) by the sum of all the amounts of the measured individual GAG forms to obtain a fraction (or proportion or relative concentration). However, the absolute concentration (or absolute level) of an individual GAG form (e.g., a sulfated GAG form) can alternatively or additionally be measured.

[0156] According to the present invention, quantitative, semi-quantitative or qualitative assessment (determination) of the levels of one or more GAG ​​forms can be performed.

[0157] Suitable methods for doing this are well known to those skilled in the art, and any of these can be used. However, a convenient way to achieve such quantification of the disaccharide composition or appropriate properties or forms of CS or HS (and separation of disaccharide forms) is to use electrophoresis, in particular capillary electrophoresis, for example capillary electrophoresis with fluorescence detection, for example capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) (e.g., Galeotti 2014, supra, or Kottler 2013, supra). An alternative method that is preferred in some embodiments is to use liquid chromatography, preferably HPLC (high performance liquid chromatography), for example SAX HPLC, or such as described in, for example, Volpi 2006 (supra), Galeotti and Volpi 2011 (supra), Volpi et al., 2014 (supra), or Volpi and Linhardt, 2010 (supra). Preferably, mass spectrometry (HPLC-MS), such as electrospray ionization mass spectrometry (ESI-MS), e.g., HPLC ESI-MS, is also used. Particularly preferred methods are outlined in the Examples. One particular method is capillary electrophoresis (e.g., capillary electrophoresis with laser-induced fluorescence detection). Another particularly preferred example would be HPLC followed by MS (HPLC-MS), e.g., HPLC ESI-MS. Preferred HPLC-MS methods are discussed elsewhere herein.

[0158] Thus, in preferred methods of the invention, the level or chemical composition of the GAG ​​or GAG profile is determined by HPLC and mass spectrometry. Preferably, the HPLC is ultra HPLC and / or the mass spectrometry is triple quadrupole mass spectrometry. In certain preferred methods, the level or chemical composition of the GAG ​​or GAG profile is determined by a combination of high performance liquid chromatography (HPLC), preferably ultra HPLC (UHPLC), and mass spectrometry, e.g., MS / MS or triple quadrupole mass spectrometry. A preferred method comprises ultra high performance liquid chromatography (UHPLC) coupled (or combined) with electrospray ionization triple quadrupole mass spectrometry.

[0159] Generally, determining GAG properties (or forms or characteristics) according to the present invention does not involve measuring GAG molecules in the exact same form as found in a subject's bodily fluid (e.g., does not involve measuring GAGs in their naturally occurring form). For example, such native or naturally occurring GAG molecules are often found in biological samples (e.g., bodily fluid samples) in the form of long sugar chains that can either be bound to proteins (also referred to herein as protein-bound GAGs or proteoglycan GAGs) or not be bound to proteins (also referred to herein as free GAGs or protein-free GAGs).

[0160] In some embodiments, the methods of the invention may include a step of processing the sample. Thus, in some embodiments, the methods of the invention may be performed on such processed samples or materials derived from such processed samples. Thus, generally, the methods of the invention are performed on samples that have been processed in some way (e.g., artificial samples rather than natural samples).

[0161] Processing steps can include, but are not limited to, extraction or purification of GAGs from a sample, e.g., via the use of a protease such as proteinase K, fragmentation, cleavage, or digestion of proteins present in the sample, e.g., as a means of separating, extracting, or removing GAGs from proteins to which they are bound, e.g., purification of GAGs using an anion exchange resin, isolation of cells from a sample, isolation of cellular components from a sample, and extraction (e.g., isolation or purification) of proteins / peptides from a sample. Thus, processing steps also include steps performed on a body fluid sample to prepare it for analysis, e.g., removal of cells or other impurities in the case of a urine sample. Processing steps can involve one or more of digestion, extraction, purification, boiling, filtration, lyophilization, fractionation, centrifugation, concentration, dilution, inactivation of interfering components, addition of reagents, derivatization, conjugation, etc. Exemplary processing steps are described in the Examples.

[0162] While certain methods of the invention may involve a step of fragmenting or cleaving or digesting proteins present in a sample (e.g., via the use of a protease such as proteinase K as a means of separating or extracting or removing GAGs from the proteins to which they are bound) and / or purifying GAGs (e.g., using an anion exchange resin), as will be apparent from the discussion elsewhere herein, certain preferred methods do not involve a step of fragmenting / cleaving / digesting proteins and / or a step of purifying GAGs (e.g., using an anion exchange resin). In particular, preferred methods in which the level and / or chemical composition of the protein-free fraction of GAGs are determined do not involve a step of fragmenting / cleaving / digesting proteins.

[0163] Generally, a GAG-containing body fluid sample obtained from a subject is subjected to at least one processing step before determining its level and / or chemical composition according to the methods of the present invention. Specifically, in methods in which the level of one or more specific sulfated or non-sulfated forms of CS or HS disaccharides is determined, the GAG ​​is preferably subjected to a processing step to obtain the disaccharide units for analysis.

[0164] In some such methods of the invention in which the level of a particular individual disaccharide form is measured, the GAG ​​(e.g., a full-length GAG molecule, or a polymeric polysaccharide chain of GAG, or a chain of repeating disaccharide units of GAG) is subjected to a processing step (e.g., a step of fragmentation or cleavage or digestion (e.g., by chemical digestion or enzymatic treatment)). Suitable methods of digestion or enzymatic treatment will be known to those of skill in the art, for example, the use of one or more GAG ​​lyase enzymes, e.g., one or more chondroitinase enzymes such as chondroitinase ABC or chondroitinase B, and / or the use of one or more heparinase enzymes such as heparinase I-II-III to obtain the disaccharide units that are then analyzed.

[0165] Other methods for determining GAG levels or composition are known in the art that can be used, however, examples include analytical techniques involving the use of antibodies against various GAG forms, such as Western blot, ELISA, or FACS, or methods involving agarose gel electrophoresis (e.g., fluorophore-assisted carbohydrate electrophoresis (FACE)) or polyacrylamide gel electrophoresis (PAGE).

[0166] In some embodiments, the level of one or more GAG ​​forms (e.g., a particular sulfated or non-sulfated form of a CS or HS disaccharide, derived by fragmentation, cleavage, or digestion from a full-length GAG molecule or a chain of repeating disaccharide units of a GAG molecule) associated with (e.g., physically associated with, complexed with, derivatized with, or labeled with) a reagent (e.g., 2-aminoacridone) being used to detect the GAG ​​form is determined. Thus, in some embodiments, the level of a complex of a GAG form with a reagent used to detect the GAG ​​form is determined. Suitable reagents for detecting particular GAG forms are discussed elsewhere herein, but include, for example, antibodies or certain fluorophores (or other detectable labels or dyes) attached to (or used to derivatize) the GAG ​​form in question to render it detectable by a fluorometer (or other detection device). Thus, purely by way of example, in some embodiments, the level of a GAG form associated with (e.g., complexed with, or derivatized by) an antibody, fluorophore, or the like can be determined. In some embodiments, the level of GAG forms associated (eg, complexed or derivatized) with 2-aminoacridone can be determined.

[0167] Certain preferred methods of the present invention include determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, thereby advantageously eliminating the need to separate or extract GAG molecules from the proteins to which they are bound. Instead, in such embodiments, the protein-free fraction (and only the protein-free fraction) of GAGs in a body fluid sample can be analyzed from the sample without any such processing to separate GAGs from proteins, e.g., by digesting the proteins. Thus, preferred methods do not include a processing step in which the sample is contacted with a proteolytic agent, such as a protease.

[0168] Other preferred methods do not include a step in which GAGs are purified from a sample based on the negative charge of the GAGs (eg, using an anion exchange resin).

[0169] Therefore, in a preferred method of the present invention, the sample has been obtained from the subject and has been subjected to processing before determining the level and / or chemical composition, The process is as follows: (a) fragmenting said one or both GAGs into disaccharide units, (b) Before (a), (i) contacting the sample with a proteolytic agent; and (ii) purifying the one or both GAGs in the sample based on the negative charge of the GAG.

[0170] A still further aspect of the present invention provides a method of screening for OA or RA in a subject having or suspected of having arthritis, the method comprising determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, determining the level and / or chemical composition of one or both of the GAGs includes determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; the sample has been obtained from the subject and subjected to processing prior to determining the level and / or chemical composition; The process is as follows: (a) fragmenting said one or both GAGs into disaccharide units, (b) Before (a), (i) contacting the sample with a proteolytic agent; and (ii) purifying the one or both GAGs in the sample based on the negative charge of the GAG.

[0171] Embodiments of other aspects of the invention described elsewhere in this specification may be applied to this aspect of the invention, mutatis mutandis (e.g., preferred GAG properties (or groups of GAG properties), preferred processing steps, preferred body fluids, etc.).

[0172] In preferred methods, said fragmenting in (a) is conveniently carried out by contacting said one or both GAGs with one or more GAG ​​lyase enzymes (e.g., as discussed elsewhere herein). For example, said fragmenting in (a) may be carried out by contacting said one or both GAGs with one or more chondroitinase enzymes and / or one or more heparinase enzymes.

[0173] In art-based methods, the contacting step of (b)(i) is conveniently carried out by contacting the sample with one or more protease enzymes, e.g., proteinase K. Accordingly, in certain preferred methods of the invention, such a step is not performed. The proteolytic agent of (b)(i) may be a protease (e.g., a non-specific protease such as proteinase K). Accordingly, certain preferred methods of the invention do not include a step of contacting the sample with a protease (e.g., a non-specific protease, e.g., proteinase K) prior to (a).

[0174] In art-based methods, the purifying step of (b)(ii) is conveniently carried out by using an anion exchange resin. Accordingly, in certain preferred methods of the invention, such a step is not performed. Accordingly, certain preferred methods of the invention do not include a step prior to (a) of purifying the one or both GAGs in the sample using an anion exchange resin.

[0175] In a preferred method of the invention, the method does not include the contacting of (b)(i).

[0176] In a preferred method of the invention, the method does not include purifying (b)(ii).

[0177] In other preferred methods of the invention, neither step (b)(i) nor (b)(ii) is performed. Thus, in particularly preferred embodiments, the method does not include the contacting of (b)(i) or the purifying of (b)(ii).

[0178] As noted above, in some embodiments, an altered (possibly increased or decreased) level of one or more of the GAG ​​signatures according to the present invention (4S CS (e.g., absolute concentration of 4S CS), NS HS (e.g., absolute concentration of NS HS and / or relative concentration of NS HS), OS HS (e.g., absolute concentration of OS HS), total concentration of CS and / or total concentration of HS) compared to a control level is indicative of either OA or RA in the subject (indicative of whether the subject has either OA or RA). One of skill in the art will be readily able to establish appropriate control levels for use according to the present invention.

[0179] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the invention that is different from (or altered (e.g., increased or decreased) compared to) a control level of the same one or more GAG ​​signatures is indicative of OA, and the control level is within (or falls within) a reference interval determined for the same sample type obtained from a group of subjects (or from a reference population) that does not have OA; or (b) A level of one or more GAG ​​signatures according to the present invention that is different from (or altered (e.g., increased or decreased) compared to) a control level of the same one or more GAG ​​signatures is indicative of RA, the control level falling within (or falling within) a reference interval determined for the same sample type obtained from a group of subjects (or from a reference population) without RA.

[0180] In some embodiments, the group (or reference population) of subjects without OA can be a group (or reference population) of subjects with RA. In some embodiments, the group (or reference population) of subjects without RA can be a group (or reference population) of subjects with OA.

[0181] The concept of a "reference interval" (which may also be referred to as a "reference range") is well known in the art, particularly in medicine and health-related fields. A reference interval is a range (or interval) of values ​​(e.g., levels) for a physiological measure established or determined for (or established or determined as characterizing or indicative of) a particular health condition in a reference population (or reference subject group).

[0182] In some cases, a reference interval is a range (or interval) of values ​​(e.g., levels) for a physiological measure established or determined for (or established or determined as characteristic of or indicative of) a particular disease (or pathological) state in a reference population having a given disease or condition.

[0183] The reference interval for a particular measurement or value (e.g., the level of one or more GAG ​​characteristics according to the present invention) defines an interval (or range) between which a certain percentage (e.g., %) of the measurement values ​​(or values) in a reference population falls. This percentage may be, for example, 80% to 99%. For example, the percentage may be 80%, 85%, 90%, or 95%. Preferably, the percentage is 95%. Typically and preferably, the percentage is the median percentage of the measurement values ​​(or values) in the reference population. By way of example, if the percentage is 95% (preferably, the median percentage is 95%), then the reference interval for a particular measurement or value (e.g., the level of one or more GAG ​​characteristics according to the present invention) is the interval (or range) between which 95% of the measurement values ​​(or values) in the reference population fall. This means that 5% of the measurement values ​​in such a reference population fall outside this reference interval. If the 95% percentage is the median percentage for the reference population (which is preferred), this means that 2.5% of the measurements (or values) are less than the lower limit of the reference interval and 2.5% of the measurements (or values) are higher than the upper limit of the reference interval. If the 80% percentage is the median percentage for the reference population, this means that 10% of the measurements (or values) are less than the lower limit of the reference interval and 10% of the measurements (or values) are higher than the upper limit of the reference interval. If the 85% percentage is the median percentage for the reference population, this means that 7.5% of the measurements (or values) are less than the lower limit of the reference interval and 7.5% of the measurements (or values) are higher than the upper limit of the reference interval. If the 90% percentage is the median percentage for the reference population, this means that 5% of the measurements (or values) are less than the lower limit of the reference interval and 5% of the measurements (or values) are higher than the upper limit of the reference interval.

[0184] The upper limit of the reference interval is commonly referred to as the upper reference limit (URL). The lower limit of the reference interval is commonly referred to as the lower reference limit (LRL). In some embodiments, the URL or LRL can be used as a cutoff or threshold level to provide an indication of OA or RA. In some embodiments, an altered level (an increased level or a decreased level, as the case may be) of one or more GAG ​​characteristics according to the invention compared to (or relative to) the URL or LRL of the reference interval is indicative of OA or RA (as appropriate).

[0185] Those skilled in the art are familiar with reference intervals, and the present inventors have found that the levels of certain GAG characteristics are useful for screening for OA and RA according to the present invention, so those skilled in the art can easily establish appropriate reference intervals (and associated URLs and LRLs) from (or for) appropriate reference populations for use according to the present invention.As mentioned above, the concept of reference intervals is well established in the art.For example, the Clinical and Laboratory Standards Institute publication "EP28-A3c Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline-Third Edition" (2010, Vol. 28, No. 30; ISBN 1-56238-682-4) provides detailed discussion and guidance in this regard.

[0186] The reference interval can be based on (or obtained from) measurements (or values) of any suitable number of subjects in a reference population (i.e., the reference population can be of any suitable size). One of skill in the art can readily select a reference population of appropriate size. Guidance regarding appropriate reference population sizes is provided in the Clinical and Laboratory Standards Institute publications mentioned above. Preferably, the reference population has at least 10 subjects. In some embodiments, there may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 200, at least 500, or at least 1000 subjects in the reference population. In some embodiments, there may be up to 50, up to 100, up to 200, or up to 500 subjects in the reference population. In some embodiments, there may be up to 10-20, 10-50, 10-100, 10-200, 10-500, or 10-1000 subjects in the reference population.

[0187] As is clear from the above, in some embodiments of the present invention that provide an indication of OA, the reference interval is the reference interval for a given GAG signature according to the present invention determined for the same sample type from a reference population group of subjects that do not have OA (for example, have RA).Also as is clear from the above, in some embodiments of the present invention that provide an indication of RA, the reference interval is the reference interval for a given GAG signature according to the present invention determined for the same sample type from a reference population group of subjects that do not have RA (for example, have OA).

[0188] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the invention that is higher than (or increased relative to) a control level of the same one or more GAG ​​signatures is indicative of OA, the control level being within (or falling within) a reference interval determined for the same sample type obtained from a group of subjects (or from a reference population) that do not have OA; or (b) A level of one or more GAG ​​signatures according to the invention that is lower than (or reduced compared to) a control level of the same one or more GAG ​​signatures indicates RA, the control level being within (or falling within) a reference interval determined for the same sample type obtained from a group of subjects (or from a reference population) without RA.

[0189] In some embodiments, the group of subjects without OA can be a group of subjects with RA. In some embodiments, the group of subjects without RA can be a group of subjects with OA.

[0190] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the invention that is higher than (or increased compared to) a control level of the same one or more GAG ​​signatures is indicative of OA, the control level being the upper reference limit of a reference interval determined for the same sample type obtained from a group of subjects (or a reference population) with RA; or (b) A level of one or more GAG ​​signatures according to the present invention that is lower than (or reduced compared to) a control level of the same one or more GAG ​​signatures is indicative of RA, the control level being the upper reference limit of the reference interval determined for the same sample type obtained from a group of subjects (or reference population) with RA.

[0191] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the invention that is lower than (or reduced compared to) a control level of the same one or more GAG ​​signatures is indicative of OA, the control level being the upper reference limit of a reference interval determined for the same sample type obtained from a group of subjects (or a reference population) with OA; or (b) A level of one or more GAG ​​signatures according to the present invention that is lower than (or reduced compared to) a control level of the same one or more GAG ​​signatures indicates RA, the control level being the lower reference limit of a reference interval determined for the same sample type obtained from a group of subjects (or reference population) with OA.

[0192] As mentioned above, the upper and lower reference limits may in some cases be used as cut-off or threshold levels, or in some cases alternatively be considered as cut-off or threshold levels, and an indication of OA or RA is reached (or provided) based on the measured level of one or more GAG ​​characteristics according to the present invention relative to (or compared with) such cut-off limits.

[0193] In some preferred embodiments, the reference interval for a particular measurement or value (e.g., the level of one or more of the GAG ​​characteristics according to the present invention) defines an interval (or range) within which 95% of the measurements (or values) in a reference population fall.

[0194] In certain alternative embodiments, a reference (or control) range can be generated based on a 95% confidence interval (e.g., 95% confidence around the mean measurement in a group of OA or RA subjects). For example, in this regard, with reference to the experimental examples herein, a reference range for the absolute concentration of 4S CS for a reference population of RA subjects can be 0.939 to 6.573 μg / mL in urine. In some embodiments, an absolute concentration of 4S CS (e.g., in urine) greater than 6.573 μg / mL can indicate OA. In some embodiments, an absolute concentration of 4S CS (e.g., in urine) less than 6.573 μg / mL can indicate RA. Other examples of reference ranges can be derived from the data in Table A herein. For the avoidance of doubt, such reference ranges based on Table A are purely exemplary, and the methods of the present invention are not limited thereto.

[0195] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the invention that is higher than a control level of the same one or more GAG ​​signatures is indicative of OA, the control level being determined in the same sample type obtained from one or more subjects (e.g., a group or population of subjects) with RA; or (b) A level of one or more GAG ​​signatures according to the present invention that is lower than a control level of the same one or more GAG ​​signatures is indicative of RA, the control level being determined in the same sample type obtained from one or more subjects (e.g., a group or population of subjects) with OA.

[0196] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the invention that is higher than a control level of the same one or more GAG ​​signatures is indicative of OA, the control level being the mean or median level determined in the same sample type obtained from a population of subjects with RA; or (b) A level of one or more GAG ​​signatures according to the invention that is lower than a control level of the same one or more GAG ​​signatures is indicative of RA, the control level being the mean or median level determined in the same sample type obtained from a population of subjects with OA.

[0197] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the invention that is higher than a control level of the same one or more GAG ​​signatures is indicative of OA, the control level being at least 20% (or at least 30%, at least 40%, at least 50%, at least 100%, or at least 200%) higher than the mean or median level determined in the same sample type from a population of subjects with RA; or (b) A level of one or more GAG ​​signatures according to the invention that is lower than a control level of the same one or more GAG ​​signatures indicates RA, the control level being at least 20% (or at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%) lower than the mean or median level determined in the same sample type obtained from a population of subjects with OA.

[0198] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the invention that is higher than a control level of the same one or more GAG ​​signatures is indicative of OA, the control level being a level determined by determining the level of the one or more GAG ​​signatures in the same sample type obtained from one or more subjects (e.g., a population of subjects) with RA and one or more subjects (e.g., a population of subjects) with OA, the control level being a cut-off level derived (or established or determined) to distinguish (or discriminate) samples from OA subjects as opposed to samples from RA subjects; or (b) A level of one or more GAG ​​signatures according to the present invention that is lower than a control level of the same one or more GAG ​​signatures is indicative of RA, the control level being a level determined by determining the level of the one or more GAG ​​signatures in the same sample type obtained from one or more subjects (e.g., a population of subjects) with RA and one or more subjects (e.g., a population of subjects) with OA, the control level being a cut-off level derived (or established or determined) to distinguish (or discriminate) samples from subjects with OA as opposed to samples from subjects with RA.

[0199] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) an altered (preferably higher) level of one or more GAG ​​signatures according to the invention compared to a control level of the same one or more GAG ​​signatures is indicative of OA, the control level being a level determined by determining the level of the one or more GAG ​​signatures in the same sample type obtained from one or more (e.g., population) reference subjects (e.g., reference subjects with RA), the control level being a cut-off level (or threshold level) derived (or established or determined) such that an altered (preferably higher) level of one or more GAG ​​signatures according to the invention compared thereto provides an indication of OA; or (b) A level of one or more GAG ​​signatures according to the present invention that is altered (preferably lower) compared to a control level of the same one or more GAG ​​signatures is indicative of RA, the control level being a level determined by determining the level of the one or more GAG ​​signatures in the same sample type obtained from one or more reference subjects (e.g., of a population) (e.g., reference subjects with OA), the control level being a cut-off level derived (or established or determined) that provides an indication of RA when the level of one or more GAG ​​signatures according to the present invention is altered (preferably lower) compared to it.

[0200] In some embodiments of the present invention, a control level, e.g., a cut-off level (or threshold level), can be derived (or determined or established) to provide a confidence level in (or for) an indication of OA or RA (as the case may be) according to the present invention, for example, a confidence level of 80%, 85%, 90% or 95% (or at least 80%, 85%, 90% or 95%) in (or for) an indication of OA or RA. Thus, in some embodiments of the present invention, a control level, e.g., a cut-off level (or threshold level), can be derived (or determined or established) to provide a confidence level in (or for) an indication of OA or RA (as the case may be) according to the present invention when an altered level (possibly increased (or higher) or decreased (or lower)) of one or more GAG ​​characteristics according to the present invention is determined (or observed) compared to the control level (e.g., cut-off or threshold level). For example, a control level, e.g., a cutoff level (or threshold level), can be derived (or determined or established) to provide a confidence level (e.g., an 80%, 85%, 90% or 95% confidence level (or at least an 80%, 85%, 90% or 95% confidence level)) that an indication (e.g., a diagnosis) of OA or RA is accurate (or correct).

[0201] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) the level of one or more GAG ​​signatures according to the invention within a range (within a range of levels) established as indicative that the sample is from an OA subject as opposed to an RA subject is indicative of OA; or (b) The level of one or more GAG ​​signatures according to the present invention within a range (within a range of levels) established as indicative that the sample is from an RA subject as opposed to an OA subject is indicative of RA.

[0202] In some such embodiments, the range in (i)(a) is a range of levels determined by determining the levels of the one or more GAG ​​signatures in the same sample type obtained from a population of subjects with RA and a population of subjects with OA, and establishing a range of levels indicative of samples being from subjects with OA but not from subjects with RA, and / or the range in (ii)(b) is a range of levels determined by determining the levels of the one or more GAG ​​signatures in the same sample type obtained from a population of subjects with RA and a population of subjects with OA, and establishing a range of levels indicative of samples being from subjects with RA but not from subjects with OA.

[0203] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) the level of one or more GAG ​​signatures according to the invention within a reference interval (or range) established (or determined) as indicative that the sample is from an OA subject is indicative of OA; or (b) The level of one or more GAG ​​signatures according to the present invention within a reference interval (or range) established (or determined) as indicating that the sample is derived from an RA subject is indicative of RA.

[0204] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the present invention established (or determined) as indicative that the sample is from an OA subject is indicative of OA; or (b) A level of one or more GAG ​​signatures according to the present invention that is established (or determined) to be indicative that the sample is derived from an RA subject is indicative of RA.

[0205] In some embodiments of the methods of the invention (e.g., screening or diagnostic or discrimination methods), (a) a level of one or more GAG ​​signatures according to the invention that has been established (or determined) to be indicative that the sample is from an OA subject rather than from an RA subject is indicative of OA; or (b) A level of one or more GAG ​​signatures according to the present invention that is established (or determined) to be indicative that the sample is from an RA subject rather than an OA subject is indicative of RA.

[0206] An altered (possibly increased or decreased) level (or composition or score) of one or more GAG ​​forms (GAG characteristics) according to the present invention includes any measurable change or alteration in the GAG ​​form (biomarker) (or score) in question when the GAG ​​form in question is compared to a control level (e.g., a reference limit or cut-off or threshold level). An altered level (or score) includes an increased or decreased level (or score). Preferably, the level (or score) is significantly altered compared to the level (or score or cut-off level) found in an appropriate control (e.g., a control sample or subject or population). More preferably, a significantly altered level or composition or score is statistically significant, preferably with a p-value of <0.05.

[0207] In some embodiments, a change in level (or score) of ≧2%, ≧3%, ≧5%, ≧10%, ≧25%, ≧50%, ≧75%, ≧100%, ≧200%, ≧300%, ≧400%, ≧500%, ≧600%, ≧700%, ≧800%, ≧900%, ≧1000%, ≧2000%, ≧5000%, or ≧10,000% compared to the level (or score) found in a suitable control sample or subject or population (i.e., when compared to a control level) may indicate the presence of OA or RA (as appropriate) according to the present invention.

[0208] An "increase" or "increased" level of one or more of the GAG ​​forms (GAG characteristics) or scores described herein includes any measurable increase or elevation of the GAG ​​form (biomarker) (or score) in question when the GAG ​​form (or score) in question is compared to a control level (or control score or cut-off level or reference limit). Preferably, the level (or score) is significantly increased compared to the level (or score or cut-off level) found in an appropriate control (e.g., a control sample or subject or population). More preferably, a significant increase in the level (or score) is statistically significant, preferably with a p-value of <0.05.

[0209] In some embodiments, an increase in the level (or score) of ≧2%, ≧3%, ≧5%, ≧10%, ≧25%, ≧50%, ≧75%, ≧100%, ≧200%, ≧300%, ≧400%, ≧500%, ≧600%, ≧700%, ≧800%, ≧900%, ≧1000%, ≧2000%, ≧5000%, or ≧10,000% compared to the level (or score) found in an appropriate control sample or subject or population (i.e., compared to a control level or control score or cut-off level) may indicate the presence of OA or RA (as appropriate) according to the present invention.

[0210] A "reduction" or "decreased" level of one or more levels of a GAG form (GAG signature) or score as described herein includes any measurable decrease or reduction in the GAG ​​form (biomarker) (or score) in question when the GAG ​​form (or score) in question is compared to a control level (or control score or cut-off level). Preferably, the level (or score) is significantly reduced compared to the level (or score or cut-off level) found in an appropriate control (e.g., a control sample or subject or population). More preferably, a significantly reduced level (or score) is statistically significant, preferably with a p-value of <0.05.

[0211] In some embodiments, a decrease in the level (or score) of ≧2%, ≧3%, ≧5%, ≧10%, ≧25%, ≧50%, ≧75%, ≧80%, ≧90%, ≧95%, or ≧99% compared to the level (or score) found in an appropriate control sample or subject or population (i.e., compared to a control level or control score or cut-off level) indicates the presence of OA or RA (as appropriate) according to the present invention.

[0212] "Control levels" are discussed elsewhere herein. A "control level" can be the level of the relevant GAG signature in a control subject or population (e.g., in a sample obtained from the control subject or population). A "control level" can also be the level of the relevant GAG signature, which level is derived from (or established based on, or calculated from) the level in a control subject or population (e.g., a sample obtained from the control subject or population). Such a population can be referred to as a reference population. Suitable control subjects (or populations) or samples for use in the methods of the present invention will be readily identified by those of skill in the art. Suitable control levels are described elsewhere herein. A control level can correspond to the level of an equivalent (corresponding) GAG form in a suitable control subject or sample or population, for example, a cutoff level or threshold level or range found in a control or reference population. A control level can also be referred to as a "reference" level. A control level can be a discrete number or a range.

[0213] A control level for comparison may be derived by testing a suitable control subject or set of control subjects (or control population), although the methods of the invention do not necessarily involve performing activity tests on control subjects as part of the methods of the invention, but will generally involve comparison with a control level previously determined from a control subject (or control population) and known to the person performing the methods of the invention.

[0214] A "control chemical composition" is a chemical composition in a control subject or population (e.g., in a sample obtained from a control subject or population). The above discussion regarding "control levels" (e.g., appropriate control subjects, control samples, control populations, etc.) can be applied mutatis mutandis to a "control chemical composition."

[0215] As described elsewhere herein, screening for OA and RA according to the present invention may involve using a score (or GAG score) or using a score (or GAG score) to express the level and / or chemical composition determined according to the present invention. In some such embodiments, an altered (e.g., increased or decreased, as the case may be) score compared to a "control score" (or cutoff or threshold level) is indicative of OA or RA (as appropriate) in the subject. The discussion elsewhere herein regarding a "control level" (e.g., a suitable control subject, control sample, control population, etc.) may apply mutatis mutandis to a "control score."

[0216] As described elsewhere herein, in some preferred methods of the invention, the methods comprise determining the level and / or chemical composition (or score based thereon) of the protein-free fraction of one or both of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS) according to the invention. In some such embodiments, an altered level and / or chemical composition of the protein-free fraction (or score based thereon) compared to the level and / or chemical composition of the protein-free fraction of the one or both GAGs in a control (e.g., a control sample or control score or cut-off level) is indicative of OA or RA (as appropriate). Discussion elsewhere herein regarding a "control level" or "control chemical composition" or "control score" (e.g., a suitable control subject, control sample, control population, etc.) applies mutatis mutandis to embodiments of the invention comprising determining the level and / or chemical composition (or score based thereon) of the protein-free fraction of one or both of the GAGs according to the invention.

[0217] The methods of the present invention can also be used to monitor the progression of OA (e.g., worsening of OA) or the progression of RA (e.g., worsening of RA). Such monitoring can be performed before, during, or after treatment of OA or RA by surgery or therapy (e.g., drug therapy). Thus, in another aspect, the present invention provides a method for monitoring the progression of OA or RA in a subject having OA or RA. In such methods for monitoring the progression of OA or RA in a subject, the level of (or a score derived or based on) one or more of the GAG ​​signatures according to the present invention indicates the progression of OA or RA (as appropriate).

[0218] Thus, in another aspect, the present invention provides a method of monitoring the progression of osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject with arthritis (OA or RA), the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample is obtained from the subject.

[0219] In embodiments in which the progression of OA is monitored, the subject is one who has OA.

[0220] In embodiments in which the progression of RA is monitored, the subject is one who has RA.

[0221] In a preferred embodiment of the method of the present invention for monitoring the progression of OA or RA, an altered level (possibly an increased level or a decreased level) of one or more of the GAG ​​characteristics of the present invention (4S CS (e.g., absolute concentration of 4S CS), NS HS (e.g., absolute concentration of NS HS and / or relative concentration of NS HS), OS HS (e.g., absolute concentration of OS HS), total concentration of CS and / or total concentration of HS) compared to a control level indicates progression of OA or RA in the subject.

[0222] In some embodiments of the method of monitoring progression according to the present invention, (a) monitoring the progression of OA, and wherein an altered (or different), preferably increased (or higher), level of one or more GAG ​​profiles according to the invention over time, compared to a control level of the same one or more GAG ​​profiles, is indicative of OA progression, wherein the control level is determined in the same sample type from one or more subjects without OA, or is determined in the same sample type from a previous sample taken from a subject whose OA progression is being monitored; or (b) The progression of RA is monitored, and an altered (or different) level, preferably a decreased (or lower) level, of one or more GAG ​​profiles according to the present invention over time, compared to a control level of the same one or more GAG ​​profiles, indicates progression of RA, the control level being determined in the same sample type obtained from one or more subjects who do not have RA, or in the same sample type obtained from a previous sample taken from a subject whose progression of RA is being monitored.

[0223] In some embodiments of the methods of monitoring the progression of OA according to the present invention, the control level may be the level of one or more GAG ​​signatures according to the present invention determined in the same sample type obtained from one or more subjects (e.g., a population of subjects (e.g., a reference population)) who do not have OA.

[0224] In some embodiments of the methods of monitoring the progression of RA according to the present invention, the control level may be the level of one or more GAG ​​signatures according to the present invention determined in the same sample type obtained from one or more subjects (e.g., a population of subjects (e.g., a reference population)) who do not have RA.

[0225] In some preferred embodiments of the method of monitoring progress according to the present invention, (a) monitoring the progression of OA, wherein an increasing level of one or more GAG ​​signatures according to the invention over time, compared to a control level of the same one or more GAG ​​signatures, indicates progression of OA, wherein the control level is determined in the same sample type from a previous sample taken from the subject whose OA progression is being monitored; or (b) The progression of RA is monitored, and a decreasing level of one or more GAG ​​signatures according to the present invention over time indicates progression of RA compared to a control level of the same one or more GAG ​​signatures, the control level being determined in the same sample type obtained from a previous sample taken from the subject whose progression of RA is being monitored.

[0226] Thus, in some embodiments of the methods of monitoring OA progression according to the present invention, the control level is a level determined in the same sample type obtained from a previous (e.g., first) sample taken from the subject whose OA progression is being monitored.

[0227] In some embodiments of the methods of monitoring the progression of RA according to the present invention, the control level is a level determined in the same sample type obtained from a previous (e.g., first) sample taken from the subject whose progression of RA is being monitored.

[0228] The level from an earlier (e.g., first) sample from a subject whose OA or RA progression (optionally) is being monitored can be considered the "baseline" level in the subject. This type of control level (i.e., the control level from an individual subject) is particularly useful in embodiments of the present invention in which continuous or periodic measurements of GAG forms in an individual are performed to explore changes in the level of GAG forms. In this regard, a suitable control level can be the individual's own baseline, stable value, null value, previous value, or dry value (optionally), as opposed to the control or cut-off level found in the entire control population. Thus, the control level of the method for monitoring the progression of OA or RA (optionally) according to the present invention can correspond to the level of the marker (GAG form) in question in the same individual subject or sample from the subject measured at an earlier time point (e.g., the "baseline" level in the subject).

[0229] In some embodiments, levels of one or more of the GAG ​​signatures according to the present invention indicate OA progression, preferably high (or higher) or increasing (or gradually increasing) levels (e.g., measured over time, e.g., by taking serial or periodic measurements) indicate OA progression (OA worsening).

[0230] In some embodiments, levels of one or more of the GAG ​​signatures according to the present invention indicate RA progression, preferably low (or lower) or decreasing (or gradually decreasing) levels (e.g., measured over time, e.g., by taking continuous or periodic measurements) indicate RA progression (RA worsening).

[0231] In a preferred method of monitoring progress according to the present invention, the body fluid sample is a urine sample (or a treated urine sample).

[0232] Preferred GAG profiles (or groups of GAG profiles) for use in connection with the methods of monitoring progression according to the invention are discussed elsewhere herein in connection with other aspects of the invention, for example, 4S CS and NS HS are typically preferred.

[0233] Sample processing steps and / or detection (or measurement etc.) methods for use in connection with the methods of monitoring progression according to the invention are discussed elsewhere herein in connection with other aspects of the invention. Indeed, unless otherwise clear from the context, features described elsewhere herein in connection with screening methods (e.g. diagnostic etc.) may also be applied to the methods of monitoring progression according to the invention.

[0234] In a further aspect, the present invention provides a method for providing information useful for monitoring the progression of osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject with arthritis, the method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample is obtained from a subject, and the information, of course, is typically the determined level of one or more of the GAG ​​properties.

[0235] The methods of the present invention can be used in the active monitoring of patients who have not undergone surgery or treatment, for example, to monitor the progression of OA or RA (if necessary) in untreated patients. Again, serial measurements allow for assessment of whether or to what extent OA or RA (if necessary) is worsening, and therefore allow for more appropriate decisions to be made regarding, for example, whether therapeutic or surgical intervention is necessary or advisable.

[0236] In another aspect, the present invention provides a method for determining the clinical severity of OA or RA (if necessary) in a subject. In such a method, the level of one or more GAG ​​forms according to the present invention in a sample (or a score derived therefrom or based thereon) shows an association with the severity of OA or RA (if necessary). Thus, the level of one or more GAG ​​forms according to the present invention can indicate the severity of OA or RA. In some embodiments, the more the level of one or more GAG ​​forms according to the present invention changes (possibly increasing or decreasing) compared to a control level (e.g., a control level described elsewhere herein), the more likely the patient is suffering from a more severe form of OA or RA (if necessary). Thus, in some embodiments, the method of the present invention can be used in selecting patients for treatment.

[0237] Serial (periodic) measurement of the levels of one or more of the GAG ​​forms (biomarkers) according to the present invention (or scores derived therefrom or calculated thereon) can also be used to monitor the severity of OA or RA (as appropriate), looking for either an increase or decrease in levels over time. Observation of altered levels (increase or decrease, as the case may be) can also be used to guide and monitor treatment before treatment or surgery, for example, before the initiation of pharmaceutical treatment or surgery, or in a "follow-up" situation during or after treatment to assess the effectiveness of treatment and look for signs of treatment failure.

[0238] The present invention also provides a method for patient or treatment selection, providing a means for distinguishing (or discriminating) patients with OA from patients with RA. Thus, alternatively, the methods of the present invention provide a method for distinguishing (or discriminating) between OA and RA. Such a method can guide appropriate treatment.

[0239] The present invention also provides a means of distinguishing (or discriminating) patients with severe OA from patients with less severe OA (e.g., identifying or providing an indication of patients with a particular clinical stage of OA (e.g., stage 1, stage 2, stage 3 or stage 4 OA)), and therefore provides a method for patient or treatment selection.

[0240] The present invention also provides a means of distinguishing (or discriminating) patients with severe RA from those with less severe RA (e.g., identifying or providing an indication of patients with a particular clinical stage of RA (e.g., stage 1, stage 2, stage 3, or stage 4 RA)), and thus provides a method for patient or treatment selection. Classification of OA or RA at a given stage may follow any definition recognized and accepted in the art. Those skilled in the art are familiar with staging systems and conventions for OA and RA.

[0241] In some embodiments, the present invention provides methods of monitoring (e.g., continuously monitoring or actively monitoring) a subject with OA or RA (e.g., a subject being treated for OA or RA). Such monitoring can guide which treatment to use, or whether no treatment should be given.

[0242] In some embodiments, patients with less severe (or early stage) OA or RA may be placed under observation or active surveillance and may not receive treatment (e.g., medication or surgery). In some embodiments, patients with severe or more severe (or later stage) OA or RA may receive treatment.

[0243] The present invention also provides a method for determining (or monitoring) the effectiveness of a therapeutic regimen being used to treat OA or RA (if desired), i.e., a method for tracking or monitoring response to treatment. In such methods, a change (possibly an increase or decrease) in the level (or score) of one or more GAG ​​signatures according to the present invention indicates the effectiveness of the therapeutic regimen being used. For example, in some embodiments, if the level (or score) of one or more GAG ​​forms according to the present invention, where an increased level (or score) indicates OA, decreases during (or after) treatment, this indicates an effective therapeutic regimen. As another example, in some embodiments, if the level (or score) of one or more GAG ​​forms according to the present invention, where a decreased level (or score) indicates RA, increases during (or after) treatment, this indicates an effective therapeutic regimen. In such methods, continuous (periodic) measurement of the level of one or more GAG ​​signatures (biomarkers) according to the present invention over time can also be used to determine the effectiveness of the therapeutic regimen being used. Similar methods can be used to provide a way to determine (or monitor) the effectiveness of surgical regimens being used to treat OA or RA.

[0244] The methods of the present invention can be performed on any suitable bodily fluid sample. Typically, the sample is obtained (removed) from a subject (e.g., as described elsewhere herein), preferably a human subject. In other aspects, the method further comprises the step of obtaining the sample from the subject.

[0245] Reference herein to "body fluid" includes reference to any fluid derived from the body of a subject. The body fluid or sample may be in the form of a liquid biopsy. A preferred body fluid according to the present invention is urine. Urine is a particularly preferred body fluid because it is an easily accessible body fluid. Thus, in a preferred embodiment, the level of a GAG signature according to the present invention is the level in a urine sample.

[0246] The term "sample" also encompasses any material obtained by processing a body fluid sample (e.g., obtained by processing a urine sample). Thus, the term "sample" includes processed samples (e.g., processed urine samples). Processing of a biological sample to obtain a test sample may involve one or more of digestion, boiling, filtration, distillation, centrifugation, lyophilization, fractionation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, derivatization, complexation, etc., as described elsewhere herein. Appropriate processing steps can be selected depending on the characteristics of the method being performed.

[0247] Any suitable method for isolating a body fluid sample (eg, a urine sample) may be used.

[0248] Any sample that can be directly or indirectly affected by OA or RA can be used. The sample (e.g., original or untreated sample) typically contains a protein-free fraction of GAGs and a protein-bound fraction of GAGs (as discussed elsewhere herein). In a preferred method of the present invention, the level and / or chemical composition of the protein-free fraction of one or both of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS) are determined. Thus, in certain preferred embodiments, the body fluid sample has been (or will be) processed so that only (or essentially only) the protein-free fraction of the GAG ​​(typically the disaccharide units derived therefrom) is subsequently analyzed (i.e., the level and / or chemical composition of the protein-free fraction of one or both of the GAGs (typically the disaccharide units derived therefrom) is subsequently determined). In other methods of the invention, the sample has been (or is to be) processed so that the total (protein-free + protein-bound) fraction or pool of said one or both GAGs is then analyzed (i.e., the level and / or chemical composition of the total (protein-free + protein-bound) fraction or pool of said one or both GAGs (typically disaccharide units derived therefrom) is then determined).

[0249] The term "sample" also encompasses any material obtained by processing a biological sample (e.g., as described above), the obtained material comprising a disaccharide unit (or population of disaccharide units) obtained by processing a GAG (e.g., as described elsewhere herein).

[0250] In some embodiments, the methods of the present invention may include a step of processing the sample. Thus, in some embodiments, the methods of the present invention may be performed on such a processed sample or material derived from such a processed sample. Thus, in some embodiments, the methods of the present invention may be performed on a processed sample. Processing steps include, but are not limited to, isolating cells from the sample, isolating cellular components from the sample, and extracting (e.g., isolating or purifying) proteins / peptides (however, because certain preferred methods of the present invention involve determining the protein-free GAG ​​fraction, extraction of proteins or removal of protein components from proteoglycans (protein-bound GAGs) present in the sample (e.g., by digesting or otherwise removing protein components) are preferably not performed in some embodiments). Processing steps may involve one or more of filtration, distillation, centrifugation, extraction, concentration, dilution, purification, inactivation of interfering components, addition of reagents, derivatization, amplification, adapter ligation, etc.

[0251] The sample can be used immediately or stored (eg, at -80°C) for later use.

[0252] The method of the present invention described herein can be carried out on any type of subject that can suffer from OA or RA.The method is generally carried out on mammals, for example, humans, primates (for example, monkeys), experimental mammals (for example, mice, rats, rabbits, guinea pigs), domestic mammals (for example, horses, cows, sheep, pigs) or domestic pets (for example, cats, dogs).Preferably, the subject is human.

[0253] In some embodiments of the present invention, the subject (e.g., a human) is a subject with arthritis. Thus, in some embodiments, the subject is a subject diagnosed with arthritis (e.g., OA or RA). In other embodiments, the subject (e.g., a human) is a subject suspected of having arthritis (e.g., OA or RA). In the method of monitoring progression according to the present invention, the subject is a subject with arthritis (having OA or having RA).

[0254] In some embodiments, methods of the present invention are provided that further include treating the OA or RA (if necessary) with therapy (e.g., drug therapy) or surgery, or by prescribing another clinical management program. Methods of treating OA and RA with therapy or surgery, or by prescribing another clinical management program, are known in the art.

[0255] In some embodiments, if the results of the methods of the invention indicate OA in a subject (or indicate progression (worsening) of OA) (e.g., if a positive indication or diagnosis of OA is made), additional steps can be performed to treat OA with therapy or surgery or to provide an appropriate clinical management program for OA. For example, if the results of the methods of the invention indicate OA in a subject (or indicate progression (worsening) of OA), the subject may be instructed (or advised) to make certain lifestyle changes (e.g., to exercise or exercise more regularly), the subject may be instructed (or advised) to lose weight (e.g., if the subject is overweight), the subject may be instructed (or advised) to wear suitable (or more suitable) footwear, and / or the subject may be instructed (or advised) to use a device to reduce joint strain. As a further example, if the results of the methods of the present invention indicate OA in the subject (or indicate progression (worsening) of OA), the subject may alternatively or additionally be prescribed a physical therapy program, and / or the subject may be prescribed pain medication, and / or the subject may undergo joint replacement surgery.

[0256] In some embodiments, if the results of the methods of the invention are indicative of RA in a subject (or indicate progression (worsening) of RA) (e.g., a positive indication or diagnosis of RA is made), additional steps can be performed to treat the RA by therapy or surgery or to provide an appropriate clinical management program for RA. For example, if the results of the methods of the invention are indicative of RA in a subject (or indicate progression (worsening) of RA), the subject can be treated with (or prescribed) a disease-modifying anti-rheumatic drug (DMARD) (such as methotrexate, leflunomide, hydroxychloroquine, or sulfasalazine) and / or a JAK inhibitor and / or analgesic and / or a steroid and / or a non-steroidal anti-inflammatory drug (NSAIDS) and / or a biologic agent (such as adalimumab, etanercept, or infliximab). As a further example, if the results of the methods of the present invention are indicative of RA in the subject (or indicate progression (worsening) of RA), the subject may alternatively or additionally be prescribed a physical therapy program and / or the subject may undergo joint replacement surgery.

[0257] In some embodiments, if the level of one or more GAG ​​signatures according to the present invention in a sample (or a score based on these levels) is altered to a certain degree compared to a control level or score (or cut-off level), a further step of administering a therapeutically effective amount of a pharmaceutical agent to the patient is performed and / or surgery is performed. Preferred degrees of alteration are discussed elsewhere herein.

[0258] In some aspects, methods of the invention are provided that further comprise the step of (if necessary) performing (or carrying out) additional diagnostic or screening procedures for OA or RA, e.g., as discussed elsewhere.

[0259] A still further aspect provides a kit for screening for OA or RA (e.g., for diagnosing the severity or progression of OA or RA, or for distinguishing between OA and RA), comprising one or more agents suitable for determining the level of one or more GAG ​​profiles (GAG forms) described herein according to the present invention in a sample. A still further aspect provides a kit for screening for OA or RA (e.g., for diagnosing or determining the severity or progression of OA or RA), comprising one or more reagents (or components) for processing a body fluid sample (e.g., urine) containing a GAG whose level and / or chemical composition is to be determined according to the present invention. In a preferred aspect, the kit is for use in a method of the present invention described herein. Preferably, the kit includes instructions for using the kit components, e.g., in screening (e.g., diagnosis) according to the present invention.

[0260] In another aspect, the present invention provides a method for determining (or detecting) the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, comprising: determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The sample is obtained from a subject having or suspected of having OA or RA.

[0261] In one aspect, the present invention provides a method for detecting (or determining) the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS), the method comprising: (a) obtaining a body fluid sample from a human patient having or suspected of having OA or RA; (b) detecting (or determining) the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in the sample; Detecting (or determining) the level and / or chemical composition of one or both of the GAGs includes determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS.

[0262] A still further aspect of the present invention provides a method for detecting (or determining) the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample, comprising: detecting (or determining) the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​properties selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; the sample has been obtained from the subject having or suspected of having OA or RA and has been subjected to processing prior to determining the level and / or chemical composition; The process is as follows: (a) fragmenting said one or both GAGs into disaccharide units, (b) Before (a), (i) contacting the sample with a proteolytic agent; and (ii) purifying the one or both GAGs in the sample based on the negative charge of the GAG.

[0263] The features and discussion herein regarding screening methods (e.g., diagnostic methods) for OA or RA, e.g., preferred GAG forms or combinations thereof for measurement, can be applied mutatis mutandis to the detection methods of the present invention.

[0264] Where the terms "comprise," "comprises," "comprising," "has," or "having," or other equivalent terms are used herein, in some more specific embodiments, these terms include the terms "consists of" or "consists essentially of," or other equivalent terms.

[0265] The invention will now be further described with reference to the following non-limiting examples. [Example]

[0266] Introduction We investigated the urinary glycosaminoglycan (GAG) profile, or GAGome, which includes chondroitin sulfate (CS) and heparan sulfate (HS) disaccharides, as a biomarker for osteoarthritis (OA) and rheumatoid arthritis (RA). In this study, we examined the GAGome of the protein-free fraction of GAGs (or free GAGome) from urine samples.

[0267] Patients and methods We conducted a prospective, single-center, case-control study. The study population represented patients with OA or RA. The inclusion criteria were: For the OA arm: diagnosis of osteoarthritis (knee pain >6 months and radiology-confirmed diagnosis); age ≥18 years; For the RA arm: diagnosis of rheumatoid arthritis (confirmed by meeting the American College of Rheumatology criteria for RA); age ≥ 18 years.

[0268] No patients were excluded. All patients signed informed consent forms. Any void spot urine samples from eligible patients were obtained at one visit in one collection cup, which were stored sequentially at -20°C until analysis. Patients were assigned to two groups depending on the inclusion arm, OA vs. RA. Patient characteristics are summarized in Table 1.

[0269] [Table 1]

[0270] We measured urinary GAGomes in a single, blinded laboratory using a standardized UHPLC-MS / MS method (D. Tamburro, S. Bratulic, S.A. Shameh, N.K. Soni, A. Bacconi, F. Maccari, F. Galeotti, K. Mattsson, N. Volpi, J. Nielsen, F. Gatto, Journal of Chromatography B, 1177 (2021) 122761). Briefly, GAGome extraction from each sample was performed according to the instructions for the Elypta MIRAM® Free Glycosaminoglycan Kit. All reagents and consumables used were provided in the kit. The method for GAGome extraction involved an enzymatic digestion assay using chondroitinase ABC and heparinase I-II-III to depolymerize GAGs in the sample into disaccharides. It should be noted that, compared to other methods described in the art in which the use of nonspecific proteases is recommended for biological fluid analysis (see Volpi et al., Nature Protocols, 9, 541-558 (2014)), the present method omitted the addition of proteases, and therefore the analysis was limited to the protein-free fraction of GAGs. It should be noted that, compared to Volpi et al., the method used in this study omitted the step of purifying GAGs using an anion exchange resin.

[0271] Subsequently, the GAG ​​disaccharides were labeled with 2-aminoacridone.

[0272] The processed samples were then injected into an ultra-high performance liquid chromatography (UHPLC) coupled to an electrospray ionization triple quadrupole mass spectrometry system (ESI-MS / MS, Waters® 6 Acquity I-class Plus Xevo TQ-S micro) for disaccharide separation and detection. Multiple reaction monitoring (MRM) analysis implemented in mass spectrometry software (Waters® 9 TargetLynx) was used to acquire peaks of GAG disaccharides (listed below) at pre-specified retention times across six transitions. We used mass spectrometry software (Waters® TargetLynx) for peak integration, calibration curve construction, and quantification.

[0273] The resulting processed data were exported in Excel format and imported into R (4.0.2) to compare the levels of each free GAGome feature between OA and RA. A two-tailed t-test was used to assess differences between groups. A p-value of <0.05 was considered statistically significant.

[0274] The measured GAG profile (GAGomes) consisted of the absolute concentrations of 17 GAG disaccharides corresponding to eight different sulfation patterns of chondroitin sulfate (CS), heparan sulfate (HS), and hyaluronic acid (HA) disaccharides. Specifically, we quantified eight CS disaccharides (0S CS, 2S CS, 6S CS, 4S CS, 2S6S CS, 2S4S CS, 4S6S CS, and Tris CS) and eight HS disaccharides (0S HS, 2S HS, 6S HS, NS HS, NS6S HS, NS2S HS, 2S6S HS, and Tris HS). The GAGome was expanded to include 22 additional dependent features: total CS and total HS concentrations as the sum of the corresponding disaccharide concentrations, CS and HS charge, two ratios (4S CS / 0S CS and 6S CS / 0S CS), and the relative concentration (or mass fraction, %) of each of the 16 CS and HS disaccharides by normalizing their absolute concentrations by the total CS and HS concentrations, respectively. Thus, for the urine samples, the GAGome consisted of 39 GAG features.

[0275] result The free GAGome features listed in Table A (features with the suffix [ug / mL] in Table A) were detected in most patients in the study (>0.1 ug / mL in at least 10 of the 20 total patients in the study). Specifically, we detected six CS disaccharides (0S CS, 6S CS, 4S CS, 2S6S CS, 2S4S CS, 4S6S CS) at concentrations greater than 0.1 μg / mL in samples obtained from at least 10 of the 20 total patients in this study, and two HS disaccharides (0S HS, NS HS) at concentrations greater than 0.1 μg / mL in samples obtained from at least 10 of the 20 total patients in this study. The free GAGome was also expanded to include additional subordinate features: total CS concentration as the sum of the eight CS disaccharide concentrations mentioned above and total HS concentration as the sum of the eight HS disaccharide concentrations mentioned above; CS and HS charge; two ratios (4S CS / 0S CS and 6S CS / 0S CS); and the relative concentrations (or mass fractions, %) of each of the six CS disaccharides mentioned above (0S CS, 6S CS, 4S CS, 2S6S CS, 2S4S CS, 4S6S CS) and two HS disaccharides (0S HS, NS HS) by normalizing their absolute concentrations by the total CS and HS concentrations, respectively.

[0276] [Table 2] In the "GAGome Features" column of Table A above, the row with [ug / mL] after the listed GAGome feature indicates the absolute concentration (micrograms / mL; μg / mL) of the listed GAGome feature. Total CS and total HS concentrations are also in μg / mL. Charged CS and charged HS are discussed elsewhere herein. Ratios (4S / 0S CS and 6S / 0S CS) are discussed elsewhere herein. Other rows for listed sulfated or non-sulfated GAGome features with or without the [ug / mL] suffix indicate the relative concentration (micrograms / micrograms total for the associated GAG group, e.g., CS) of the listed GAGome feature (also considered "mass fraction" - relative concentrations are also explained elsewhere herein). "CI" = confidence interval.

[0277] We observed statistically significant differences (p<0.05) in the levels of 4S CS (μg / mL) and NS HS (μg / mL), with samples from OA patients exhibiting approximately two-fold higher concentrations of these disaccharides compared to RA patients. The data also indicate other differences between OA and RA (p<0.1), including increased 0S HS (μg / mL), total CS, and total HS concentrations in OA versus RA. While sample size was likely insufficient to achieve statistical significance for the increases in 0S HS (μg / mL), total CS, and total HS concentrations observed in OA versus RA, a clear trend toward increased concentrations of these GAGome features was observed.

[0278] In conclusion, this data clearly supports the notion that OA and RA exhibit different urinary GAGome profiles, with some disaccharides having different concentrations between the groups (i.e., between OA and RA). These results indicate that the urinary GAGome profiles according to the present invention can be used as diagnostic or screening biomarkers for OA or RA. Furthermore, the biomarkers according to the present invention can be used to distinguish between OA and RA, for example, by establishing reference intervals around one or more disaccharides identified herein as different in OA versus RA.

Claims

1. 1. A method for distinguishing between osteoarthritis (OA) and rheumatoid arthritis (RA) in a subject having or suspected of having arthritis, said method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a urine sample; determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or more GAG ​​characteristics selected from the group consisting of: (i) 4S CS, (ii) NS HS, (iii) 0S HS, (iv) total concentration of CS, and (v) total concentration of HS; The method, wherein the sample is obtained from the subject.

2. (a) the level of the one or more GAG ​​signatures is higher than a control level of the same one or more GAG ​​signatures, and the control level is determined in the same sample type obtained from one or more subjects with RA; or (b) the level of the one or more GAG ​​signatures is lower than a control level of the same one or more GAG ​​signatures, which is indicative of RA, and the control level is determined in the same sample type obtained from one or more subjects with OA.

3. (a) the level of the one or more GAG ​​signatures differing from a control level of the same one or more GAG ​​signatures is indicative of OA, and the control level falls within a reference interval determined for the same sample type obtained from a group of subjects without OA; or (b) The method of claim 1, wherein the level of the one or more GAG ​​signatures differs from a control level of the same one or more GAG ​​signatures, and the control level is within a reference interval determined for the same sample type obtained from a group of subjects without RA.

4. 4. The method of claim 1, wherein determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or both of the GAG ​​characteristics: 4S CS and NS HS.

5. Determining the level and / or chemical composition of one or both of the GAGs (i) absolute concentration of 4S CS; (ii) absolute concentration of NS HS and / or relative concentration of NS HS; (iii) absolute concentration of OS HS; (iv) the total concentration of CS, and (v) total concentration of HS.

6. 6. The method of any one of claims 1 to 5, wherein determining the level and / or chemical composition of one or both of the GAGs comprises determining the level of one or both GAG properties selected from the group consisting of absolute concentration of 4S CS and absolute concentration of NS HS.

7. 7. The method of any one of claims 1 to 6, wherein the method comprises determining the level of more than one of the GAG ​​signatures, preferably the method comprises determining the level of two or more, three or more, four or more or all of the GAG ​​signatures.

8. 8. The method of any one of claims 1 to 7, wherein determining the level and / or chemical composition comprises determining the level and / or chemical composition of the protein-free fraction of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS).

9. 9. The method of any one of claims 1 to 8, wherein the sample has been obtained from the subject and subjected to at least one processing step before determining the level and / or chemical composition.

10. 10. The method of any one of claims 1 to 9, wherein the level of one or more specific sulfated or non-sulfated forms of CS or HS disaccharides is determined and the GAGs are or have been subjected to processing steps to obtain the disaccharide units for analysis.

11. 11. The method of claim 9 or 10, wherein the at least one processing step does not include contacting the sample with a proteolytic agent.

12. the sample has been obtained from the subject and subjected to processing prior to determining the level and / or chemical composition; The process (a) fragmenting said one or both GAGs into disaccharide units; (b) before (a), (i) contacting the sample with a proteolytic agent; and The method of any one of claims 1 to 11, which does not include at least one of (ii) purifying the one or both GAGs in the sample based on the negative charge of the GAG.

13. 13. The method of claim 12, wherein the method does not include the contacting of (b)(i) or the purifying of (b)(ii).

14. 14. The method of claim 12 or 13, wherein the fragmenting in (a) is carried out by contacting the one or both GAGs with one or more GAG ​​lyase enzymes.

15. 15. The method of claim 14, wherein the one or more GAG ​​lyase enzymes are one or more chondroitinase enzymes and / or one or more heparinase enzymes.

16. 16. The method of any one of claims 12 to 15, wherein the purifying in (b)(ii) is carried out using an anion exchange resin.

17. 17. The method of any one of claims 1 to 16, wherein the level or chemical composition of the GAG ​​profile is determined by HPLC and mass spectrometry.

18. 18. The method of claim 17, wherein the HPLC is ultra HPLC and / or the mass spectrometry is triple quadrupole mass spectrometry.

19. The method of any one of claims 1 to 18, wherein the subject has arthritis.

20. The method of any one of claims 1 to 19, wherein the subject is a human.

21. 1. A method for monitoring the progression of osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject with arthritis, said method comprising: determining the level and / or chemical composition of one or both of the glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) in a urine sample; Determining the level and / or chemical composition of one or both of the GAGs (i) 4S CS; (ii) NS HS; (iii) 0S HS, (iv) the total concentration of CS, and (v) total concentration of HS, wherein the sample is obtained from the subject.

22. (a) monitoring the progression of OA, wherein an altered level of the one or more GAG ​​signatures over time compared to a control level of the same one or more GAG ​​signatures indicates progression of OA, wherein the control level is determined in the same sample type obtained from one or more subjects who do not have OA, or is determined in the same sample type obtained from a previous sample taken from the subject whose progression of OA is being monitored; or (b) monitoring the progression of RA, wherein altered levels of the one or more GAG ​​signatures over time compared to control levels of the same one or more GAG ​​signatures indicate progression of RA, wherein the control levels are determined in the same sample type obtained from one or more subjects who do not have RA or in the same sample type obtained from a previous sample taken from the subject whose progression of RA is being monitored.

23. Method according to claim 21 or 22, wherein the method has the features according to any one of claims 4 to 20.

Images