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

Abstract

The present invention relates to a method of screening for osteoarthritis (OA) and rheumatoid arthritis (RA) in a subject suffering from arthritis or suspected of suffering from arthritis and a method of discriminating between OA and rheumatoid arthritis RA comprising determining the level of one or more glycosaminoglycan (GAG) properties according to the invention in a sample of bodily fluid. The invention also provides methods for monitoring OA or RA progression in a subject suffering from arthritis.

Description

Technical Field

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

[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 degeneration of cartilage that cushions the bone ends in joints. OA is sometimes referred to as "wear-and-tear" arthritis. OA is a condition that typically causes joint pain, stiffness, and swelling. 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 RA, the immune system attacks the cells that line the joints. Like OA, RA is also a condition that typically causes joint pain, stiffness, and swelling.

[0005] Treatments or other clinical management strategies for RA and OA are generally different (i.e., RA is different from OA). For example, a subject with RA may be prescribed disease-modifying antirheumatic drugs (DMARDs). DMARDs are not routinely prescribed for OA because in OA, DMARDs do not provide clinically significant pain relief.

[0006] It is evident from the above discussion that although OA and RA are two types of arthritis and both are typically characterized by having common symptoms (e.g., joint pain, stiffness, and swelling), OA and RA are different conditions, having different underlying causes, and are treated differently clinically (e.g., with different treatments or other clinical management strategies).

[0007] Since OA and RA have common symptoms with each other, it may be difficult for clinicians to determine whether an arthritis patient or a suspected arthritis patient has OA or RA using existing screening or diagnostic methods (e.g., radiological imaging methods), i.e., it may be difficult to distinguish OA from RA.

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

[0009] Circulating biomarkers are molecules that can be measured in readily available body fluids (e.g., urine) of an individual, and the levels thereof can be used to assist, for example, in screening for diseases, diagnosing diseases, and monitoring disease progression.

[0010] What is needed in the art are new methods for screening for OA and RA (e.g., for differentiating OA from RA). The identification of novel biomarkers for OA and RA may have potential clinical significance for a large number of patients and would be an important clinical advancement. Here, the present inventors provide methods employing certain markers that are advantageously used for screening for OA and RA (e.g., differentiating OA from RA). Advantageously, such methods are non-invasive and can be performed on readily available samples. Summary of the Invention

[0011] Surprisingly and advantageously, the present inventors have identified that, compared to samples from RA patients, in body fluid samples from OA patients, the levels and / or the chemical composition of certain glycosaminoglycans (GAGs) are different. These differential levels of GAG CS or HS or the differential chemical composition of GAG CS or HS (GAG profile), as characterized by the differential level of certain GAG properties according to the present invention, can serve as biomarkers for screening for OA and RA (e.g., for differentiating OA from RA). Apparently, the discovery that OA and RA screening (e.g., differentiating OA from RA) can be performed using readily available body fluid samples (e.g., urine) from a subject is highly advantageous.

[0012] Accordingly, in one aspect, the present invention provides a method for screening for osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject having arthritis or suspected of having arthritis, the method comprising:

[0013] determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample,

[0014] wherein 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:

[0015] (i) 4S CS,

[0016] (ii) NS HS,

[0017] (iii) 0S HS,

[0018] (iv) the total concentration of CS; and

[0019] (v) the total concentration of HS,

[0020] wherein the sample has been obtained from the subject.

[0021] In some embodiments, the method of the invention comprises determining the level of 4S CS.

[0022] In some embodiments, the method of the invention comprises determining the level of NS HS.

[0023] In some embodiments, the method of the invention comprises determining the level of 0S HS.

[0024] In some embodiments, the method of the invention comprises determining the total concentration of CS.

[0025] In some embodiments, the method of the invention comprises determining the total concentration of HS.

[0026] In some embodiments, the method of the invention comprises determining the level of 4S CS or NS HS.

[0027] In some embodiments, the method of the invention comprises determining the levels of 4S CS and NS HS.

[0028] In some embodiments, the method of the invention comprises determining the level of one or more GAG properties selected from the group consisting of:

[0029] (i) the absolute concentration of 4S CS,

[0030] (ii) the absolute concentration and / or relative concentration of NS HS,

[0031] (iii) the absolute concentration of 0S HS,

[0032] (iv) the total concentration of CS; and

[0033] (v) the total concentration of HS.

[0034] The terms "absolute concentration", "relative concentration" and "total concentration" are discussed elsewhere herein.

[0035] Unless otherwise specified from the context, the term "one or more" includes "all".

[0036] In some embodiments, the method of the invention comprises determining the absolute concentration of 4S CS.

[0037] In some embodiments, the method of the present invention includes determining the absolute concentration of NS HS.

[0038] In some embodiments, the method of the present invention includes determining the relative concentration of NS HS.

[0039] In some embodiments, the method of the present invention includes determining the absolute concentration of 0S HS.

[0040] In some embodiments, the method of the present invention includes determining the total concentration of CS.

[0041] In some embodiments, the method of the present invention includes determining the total concentration of HS.

[0042] In some embodiments, the method of the present invention includes determining the absolute concentration of 4S CS or the absolute concentration of NS HS.

[0043] In some embodiments, the method of the present invention includes determining the absolute concentrations of 4S CS and NS HS.

[0044] Thus, 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 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.

[0045] Unless otherwise explicitly stated from the context, a reference to one or more GAG properties (or GAG forms or GAG characteristics or GAGome characteristics or GAGome properties) "according to this invention" or "according to the present invention" (or equivalent phrases) refers to 4S CS (preferably, the absolute concentration of 4S CS), NS HS (preferably, the absolute concentration and / or the relative concentration of NS HS, more preferably, the absolute concentration of NS HS), 0S HS (preferably, the absolute concentration of 0S HS), the total concentration of CS, and / or the total concentration of HS. Particularly preferred GAG properties "according to this invention" or "according to the present invention" are 4S CS (preferably, the absolute concentration of 4S CS) and / or NS HS (preferably, the absolute concentration of NS HS).

[0046] In a preferred method of the present invention, compared to a control level, a change (either an increase or a decrease, as appropriate) in the level of one or more GAG properties according to the present invention (4SCS (e.g., the absolute concentration of 4S CS), NS HS (e.g., the absolute concentration and / or relative concentration of NS HS), 0SHS (e.g., the absolute concentration of 0S HS), the total concentration of CS and / or the total concentration of HS) indicates OA or RA in the subject (indicating whether the subject has OA or RA).

[0047] In certain methods of the present invention, compared to a control level, a change (e.g., an increase or a higher level) in the level of one or more GAG properties according to the present invention (4SCS (e.g., the absolute concentration of 4S CS), NS HS (e.g., the absolute concentration and / or relative concentration of NS HS), 0SHS (e.g., the absolute concentration of 0S HS), the total concentration of CS and / or the total concentration of HS) indicates OA in the subject (indicating whether the subject has OA).

[0048] In certain methods of the present invention, compared to a control level, a change (e.g., a decrease or a lower level) in the level of one or more GAG properties according to the present invention (4SCS (e.g., the absolute concentration of 4S CS), NS HS (e.g., the absolute concentration and / or relative concentration of NS HS), 0SHS (e.g., the absolute concentration of 0S HS), the total concentration of CS and / or the total concentration of HS) indicates RA in the subject (indicating whether the subject has RA).

[0049] One skilled in the art will be able to readily establish an appropriate control level for use according to the present invention. Certain appropriate control levels and how to achieve an indication of OA or RA (discrimination between possible OA and possible RA) are discussed elsewhere herein.

[0050] In some embodiments of the method of the present invention, a level within an established (or given or appropriate) reference range (or reference interval) of one or more of the GAG properties according to the present invention (4S CS (e.g., the absolute concentration of 4S CS), NS HS (e.g., the absolute concentration and / or relative concentration of NS HS), 0S HS (e.g., the absolute concentration of 0S HS), the total concentration of CS and / or the total concentration of HS) or a level that changes (either an increase or a decrease, as appropriate) relative to an established (or given or appropriate) reference range (or reference interval) indicates OA or RA in the subject (indicating whether the subject has OA or RA).

[0051] A technician will be able to readily establish an appropriate reference range (or reference interval) for use in accordance with the present invention. Certain appropriate reference ranges (or reference intervals) and how such reference ranges can be used to arrive at an indication of OA or RA are discussed elsewhere herein.

[0052] In a preferred method of the present invention, both the level and the chemical composition are determined. In other preferred methods of the present invention, the chemical composition alone is determined, or in other preferred methods, the level (total level or total concentration) of CS and / or HS alone is determined.

[0053] The method of the present invention includes determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) of glycosaminoglycan (GAG) 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 levels and / or chemical compositions of chondroitin sulfate (CS) and heparan sulfate (HS) are determined.

[0054] As described elsewhere herein, in some preferred embodiments of the method of the present invention, the method includes determining the level and / or chemical composition of the protein-free fraction of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) of glycosaminoglycan (GAG) 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 levels and / or chemical compositions of the protein-free fractions of chondroitin sulfate (CS) and heparan sulfate (HS) are determined.

[0055] Although in some embodiments of the method of the present invention, the method includes determining the level and / or chemical composition of the protein-free fraction of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) of glycosaminoglycan (GAG), it is not necessary to specifically determine the level and / or chemical composition of the "protein-free fraction" in all aspects of the present invention.

[0056] Glycosaminoglycans (GAGs) are sugar-containing molecules that can attach to proteins at serine residues (i.e., can form part of a proteoglycan). They are formed from linear or unbranched monosaccharides (i.e., polysaccharides) that can be sulfated. Heparan sulfate (HS), chondroitin sulfate (CS), keratan sulfate (KS), hyaluronic acid (HA), and heparin are common types of GAGs, where HS and CS are examples of sulfated GAGs. Different types of GAGs are distinguished by different repeating disaccharide units.

[0057] When linked or attached to proteins, CS and HS are GAGs that share a common biosynthetic pathway in their linkage to the core protein, but they then differ in their polymerization, as the CS repeating disaccharide consists of repeating N-acetylgalactosamine (GalNAc) and glucuronic acid residues (GlcA), while the repeating disaccharide in HS generally consists of repeating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) residues. Each monosaccharide is attached by specific enzymes, allowing for multilevel regulation of GAG synthesis.

[0058] Although GAGs can attach to proteins, i.e., they can be in protein-bound or proteoglycan form, GAGs can also exist in a "free" form, i.e., they can also be in non-protein-bound or non-proteoglycan form. Such "free" forms of GAGs are referred to herein as "protein-free GAGs".

[0059] Thus, in body fluids (or body samples), there are typically a protein-free fraction (or protein-free pool) of GAGs and a protein-bound fraction (or protein-bound pool) of GAGs. The two fractions together (i.e., the protein-free fraction plus the protein-bound fraction) can be referred to as the total GAG fraction or total GAG pool.

[0060] In a preferred method of the present invention, the level or composition of the protein-free fraction of one or both of GAG chondroitin sulfate (CS) and heparan sulfate (HS) in a body fluid sample is determined.

[0061] The protein-free fraction GAGs for analysis (or the disaccharide units derived therefrom as discussed elsewhere herein) can be obtained by any suitable means.

[0062] As discussed elsewhere herein, a body fluid sample is typically processed prior to analysis. Such processing generally involves subjecting the GAG to a processing step to obtain disaccharide units for analysis. Such processing steps generally involve contacting the sample (or the GAG in the sample) with an enzyme that digests (or fragments) the GAG into disaccharide units (e.g., a GAG lyase such as chondroitinase or heparinase). Without wishing to be bound by theory, such enzymes cannot access protein-bound GAGs, but act only (or substantially only) on protein-free GAGs (or use them as substrates). If a proteoglycan, which is a protein to which a GAG is bound or attached, is contacted with a proteolytic agent (e.g., a protease such as proteinase K), its protein component is digested by the proteolytic agent and the protein-bound GAG is freed or released, meaning that the protein-bound GAG becomes (or is converted to) a "free" or "released" form, which is then used to be digested (or fragmented) by an enzyme such as a GAG lyase. As in certain preferred methods of the present invention, it is specifically determined the level and / or composition of the protein-free fraction (or native protein-free fraction) of one or both of GAG CS and HS, and thus the preferred methods of the present invention do not include contacting the sample with a proteolytic agent (e.g., a protease such as proteinase K). Thus, omitting the proteolytic agent during the processing of the sample for analysis is one way to obtain (or only obtain or substantially only obtain) the protein-free fraction of the GAG (or disaccharides derived therefrom subsequently) for analysis. Protein-bound GAGs (proteoglycan GAGs) that have been (or become) "free" or "released" by the action of a proteolytic agent (e.g., a protease) are not protein-free GAGs according to the present invention.

[0063] Thus, protein-free GAG (or the protein-free fraction of GAG) is GAG (or a fraction of GAG) that has been (or is native) free (i.e., not protein-bound) in the absence (or without) of a sample that has been treated with a proteolytic agent (e.g., a protease). In other words, protein-free GAG (or the protein-free fraction of GAG) is GAG (or a fraction of GAG) that is free (i.e., not protein-bound) in the original or initial or untreated sample. For example, protein-free GAG (or the protein-free fraction of GAG) can be GAG (or a fraction of GAG) present in a sample (e.g., the original or untreated sample) and sensitive to digestion (or fragmentation) into disaccharide units as described elsewhere herein using an enzyme such as a lyase, or prone to such a process, or available for such a process (e.g., as a substrate for such a process).

[0064] As indicated above, protein-free GAG (or the protein-free fraction of GAG) can be considered the non-protein-bound form (or non-protein-bound fraction) of GAG or the non-proteoglycan form (or non-proteoglycan fraction) of GAG. In other words, protein-free GAG (or the protein-free fraction of GAG) is GAG that does not modify proteoglycans in the original or initial or untreated sample.

[0065] 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 chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample according to the present invention, wherein the sample is subjected to treatment before determining the level and / or composition, and wherein the treatment does not include contacting the sample with a proteolytic agent (e.g., a protease such as proteinase K) or other agents that can release protein-free GAG from protein-bound GAG.

[0066] From another perspective, in some preferred embodiments, the method comprises determining the level and / or chemical composition of the protein-free fraction of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample according to the present invention, wherein the sample is subjected to treatment before determining the level and / or composition, and wherein the treatment does not include contacting the sample with a proteolytic agent (e.g., a protease such as proteinase K) or other agents that can release GAG (or GAG chains) from proteoglycans.

[0067] 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 chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample according to the present invention, wherein the sample has been obtained from the subject and has been subjected to treatment before determining the level and / or chemical composition, wherein the treatment

[0068] (a) comprises fragmenting the one or two GAGs into disaccharide units (e.g., as described elsewhere herein); and

[0069] (b) does not include contacting the sample with an agent capable of releasing GAG (or GAG chains) from proteoglycans prior to (a).

[0070] 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 chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample according to the present invention, wherein the sample has been obtained from the subject and has been subjected to treatment before determining the level and / or chemical composition, wherein the treatment

[0071] (a) Comprising fragmenting the one or two GAGs into disaccharide units (e.g., as described elsewhere herein); and

[0072] (b) Not including contacting the sample with a proteolytic agent prior to (a).

[0073] As indicated above, protein-bound GAG (or the protein-binding fraction of GAG) can be considered proteoglycan GAG (or the proteoglycan fraction of GAG). From another perspective, protein-bound GAG (or the protein-binding fraction of GAG) can be considered GAGs that generally require contact of the protein to which they are bound with a proteolytic agent (e.g., a protease such as a non-specific protease) in order for them to be (or become) free or released.

[0074] In some other embodiments of the methods of the present invention, the levels and / or chemical compositions (i.e., protein-free GAG plus protein-bound GAG) according to the present invention of the intact fraction (or intact pool) of one or both of GAG CS and HS in a body fluid sample can be determined. In such embodiments, the sample is typically contacted with a proteolytic agent during the processing of the sample.

[0075] As used herein, the "level" of HS or CS generally refers to the total level or amount (e.g., concentration) of HS or CS present in the sample. The levels of CS and / or HS in the 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 are gel electrophoresis, such as agarose gel electrophoresis (e.g., FACE, fluorophore-assisted carbohydrate electrophoresis) or mass spectrometry or liquid chromatography (e.g., HPLC, optionally in combination with mass spectrometry (HPLC-MS)). Preferred methods involve high-performance liquid chromatography (HPLC) in combination with mass spectrometry, preferably ultra-HPLC (UHPLC), such as MS / MS or triple quadrupole mass spectrometry. Preferred methods include ultra-high performance liquid chromatography (UHPLC) coupled with an electrospray ionization triple quadrupole mass spectrometry system.

[0076] Conveniently, these levels can be measured as concentrations (e.g., true or absolute levels or concentrations), such as measured in micrograms per milliliter (µg / ml). However, again, any suitable measure of level can be used.

[0077] In exemplary methods of the present invention, the levels of HS and / or CS are determined separately or individually (e.g., the levels of the protein-free fractions of HS and / or CS). In other words, these methods do not involve measuring the total GAG level in a sample or the total level of all GAGs present in combination (e.g., in a protein-free GAG fraction), but rather involve measuring the level of one or more of the individual GAGs HS or CS.

[0078] In certain embodiments, the levels (e.g., total levels or concentrations) of CS and / or HS (e.g., the protein-free fractions of CS and / or HS) can be determined in a body fluid sample (e.g., a urine sample).

[0079] The individual monosaccharide units that make up CS and HS can have different sulfation patterns with respect to the position of sulfate molecules and the amount / number of sulfate molecules. For CS, sulfation most commonly occurs at the 2-position of GlcA and at one or more of the 4- and 6-positions of GalNAc. For HS, sulfation can occur at the 2-position of GlcA after epimerization to IdoA (iduronic acid), at the 3- and 6-positions of GlcNAc, and at the N-sulfation of GlcNAc, among one or more of these positions. Thus, each individual disaccharide in a GAG chain can have 0 (i.e., unsulfated), 1, 2, 3, or 4 (only in HS) sulfated forms, and this in turn results in GAG chains having different overall chemical compositions with respect to sulfation levels and specific disaccharide sulfation patterns.

[0080] As described elsewhere herein, preferred embodiments of the present invention relate to 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 GAG and the disaccharide sulfation composition of GAG. In particular, the term includes the determination of one or more specific forms (e.g., sulfated forms) of the disaccharides that make up CS or HS GAG. In other words, the term "chemical composition" refers to the amount or level of one or more of the various sulfated and / or unsulfated forms of CS or HS disaccharides, as well as, for example, some other property of the individual GAGs present, such as the total HS or CS GAG level, or other properties related to GAG sulfation, such as HS charge or CS charge, as further described elsewhere herein. Such chemical compositions analyzed or determined in the present invention may also be referred to herein as GAG profiles, GAG forms, GAG signatures, GAG properties, GAGomes, GAGome signatures.

[0081] 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 in the sulfated form) of the disaccharides that make up CS and / or HS.

[0082] It is evident from the above discussion that, according to the present invention, it is necessary to determine the level of one or more GAG properties 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. Thus, according to the present invention and the present disclosure, determining the "chemical composition" must include determining the level of one or more GAG properties selected from the group consisting of: 4S CS, NS HS, 0S HS, the total concentration of CS, and the total concentration of HS. The level of one or more other (additional) GAG properties may be additionally determined (or measured) (e.g., as described herein), but according to the present invention and the present disclosure, it is the level of one or more GAG properties selected from the group consisting of: 4S CS, NS HS, 0S HS, the total concentration of CS, and the total concentration of HS that permits an indication (e.g., diagnosis) of OA or RA to be made (or achieved).

[0083] For CS, there are 8 major sulfated and unsulfated forms (sulfation patterns, disaccharide sulfated forms), which are: 0S CS (also known as unsulfated CS or CS O unit), 2S CS (also known as chondroitin-2-sulfate), 4S CS (also known as chondroitin-4-sulfate or CS A unit), 6S CS (also known as chondroitin-6-sulfate or CS C unit), 2S4SCS (also known as chondroitin-2-4-sulfate), 2S6S CS (also known as chondroitin-2-6-sulfate or CS D unit), 4S6SCS (also known as chondroitin-4-6-sulfate or CS E unit), and Tris CS (also known as chondroitin-2-4-6-sulfate or trisulfated CS).

[0084] Each of the above is a form of CS GAG (CS GAG form or property or characteristic or GAGome characteristic) that can be measured in the method of the present invention. One or more of these forms can be measured, e.g., up to 8 sulfated forms of these can be measured, e.g., 1, 2, 3, 4, 5, 6, 7, or all 8. In some embodiments, it is preferred to measure all 8 of these sulfated forms. In some embodiments, it is preferred to measure or at least measure the CS sulfated forms 0S CS, 6S CS, 4S CS, 2S6S CS, 2S4S CS, 4S6S CS.

[0085] Another GAG property of CS that can be measured in the methods of the present invention is the total concentration of CS (which may also be referred to herein as CS tot or Tot CS or total CS) or the total level of CS. This is typically measured as a concentration, such as an absolute concentration, for example in μg / ml, as described elsewhere herein. In some embodiments, the total CS is measured by summing the levels of all of the CS disaccharide forms (8 major sulfated and non-sulfated forms) listed above. Thus, the total CS can be the sum of the levels of all of the CS disaccharide forms (8 major sulfated and non-sulfated forms) listed above. In some embodiments, the total CS is measured by summing the levels of all CS disaccharide forms that are analytically detectable (or levels above that) or levels that have been selected as a minimum threshold level (a minimum threshold level that is considered to be analytically detectable or of analytical significance, e.g., the limit of detection or lower limit of quantification for such CS disaccharide forms) or levels above that. For example, the total CS can be the sum of all CS disaccharide forms measured at a given analytically detectable level or above that level, or at a selected minimum threshold level or above that level (e.g., at a concentration > 0.1 μg / ml). Levels of CS disaccharide forms below a given analytically detectable level or below the minimum threshold level will generally provide only a negligible (or trace) contribution to the total CS concentration and thus, if desired, such CS disaccharide forms can be excluded from the summation of CS disaccharide forms performed to determine or measure the total CS concentration.

[0086] In embodiments of the present invention in which the total concentration of CS is measured as one of the GAG properties, it may be preferred to measure at least one other GAG property or CS property according to the present invention, such as a property 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, it is preferred to measure one or more CS GAG properties according to the present invention.

[0087] "Loaded CS" is another form or property of GAG that can be measured in the present invention, for example, as part of the GAG profile. "Loaded CS" refers to the total fraction of sulfated disaccharides of CS, that is, the fraction of sulfated disaccharides of CS present, measured, or detected in the sample relative to the total CS disaccharides present, measured, or detected in the sample (i.e., sulfated CS disaccharides / sulfated + unsulfated CS disaccharides). In some embodiments, "loaded CS" refers to the weighted sum of the concentrations of all CS disaccharides divided by the total CS, where the weight is the count of sulfonyl groups in the disaccharide, i.e., 0 for 0S CS, 1 for 4S CS, 6S CS, and 2S CS, 2 for 2S6SCS, 4S6S CS, and 2S4S CS, and 3 for Tris CS (and this is the definition of "loaded CS" used in conjunction with the term "loaded CS" in the Examples section herein).

[0088] Since the measurement of "loaded 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 property or CS property.

[0089] In some embodiments, it is preferred to measure up to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) or all 8 of the sulfated forms of CS (i.e., sulfated and unsulfated forms). In some embodiments, loaded CS may additionally be measured. In a preferred embodiment, at least one (or at least 2, 3, 4, 5, 6, 7, or 8) sulfated forms of CS are measured.

[0090] In some embodiments, one or more (or all) of the following GAG properties can be measured or determined: the relative level of 4S CS relative to 6S CS (e.g., the ratio 4S CS / 6S CS or the inverse ratio 6S CS / 4S CS), the relative level of 6S CS relative to 0S CS (e.g., the ratio 6S CS / 0S CS or the inverse ratio 0S CS / 6S CS), or the relative level of 4S CS relative to 0S CS (e.g., the ratio 4S CS / 0S CS or the inverse ratio 0S CS / 4S CS). In some embodiments, the relative level of 4S CS relative to 6S CS (e.g., the ratio 4S CS / 6S CS or the inverse ratio 6S CS / 4S CS) is not measured or determined.

[0091] For HS, there are eight major sulfated and unsulfated forms (sulfation patterns, disaccharide sulfated forms), which are: 0S HS (also known as unsulfated HS), 2S HS (which is sulfated at the 2-position of GlcA), NS HS (which is sulfated at the N-position of GlcNAc), 6S HS (which is sulfated at the 6-position of GlcNAc), 2S6S HS (which is sulfated at the 2-position of GlcA and the 6-position of GlcNAc), NS6S HS (which is sulfated at the 6-position and N-position of GlcNAc), NS2S HS (which is sulfated at the 2-position of GlcA and the N-position of GlcNAc), Tris HS (which is sulfated at the 2-position of GlcA, the 6-position and N-position of GlcNAc, also known as trisulfated HS). Note that sulfation at the 3-position of GlcNAc is also possible, but is rarely observed.

[0092] Each of the above is a form of HS GAG (HS GAG form or property or characteristic or GAGome characteristic) that can be measured or determined in the methods of the present invention. However, due to its rarity, in a preferred embodiment of the present invention, the sulfated form having sulfation at the 3-position of GlcNAc is not measured. Thus, in the methods of the present invention, one or more (or all) of these nine (or preferably eight) forms can be measured, for example, up to nine (or preferably up to eight) of these sulfated forms can be measured, such as 1, 2, 3, 4, 5, 6, 7, 8, or all 9. In some embodiments, it is preferred to measure all eight of these sulfated forms (excluding the sulfated form having sulfation at the 3-position of GlcNAc).

[0093] Another GAG property of HS that can be measured in the methods of the present invention is the total concentration of HS (which may also be referred to herein as HS tot or Tot HS or total HS) or the total level of HS. This is typically measured as a concentration, such as an absolute concentration, for example in μg / ml, as described elsewhere herein. In some embodiments, the total HS is measured by summing the levels of all measured HS disaccharide forms listed above (preferably, the 8 major sulfated and unsulfated forms, i.e., excluding the rare sulfated form having sulfation at the 3-position of GlcNAc). Thus, the total HS can be the sum of the levels of all measured HS disaccharide forms listed above (preferably, the sum of the 8 major sulfated and unsulfated forms, i.e., excluding the rare sulfated form having sulfation at the 3-position of GlcNAc). In some embodiments, the total HS is measured by summing the levels of all HS disaccharide forms measured at an analytically detectable level (or levels above that level) or at a level (or levels above that level) that has been selected as a minimum threshold level (the minimum threshold level considered to be analytically detectable or of analytical significance, e.g., the limit of detection or lower limit of quantification for such HS disaccharide forms). For example, the total HS can be the sum of all HS disaccharide forms measured at a given analytically detectable level or levels above that level, or at a selected minimum threshold level or levels above that level (e.g., at a concentration >0.1 μg / ml). The levels of HS disaccharide forms below a given analytically detectable level or below the minimum threshold level will generally provide only a negligible (or trace) contribution to the total HS concentration and can thus be excluded from the summation of HS disaccharide forms performed to determine or measure the total HS concentration, if desired.

[0094] In embodiments of the present invention in which the total concentration of HS is measured as one of the GAG properties, it may be preferred to measure at least one other GAG property or HS property according to the present invention, such as a property that is not based on the total level of other individual GAGs present (e.g., not total CS). In some embodiments, the total concentration of HS is not measured. In some embodiments, it is preferred to measure one or more HS GAG properties according to the present invention.

[0095] "Loaded HS" is another form or property of GAG that can be measured in the present invention, for example, as part of the GAG profile. "Loaded HS" refers to the total fraction of sulfated disaccharides of HS, i.e., the fraction of sulfated disaccharides of HS present or measured in the sample relative to the total HS disaccharides present or measured in the sample (i.e., sulfated HS disaccharides / sulfated + unsulfated HS disaccharides). In some embodiments, "loaded HS" refers to the weighted sum of the concentrations of all HS disaccharides divided by the total HS, where the weight is the count of sulfonyl groups in the disaccharide, i.e., 0 for 0S HS, 1 for NS HS, 6S HS, 2S HS, 2 for 2S6S HS, NS6S HS, and NS2S HS, and 3 for Tris HS (and this is the definition of "loaded HS" used in conjunction with the term "loaded HS" in the Examples section herein).

[0096] Since the measurement of "loaded HS" depends on the measurement of other properties, namely the measurement of sulfated and unsulfated HS disaccharides, this property is not referred to herein as an independent GAG property or HS property.

[0097] In some embodiments, it is preferred to measure at most 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) or all 8 of the major sulfated forms of HS (i.e., the sulfated and unsulfated forms excluding the sulfated forms that would have sulfation at the 3-position of GlcNAc as listed above), as well as total HS. In some embodiments, loaded HS may additionally be measured. In a preferred embodiment, at least one (or at least 2, 3, 4, 5, 6, 7, or 8) HS sulfated forms are measured.

[0098] In some embodiments, 8 major sulfated and unsulfated HS forms, total HS, 8 major sulfated and unsulfated CS forms, and total CS are measured, i.e., 18 GAG properties.

[0099] In some embodiments, 8 major sulfated and unsulfated HS forms and 8 major sulfated and unsulfated CS forms are measured, i.e., 16 GAG properties.

[0100] In some embodiments, the level and / or chemical composition of hyaluronic acid (HA) in a body fluid sample may additionally be determined. Hyaluronic acid (HA) is typically unsulfated. Thus, when measuring HA according to the present invention, the level (total level or total concentration) of HA (which may also be referred to as total HA) is typically and preferably measured. This is typically measured as a concentration, e.g., in μg / ml, as described elsewhere herein. In some embodiments, HA is not measured.

[0101] In some embodiments, eight major sulfated and unsulfated HS forms, total HS, eight major sulfated and unsulfated CS forms, total CS, and total HA are measured, i.e., 19 GAG properties.

[0102] CS (total CS) and HS (total HS) are typically measured in absolute concentration, e.g., in μg / ml, as described elsewhere herein.

[0103] The various CS sulfated forms and HS sulfated forms can also be measured in absolute concentration, 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 absolute concentration of the given CS sulfated form or the given HS sulfated form. In some embodiments, the absolute level or absolute concentration is preferred.

[0104] However, the various CS sulfated forms and HS sulfated forms can alternatively or additionally be measured in relative concentration (or relative level). 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 relative concentration.

[0105] Thus, the "level" or "concentration" of a GAG sulfated form can be its absolute concentration or its relative concentration. In some embodiments, both the absolute concentration and the relative concentration of one or more sulfated GAG forms are measured (or determined).

[0106] "Relative concentration" can be considered the mass fraction (e.g., in %) 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).

[0107] Thus, the relative concentration of a given CS sulfated form can be considered the mass fraction (e.g., in %) of the given CS sulfated form, obtained (or calculated or determined) by normalizing its absolute concentration with the total CS concentration.

[0108] The relative concentration of a given HS sulfated form can be considered the mass fraction (e.g., in %) of the given HS sulfated form, obtained (or calculated or determined) by normalizing its absolute concentration with the total HS concentration.

[0109] Relative concentration can be expressed as a percentage (%).

[0110] Thus, in some embodiments, the level (or concentration) of a given CS sulfation form or a given HS sulfation form can be the absolute concentration and / or relative concentration of the given CS sulfation form or the given HS sulfation form. In some embodiments, the level (or concentration) of a given CS sulfation form or a given HS sulfation form is the absolute concentration of the given CS sulfation form or the given HS sulfation form. In some embodiments, the level (or concentration) of a given CS sulfation form or a given HS sulfation form is the relative concentration of the given CS sulfation form or the given HS sulfation form.

[0111] The relative concentration can alternatively be regarded as a "fraction" or "mass fraction" or "proportion" or "relative measurement result", as discussed below.

[0112] As discussed above, GAG properties or GAG forms (e.g., disaccharide sulfation forms (excluding total CS or total HS)) can be measured as fractional size or fraction or mass fraction (e.g., µg / µg) or proportion or relative measurement result, rather than as absolute levels or concentrations, e.g., given as a value less than 1 or normalized to 1 based on the level (or in %) of all sulfation forms (or all major sulfation forms) of the relevant GAG class measured in the sample. In other words, the level of each of the desired sulfation forms is measured independently and then normalized to 1. In other words, the level of each of the desired sulfation forms is measured independently and then its mass fraction or volume fraction or mole fraction is calculated. These fractions can also be expressed as percentages. In other words, these fractions can also be normalized to 100. For example, in some embodiments, the fractional size of a given sulfated CS form or unsulfated CS form can be determined by measuring the level of the given sulfated CS form or unsulfated CS form and dividing it by the sum of the levels of all (or all of the major sulfation forms) of the sulfated CS forms and unsulfated CS forms measured (or present) in the sample. In some embodiments, the fractional size of a given sulfated HS form or unsulfated HS form can be determined by measuring the level of the given sulfated HS form or unsulfated HS form and dividing it by the sum of the levels of all of the sulfated HS forms (or major sulfation forms) and unsulfated HS forms measured (or present) in the sample. When calculating such fractions, it is preferred to measure at least the major sulfation forms of CS or HS so that the fraction of a particular individual sulfation form can be normalized to 1.

[0113] Relative measurement results may be easier to interpret. For example, a measurement result of 0.6 for 0S HS indicates that 60% of the measured HS disaccharides are unsulfated. However, absolute levels can also be measured. In fact, in some embodiments, it is preferred to measure the absolute concentration of one or more sulfation forms.

[0114] In some preferred embodiments of the present invention, the disaccharide composition (e.g., a specific sulfation pattern (e.g., sulfated form)) of one or more (or all) of the disaccharides that make up CS and / or HS is measured or determined. In some embodiments, one or more (or all) of the sulfation properties or forms (or GAG characteristics or properties or GAGome characteristics or properties) of 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 methods can be used. However, a convenient method for achieving such quantification of the disaccharide composition or the appropriate properties or forms of CS or HS (and separation of the disaccharide forms) is to use electrophoresis, particularly capillary electrophoresis, such as capillary electrophoresis with fluorescence detection, such as capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). An alternative method is liquid chromatography, preferably HPLC (high-performance liquid chromatography), such as SAX HPLC. Preferably, mass spectrometry (e.g., HPLC-MS) is also used, such as electrospray ionization mass spectrometry (ESI-MS). Alternatively, mass spectrometry can be used without chromatography, such as liquid chromatography. One example is capillary electrophoresis with laser-induced fluorescence detection. Another example is HPLC ESI-MS. Preferred methods involve high-performance liquid chromatography (HPLC) combined with mass spectrometry, preferably ultra-HPLC (UHPLC), such as MS / MS or triple quadrupole mass spectrometry. Preferred methods include ultra-high-performance liquid chromatography (UHPLC) coupled with electrospray ionization triple quadrupole mass spectrometry. Particularly preferred methods are outlined in the examples.

[0115] In some methods of the present invention in which the levels of one or more individual disaccharide forms are measured, the GAG is subjected to a processing step, such as a fragmentation or cleavage or digestion step, such as by chemical digestion or enzymatic treatment, in order to obtain disaccharide units, which are then analyzed. The enzyme can be a GAG lyase, such as chondroitinase or 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 are used, preferably chondroitinase ABC and heparinase I-II-III.

[0116] In some methods of the present invention, the GAG in a sample is subjected to an extraction step (e.g., using a proteolytic agent such as a protease, such as a non-specific protease, such as proteinase K) and / or a purification step (e.g., using an anion exchange resin (or other means of purifying GAG based on its negative charge)).

[0117] However, in the preferred method of the present 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 a preferred embodiment of the present invention, when determining the level and / or composition of the protein-free fraction of one or more GAGs, such a protein digestion (extraction) step is not performed. In other words, the method does not involve a processing step in which a sample is contacted with a proteolytic agent (such as a protease, e.g., proteinase K). As discussed elsewhere herein, omitting the processing step in which a sample is contacted with a proteolytic agent means that a specific analysis can be performed on the protein-free fraction of GAGs.

[0118] In other preferred embodiments of the present invention, the method does not involve a processing step in which GAGs (e.g., one or both of GAG CS and HS) are purified from a sample based on the negative charge of the GAG (e.g., using an anion exchange resin). Without wishing to be bound by theory, omitting the processing step in which GAGs (e.g., one or both of GAG CS and HS) are purified from a sample based on the negative charge of the GAG (e.g., using an anion exchange resin) simplifies the method and can improve the efficiency of GAG yield obtained during the processing of body fluid samples.

[0119] In the preferred method of the present invention, the method does not involve a processing step in which a sample is contacted with a proteolytic agent (such as 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 a sample based on the negative charge of the GAG (e.g., using an anion exchange resin (or other means of purifying GAG based on the negative charge of the GAG)).

[0120] In some methods of the present invention, GAGs in a sample (e.g., various different forms of GAGs in the sample) are subjected to separation and / or quantification steps as described elsewhere herein. For example, as discussed elsewhere herein, HPLC combined with mass spectrometry can be used in a preferred embodiment. Particularly preferred methods include ultra-high performance liquid chromatography (UHPLC) coupled with electrospray ionization triple quadrupole mass spectrometry.

[0121] Other methods that can be used are known in the art. However, examples are analytical techniques involving the use of antibodies against various forms of GAGs, such as techniques such as Western blotting, ELISA, or FACS, or methods involving agarose gel electrophoresis (e.g., fluorophore-assisted carbohydrate electrophoresis (FACE)) or polyacrylamide gel electrophoresis (PAGE).

[0122] In any embodiment involving determining the level of one or more GAG forms from a certain 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 the listed GAG forms (or GAG properties or GAG features or GAGome features) according to the present invention can be determined.

[0123] In some embodiments, the level of a single GAG form (GAG property or GAG feature or GAGome feature) is determined.

[0124] In other embodiments of the present invention, the levels of more than one GAG form (GAG property) are determined (e.g., determining 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). The so-called "more than one" means 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, etc. In some embodiments, for any list of markers or GAG properties provided herein, all the markers or GAG properties are measured (or determined). Moreover, the levels of each and every possible combination in the GAG forms can be determined.

[0125] Thus, in some embodiments, a multi-marker method is performed. Determining the levels of multiple GAG forms (biomarker multiplexing) among the GAG forms according to the present invention can improve the accuracy of screening (e.g., diagnosis).

[0126] Thus, although the markers (GAG forms or GAG properties or features or GAGome features) according to the present invention can be used alone in the methods of the present invention, they can also be used in combination, for example, in the form of a multi-marker assay.

[0127] In some embodiments, the level of a single GAG form (GAG property) according to the present invention is used as a basis for screening for OA or RA. For example, in some embodiments, OA can be distinguished (or differentiated) from RA based on the level of a single GAG form according to the present invention (i.e., based on the total concentration level of 4SCS, NS HS, 0S HS, CS, or the total concentration level 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 a basis for screening for OA or RA. For example, OA can be distinguished (or differentiated) from RA based on the levels of more than one GAG form according to the present invention (i.e., based on the levels of more than one (e.g., all) of the total concentrations of 4S CS, NS HS, 0S HS, CS, or the total concentration of HS, e.g., the levels of any subgroup of these GAG forms as discussed elsewhere herein).

[0128] In some embodiments, in the case where a form of GAG or a group (or subgroup or subset) of forms of GAG according to the present invention is used as a basis for screening for OA or RA (e.g., to discriminate or distinguish OA from RA), the levels of one or more (or all) of the other forms of GAG (or GAG properties) described herein may additionally be determined or measured.

[0129] Based on the observed changes in the levels of the various GAG properties according to the present invention, if desired, scoring methods, scoring systems, markers, or formulas may be designed that use such levels of the various forms of GAG in order to obtain an indication, e.g., in the form of a value or a score, which can then be used for screening (e.g., diagnosis, etc.). Based on one or more individual GAG features according to the present invention (or including such individual GAG features), appropriate scoring systems and parameters to be measured (e.g., forms of GAG) can be readily designed.

[0130] In some embodiments according to the present invention, the chemical composition may be represented by a score (or GAG score) based on the measured levels (or derived or calculated by using) of one or more (preferably more than one) of the GAG properties according to the present invention.

[0131] Thus, the score may be based on one or more (or all) of the measured (or determined) GAG properties (or derived or calculated or accounted for by using) selected from the group consisting of: 4S CS, NS HS, 0S HS, total CS, and total HS.

[0132] In some embodiments, the score may be based on the measured levels (or derived or calculated or accounted for by using) of one or more GAG properties in more than one type of body fluid sample. In some such embodiments, the GAG properties measured in each type of body fluid may be the same or they may be different.

[0133] In some embodiments, a person skilled in the art may design appropriate thresholds or cut-off values for use with the score (e.g., which are used to declare a sample as OA or RA, as the case may be (or indicate likely OA or likely RA)). By way of example, the cut-off value (or threshold) may be calculated based on an ROC curve. In some cases, the maximum selected rank statistic may be used to identify the cut-off value (or cut-off score).

[0134] In some embodiments, screening (e.g., diagnosis, etc.) may be performed by the process of comparing a given score of a sample (or subject) to be screened (e.g., diagnosed) with a threshold or cut-off 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 cut-off value.

[0135] Any scoring method, scoring system, biomarker, or formula that includes any suitable combination of the GAG properties according to the present invention can be used to obtain an indication, for example, in the form of a value or score, which can then be used for screening (e.g., diagnosing) OA or RA. For example, the method, etc. can be an algorithm that includes any suitable combination of the GAG properties according to the present invention as input to, for example, perform pattern recognition on a sample to obtain an indication, for example, in the form of a value or score, which can then be used for screening (e.g., diagnosing) OA or RA. Non-limiting examples of such algorithms include machine learning or deep learning algorithms (algorithm classifiers) that implement classification, such as linear classifiers (e.g., Fisher linear discriminant, logistic regression, naive Bayes classifier, perceptron); support vector machines (e.g., least squares support vector machine); quadratic classifiers; kernel estimation (e.g., k-nearest neighbor); boosting; decision trees (e.g., random forest); neural networks; learning vector quantization.

[0136] The use of such classifiers (e.g., machine learning classifiers, e.g., random forest classifiers) will be within the skill of those in the art.

[0137] In some embodiments, a multivariable logistic regression model can be used to calculate the GAG score.

[0138] 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 a level and / or chemical composition according to the present invention. As described elsewhere herein, in some such embodiments, a change in the score (e.g., an increase or decrease, depending on the circumstances) as compared to a control score (or cut-off level or threshold level) indicates OA or RA (as appropriate) in the subject.

[0139] As discussed above, the present invention provides a method for screening a subject for OA or RA. The present invention also provides a method for diagnosing OA or RA in a subject. Thus, the method for screening for OA or RA according to the present invention can be used, for example, to diagnose OA or RA.

[0140] Thus, in one aspect, the present invention provides a method for diagnosing OA or RA in a subject. In some embodiments, if the level of one or more GAG properties according to the present invention in a sample changes (an increase or decrease, depending on the circumstances) as compared to an appropriate control level (e.g., as discussed elsewhere herein), a positive diagnosis (i.e., the presence of OA or RA, as appropriate) is made.

[0141] Accordingly, on the other hand, the present invention provides a method for diagnosing osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject having arthritis or suspected of having arthritis, the method comprising:

[0142] determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS), which are glycosaminoglycans (GAGs), in a body fluid sample,

[0143] wherein 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) the total concentration of CS, and (v) the total concentration of HS,

[0144] wherein the sample has been obtained from the subject.

[0145] The embodiments of the screening method of the present invention described elsewhere herein are applicable, mutatis mutandis, to this aspect of the present invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0146] The screening (e.g., diagnostic) method according to the present invention can be used to discriminate (or distinguish or differentiate) OA from RA in a subject having arthritis or suspected of having arthritis.

[0147] Accordingly, on the other hand, the present invention provides a method for discriminating (or distinguishing or differentiating) OA from RA in a subject having arthritis or suspected of having arthritis, the method comprising:

[0148] determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS), which are glycosaminoglycans (GAGs), in a body fluid sample,

[0149] wherein 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) the total concentration of CS, and (v) the total concentration of HS,

[0150] wherein the sample has been obtained from the subject.

[0151] The embodiments of the screening method of the present invention described elsewhere herein are applicable, mutatis mutandis, to this aspect of the present invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0152] In a preferred aspect, the present invention provides a method for differentiating (or distinguishing or discriminating) osteoarthritis (OA) from rheumatoid arthritis (RA) in a subject having arthritis or suspected of having arthritis, the method comprising:

[0153] determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a urine sample,

[0154] wherein 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) the total concentration of CS, and (v) the total concentration of HS,

[0155] wherein the sample has been obtained from the subject. Embodiments of the screening methods of the present invention described elsewhere herein are applicable, mutatis mutandis, to this aspect of the present invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred control levels, etc.).

[0156] The levels of one or more GAG properties according to the present invention can be used for the differential diagnosis of OA and RA (OA versus RA), or as part of the differential diagnosis process for OA and RA (OA versus RA).

[0157] The concept of "differential diagnosis" is well known in the art. Briefly, differential diagnosis can be considered the process of differentiating between two or more diseases (or disorders) that may cause a subject's symptoms. Thus, when a subject's symptoms match those that may be caused by more than one disorder, the differential diagnosis process can be used.

[0158] Accordingly, in another aspect, the present invention provides a method for the differential diagnosis of OA or RA (OA versus RA) in a subject having arthritis or suspected of having arthritis, the method comprising:

[0159] determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample,

[0160] wherein 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) the total concentration of CS, and (v) the total concentration of HS,

[0161] wherein said sample has been obtained from said subject.

[0162] Embodiments of the screening methods of the invention described elsewhere herein 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.).

[0163] As described elsewhere herein, screening (e.g., diagnostic) methods according to the invention can be used in combination with other diagnostic tests or methods.

[0164] Thus, in another aspect, the invention provides a method of confirming (or validating) a previous diagnosis of OA or RA in a subject, the method comprising:

[0165] determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample,

[0166] wherein said determining the level and / or chemical composition of one or both of said 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,

[0167] wherein said sample has been obtained from said subject.

[0168] Embodiments of the screening methods of the invention described elsewhere herein 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.).

[0169] In another aspect, the invention provides a method of screening for osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject having arthritis or suspected of having arthritis, the method comprising:

[0170] 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,

[0171] wherein said sample has been obtained from said subject.

[0172] Embodiments of the screening methods of the invention described elsewhere herein can be adapted, 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.).

[0173] In another aspect, the invention provides a method of differentiating osteoarthritis (OA) from rheumatoid arthritis (RA) in a subject having arthritis or suspected of having arthritis, the method comprising:

[0174] 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 sample of a body fluid (e.g., urine) obtained from the subject;

[0175] wherein the sample has been obtained from the subject. Embodiments of the screening methods of the invention described elsewhere herein can be adapted, 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.).

[0176] In another aspect, the invention provides a method of diagnosing osteoarthritis (OA) in a subject having arthritis or suspected of having arthritis, the method comprising:

[0177] determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample;

[0178] wherein 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;

[0179] wherein the sample has been obtained from the subject.

[0180] Embodiments of the screening methods of the invention described elsewhere herein can be adapted, 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.).

[0181] In another aspect, the present invention provides a method of providing information for screening for osteoarthritis (OA) or rheumatoid arthritis (RA) (e.g., diagnosing, differentiating, discriminating OA or RA, or confirming a diagnosis of OA or RA) in a subject having or suspected of having arthritis, the method comprising:

[0182] determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample,

[0183] wherein 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,

[0184] wherein the sample has been obtained from the subject. The information is of course typically the level of determination (or measurement) of one or more of the GAG properties.

[0185] Embodiments of the screening methods of the present invention described elsewhere herein can be adapted, mutatis mutandis, to this aspect of the present invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0186] Yet another aspect of the present invention provides a method of screening for OA or RA (e.g., diagnosing, differentiating, discriminating OA or RA, or confirming a diagnosis of OA or RA) in a subject, the method comprising: analyzing disaccharide units of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) that have been derived from a body fluid sample, wherein the method comprises levels of one or more GAG properties according to the present invention. Embodiments of the screening methods of the present invention described elsewhere herein can be adapted, mutatis mutandis, to this aspect of the present invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0187] Another aspect of the present invention provides a method for screening a subject for OA or RA (e.g., for diagnosis, differential diagnosis, differentiating OA or RA, or confirming a diagnosis of OA or RA), the method comprising: analyzing a population of disaccharide units consisting essentially of disaccharide units that have been 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, wherein the method comprises determining the level of one or more GAG properties according to the present invention. Embodiments of the screening methods of the present invention described elsewhere herein are applicable, mutatis mutandis, to this aspect of the present invention (e.g., preferred GAG forms (or groups of GAG forms), preferred processing steps, preferred body fluids, preferred control levels, etc.).

[0188] Unless otherwise explicitly stated from the context, features and discussions herein regarding methods for screening for OA or RA (e.g., regarding preferred GAG properties or combinations thereof, or measured scores, preferred processing steps, preferred body fluids, preferred control levels, etc.) are applicable, mutatis mutandis, to other related methods of the present invention (methods for diagnosing, differentially diagnosing, differentiating OA or RA, or confirming a diagnosis of OA or RA, methods for providing information for OA or RA, etc.).

[0189] In some embodiments, the present invention provides for the combined use of the methods of the present invention (e.g., screening, diagnostic, or progression-determining methods as described herein) 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, scans or X-rays), or magnetic resonance imaging (MRI scans), or ultrasound imaging, or histological assessment (e.g., using a biopsy), or established criteria (e.g., for diagnosis) for indicating OA or RA). By way of example, with respect to 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., for RA, the American Rheumatism Association for RA criteria) can be used in combination with the methods of the present invention. With respect to OA, the KL (Kellgren-Lawrence) system (or KL scoring system) can be used in combination with the methods of the present invention. The KL system is a well-known radiographic classification system for OA.

[0190] Thus, for example, the methods of the present invention can be used to confirm a diagnosis of OA or RA in a subject (confirm a previous diagnosis). In some embodiments, the methods of the present invention are used alone.

[0191] The levels of the GAG forms discussed can be determined or measured by analyzing a sample that has been obtained or removed from a subject by appropriate means. The determination is typically performed in vitro.

[0192] The level of one or more of the GAG forms in the sample can be measured (determined) by any suitable assay or technique or method, many of which are well-known and documented in the art. Electrophoresis (e.g., agarose gel electrophoresis or capillary electrophoresis (especially 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 of the GAG forms according to the invention. Liquid chromatography, especially HPLC (high performance liquid chromatography) in combination with mass spectrometry (MS), is a preferred technique for measuring (determining) the level of one or more of the GAG forms according to the invention.

[0193] Suitable electrophoresis (e.g., capillary electrophoresis) and liquid chromatography methods (e.g., HPLC techniques) for GAG form analysis, as well as appropriate mass spectrometry methods (and related data processing techniques), are well-known and documented in the art.

[0194] One method that can be used in the present invention is capillary electrophoresis with laser-induced fluorescence detection, CE-LIF (e.g., as described by Galeotti et al., 2014, Electrophoresis, Vol. 35, pp. 811-818; and Kottler et al., 2013, Electrophoresis, Vol. 34, pp. 2323-2336). HPLC combined with post-column derivatization and fluorescence detection can also be used, e.g., as described by Volpi, 2006, Curr Pharm Des, Vol. 12, pp. 639-658. HPLC combined with ESI-MS (electrospray ionization mass spectrometry) can also be used, e.g., as described by Volpi and Linhardt, 2010, Nature protocols, Vol. 5, pp. 993-1004. Fluorescence detection can also be used, e.g., as described by Galeotti and Volpi, 2011, Anal Chem, Vol. 83, pp. 6770-6777 or Volpi et al., 2014, NatureProtocols, Vol. 9, pp. 541-558. Agarose gel electrophoresis can also be used, e.g., FACE (fluorophore-assisted carbohydrate electrophoresis), as described by Volpi and Maccari, 2006, Analyt Technol Biomed Life Sci, 834:1-13; and Volpi and Maccari, 2002, Electrophoresis, Vol. 23, pp. 4060-4066).

[0195] Particularly preferred methods for determining the level of one or more GAG forms in a GAG form in a sample are described in the examples herein. Thus, preferred methods can involve high performance liquid chromatography (HPLC), preferably ultra-HPLC (UHPLC), combined with mass spectrometry, preferably 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. Examples of such methods are described in Tamburro et al. (Journal of Chromatography B, 1177 (2021) 122761).

[0196] Certain methods of sample preparation (or processing) (e.g., GAG extraction and purification) are also known in the art and have been described, e.g., Volpi and Maccari, 2005, Biomacromolecules, Vol. 6, pp. 3174-3180 and ClinChim Acta, Vol. 356, pp. 125-133, Coppa et al., 2011, Glycobiology, Vol. 21, pp. 295-303. Such reported art-based sample preparation (or processing) methods involve protease treatment (protease extraction) and purification steps based on the use of anion exchange resins. In some methods of the present invention, such protease treatment steps and / or purification steps using anion exchange resins may be carried out. However, as discussed elsewhere herein, in the preferred methods, the protease treatment step and / or the purification step using anion exchange resins are not carried out. In particular, in the preferred methods, when analyzing the protein-free fraction of GAG, then the protease treatment step is not carried out.

[0197] In some embodiments, HPLC and mass spectrometry (and related data processing techniques) are used to obtain the fraction of the level of one or more specific GAG forms (e.g., sulfated or unsulfated disaccharide forms) in a sample compared to the total amount. For example, after sample preparation, enzymes can be used to digest the GAG, separated in an HPLC column and characterized using MS. As described elsewhere herein, the sum of all amounts of the individual GAG forms measured can be conveniently normalized (i.e., divided) by the amount of one or more individual GAG forms (e.g., specific sulfated or unsulfated disaccharide forms) to produce a fraction (or ratio or relative concentration). Alternatively or additionally, the absolute concentration (or absolute level) of the individual GAG forms (e.g., the sulfated form of GAG) can be measured.

[0198] According to the present invention, the level of one or more GAG forms in the GAG form can be quantitatively, semi-quantitatively or qualitatively evaluated (determined).

[0199] The appropriate methods for performing this operation are well known to those skilled in the art, and any one of these methods can be used. However, a convenient method for achieving such quantification of the disaccharide composition or the appropriate properties or forms of CS or HS (and the separation of the disaccharide forms) is to use electrophoresis, particularly capillary electrophoresis, such as capillary electrophoresis with fluorescence detection, such as capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) (e.g., as described in Galeotti, 2014 (supra) or Kottler, 2013 (supra)). An alternative method preferred in some embodiments is to use liquid chromatography, preferably HPLC (high-performance liquid chromatography), such as SAX HPLC or as described in Volpi, 2006 (supra), Galeotti and Volpi, 2011 (supra), Volpi et al., 2014 (supra) or Volpi and Linhardt, 2010 (supra). Preferably, mass spectrometry (HPLC-MS) is also used, such as electrospray ionization mass spectrometry (ESI-MS), such as HPLC ESI-MS. Particularly preferred methods are outlined in the examples. An example of a specific method is capillary electrophoresis (e.g., capillary electrophoresis with laser-induced fluorescence detection). Another particularly preferred example would be MS after HPLC (HPLC-MS), such as HPLC ESI-MS. The preferred HPLC-MS methods are discussed elsewhere herein.

[0200] Thus, in a preferred method of the present invention, the level or chemical composition or GAG properties of the GAG are 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 or GAG properties of the GAG are determined by high-performance liquid chromatography (HPLC), preferably ultra-HPLC (UHPLC), in combination with mass spectrometry (e.g., MS / MS or triple quadrupole mass spectrometry). Preferred methods include ultra-high-performance liquid chromatography (UHPLC) coupled (or combined) with electrospray ionization triple quadrupole mass spectrometry.

[0201] Generally speaking, the determination of GAG properties (or forms or characteristics) according to the present invention does not involve the measurement of GAG molecules in exactly the same form as found in the body fluids of a subject (e.g., does not involve the measurement of naturally occurring forms of GAG). For example, such natural or naturally occurring GAG molecules are typically found in biological samples (e.g., body fluid samples) in the form of long sugar chains, which may be attached to proteins (also referred to herein as protein-bound GAG or proteoglycan GAG) or not attached to proteins (also referred to herein as free GAG or protein-free GAG).

[0202] In some embodiments, the methods of the present invention may include the step of processing a sample. Thus, in some embodiments, the methods of the present invention may be performed on such processed samples or materials derived from such processed samples. Thus, generally speaking, the methods of the present invention are performed on samples that have been processed in some way (e.g., they are artificial samples rather than natural samples).

[0203] The processing step may include, but is not limited to, steps of extracting or purifying GAG from the sample, fragmenting or lysing or digesting proteins present in the sample (e.g., as a means of separating or extracting or removing GAG from the proteins to which they are attached, e.g., by using a protease such as proteinase K), purifying GAG (e.g., using an anion exchange resin), separating cells from the sample, separating cell components from the sample, extracting (e.g., separating or purifying) proteins / peptides from the sample. Thus, the processing step also includes steps performed on a body fluid sample to prepare the body fluid sample for analysis, e.g., in the case of a urine sample, removal of cells or other impurities may be performed. The processing step may involve one or more of digestion, extraction, purification, boiling, filtration, lyophilization, fractionation, centrifugation, concentration, dilution, inactivation of interfering components, addition of reagents, derivatization, complexation, etc. Exemplary processing steps are described in the examples.

[0204] Although in certain methods of the present invention, steps of fragmenting or lysing or digesting proteins present in the sample (e.g., as a means of separating or extracting or removing GAG from the proteins to which they are attached, e.g., by using a protease such as proteinase K) and / or purifying GAG (e.g., using an anion exchange resin) may be performed, as is apparent from the discussion elsewhere herein, in certain preferred methods, the steps of fragmenting / lysing / digesting proteins and / or purifying (e.g., using an anion exchange resin) are not performed. In particular, in the preferred methods in which the level and / or chemical composition of the protein-free fraction of GAG is determined, the steps of fragmenting / lysing / digesting proteins are not performed.

[0205] Generally speaking, prior to determining the level and / or chemical composition according to the methods of the present invention, a GAG-containing body fluid sample obtained from a subject is subjected to at least one processing step. In particular, in the methods in which the level of one or more of the specific sulfated or non-sulfated forms of CS or HS disaccharides is determined, preferably the GAG is subjected to a processing step to obtain disaccharide units for analysis.

[0206] In some such methods of the invention in which the levels of certain individual disaccharide forms are measured, a GAG (e.g., a full-length GAG molecule), or a polymeric polysaccharide chain of a GAG, or a chain of repeating disaccharide units of a GAG is subjected to a processing step, such as a fragmentation or cleavage or digestion step, 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, such as using one or more GAG lyases, such as one or more chondroitinases such as chondroitinase ABC or chondroitinase B, and / or using one or more heparinases such as heparinase I-II-III, in order to obtain disaccharide units, which are then analyzed.

[0207] Other methods for determining the level or composition of GAGs are known in the art. However, examples are analytical techniques involving the use of antibodies against various GAG forms, such as techniques such as Western blotting, ELISA, or FACS, or methods involving agarose gel electrophoresis (e.g., fluorophore-assisted carbohydrate electrophoresis (FACE)) or polyacrylamide gel electrophoresis (PAGE).

[0208] In some embodiments, the level of one or more GAG forms (e.g., specific sulfated or non-sulfated forms of CS or HS disaccharides, which are derived from full-length GAG molecules or chains of repeating disaccharide units of GAG molecules, e.g., by fragmentation, cleavage, or digestion) associated (e.g., physically associated with, complexed with, derivatized with, or labeled with) with a reagent (e.g., 2-aminoacridone) for detecting GAG forms is determined. Thus, in some embodiments, the level of a complex of a GAG form and a reagent for detecting GAG forms is determined. Reagents suitable for detecting specific GAG forms are discussed elsewhere herein, but include antibodies or some fluorophore (or other detectable label or dye) attached to (or used for derivatizing) the GAG form being discussed, e.g., such that it is detectable by a fluorometer (or other detection device). Thus, by way of pure example, in some embodiments, the level of a GAG form associated (e.g., complexed with or derivatized with) with an antibody or fluorophore, etc., can be determined. In some embodiments, the level of a GAG form associated (e.g., complexed with or derivatized with) with 2-aminoacridone can be determined.

[0209] Since certain preferred methods of the present invention include the step of determining the level and / or chemical composition of the protein-free fraction of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) among glycosaminoglycans (GAGs) in a body fluid sample, it is advantageously not necessary to separate or extract them from the proteins to which the GAG molecules are attached. Instead, in such embodiments, the protein-free fraction (only the protein-free fraction) of GAGs in the body fluid sample can be analyzed from the sample without any such treatment to separate GAGs from proteins, such as by digesting the proteins. Thus, the preferred methods do not include a treatment step in which the sample is contacted with a proteolytic agent such as a protease.

[0210] Other preferred methods do not include the step of purifying GAGs from the sample based on the negative charge of the GAGs (e.g., using an anion exchange resin).

[0211] Thus, in the preferred methods of the present invention, the sample has been obtained from the subject and has been subjected to treatment before determining the level and / or chemical composition,

[0212] wherein the treatment

[0213] (a) includes fragmenting the one or two GAGs into disaccharide units; and

[0214] (b) does not include at least one of the following operations before (a):

[0215] (i) contacting the sample with a proteolytic agent; and

[0216] (ii) purifying the one or two GAGs in the sample based on the negative charge of the GAGs.

[0217] Another aspect of the present invention provides a method for screening a subject with arthritis or a patient suspected of having arthritis for OA or RA, the method including determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) among glycosaminoglycans (GAGs) in a body fluid sample,

[0218] wherein 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) the total concentration of CS, and (v) the total concentration of HS,

[0219] wherein the sample has been obtained from the subject and has been subjected to treatment before determining the level and / or chemical composition,

[0220] wherein the treatment

[0221] (a) includes fragmenting the one or two GAGs into disaccharide units; and

[0222] (b) does not include at least one of the following operations prior to (a):

[0223] (i) contacting the sample with a proteolytic agent; and

[0224] (ii) purifying the one or two GAGs in the sample based on the negative charge of the GAG.

[0225] Embodiments of other aspects of the invention described elsewhere herein can be applied, with necessary modifications, to this aspect of the invention (e.g., preferred GAG properties (or groups of GAG properties), preferred processing steps, preferred body fluids, etc.).

[0226] In a preferred method, the fragmentation in (a) is conveniently carried out by contacting the one or two GAGs with one or more GAG lyases (e.g., as discussed elsewhere herein). For example, the fragmentation in (a) can be carried out by contacting the one or two GAGs with one or more chondroitinases and / or one or more heparinases.

[0227] In methods based on those in the art, the contacting step in (b)(i) is conveniently carried out by contacting the sample with one or more proteases (e.g., proteinase K). Thus, in certain preferred methods of the invention, this step is not carried out. The proteolytic agent in (b)(i) can be a protease (e.g., a non-specific protease such as proteinase K). Thus, certain preferred methods of the invention do not include the step of contacting the sample with a protease (e.g., a non-specific protease such as proteinase K) prior to (a).

[0228] In methods based on those in the art, the purification step in (b)(ii) is conveniently carried out by using an anion exchange resin. Thus, in certain preferred methods of the invention, this step is not carried out. Thus, certain preferred methods of the invention do not include the step of purifying the one or two GAGs in the sample using an anion exchange resin prior to (a).

[0229] In a preferred method of the invention, the method does not include the contacting in (b)(i).

[0230] In a preferred method of the invention, the method does not include the purification in (b)(ii).

[0231] In other preferred methods of the invention, neither step (b)(i) nor (b)(ii) is carried out). Thus, in a particularly preferred embodiment, the method does not include the contacting in (b)(i) or the purification in (b)(ii).

[0232] As mentioned above, in some embodiments, compared to a control level, a change (either an increase or a decrease, as appropriate) in the level of one or more GAG properties (4S CS (e.g., the absolute concentration of 4S CS), NS HS (e.g., the absolute concentration and / or relative concentration of NS HS), 0S HS (e.g., the absolute concentration of 0S HS), the total concentration of CS, and / or the total concentration of HS) according to the present invention indicates OA or RA in the subject (indicating whether the subject has OA or RA). A person skilled in the art will be able to readily establish an appropriate control level for use in accordance with the present invention.

[0233] In some embodiments of the methods of the present invention (e.g., screening or diagnostic or discriminatory methods),

[0234] (a) A difference in the level of one or more GAG properties according to the present invention from a control level of the same one or more GAG properties (or a change (e.g., an increase or a decrease) compared to the control level) indicates OA, where the control level is within (or included in) a reference interval determined for the same sample type in a group of subjects (or reference population) who have never had OA; or

[0235] (b) A difference in the level of one or more GAG properties according to the present invention from a control level of the same one or more GAG properties (or a change (e.g., an increase or a decrease) compared to the control level) indicates RA, where the control level is within (or included in) a reference interval determined for the same sample type in a group of subjects (or reference population) who have never had RA.

[0236] In some embodiments, the group of subjects (or reference population) who have never had OA can be the group of subjects (or reference population) who have RA. In some embodiments, the group of subjects (or reference population) who have never had RA can be the group of subjects (or reference population) who have OA.

[0237] The concept of a "reference interval" (which may also be referred to as a "reference range") is well known in the art, particularly in the medical and health-related fields. A reference interval is a range (or interval) of values (e.g., levels) of a physiological measurement that has been established or determined (or has been established or determined to characterize or indicate) a particular health state for a reference population (or reference group of subjects).

[0238] In some cases, a reference interval is a range (or interval) of values (e.g., levels) of a physiological measurement that has been established or determined (or is established or determined to characterize or indicate a particular disease (or disorder) state) for a reference population with a given disease or disorder.

[0239] A reference interval for a particular measurement result or value (e.g., the level of one or more GAG properties according to the present invention) defines an interval (or range within which) a particular percentage (e.g., %) of the measurement results (or values) of the reference population fall. The percentage can be, for example, from 80% to 99%. For example, the percentage can be 80%, 85%, 90% or 95%. Preferably, the percentage is 95%. Generally and preferably, the percentage is the central percentage of the measurement results (or values) of the reference population. For example, when the percentage is 95% (preferably, the central percentage is 95%), the reference interval for a particular measurement result or value (e.g., the level of one or more GAG properties according to the present invention) is the interval (or range within which) 95% of the measurement results (or values) of the reference population fall. This means that 5% of the measurement results of such a reference population will be outside the reference interval. When the percentage 95% is the central percentage of the reference population (which is preferred), this means that 2.5% of the measurement results (or values) will be less than the lower limit of the reference interval and 2.5% of the measurement results (or values) will be greater than the upper limit of the reference interval. When the percentage 80% is the central percentage of the reference population, this means that 10% of the measurement results (or values) will be less than the lower limit of the reference interval and 10% of the measurement results (or values) will be greater than the upper limit of the reference interval. When the percentage 85% is the central percentage of the reference population, this means that 7.5% of the measurement results (or values) will be less than the lower limit of the reference interval and 7.5% of the measurement results (or values) will be greater than the upper limit of the reference interval. When the percentage 90% is the central percentage of the reference population, this means that 5% of the measurement results (or values) will be less than the lower limit of the reference interval and 5% of the measurement results (or values) will be greater than the upper limit of the reference interval.

[0240] The upper limit of a reference interval is generally referred to as the reference upper limit (URL). The lower limit of a reference interval is generally referred to as the reference lower limit (LRL). In some embodiments, the URL or LRL can be used with a cut-off or threshold level to provide an indication of OA or RA. In some embodiments, a change (either an increase or a decrease in level, depending on the circumstances) in the level of one or more GAG properties according to the present invention compared to (or relative to) the URL or LRL of the reference interval indicates OA or RA, as the case may be.

[0241] Those skilled in the art are familiar with reference intervals, and now the present inventors have found that certain levels of GAG properties can be used to screen for OA and RA according to the present invention. Those skilled in the art will be able to readily establish appropriate reference intervals (and associated URLs and LRLs) based on (or with respect to) an appropriate reference population for use in accordance with the present invention. As mentioned above, the concept of reference intervals has been 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 (EP28-A3c: Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline – Third Edition)" (2010, Volume 28, Number 30; ISBN 1-56238-682-4) provides a detailed discussion and guidance on this point.

[0242] A reference interval can be based on measurements (or values) obtained from (or of) any appropriate number of subjects in a reference population (i.e., the reference population can have any appropriate size). A person skilled in the art can readily select a reference population of appropriate size. Guidance on the appropriate size of a reference population is provided in the Clinical and Laboratory Standards Institute publication mentioned above. Preferably, the reference population has at least 10 subjects. In some embodiments, there can 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 can be at most 50, at most 100, at most 200, or at most 500 subjects in the reference population. In some embodiments, there can be from 10 to 20, 10 to 50, 10 to 100, 10 to 200, 10 to 500, or 10 to 1000 subjects in the reference population.

[0243] As is apparent from the foregoing, in some embodiments of the present invention that provide an indication of OA, the reference interval is a reference interval determined for the same sample type from a reference population group of subjects who have never had OA (e.g., have RA) for a given GAG property of the present invention. Also as is apparent from the foregoing, in some embodiments of the present invention that provide an indication of RA, the reference interval is a reference interval determined for the same sample type from a reference population group of subjects who have never had RA (e.g., have OA) for a given GAG property of the present invention.

[0244] In some embodiments of the methods of the present invention (e.g., screening or diagnostic or discriminatory methods),

[0245] (a) A level of one or more GAG properties according to the present invention that is higher than a control level of the same one or more GAG properties (or elevated compared to the control level) indicates OA, wherein the control level is within (or included in) a reference interval determined for the same sample type from a group of subjects (or reference population) who have never had OA; or

[0246] (b) A level of one or more GAG properties according to the present invention that is lower than a control level of the same one or more GAG properties (or decreased compared to the control level) indicates RA, wherein the control level is included within (or included in) a reference interval determined for the same sample type from a group of subjects (or reference population) who have never had RA.

[0247] In some embodiments, the group of subjects who have never had OA can be a group of subjects who have RA. In some embodiments, the group of subjects who have never had RA can be a group of subjects who have OA.

[0248] In some embodiments of the methods of the present invention (e.g., screening or diagnostic or discriminatory methods),

[0249] (a) A level of one or more GAG properties according to the present invention that is higher than a control level of the same one or more GAG properties (or elevated compared to the control level) indicates OA, wherein the control level is the upper reference limit of a reference interval determined for the same sample type from a group of subjects (or reference population) who have RA; or

[0250] (b) A level of one or more GAG properties according to the present invention that is lower than a control level of the same one or more GAG properties (or decreased compared to the control level) indicates RA, wherein the control level is the upper reference limit of a reference interval determined for the same sample type from a group of subjects (or reference population) who have RA.

[0251] In some embodiments of the method of the present invention (e.g., screening or diagnostic or discriminatory methods),

[0252] (a) A level of one or more GAG properties according to the present invention being lower than a control level of the same one or more GAG properties (or decreased compared to the control level) indicates OA, where the control level is the upper reference limit of a reference interval that has been determined for the same sample type obtained from a group of subjects with OA (or reference population); or

[0253] (b) A level of one or more GAG properties according to the present invention being lower than a control level of the same one or more GAG properties (or decreased compared to the control level) indicates RA, where the control level is the lower reference limit of a reference interval that has been determined for the same sample type obtained from a group of subjects with OA (or reference population).

[0254] As described above, the upper reference limit and the lower reference limit can in some cases be used as or in some cases can alternatively be regarded as cut-off levels or threshold levels, where an indication of OA or RA is achieved (or provided) based on the measured level of one or GAG properties according to the present invention relative to (or compared to) such cut-off limit.

[0255] In some preferred embodiments, the reference interval of a particular measurement result or value (e.g., the level of one or more GAG properties among the GAG properties according to the present invention) defines an interval (or range within which) 95% of the measurement results (or values) of the reference population fall.

[0256] In certain alternative embodiments, a reference (or control) range can be generated based on a 95% confidence interval (e.g., 95% confidence around the average measurement result of the OA or RA subject group). For example, in this regard and referring to the experimental examples herein, in urine, the reference range for the absolute concentration of 4S CS in the reference population of RA subjects can be from 0.939 µg / ml to 6.573 µg / ml. In some embodiments, an absolute concentration of 4S CS higher than 6.573 µg / ml (e.g., in urine) can indicate OA. In some embodiments, an absolute concentration of 4S CS lower than 6.573 µg / ml (e.g., in urine) 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 method of the present invention is not limited thereto.

[0257] In some embodiments of the method of the present invention (e.g., screening or diagnostic or discriminatory methods),

[0258] (a) A level of one or more GAG properties according to the present invention being higher than a control level of the same one or more GAG properties indicates OA, wherein the control level has been determined in the same sample type obtained from one or more subjects (e.g., a group or population of subjects) suffering from RA; or

[0259] (b) A level of one or more GAG properties according to the present invention being lower than a control level of the same one or more GAG properties indicates RA, wherein the control level has been determined in the same sample type obtained from one or more subjects (e.g., a group or population of subjects) suffering from OA.

[0260] In some embodiments of the method of the present invention (e.g., a screening or diagnostic or discriminating method),

[0261] (a) A level of one or more GAG properties according to the present invention being higher than a control level of the same one or more GAG properties indicates OA, wherein the control level is the average or median level determined in the same sample type obtained from a population of subjects suffering from RA; or

[0262] (b) A level of one or more GAG properties according to the present invention being lower than a control level of the same one or more GAG properties indicates RA, wherein the control level is the average or median level determined in the same sample type obtained from a population of subjects suffering from OA.

[0263] In some embodiments of the method of the present invention (e.g., a screening or diagnostic or discriminating method),

[0264] (a) A level of one or more GAG properties according to the present invention being higher than a control level of the same one or more GAG properties indicates OA, wherein the control level is a level that is at least 20% (or at least 30%, at least 40%, at least 50%, at least 100% or at least 200%) higher than the average or median level determined in the same sample type obtained from a population of subjects suffering from RA; or

[0265] (b) A level of one or more GAG properties according to the present invention being lower than a control level of the same one or more GAG properties indicates RA, wherein the control level is a level that is 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 average or median level determined in the same sample type obtained from a population of subjects suffering from OA.

[0266] In some embodiments of the method of the present invention (e.g., a screening or diagnostic or discriminating method),

[0267] (a) A level of one or more GAG properties according to the present invention that is higher than a control level of the same one or more GAG properties indicates OA, wherein the control level is a level determined by determining the level of the one or more GAG properties in the same sample type obtained from one or more subjects (e.g., a group of subjects) with RA and from one or more subjects (e.g., a group of subjects) with OA, and wherein the control level is a cut-off level that has been derived (or established or determined) to distinguish (or discriminate) a sample from an OA subject from a sample from an RA subject; or

[0268] (b) A level of one or more GAG properties according to the present invention that is lower than a control level of the same one or more GAG properties indicates RA, wherein the control level is a level determined by determining the level of the one or more GAG properties in the same sample type obtained from one or more subjects (e.g., a group of subjects) with RA and from one or more subjects (e.g., a group of subjects) with OA, and wherein the control level is a cut-off level that has been derived (or established or determined) to distinguish (or discriminate) a sample from a subject with OA from a sample from a subject with RA.

[0269] In some embodiments of the methods of the present invention (e.g., screening or diagnostic or discrimination methods),

[0270] (a) A change (preferably higher) in the level of one or more GAG properties according to the present invention compared to a control level of the same one or more GAG properties indicates OA, wherein the control level is a level determined by determining the level of the one or more GAG properties in the same sample type obtained from one or more reference subjects (e.g., reference subjects with RA) (e.g., a group of reference subjects), and wherein the control level is a cut-off level (or threshold level) that has been derived (or established or determined) to provide an indication of OA when the level of one or more GAG properties according to the present invention changes (preferably higher) compared to the control level; or

[0271] (b) A change (preferably lower) in the level of one or more GAG properties according to the present invention compared to a control level of the same one or more GAG properties indicates RA, wherein the control level is a level determined by determining the level of the one or more GAG properties in the same sample type obtained from one or more reference subjects (e.g., reference subjects with OA) (e.g., a group of reference subjects), and wherein the control level is a cut-off level that has been derived (or established or determined) to provide an indication of RA when the level of one or more GAG properties according to the present invention changes (preferably lower) compared to the control level.

[0272] In some embodiments of the present invention, a control level, such as a cut-off level (or threshold level), can be derived (or determined or established) to provide a confidence level for an indication (or for the indication) of OA or RA (as appropriate) according to the present invention, such as an (or at least) 80%, 85%, 90% or 95% confidence level for the indication (or for the indication) of OA or RA. Thus, in some embodiments of the present invention, when it is determined (or observed) that the level of one or more GAG properties according to the present invention changes (increases (or is higher) or decreases (or is lower) as appropriate) compared to a control level, such as a cut-off level or threshold level, a control level, such as a cut-off level (or threshold level), can be derived (or determined or established) to provide a confidence level for an indication (or for the indication) of OA or RA (as appropriate) according to the present invention. For example, a control level, such as a cut-off level (or threshold level), can be derived (or determined or established) to provide a confidence level (such as an (or at least) 80%, 85%, 90% or 95% confidence level) that an indication (such as a diagnosis) of OA or RA is accurate (or correct).

[0273] In some embodiments of the method of the present invention (such as a screening or diagnostic or discriminatory method),

[0274] (a) The level of one or more GAG properties according to the present invention indicates OA within a range (within the level range) that has been established to indicate that a sample is from an OA subject rather than from an RA subject; or

[0275] (b) The level of one or more GAG properties according to the present invention indicates RA within a range (within the level range) that has been established to indicate that a sample is from an RA subject rather than from an OA subject.

[0276] In some such embodiments, (i) the range of (a) is a level range determined by determining the levels of the one or more GAG properties in the same sample type obtained from a group of subjects with RA and from a group of subjects with OA and establishing a level range that indicates that a sample is from a subject with OA rather than from a subject with RA; and / or (ii) the range of (b) is a level range determined by determining the levels of the one or more GAG properties in the same sample type obtained from a group of subjects with RA and from a group of subjects with OA and establishing a level range that indicates that a sample is from a subject with RA rather than from a subject with OA.

[0277] In some embodiments of the method of the present invention (such as a screening or diagnostic or discriminatory method),

[0278] (a) The level of one or more GAG properties according to the present invention indicates OA within a reference interval (or range) that has been established (or determined) to indicate that the sample is from an OA subject; or

[0279] (b) The level of one or more GAG properties according to the present invention indicates RA within a reference interval (or range) that has been established (or determined) to indicate that the sample is from an RA subject.

[0280] In some embodiments of the methods of the present invention (e.g., screening or diagnostic or discriminating methods),

[0281] (a) The level of one or more GAG properties according to the present invention that has been established (or determined) to be a level indicating that the sample is from an OA subject indicates OA; or

[0282] (b) The level of one or more GAG properties according to the present invention that has been established (or determined) to be a level indicating that the sample is from an RA subject indicates RA.

[0283] In some embodiments of the methods of the present invention (e.g., screening or diagnostic or discriminating methods),

[0284] (a) The level of one or more GAG properties according to the present invention that has been established (or determined) to be a level indicating that the sample is from an OA subject rather than an RA subject indicates OA; or

[0285] (b) The level of one or more GAG properties according to the present invention that has been established (or determined) to be a level indicating that the sample is from an RA subject rather than an OA subject indicates RA.

[0286] A change (increase or decrease, as appropriate) in the level (or composition or score) of one or more GAG forms (GAG properties) in the GAG forms (GAG properties) according to the present invention includes any measurable change or variation in the GAG form (biomarker) (or score) being discussed when the GAG form being discussed is compared to a control level (e.g., a reference limit or cut-off level or threshold level). The change in level (or score) includes an increase or decrease in level (or score). Preferably, the level (or score) is significantly changed 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, the significant change in level or composition or score is statistically significant, preferably with a p-value < 0.05.

[0287] In some embodiments, a change in a 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., when compared to a control level) may indicate the presence of OA or RA, as appropriate, according to the present invention.

[0288] An "increase" or "elevated" level of one or more of the GAG forms (GAG properties) or scores as described herein includes any measurable increase or rise in the GAG form (biomarker) (or score) under discussion when 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, the significant increase in the level (or score) is statistically significant, preferably with a p-value < 0.05.

[0289] In some embodiments, an increase in a 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., when 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.

[0290] A "decrease" or "reduced" level of one or more of the GAG forms (GAG properties) or scores as described herein includes any measurable decrease or drop in the GAG form (biomarker) (or score) under discussion when compared to a control level (or control score or cut-off level). Preferably, the level (or score) is significantly decreased 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, the significant decrease in the level (or score) is statistically significant, preferably with a p-value < 0.05.

[0291] In some embodiments, a reduction in a level (or score) of ≥2%, ≥3%, ≥5%, ≥10%, ≥25%, ≥50%, ≥75%, ≥80%, ≥90%, ≥95%, ≥99% compared to the level (or score) found in an appropriate control sample or subject or population (i.e., when 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 invention.

[0292] "Control level" is discussed elsewhere herein. A "control level" may be the level of a relevant GAG property in a control subject or population (e.g., in a sample obtained from a control subject or population). A "control level" may be the level of a relevant GAG property, where the level is derived from (or is based on or is calculated according to) the level in a control subject or population (e.g., in a sample obtained from a control subject or population). Such a population may be referred to as a reference population. A person of ordinary skill in the art will readily identify an appropriate control subject (or population) or sample for use in the methods of the invention. Suitable control levels are described elsewhere herein. A control level may correspond to the level of an equivalent (corresponding) GAG form in an appropriate control subject or sample or population, e.g., may correspond to a cut-off or threshold level or range found in a control or reference population. A control level may also be referred to as a "reference" level. A control level may be a discrete number or a range.

[0293] Although a control level for comparison may be derived by testing an appropriate control subject or a group of control subjects (or control population), the methods of the invention will not necessarily involve the active testing of a control subject as part of the methods of the invention, but will generally involve comparison to a control level that has been previously determined from a control subject (or control population) and is known to the person performing the methods of the invention.

[0294] "Control chemical composition" is the 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 level" (e.g., appropriate control subjects, control samples, control populations, etc.) applies mutatis mutandis to "control chemical composition".

[0295] 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 represent a level and / or chemical composition determined according to the present invention. In some such embodiments, a change in the score (e.g., an increase or decrease, depending on the circumstances) as compared to a "control score" (or cut-off level or threshold level) indicates OA or RA (as the case may be) in the subject. The discussion elsewhere herein regarding "control levels" (e.g., appropriate control subjects, control samples, control populations, etc.) is applicable, mutatis mutandis, to "control scores".

[0296] As described elsewhere herein, in some preferred methods of the present invention, the method comprises determining the level and / or chemical composition (or a score based thereon) of the protein-free fraction of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) GAGs according to the present invention. In some such embodiments, a change in the level and / or chemical composition (or a score based thereon) of the protein-free fraction as compared to the level and / or chemical composition of one or both of said GAGs in a control (e.g., a control sample or a control score or cut-off level) indicates OA or RA (as the case may be). The discussion elsewhere herein regarding "control levels" or "control chemical compositions" or "control scores" (e.g., appropriate control subjects, control samples, control populations, etc.) is applicable, mutatis mutandis, to embodiments of the present invention that comprise determining the level and / or chemical composition (or a score based thereon) of the protein-free fraction of one or both of said GAGs according to the present invention.

[0297] The methods of the present invention can also be used to monitor OA progression (e.g., OA worsening) or RA progression (e.g., RA worsening). Such monitoring can be carried out before, during, or after treating OA or RA by surgery or therapy (e.g., pharmaceutical therapy). Thus, in another aspect, the present invention provides a method for monitoring OA or RA progression in a subject suffering from OA or RA. In such methods for monitoring OA or RA progression in a subject, the level (or a score derived therefrom or based thereon) of one or more GAG properties according to the present invention indicates OA or RA progression (as the case may be).

[0298] Thus, in another aspect, the present invention provides a method for monitoring osteoarthritis (OA) or rheumatoid arthritis (RA) progression in a subject suffering from arthritis (OA or RA), the method comprising:

[0299] determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample,

[0300] 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) the total concentration of CS, and (v) the total concentration of HS,

[0301] wherein the sample has been obtained from the subject.

[0302] In an embodiment of monitoring OA progression, the subject is a subject with OA.

[0303] In an embodiment of monitoring RA progression, the subject is a subject with RA.

[0304] In a preferred embodiment of the method of monitoring OA or RA progression according to the present invention, compared with a control level, a change (either an increase or a decrease depending on the case) in the level of one or more GAG properties (4S CS (e.g., the absolute concentration of 4S CS), NS HS (e.g., the absolute concentration and / or relative concentration of NSHS), 0S HS (e.g., the absolute concentration of 0S HS), the total concentration of CS, and / or the total concentration of HS) among the GAG properties according to the present invention indicates the progression of OA or RA in the subject.

[0305] In some embodiments of the method of monitoring progression according to the present invention

[0306] (a) Monitoring OA progression, and a change (or difference) over time in the level of one or more GAG properties according to the present invention compared with a control level of the same one or more GAG properties, preferably an increase (or higher), indicates OA progression, wherein the control level has been determined in the same sample type obtained from one or more subjects who have never had OA, or has been determined in the same sample type obtained from an earlier sample taken from the subject whose OA progression is being monitored; or

[0307] (b) Monitoring RA progression, and a change (or difference) over time in the level of one or more GAG properties according to the present invention compared with a control level of the same one or more GAG properties, preferably a decrease (or lower), indicates RA progression, wherein the control level has been determined in the same sample type obtained from one or more subjects who have never had RA, or has been determined in the same sample type obtained from an earlier sample taken from the subject whose RA progression is being monitored.

[0308] In some embodiments of the method of monitoring OA progression according to the present invention, the control level can be the level of one or more GAG properties according to the present invention determined in the same sample type obtained from one or more subjects who have never had OA (e.g., a group of subjects (e.g., a reference subject group)).

[0309] In some embodiments of the method of monitoring RA progression according to the present invention, the control level can be the level of one or more GAG properties according to the present invention determined in the same sample type obtained from one or more subjects who have never had RA (e.g., a group of subjects (e.g., a reference subject group)).

[0310] In some preferred embodiments of the method of monitoring progression according to the present invention,

[0311] (a) monitoring OA progression, and an increase over time in the level of one or more GAG properties according to the present invention compared to a control level of the same one or more GAG properties indicates OA progression, wherein the control level has been determined in the same sample type obtained from an earlier sample taken from the subject whose OA progression is being monitored; or

[0312] (b) monitoring RA progression, and a decrease over time in the level of one or more GAG properties according to the present invention compared to a control level of the same one or more GAG properties indicates RA progression, wherein the control level has been determined in the same sample type obtained from an earlier sample taken from the subject whose RA progression is being monitored.

[0313] Thus, in some embodiments of the method of monitoring OA progression according to the present invention, the control level is the level determined in the same sample type obtained from an earlier (e.g., first) sample taken from the subject whose OA progression is being monitored.

[0314] In some embodiments of the method of monitoring RA progression according to the present invention, the control level is the level determined in the same sample type obtained from an earlier (e.g., first) sample taken from the subject whose RA progression is being monitored.

[0315] Levels from an earlier (e.g., first) sample from a subject whose OA or RA progression (as appropriate) is being monitored can be considered the "baseline" level of the subject. This type of control level (i.e., a control level from an individual subject) is particularly useful for embodiments of the present invention in which serial or periodic measurements of GAG forms in an individual are made to look for changes in the levels of the GAG forms. In this regard, an appropriate control level can be the individual's own baseline value, steady value, zero value, previous value, or dry value (as appropriate), rather than a control or cut-off level found in a general control population. Alternatively, a control level for a method of monitoring OA or RA (as appropriate) progression according to the present invention can correspond to the level of the marker (GAG form) being discussed measured at an earlier time point in the same individual subject or in a sample from said subject (e.g., the "baseline" level of the subject).

[0316] In some embodiments, levels of one or more GAG properties according to the present invention indicate OA progression, preferably where the level is high (or higher) or elevated (or continuously elevated) (e.g., as measured over time, e.g., by making serial or periodic measurements) indicates OA progression (OA worsening).

[0317] In some embodiments, levels of one or more GAG properties according to the present invention indicate RA progression, preferably where the level is low (or lower) or decreased (or continuously decreased) (e.g., as measured over time, e.g., by making serial or periodic measurements) indicates RA progression (RA worsening).

[0318] In a preferred method of monitoring progression according to the present invention, the body fluid sample is a urine sample (or a processed urine sample).

[0319] Preferred GAG properties (or groups of GAG properties) for use in conjunction with a method of monitoring progression according to the present invention are discussed elsewhere herein in connection with other aspects of the present invention. For example, 4S CS and NS HS are generally preferred.

[0320] Sample processing steps and / or detection (or measurement methods, etc.) for use in conjunction with a method of monitoring progression according to the present invention are discussed elsewhere herein in connection with other aspects of the present invention. In fact, unless otherwise explicitly stated from the context, features described elsewhere herein in connection with screening methods (e.g., diagnosis, etc.) are also applicable to the method of monitoring progression of the present invention.

[0321] In another aspect, the present invention provides a method of providing information for monitoring the progression of osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject suffering from arthritis, the method comprising:

[0322] Determine the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS), which are glycosaminoglycans (GAGs), in a body fluid sample,

[0323] wherein 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) the total concentration of CS, and (v) the total concentration of HS,

[0324] wherein the sample has been obtained from the subject. The information is of course typically the determined level of one or more of the GAG properties.

[0325] The methods of the invention can be used to actively monitor patients who have not undergone surgery or therapy, for example to monitor the progression of OA or RA (as appropriate) in untreated patients. Similarly, serial measurements can allow assessment of whether OA or RA (as appropriate) is worsening or the extent to which OA or RA is worsening, thus for example allowing more rational decisions to be made about whether treatment or surgical intervention is necessary or desirable.

[0326] In another aspect, the invention provides a method for determining the clinical severity of OA or RA (as appropriate) in a subject. In such methods, the level (or a score derived from or based on it) of one or more of the GAG forms according to the invention in the sample shows an association with the severity of OA or RA (as appropriate). Thus, the level of one or more of the GAG forms according to the invention can indicate the severity of OA or RA. In some embodiments, the more the level of one or more of the GAG forms according to the invention changes (increases or decreases as appropriate) compared to a control level (e.g., a control level as described elsewhere herein), the greater the likelihood of a more severe form of OA or RA (as appropriate). Thus, in some embodiments, the methods of the invention can be used to select patients for treatment.

[0327] Serial (periodic) measurements of the levels of one or more GAG forms (biomarkers) in accordance with the present invention (biomarkers), or a score derived from or calculated based thereon, can also be used to monitor the severity of OA or RA, as the case may be, by observing whether the levels increase or decrease over time. Observation of a change in the level (increase or decrease, as the case may be) can also be used to guide and monitor therapy (or other clinical decisions), for example, in the case of "watchful waiting" before treatment or surgery (e.g., before the start of drug therapy or surgery) or during or after treatment, to evaluate the effectiveness of the treatment and to look for signs of treatment failure.

[0328] The present invention also provides a method of patient selection or treatment selection, as it provides a means of differentiating (or discriminating) patients with OA from patients with RA. Thus, from another perspective, the method of the present invention provides a method for differentiating (or discriminating) OA from RA. Such methods can guide appropriate treatment.

[0329] The present invention also provides a method of patient selection or treatment selection, as it provides a means of differentiating (or discriminating) patients with severe OA from patients with less severe OA (e.g., identifying or providing an indication of patients with OA at a particular clinical stage (e.g., stage 1, 2, 3, or 4 OA)).

[0330] The present invention also provides a method of patient selection or treatment selection, as it provides a means of differentiating (or discriminating) patients with severe RA from patients with less severe RA (e.g., identifying or providing an indication of patients with RA at a particular clinical stage (e.g., stage 1, 2, 3, or 4 RA)). Classification of OA or RA at a given stage can be made according to any definition recognized and accepted in the art. Those skilled in the art are familiar with the staging systems and conventions for OA and RA.

[0331] In some embodiments, the present invention provides a method of monitoring (e.g., continuously monitoring or actively surveilling) 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 treatment should not be given.

[0332] In some embodiments, patients with less severe (or early) OA or RA can be placed under watchful waiting or active surveillance and treatment (e.g., drug therapy or surgery) may not be given. In some embodiments, patients with severe or more severe (or late) OA or RA can be given treatment.

[0333] The present invention also provides a method for determining (or monitoring) the efficacy of a treatment regimen for treating OA or RA, as the case may be (in other words, tracking or monitoring the response to treatment). In such methods, a change (either an increase or a decrease, as the case may be) in the level (or score) of one or more GAG properties according to the GAG properties of the present invention indicates the efficacy of the treatment regimen being used. For example, in some embodiments, if an increase in the level (or score) indicates a decrease in the level (or score derived from (or based on)) one or more GAG forms according to the GAG forms of the present invention for OA during (or after) the therapy, this indicates that the treatment regimen is effective. As another example, in some embodiments, if a decrease in the level (or score) indicates an increase in the level (or score derived from (or based on)) one or more GAG forms according to the GAG forms of the present invention for RA during (or after) the therapy, this indicates that the treatment regimen is effective. In such methods, continuous (periodic) measurement of the level of one or more GAG properties (biomarkers) according to the GAG properties of the present invention can also be used to determine the efficacy of the treatment regimen being used. Similar methods can be used to provide a method for determining (or monitoring) the efficacy of a surgical regimen for treating OA or RA.

[0334] The methods of the present invention can be performed on any suitable body fluid sample. Generally, the sample has been obtained (removed) from a subject (e.g., as described elsewhere herein), preferably a human subject. In other aspects, the method further includes the step of obtaining a sample from the subject.

[0335] As used herein, the term "body fluid" includes reference to all fluids derived from a subject's body. The body fluid or sample can 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 obtainable body fluid. Thus, in a preferred embodiment, the level of the GAG property according to the present invention is the level in a urine sample.

[0336] The term "sample" also encompasses any material derived by processing a body fluid sample (e.g., derived by processing a urine sample). Thus, the term "sample" includes a processed sample (e.g., a processed urine sample). Processing a biological sample to obtain a test sample can involve one or more of the following: 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 according to the characteristics of the method being performed.

[0337] Any suitable method for separating a body fluid sample (e.g., a urine sample) can be employed.

[0338] Any sample that may be directly or indirectly affected by OA or RA can be used. Samples (e.g., raw or untreated samples) typically contain a protein-free fraction of GAG and a protein-bound fraction of GAG (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 chondroitin sulfate (CS) and heparan sulfate (HS) of GAG is determined. Thus, in certain preferred embodiments, the body fluid sample has been processed (or is processed) such that subsequently only (or substantially only) the protein-free fraction of the GAG (usually the disaccharide units derived therefrom) is analyzed (i.e., is processed such that subsequently the level and / or chemical composition of the protein-free fraction of the one or two GAGs (usually the disaccharide units derived therefrom) is determined). In other methods of the present invention, the sample has been processed (or is processed) such that subsequently the intact (protein-free plus protein-bound) fraction or pool of the one or two GAGs is analyzed (i.e., is processed such that subsequently the level and / or chemical composition of the intact (protein-free plus protein-bound) fraction or pool of the one or two GAGs is determined).

[0339] The term "sample" also encompasses any material derived by processing a biological sample (e.g., as described above). Derived materials include disaccharide units (or populations of disaccharide units) derived by processing GAG (e.g., as described elsewhere herein).

[0340] In some embodiments, the method of the present invention may include the step of processing a sample. Thus, in some embodiments, the method of the present invention may be performed on such processed samples or materials derived from such processed samples. Thus, in some embodiments, the method of the present invention may be performed on samples that have been processed. The processing step includes, but is not limited to, separating cells from the sample, separating cell components from the sample, and extracting (e.g., separating or purifying) proteins / peptides (although as certain preferred methods of the present invention involve determining the protein-free GAG fraction, in some embodiments, it is preferably not necessary to extract proteins or remove protein components from proteoglycans (protein-bound GAGs) present in the sample, e.g., by digestion or otherwise removing protein components). The processing step 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.

[0341] The sample can be used immediately or can be stored for later use (e.g., stored at -80 °C).

[0342] The methods of the invention described herein can be performed on any type of subject who can suffer from OA or RA. The methods are generally performed on mammals (e.g., humans, primates (e.g., monkeys), laboratory mammals (e.g., mice, rats, rabbits, guinea pigs), domestic mammals (e.g., horses, cows, sheep, pigs) or domestic pets (e.g., cats, dogs)). Preferably, the subject is a human.

[0343] In some embodiments of the invention, the subject (e.g., a human) is a subject suffering from arthritis. Thus, in some embodiments, the subject is a subject who has been 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 progress according to the invention, the subject is a subject suffering from arthritis (suffering from OA or suffering from RA).

[0344] In some aspects, there are provided methods of the invention that further comprise the step of treating OA or RA, as appropriate, by therapy (e.g., pharmaceutical therapy) or surgery or other clinical management regimens. Methods of treating OA and RA by therapy or surgery or by prescribing another clinical management regimen are known in the art.

[0345] In some embodiments, if the results of the methods of the invention indicate OA in the subject (or indicate OA progression (deterioration)) (e.g., if a positive indication or diagnosis of OA is made), then additional steps of treating the OA by therapy or surgery or providing a clinical management regimen suitable for OA can be performed. For example, if the results of the methods of the invention indicate OA in the subject (or indicate OA progression (deterioration)), then the subject can be instructed (or advised) to make certain lifestyle changes (e.g., exercise or exercise more regularly), the subject can be instructed (or advised) to lose weight (e.g., if the subject is overweight), the subject can be instructed (or advised) to wear appropriate (or more appropriate) footwear, and / or the subject can be instructed (or advised) to use a device to reduce strain on the joints. As another example, if the results of the methods of the invention indicate OA in the subject (or indicate OA progression (deterioration)), then alternatively or additionally a physical therapy regimen can be prescribed for the subject, and / or pain medications can be prescribed for the subject, and / or the subject may be a candidate for joint replacement surgery.

[0346] In some embodiments, if the results of the methods of the invention indicate RA in a subject (or indicate progression (deterioration) of RA) (e.g., make a positive indication or diagnosis of RA), then an additional step of treating the RA by therapy or surgery or providing a clinical management program suitable for RA may be carried out. For example, if the results of the methods of the invention indicate RA in a subject (or indicate progression (deterioration) of RA), then the subject may be treated with (or prescribed) the following: disease-modifying antirheumatic drugs (DMARDs) (such as methotrexate, leflunomide, hydroxychloroquine or sulfasalazine), and / or JAK inhibitors, and / or painkillers, and / or steroids, and / or non-steroidal anti-inflammatory drugs (NSAIDs), and / or biologics (such as adalimumab, etanercept or infliximab). As another example, if the results of the methods of the invention indicate RA in a subject (or indicate progression (deterioration) of RA), then alternatively or additionally a physical therapy program may be prescribed to the subject, and / or the subject may be a candidate for joint replacement surgery.

[0347] In some embodiments, if the level (or a score based on these levels) of one or more GAG properties according to the invention in a sample changes by a certain degree compared to a control level or score (or cut-off level), then an additional step of administering a therapeutically effective amount of a pharmaceutical formulation to the patient and / or performing surgery is carried out. The preferred degree of change is discussed elsewhere herein.

[0348] In some aspects, provided are methods of the invention that further include the step of performing (or carrying out) additional diagnostic or screening procedures for OA or RA, as appropriate, e.g., as discussed elsewhere.

[0349] In another aspect, provided is a kit for screening for OA or RA (e.g., for diagnosis or for determining the severity or progression of OA or RA or for differentiating OA from RA), the kit comprising one or more agents adapted to determine the level of one or more GAG properties (GAG forms) according to the invention described herein in a sample. In another aspect, provided is a kit for screening for OA or RA (e.g., for diagnosis or for determining the severity or progression of OA or RA), the kit comprising one or more reagents (or components) for processing a body fluid sample (e.g., urine) containing GAGs whose levels and / or chemical composition are determined according to the invention. In a preferred aspect, the kit is for use in the methods of the invention as described herein. Preferably, the kit comprises instructions for use of the kit components, e.g., in a screening (e.g., diagnosis) according to the invention.

[0350] In another aspect, the present invention provides a method for determining (or detecting) the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample,

[0351] wherein 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) the total concentration of CS, and (v) the total concentration of HS,

[0352] wherein the sample has been obtained from a subject having or suspected of having OA or RA.

[0353] In one aspect, the present invention provides a method for detecting (or determining) the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs), the method comprising:

[0354] (a) obtaining a body fluid sample from a human patient having or suspected of having OA or RA; and

[0355] (b) detecting (or determining) the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in the sample,

[0356] wherein 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) the total concentration of CS, and (v) the total concentration of HS.

[0357] Yet another aspect of the present invention provides a method for detecting (or determining) the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS) glycosaminoglycans (GAGs) in a body fluid sample,

[0358] wherein 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) the total concentration of CS, and (v) the total concentration of HS,

[0359] wherein the sample has been obtained from the subject having or suspected of having OA or RA and has been processed prior to determining the level and / or chemical composition,

[0360] wherein said treatment

[0361] (a) includes fragmenting said one or two GAGs into disaccharide units; and

[0362] (b) does not include at least one of the following operations before (a):

[0363] (i) contacting said sample with a proteolytic agent; and

[0364] (ii) purifying said one or two GAGs in said sample based on the negative charge of said GAG.

[0365] The features and discussions herein regarding methods for screening for OA or RA (e.g., diagnostic methods, etc.) (e.g., regarding preferred GAG forms or combinations thereof for measurement) can be applied to the detection method of the present invention with necessary modifications.

[0366] When the terms "comprising", "including", "containing", "having", or "with" or other equivalent terms are used herein, then in some more specific embodiments, these terms include the terms "consisting of" or "consisting essentially of" or other equivalent terms. Detailed Description

[0367] The present invention will be further described with reference to the following non-limiting examples:

[0368] Example

[0369] Introduction

[0370] We studied the urinary glycosaminoglycan (GAG) profile or GAGome, which contains chondroitin sulfate (CS) disaccharides and heparan sulfate (HS) disaccharides as biomarkers for osteoarthritis (OA) and rheumatoid arthritis (RA). In this study, the GAGome (or free GAGome) of the protein-free fraction of GAGs in urine samples was studied.

[0371] Patients and Methods

[0372] We conducted a prospective single-center case-control study. The study population consisted of patients with OA or RA. The inclusion criteria were:

[0373] For the OA group: diagnosed with osteoarthritis (knee pain > 6 months and radiologically confirmed); age ≥ 18 years;

[0374] For the RA group: diagnosed with rheumatoid arthritis (confirmed by meeting the American College of Rheumatology RA criteria); age ≥ 18 years.

[0375] No patients were excluded. All patients signed the informed consent form. Any on-site voided urine samples from eligible patients were obtained in a single collection cup during a single visit and were subsequently stored at -20 °C until analysis. Patients were divided into two groups according to the included OA and RA groups. Patient characteristics are summarized in Table 1.

[0376] Table 1: Characteristics of the study population

[0377]

[0378] We used 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) in a single-blind laboratory to measure the urine GAGome. Briefly, the GAGome extraction from each sample was performed according to the instructions of the Elypta MIRAM ® Free GAG kit. All reagents and consumables used were included in the kit. The method used for GAGome extraction included an enzymatic digestion assay using chondroitinase ABC and heparinase I-II-III to depolymerize GAG in the sample into disaccharides. Note that compared with other methods described in the art (see: Volpi et al., Nature Protocols, Vol. 9, pp. 541-558 (2014)) (where the use of non-specific proteases for biofluid analysis is recommended), this method omits the addition of proteases and thus limits the analysis to the protein-free fraction of GAG. Note that compared with Volpi et al., the method used in this study omits the step of purifying GAG using anion exchange resin.

[0379] Subsequently, the GAG disaccharides were labeled with 2-aminoacridone. Then the treated samples were injected into an ultra-high performance liquid chromatography (UHPLC) coupled with 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. The peaks of GAG disaccharides were obtained at the predetermined retention times of six transitions using multiple reaction monitoring (MRM) analysis implemented in the mass spectrometry software (Waters ® 9 TargetLynx). We used the mass spectrometry software (Waters® Perform peak integration, calibration curve construction, and quantification using TargetLynx).

[0380] We exported the results in Excel format, i.e., the processed data, and imported it into R (4.0.2) for comparing the levels of each free GAGome feature between OA and RA. A two-sided t-test was used to evaluate the differences between groups. A p-value < 0.05 was considered statistically significant.

[0381] The measured GAG spectrum (GAGome) consists of the absolute concentrations of 17 GAG disaccharides, corresponding to 8 different sulfation patterns of chondroitin sulfate (CS) and heparan sulfate (HS), and hyaluronic acid (HA) disaccharides. Specifically, we quantified 8 CS disaccharides (0S CS, 2S CS, 6S CS, 4S CS, 2S6S CS, 2S4S CS, 4S6S CS, Tris CS) and 8 HS disaccharides (0S HS, 2S HS, 6S HS, NS HS, NS6S HS, NS2S HS, 2S6S HS, Tris HS). The extended GAGome includes an additional 22 dependent features: the total CS and total HS concentrations as the sum of the corresponding disaccharide concentrations, CS and HS loads, two ratios (4SCS / 0S CS and 6S CS / 0S CS), and the relative concentration (or mass fraction, in %) of each of the 16 CS and HS disaccharides by normalizing their absolute concentrations with the total CS and HS concentrations, respectively. Thus, for urine samples, the GAGome consists of 39 GAG features.

[0382] Results

[0383] In most patients of this study, the free GAGome features listed in Table A (features with the suffix [ug / ml] in Table A) were detected (in at least 10 out of a total of 20 patients in this study, the free GAGome feature > 0.1 ug / mL). Specifically, six CS disaccharides (0S CS, 6S CS, 4S CS, 2S6S CS, 2S4S CS, 4S6S CS) with a concentration > 0.1 µg / ml were detected in samples obtained from at least 10 out of a total of 20 patients in this study, and two HS disaccharides (0S HS, NS HS) with a concentration > 0.1 µg / ml were detected in samples obtained from at least 10 out of a total of 20 patients in this study. The free GAGome was also extended to include additional dependent features: the total CS concentration as the sum of the concentrations of the above-mentioned 8 CS disaccharides and the total HS concentration as the sum of the concentrations of the above-mentioned 8 HS disaccharides, the CS and HS loads, two ratios (4S CS / 0S CS and 6S CS / 0S CS), and the relative concentration (or mass fraction, in %) of each of the above-mentioned six CS disaccharides (0S CS, 6S CS, 4S CS, 2S6S CS, 2S4S CS, 4S6S CS) and two HS disaccharides (0S HS, NS HS), which was calculated by normalizing their absolute concentrations with the total CS and HS concentrations, respectively.

[0384] Table A

[0385]

[0386] A statistically significant difference (p < 0.05) in the levels of 4S CS (µg / ml) and NS HS (µg / ml) was observed, where samples from OA patients showed a concentration of these disaccharides approximately 2 times higher than that of RA patients. This data also pointed out other differences (p < 0.1) between OA and RA, including an increase in 0S HS (µg / ml), the total concentration of CS, and the total concentration of HS in OA compared to RA. Although the sample size may not be sufficient to meet statistical significance in the case of observing an increase in 0S HS (µg / ml), the total concentration of CS, and the total concentration of HS in OA compared to RA, an obvious increasing trend of these GAGome features was observed.

[0387] In summary, the data clearly support the view that OA and RA exhibit different urinary GAGome profiles, with several 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. In addition, the biomarkers according to the present invention can be used to distinguish OA from RA by, for example, establishing reference intervals around one or more disaccharides that have been identified herein as being different in OA and RA.

Claims

1. A method for differentiating osteoarthritis (OA) and rheumatoid arthritis (RA) in a subject with arthritis or suspected of having arthritis, the method comprising: determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS), which are glycosaminoglycans (GAGs), in a urine sample, wherein 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) the total concentration of CS, and (v) the total concentration of HS, wherein the sample has been obtained from the subject.

2. The method according to claim 1, wherein (a) a level of one or more of the GAG properties that is higher than a control level of the same one or more GAG properties indicates OA, wherein the control level has been determined in the same sample type obtained from one or more subjects with RA; or (b) a level of one or more of the GAG properties that is lower than the control level of the same one or more GAG properties indicates RA, wherein the control level has been determined in the same sample type obtained from one or more subjects with OA.

3. The method according to claim 1, wherein (a) a level of one or more of the GAG properties that is different from a control level of the same one or more GAG properties indicates OA, wherein the control level is within a reference interval determined for the same sample type obtained from a group of subjects who have never had OA; or (b) a level of one or more of the GAG properties that is different from a control level of the same one or more GAG properties indicates RA, wherein the control level is within a reference interval determined for the same sample type obtained from a group of subjects who have never had RA.

4. The method according to any one of claims 1 to 3, 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 following GAG properties: 4S CS and NSHS.

5. The method according to any one of claims 1 to 4, wherein 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) the absolute concentration of 4S CS, (ii) the absolute concentration and / or relative concentration of NS HS, (iii) the absolute concentration of 0S HS, (iv) the total concentration of CS; and (v) the total concentration of HS.

6. The method according to 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 two GAG properties selected from the group consisting of the absolute concentration of 4S CS and the absolute concentration of NS HS.

7. The method according to any one of claims 1 to 6, wherein the method comprises determining the levels of more than one GAG property among the GAG properties, preferably the method comprises determining the levels of two or more, three or more, four or more or all of the GAG properties.

8. The method according to any one of claims 1 to 7, wherein the determining the level and / or chemical composition is 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. The method according to any one of claims 1 to 8, wherein the sample has been obtained from the subject and has been subjected to at least one processing step before determining the level and / or chemical composition.

10. The method according to any one of claims 1 to 9, wherein the level of one or more specific sulfated or non-sulfated forms of CS disaccharide or HS disaccharide is determined, and wherein the GAG has been or is subjected to a processing step to obtain disaccharide units for analysis.

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

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

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

14. The method according to claim 12 or claim 13, wherein the fragmentation of (a) is carried out by contacting the one or two GAGs with one or more GAG lyases.

15. The method according to claim 14, wherein the one or more GAG lyases are one or more chondroitinases and / or one or more heparinases.

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

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

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

19. The method according to any one of claims 1 to 18, wherein the subject is a subject suffering from arthritis.

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

21. A method for monitoring the progression of osteoarthritis (OA) or rheumatoid arthritis (RA) in a subject suffering from arthritis, the method comprises: determining the level and / or chemical composition of one or both of chondroitin sulfate (CS) and heparan sulfate (HS), which are glycosaminoglycans (GAGs), in a urine sample, wherein 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) the total concentration of CS; and (v) the total concentration of HS, wherein the sample has been obtained from the subject.

22. The method according to claim 21, wherein (a) monitoring the progression of OA, and a change over time in the level of one or more of the GAG properties compared to a control level of the same one or more GAG properties indicates progression of OA, wherein the control level has been determined in the same sample type obtained from one or more subjects who have never had OA, or has been determined in the same sample type obtained from an earlier sample taken from the subject whose progression of OA is being monitored; or (b) monitoring the progression of RA, and a change over time in the level of one or more of the GAG properties compared to the control level of the same one or more GAG properties indicates progression of RA, wherein the control level has been determined in the same sample type obtained from one or more subjects who have never had RA, or has been determined in the same sample type obtained from an earlier sample taken from the subject whose progression of RA is being monitored.

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

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