WO2013067358A2 - Detection of osteoarthritis - Google Patents

Abstract

Methods for determining osteoarthritis severity, risk, progression and methods of treatment using biomarkers in biological samples from a patient.

Description

DETECTION OF OSTEOARTHRITIS

BACKGROUND

[0001] Osteoarthritis (OA) is degenerative joint disease that leads to ermanent joint damage and is a m lti-fectcrial complex disease. The diar hrodia.! joint's diseased tissues produce local low grade inflammation wise!- eventually leads to chronic pain, loss of cartilage and function- These findings have been shown by studying the localized tissues- There is QBE ently great interest in. the field of OA to identify biomarkers that provide a method for earlier diagnosis and dentify patients at higher risk for disease progression. However,, there are many obstacles to studying OA, mchsding heterogeneity in etiology, f iabil ty in progression of disease, and long time periods to see structural changes,

[0002] Accordingly,, the inventors have identified a need in the art to detect biomarkers in serum or plasma that are related to OA so that OA can be more readily or effectively diagnosed and staged, risk for developing OA can be more readily or effectively assessed, and patients who are respooders and non-respoiiders t OA theiapy can be more readily or effectively identified.

SUMMARY

[0003] In one aspect,, the disclosure relates to a method for predictin the progression of osteoarthritis is. a patient. The method includes determini an. amount of a

etailoprc4einase in a biological sample from the patient, comparing the amount of the aietalloproteinase in. the sample to a threshold concentration, mid predicting me progression of osteoarthritis when the concentration of the metalloproteinase in me sample is above the tfareshold concemxation.

[0004] In another aspect,, the disclosure relates to a. method for identifying patients at risk for progression of osteoarthritis. The method includes determining an amount of a metalloprotdnase in a biological sample from the patient, comparing the amount of the aietalloproteinase in. the sample to a threshold concentration, and identifying that the

I patient will have a progression of osteoarthritis when the concentration of ilie

metaJloproieinase: m the sample is above ilie threshold concentration.

[0005] Frsiher, the disclosure relates to a method for predicting the progression of osteoarthritis in a pattest. The metho includes determining an amount of a

metalloproieinase in a bi ological sample from the patient, comparing the amount of the metailopi^"oiei-iase m the patient sample to a reference sample, and predicting the

progression of osteoarthritis in. a patient if the amount of inetalioproieinase in the biological sample is above the amount of the metalloproteinase in the reference sample.

[0006] Still further, the disclosure relates to a method for diagnosing osteoarthritis in a patient The method includes determining as amount of a metaHoproteinase in a biological sample from the patient, comparing the amount of a metaUoproteiaase in the sample to a threshold concentration representing the concentration, in a non-osteoarthritic population, and deteaniniag that the patient has osteoarthriti s when tlie concentration of a melalloproteiiiase in the sample is above the threshold concentration.

[0007] In yet another aspect the disclosure relates to a method for detecting osteoarthritis in a patient The method i clud detecting an amount of metalloprote nase in a biological sample fiom the patient and correlating the amou t of metaMoproteinase to osteoarthriti in Sh patient.

[0O0S] In various aspects of the disclosure, the melalloproteinase is m trix melalloproteinase (MMP-9), for example total MMP-9.

[0009] For instance, the disclosure relates to a method of determining tlie progr ession of osteoarthritis in a patient. The method includes determining an amount of MMP-9 in a first biological sample from a patient at a first time point; determining an amount of MMP-9 in a second biological sample from a patient at a second time point; comparing the amount of MMP-9 in first sample to the amount of MMP-9 in the second sample;, and determining that osteoarthritis has progressed, in the patient by determining that the amount of MMP-9 in the seco d sample is greater than tlie amount of MMP-9 in the second sample.. [0010] In oilier aspects, the disclosure is related to methods of determining a coerce of treatment for arthritis is. a patient and methods of identifying patients with worsening

BRIEF DESCRIPTIO OF THE FIGURES

[DDI 1] Figure ί is a receiver operating characteristic (ROC) carve that sho s tMMP- 9 levels correlate with KL scores sad predict radiographic severity of S OA. For radiographic everity;, standardized semi-Sexed radiographs were scored for overall Kellgren and Lawrence (KL) _.grade. Biotnaarker levels were evaluated ^'between KL scores of 1, 2 with those &h scores of 3, 4. Total MMF-9 positively correlated with KL score both at baseline (r=0.220; p<0..03€) and 24 months (r=G.249; ρ Β.015).

[0012] Figure- 2. is an ROC carve that shows tMMP-9 correlates with the level of dichotomized JSW medial (0.2 mm) for 24-niontli radiographic progression of SKOA. Standardized semi-flexed radiographs were scored for joint space width (JSW).

Progression of knee OA was defined as a cliaage between baseline and 24 month visit in. narrowest joint space width (JSW) in signal knee . Total MMP-9 correlated with

radiographic severity (p<Q..0oT) at baseline, and with progressive joint space narrowing (r= 0.228; p<0.015).

[0 13] Figure 3 is a ROC carve that shows that tMMP-9 correlates with the level of dichotomized progression of JSW medial (0.6 mm) for 24-monih radiographic

progression of SKO A.. Standardized seaii-flexed radiographs were scored tor joint space width (JSW), Progression of knee OA was defined as a chang between baseline and .24 month visit in narrowest joint space width (JSW) in signal knee. Total MMP-9 correlated wife radiographic seventy (p<Q.Q6l) at baseline, and with progressive joint space narrowing (i= 0.228; p<G.M5).

DESCRIPTION

[0014] Unless otherwise defined, the technical and scientific terms nsed herein have the same meaning as commonly understood by one of ordinary skill in the art. Expansion and clarification of some terms are provided herein. All publications, patent ap l ca ions, patents and other references mentioned herein, if not otherwise indicated, are explicitly mcorporated by reference.

[0015] As used herein, the singular foams "a," "an.", and "the" inclode plsral referents unless the context clearly dictates otherwise.

[0016] As used herein, the term "sBbjeet" refers to a mammal that can be afflicted by a osteoarthritis, hut may or may not have ssdh a disease. Typically, the terms "subject" and "patient:" are used herein interchangeably, la various embodiments, the subject is a human..

[0017] As used herein, the term "sample" is taken broadly to include any sample suitable for the methods described herein. Typically, the sample blood, serum, or plasma.

[00 IS] As used herein, the term "healthy volunteer average conceutraiioris" refers to the average concentration of the various bio-markers described herein for at least two subjects who do not have OA. Preferably, average concentration values are calculated from biornarker concentrations measured in larger groups of OAs. Healthy volunteer W¹) average concentrations are provided herein, bat one of skill in the art may also measure biomarker concentrations n one or more populations of subjects lacking RA utilizing an apparatus capable of sensitively measuring the concentrations ofbiomar&ers described herein and calculating the average values for each biomarker in such HV populations.

[0 19] As used herein, the term "therapy" refers to the administration of any medical treatment (&.g_*, pharmaceuticals) or mfcerventional treatment (e.g., surgery) to affect RA or the biomarkers relevant to- EA described herein.

[0020] As used herein, the term "CV" refers to the coefficient: of variance.

[0021] As used herein, the term "average CY^* refers to average of the coefficient of variance obtained for all samples tested in triplicate.

[0022] In one aspect the disclosure is directed to the use of plasma proteomics of matrix metalioproteinases that can serve as biomarkers- for delecting OA severity and progression in patients. In various aspects, the disclosure is directed to a highly sensitive assay that can be used to measure these biomarkers, 111 partieiilar including plasma levels of MMP-2 and MMP-9, for predicting OA seventy and pogression.

[0023] Plainer, differences in matrix metalloproie iase coneeniratkms can be used to determined between various stages of OA progression.

[0024] The disclosure provides for the use of a highly sensitive Immunoassay System to measure metailo roteiriases in lood plasma obtained from OA patients and healthy volunteer subjects, and describes the differences in biomaiker concentrations between these two study groups.

[0025] In one aspect,, the measurement of differences in the biornarker

concentrations, either up- or down-regulated, singly or in combination, is OA patients at a particular time point, for example 24 months, versus baseline provides opportunities for better simpler, earlier, faster) disease diagnosis, disease staging, risk ciassifka&sa, disease progression, disease severity and/or identification of therapy responders/non- respoaders.

[0026] Osteoarthritis Biomarkers

[DD27] The use of a biomarker tiiat can predict: the rapid disease progression in O A will facilitate the identification of patients that will have a worsening of disease. This is important for both patient management as well as drag development. There is currently great interest in the field of OA to identify biomarkers that provide a method for earlier diagnosis sad identify patients at higher risk for disease progression.

[0028] One set of proteins that are of interest as biomarkers for OA are the

metalloprotemases. Certain studies hav shown that some of ike metaJloproteinases, including MP- 2, 9.and 1.3, are over expressed in OA cartilage and synovium, as well as downregolation of tissue inhibitors of Matrix metalloproteinases (ΙΊΜΡ). See.,

Opdenakker, etaL, Cytokme Res. 10:317-324 (1991). The group ofmetdioproteinases includes the 72- and 92-kD type∑V collagenases which, is mammals, degrad the coilagens of the extracellular matrix. Other members of mis gr oup include interstitial coHageaase (MMP-i) and siromelysin (MMP-3). The 72 -kD type IV coBagenase, or MMP-2, is secreted from normal skin fibroblasts. MMP-2 is also known as gelataase. 72-kD (Nagase, H., et al., Matrix Suppl. 1 :4 1-42.4 (1 92). It has been suggested that MP-2 plays a role in early skeletal development and bone cell growth and pmfifeiafeoa. Mosig, etsL, Hum. Molec. Genet 16:1113-1123 (2607).

[DD29] The 92-kD eoilagenase, or MMP-9, is prodoced by norma! alveolar macrophages and granulocytes. MMP-9 is also known as 92-kD gelatinase, type V eollagenase, and gelatinase-9. MMP-9 is p esent in most cases of inflammatory

response . Using substrate conversion assays, increased levels of MMP-9 have been detected in arthritis patient synovial Said and in multiple sclerosis patient ceiebrospinai Said,, respectively. Opdeaakker (1991) and Gijbeis, etah, J. Newoi munol 41(1)29-34 (1992). in contrast to the changes in MMP-9 expression, MMP-2 and tissue inhibitor of metalloproteniase-1 (TI P-i) were constitutive! * expressed, and the latter did not oppose the MMP-9 activity.

[0030] Stracturalty, MMP-9 maybe be divided into fee disti ct domains: a prodomain which is cleaved upon activation, a gelatin binding domain consisting of three contiguous Sbronectin type II units, a catalytic domain containing the zinc binding site, a proline rich linker region, and a carhoxyi terminal hemopessn-like domain. The

prodomain is cleaved into the following two snbimits: 67 kDa matrix metalJoproteinase- 9, and an 82 kDa matrix metalloprotei&ase-9.

[0031] According to the one aspect of the disclosure, differences in cytokines, cheiaokmes and matrix metallopioteinase concentrations can be deteenined between various patients at different stages of OA pr ogression with highly sensitive assays.

Measuring these concentrations in a patient sampl allows for predicting disease progression for OA. In one embodiment, the biomarker detected is matrix

aielalloproteinase-9 (MMP-9), also known as gelatinase-9. In another embodiment, the MMP-9 molecule comprises SEQ ID £½. 1. In certain embod&nenis, the biomarker detected is pro-MMP-9. In another embodiment, the pro-MMP-9 molecule comprises SEQ ID No. 2. In another embodiment the biomarker detecte is gelatmase-2 (MMP-2). m certam embodiments, the MMP-2 molecale comprises SEQ H> No. 3. Table 5 at the end of the specification contains sequence information. [0032] In one aspec the disclosure is directed to methods for detectin an amount of metallopr oteinase, far example total MMP-9 (tMMP-9), in a biological sample ffom a patient. I one embodiment, the amount of metallopfoteiiiase in a patient's biological sample is compared to the progression or stage of OA in the patient. I a certain embodiment, the metaMoproteinase is MMP-9. In certain embodiments, the OA is symptomatic knee osteoarthritis (S OA). In other embodiments, the OA is present in the knee, hand, wrist, arm, elbow, shoulder, foot, ankle, leg (shin), kn.ee, thigh, hip, pelvis or spine.

[0033] The disclosure is also directed to methods for predicting the progression of osteoarthritis in a patient comprising dete nnmg an amount of a metalloproteinase i a biological sample from the patient, comparing the amount of the metalloproteinase in ie sample to a threshold concentration, and predicting the progression of osteoarthritis when the concentration of the meialloproteaiase in the sample is above the threshold concentration, hi one embodiment, the metaJloprotemase is MMP-9. hi some embodiments, me threshold concentration is predetermined. In other embodiments, the threshold concentration is calculated from me concentration of metalloprotemase in biological samples taken from the same patient at earlier time points.

[0034] The disclosure is also directed to methods for predicting the progression of osteoarthritis in a patient comprising determining an amount of a metalloproteinase in a biological sample fom the patient, comparing the amount of the meiasllopro emase in tiie sample to a reference sample, and predicting the progression of osteoarthritis in a patient if the amount of metai!opmtemase in the biological sample is above the amoant of the metalloprotemase in the reference sample, in som embodiments, tiie reference sample can be from the same patient, in other emtodiments, tiie reference sample can be from a different patient. In certain embodiments, the reference sample can be from a blood sample taken at an earlier time point, for exampl ix months, 12 months, IS months, or 24 months, before tiie test biological sample. The reference sample can be from a patient with or without OA.

[0035] The disclosure is also directed to a method for identifying patients at risk for progression of osteoarthritis comprising determining an amount of a metaUoproteinase in a biological sample from the patient, comparing the amount of the inetaUoproteinase in the sample to a threshold concentration, and identifying that the patient is at risk for progression of osteoarthritis when me concentration of the metailoproteinase in the sample is above the threshold concentration, la. other embodiments, tiie methods can be vised to identify patients, whose OA is likely to worsen over time.

[QG36] Jn another^' embodiment, the disclosure is directed to methods for detecting osteoarthriti ia a. patient. The methods include detecting an amount of MMP-9 in a biological sample from the patient and determining osteoarthritis in the patient ased apon the amosst of MMP-9 in the sample. For example, the amount of MMP-9 in the sample can be compare to a threshold concentration, such as me concentration of MMP- 9 in a population of healthy volunteer s.

[0037] The disclosure is also directed to the use of a highly sensiti e Immu oassay System to measure cytokines and other biomarkers in blood plasma obtained from OA patients and/or healthy volunteer (HV) control patients. The differences in biomarker concentrations that are discovered between the two patient groups are described. In certain embodiments,, the amount of biomarker in a patient with OA is determined and compared with th amount of the same biomarker in a HV.

[0038] In another aspect the disclosure is directed to methods for diagnosing osteoarthritis in a patient. The method includes determining an amount of MMP-9 in a biological sample from the patient, comparing the amount of MMP-9 in the sample to a threshold concentration representing the concentration in a population of healthy vokmteexs and determining that the patient has osteoarthritis when the concentration of MMP-9 hi the sample is above the threshold concentration. In other embodiments, other metalloproteinases can be detected.

[0039] Instruments and Systems Suitable for Highly Sensitive Analysis of

Metalloproteinases

[0040] In one embochmeni, the amount of a metailoproteisase in a sample is a determined using a single molecule detection system, for example, the E8LENNA^® Immunoassay System (S gules, inc., Alameda, California). See U.S. Patent Nos.

7,838,250 and 7,572,640, which are incorporate by reference herein in their entirety

S Another example of a s stem- for the highly sensitive detection of metalloproteniases is described in U.S. Patent No, 7,914,734, which, is incorporated by reference herein in its entirety.

[3041] In one aspect,, tlie methods described herds utilize as analyzer system capable of detecting a single particle in a sample. In one embodiment, the analyzer" systeni is capable of single particle detection of a fl oresce ly labeled particle wherein me analyzer system detects energy emitted by an excited fluorescent label in response to exposure by an electromagnetic radiation source when the single particle is present in an mterrogation space. In some embodiments, tlie interrogation space is defined within a capillary flow cell fetidly connected to the sampling system of the analyzer system. In. a former embodiment of the analyzer system, the single particle moves through the interrogation space of the capillary flow cell by means of a motive force.. See, e,,g_r U.S. Patent . 7,572,640. In another embodiment, the interrogation spac is moved mroug the sample. S , e.g., U.S. Patent No. 7,914,734.

[0042] In another embodiment of me analyzer system, an automati sampling system may be included i the analyzer system for mtroducing the sample into th analyzer system. In another embodiment of the analyzer system, a sample preparation system may be included i the analyzer system for preparing a sample. In a farmer embodiment, the analyzer system may contain a sample recovery system for recovering at least a portion of & sample after analysis is complete.

[0043] In one aspect, the analyzer system consists of an. electromagnetic radiation source for exciting a single particle labeled with a fluorescent label. In one embodiment, the electromagnetic radiation source of the analyzer system is a. laser. In a farther embodiment, the electromagnetic radiation source is a continuous wave laser.

[0044] In an exemplary embodiment, the electromagnetic radiation source excites a fluorescent moiety attached to a. label as the label passes through the interrogation space of the capillary flow cell. In some embodiments, me fluorescent label moiety includes one or more fluorescent dye molecules. In some- embodiments, the fluorescent label moiety is a quantum dot [0045] When me interrogation space is a capillary flow cell a label is ex osed to electrosiaigjieiic radiation when the Iabei passes through an inierrogatio-i space. The mterrogation space is typically fJoidly connected to a sampling system. In some embodiments ίίιε label passes through the ½ierroga;iion space of die capillary So cell due to a motive force to advance ilie label through the analyzer s stem. The iaierrogation space is positioned such thai it receives electromagnetic radiation emitted from the radiation source, In some embodiments., tite sampling system is an automated sampling system capable of sampling a plurality of samples ithout intervention from a human operator.

[0046] The label passes through the terrogaiion space and emits a detectable aaioaat of energy when excited by the electromagnetic radiation scarce., m one embodiment, an electromagnetic radiation detector is operably connected to the interrogation space. The electromagnetic radiation detector is capable of detecting me energy emitted by the label , aj ,₅ b the fluorescent moiety of the label.

[0047] In a farther embodiment of the analyzer system, the system form r includes a sample preparation mechanism, where a sample maybe partially or completely prepared for analysis by the analyze? system, in some embodiments of the analyzer system, the sample is discarded after it i analyzed by the system, in oilier embodiments, the analyzer system farther includes a sample recovery mechanism whereby at least a portion, or alternatively all or substantially all, of the sample may be recover d after analysis. In such an embodiment, the sample can be returned to the origin of the sample. Ih some embodiments , the sample can be retained to microliter wells on a sample microtiter plate. The .analyzer system typically farmer consists of a data acquisition system for collecting and reporting me detected signal.

[DIMS] Sample Preparation

[0049] In certain, embodiments, the patient sample must be prepared for analysis according to the method of the disclosure.

[0050] In general, any method of sample preparation ma be used that produces a label corresponding to a metailoproteisase to be measured,, where the label is detectable in the instruments described herein. As is known in the art., sample preparation in which a label is added to one or more particles may be performed in a homogeneous or heterogeneous format In some embodiments, the sample preparation is formed in a homogenous formal In analyzer system employing a homogenous, format, unbound label is sot removed from the sample. See, e.g., U.S. Patent Nos. 7,838,250. 7,572,640, and 7.914,734. In some embodiments, fee particle or particles of interest are labeled by addition of labeled antibody or antibodies that bind to the particle or particles of interest.

[0051] Antibodies

[3052] In one aspect of the disclosure, the biomarker to be measured, such as MMP- 9. can be joined with a binding partner. In certain embodiments, the binding partner is an antibody.

[0053] Any suitable binding partner with the requisite specificity for the form of molecule, a marker, to be detected can be used. If the molecule, a marker, has several different forms, various specificities of binding partners are possible. Suitable binding partners are known is the art and include antibodies, aptamers, lectins, and receptors-. A useful and versatile type of binding partner is an antibody.

[0054] In one aspect of the disclosure, the amount of MMP-9 is determined by contacting the biological sample with an antibody specific for MMP-9 and determining the amount of specific binding between the antibody and MMP-9 in the sample.

[0055] In some embodiments, the binding partner is an antibody specific for a molecule to be detected. The term "antibody," as used herein, is a broad term and is used in its ordinary sense, including, without limitation, to refer to nafara!y occurring antibodies as well as ncsi-natoraliy occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen- binding fragments thereof. It will be appreciated that the choice of epitope or region of the molecule to which the antibody is raised will determine its specificity, e.g., for various forms of the molecule, if present, or for total all, or substantially all, of the molecule).

[0056] Metiiods for producing antibodies are well-established. One skilled in the art will recognize that many procedures are available for the production of antibodies, for

I I example., as described in Antibodies, A Laboratory Manual, Ed Harlow and David Lane,. Cold Spring Harbor Laboratory (1988), Cold Spring. Harbor, N.Y. One skilled is the art will also appreciate that binding fragments r" Fab fragments that mimic antibodies can be prepared from genetic nifonnation by varioos procedures (Antibody Engineering: A Practical Approach (Bonebaeck, C, ed.}., 1995, Oxford Umversity Press, Oxford; J. Irnintinol. 149, 3914-3920 (1992)). Monoclonal and polyclonal antibodies to molecules. e.g. , proteins, and markers also commercially available ( and D Systems, Minneapolis, Minn. USA; HyTest Ltd.., Turku Finland;, Abeam Inc., Cambridge, Mass., USA, life Diagnostics, Inc., West Chester, Pa., USA;, Fitzgerald industries international. Inc., Concord Mass. USA: BiosPacific, Emeryville, Calif. USA).

[0057] In some embodiments, the antibody is a polyclonal antibody, in other embodiments, the antibody is a monoclonal antibody.

[0058] Capture binding partners and detection binding partner pairs, capture and detection antibody pairs, can be used in embodiments of the disclosure. Thus, in some embodiments, a heterogeneous assay protocol is used in which, typically, two binding partners, &g_*, two antibodies, are used. One binding partner is a capture partner, usually immobilized on a solid support, and the other binding partner is a detection binding partner, typically with a detectable label attached. Such antibody pairs are available from several commercial sources, such as BiosPacific_.,, Emeryville, Calif. Antibody pairs can also be designed and prepared by methods well-known in the art. Compositions of the disclosure include antibody pairs wherein one member of the antibody pair is a label as described herein, and the other member is a capture antibody.

[0059] Antibodies to metalloproteinases are well characterized in the field of the disclosure. For example, expression of MMP-9 was detected in different pituitary tumor types using a monoclonal antibody to investigate whether it plays a role in allowing angiogenesis and invasion. Turner, @t aL₇. J. Clin. Endocr. Metab. 85:2931-2935 (2000). Antibodies to metaSoproteinases are available from a variety of commercial and noncommercial sources. The disclosure is not limited to any of the particular antibodies provided, for exemplary purposes.

[0060] Systems for Detection [0061] As noted above, the diagnostic/prognostic methods described herein generally mvolve the determination of the amount of bioinaiker related to OA from one o-r a set of samples from a subject Det^minatio of concentrations of biomsrker related to OA in the practice of the methods can he performed using any suitable apparatus or system that: allow for the detection levels described herein. Sm, s.g._f U.S. Patent Nos. 7,838,250, 7,572,640, and 7,914,734. These patents describe nistruments, reagents and methods for measuring analyses at levels to cany oat the methods of the disclosure and thus identify those patients wit biomarker levels related to OA progression.

[0062] Detection and Characterization of OA

[0063] OA is typicaily measured -using a radiograph, oi" x-ray. A radiograph is a noninvasive medical test that helps physicians diagnose and treat medical conditions .

Imaging with, x-rays mvoives exposing a pari of the body to a small dose of ionizing radiation to produce pictures of the inside of the body. X-rays are the oldest and most frequently used ions of medical imaging, and axe well known in the art .

[0064] To determine OA progression, OA patient radiographs are scored for specific indicators which are well known in the art. in certain embodiments, one or more of the following indicators of OA progressio are scored: (I) overall KeHgreo and Lawrence (KL) grade;; (2) joint space width (JSW); (3) osteophytes and (4) subchondral sclerosis.

[0065] Other indications include measuring bone marrow lesion -volume, where the amosnt of bone marro w lesion (BML) is quantified on magnetic resonance imaging. (MRI using computer and/or manual methods.

[0066] The Kellgren-Lawrence score (KL score) for OA patient radiographs is a common metric of OA progression, traditionally used to assess the severity of radiographic kne OA. This categorical scale incorporates important radiographic features of OA such as joint space narrowing and osteophyte development, into one scale of increasing severity. The KL score is rated on a scale of 1-4, dependent on overall global severity of OAs: Grade I : do btfol narrowing of joint space and possible osteophyte lipping; Grade 2: definite osteophytes and possible narrowing of joint space; Grade 3: moderate multiple osteophytes, definite narrowing of joint space and some bone sclerosis^* Grade 4: large osteophytes, marked narrowing of joint space, sevens bone sclerosis.

[0067] The measurement of radiogisphic joist space width (JSW) is the most accepted and widely-used method of assessing OA progression. JSW is a component of ike KL score.,, bat in some embodiments it can be assessed separately. JSW is expected to decrease as KL score increases. JSW is measured (in mm) at the narrowest portion of the joint space via electronic calipers and a medical monitor. If the JS W decreases, the patient is espeiiencii-g joint space narrowing (JSN). in one embodiment,, the disclosure generates a JSN value for OA atie s based on their radiographs. To date, JSN has remained the primary outcome fey which OA progression is tested. Narrowing joint space indicates cartilage loss and worsening osteoarthritis.

[0068] In some embodiments, a patient's osteophyte score is measured. Osteophytes are thought to stabilize as osteoarthiitic joint, thereby preventing structural progression. However, osteophyte ar also associated with an increased risk of structural progression. Osteophyte score guides are known m the art;, such as the- scale from Osteoarthritis

Research Society international Atlas (OARSI). Osteophyte score can also be graded oa a semi quantitative scale, prov ing a 0-5 rating. Sm Felson, et at . Rheumatology, 44:100—104 (200:5). Several regions can be scored, fbr osteophytes, including th medial and lateral femoral condyles (MFC and LFC) and the medial and lateral tibial plateaus (MTP and LTP). Other regions can be measured as well depending on the OA bod part of interest.

[0069] Subchondral sclerosis can also be quantified as a marker of OA progression. Sabchoiidral sclerosis is defined as increased bone density or thickening in the

subchondral, layer. This often occurs wife progressive osteoarthritis and shows u on x-rays as abnormally white (dense) bone along the joint line. Subchondral sclerosis is quantified using a score from 0-5. See Swageriy, et ah. Am. Fam. Physician, 6 {2):2?9-28? (20€ί).

[0070] In some embodiments, the OA score of a patient is a composite of JSW, KLS and osteocytes. Si other embodiments, the OA score is one or mor of any of the parameters described. [0071] In one aspec the disclosure is directed to methods of determining the progress-OS of osteoarthritis in a patient The method includes ^emti i g .as amount of a mefalloprotsinase iii a first biological sample from a patient at a first time point;

determining an amount of the metailoprateinase in a second biological sample frorn a patient at a second time point; co£npaiing the amount of the metallopofeinase in first sample to tlie amount of the nieiaJloproteinase m me second sample; and determining mat osteoar ritis lias progressed in the patient by determining mat the amount of the metalloproteinase ill the second sample is greater than, the amount of the

metalloproteinase in me second sample.

[0072] In one aspect tlie disclosure is directed to a method of determining a. coarse of treatment for arthritis in a patient. The method includes determining an amount of MMP- 9 in a first biological sample from a patient at a first time point; determining an amount of MMP-9 in a second biological sample from a patient at a second time point;

comparing the amoont of MMP-9 m first sample to the amosnt of MMP-9 in the second sample; and determining that whether osteoarthritis has progressed in the patient by determining whether the amount of MMP-9 in the second sample is gr eater man the amoB&t of MMP-9 in the second sample. If OA progression is determined, the appropriate coarse of treatment can be determined and prescribed. Treatment far G is known to those of skill in the art.

[0073] In one aspect, the disclosure is directed to a method, of identifying patients at risk for the progression of osteoarthritis. The method includes determining an. amount of MMP-9 in a first biological sample from a patient at a first time point: determining an amoB&t of MMP-9 in a second biological sample from a patient at a second time point; comparing the amoont of MMP-9 in. first sample to the amosnt of MMP-9 in the second sample; and determining that the patient has an increased risk of osteoarthritis

progression by determining that the amount of MMP-9 in the second sample is gr eater than me amount of MMP-9 in the second sample.

[0074] In one aspect, the progression of osteoarthritis is measured in a patient In certain embodiments of the disclosure_., progression, of knee OA is defined as a change between the JSW at baseline (0 month) and at a second, time point, for example, 6, 12_., 18 o-r 24 months. In another embodiment, the disclosure relates to the generation of a joist space narrowing (JSN) value for OA patient radiographs. JSN is calculated by measuring the JSW at baseline (0 months), and then the JSW at some later time point

[0075] In another e lrodiment, the KL score is used, to calculate OA progression. The KL score is determined at baseline (Q months), and then the KL at some later time point. OA progression is shown if the KL score increases. hi certain embodiments,, additional parameters can be used to determine OA progression.

[۩76] In one aspect of the disclosure, biomarke s in an OA patient plasma sample are detected and compared to OA progression parameters. In one em o ime t the biomarker is a metaMoproteinase. In arious embodiments, the inetal!optoteinase is MMP-9, total MMP-9, pro-MMF-9 or a combination h reof. In another embodimeiii:, the biomarker is MMP-2. OA progression scores are compared to the amount of biomarker in the patient sample, and the two values are analysed for an association. Any accepted statistical analysis can be used to establish an association, soch as for example a students t-test

[0077] In one embodiment of fee disclosure, elevated levels of total MMP-9 are related, w th OA progression. In another embodiment of the disclosure, ele vated lewis of total MMP-9 can. predict an increased risk of progressive joint space narrowing (JSN). in another embodiment of the disclosure, elevated levels of total MMP-9 are related with radiographic severity of SKOA. These observations confirm that total MMP-9 is a prognostic biomarker for OA.

Examples

[0078] A study was designed to test the hypothesis that plasma levels of certain metailoproteiiiases can predict OA sever ty and progression, i 50 symptomatic knee O (SKOA) patients were recruited as part of a 24 month study. These SKOA patients met the American College of Rheumatology (ACR) criteria for knee OA. The AC as developed diagnostic criteria for grading progression,, remission, and fiHictioiial status of OA at various sites, including the hip, the knee, and the hand. See Altaian, et aL,

Arthritis Rheum 29:1039-49 (1 86), which is incorporated by reference in its entirety. The ACR guidelines traditionally define idio athic OA of the lai.ee as. reo a mg (1) knee pais plus osteophytes on radiographs and (2) at least one of the foJlowmg patient age older than 50 years; morning stiffness lasting 30 annates or less; or crepitus oa motion. Qtixer ACR guidelines have reduced the age requirement to 40 years or older. The patient cohort had a mean age of 63.09 ± 1.0.3, and a mesa BMI of 26.3 ± 3.6. Other parameters, mcluding well kn w pain scores raclodrag the Visual Analogue Scale (VAS) and the Western. Ontario and McMaster Uaiversifies Arthritis Index (WOMAC) pain score, were collected from each patient. Selected clinical diaractensii.es of the OA patieats tested .are smnmarized in Ta le 1.

Table 1

Summary of 'Clinical Characteristics of OA Patients

[0079] All human serum specimens used in this study were obtained under IRB approval and informed consent All specimens were collected uader protocol, which included acting time of blood collection into serum, tubes, separation of serum from cells and storing of resulting se um at -7CPC These patieats were followed for 24 months, and blood samples were taken every 6 months.

[9086] For each patient. X-ray radiographs of each, knee were taken at the first visit (baseline, 0 months) and 24 months. Standardized semi-flexed radiographs were scored for overall ellgren and Lawrence (KL) grade, osteophytes, joint space width. (JSW) and subchondral, sclerosis, by the same radiologist. Scores for all patieats are shown in Table 6 (baseline scores) and Table 7 (24 month, scores). [0081] Progression of knee OA was quantified by composing ilie change in scores between baseline and 24 inoi- i visit in ilie indicated signal knee. Scores were calculated as described, end as is well knows in fe ait by a skilled rheaniaiologist. The progression scoring for all patients is shown in Table S.

[0082] To assay for hiomarker levels, patient Mood samples were collected at baseline and every 6 months. Plasma samples from visit 18 month of 128 OA patients (37% male) were assayed for MMP-2 and MMP-9 using the highly sensitive ΕΚΕΝ Ά* ImmtiBoassay System. (Singulex, hie). Frozen serum and plasma samples were thawed and centrifuged 10 minutes at room temperature at 13 rpm. Clarified serum/plasma was removed carefbUy to avoid taking any possible; pellets or floaters and pat into fresh tabes.

[0083] Table 2 identifies the amibodi.es used in the assays, which were obtained from &D Systems (Minneapolis,. Minnesota).

Table 2

[0084] Btetecboa antibodies were conjugated to one or more floors, &g._A ALEXA FLUOR 647, life Technologies Corp.). The standard was recombinant human MMP-9 (R&D Systems, 9Π-ΜΡ), diluted as necessary m TBS, 3% BSA, SmM EDTA, and 0.1% Sodio n Aside.

[0085] Capture antibodies were bound to Streptavidm CI micxoparticles (MP^'s) obtained from Dynal (65 5.01 -03 , 10 mg ml stock).

[0086] Buffers used in the assay nclede: 1GX borate buffer saline Triton Buffer' (BBST wash buffer; 0.07 M bork acid, 0.03 M borax, 1.5 M sodium chloride, 10% Triton X- 100, pH 8.3); assay buffer (TBS (0.1M Tos, 0.15M NaC% blocking reagent (from, e.g., Scantsbodtes.. Santee, CA), 0.1%BSA, 0J%Triton, 25mM EDTA, and 5.1 % Sodiuin Aztde, stored at 4C); and elution buffer (0^:.1M glycine, 0.02% Triton X-100, pH 2.8, stored at 4°C).

[0087] Mrcropaiticle (MP) coating. 100 ul of the MPs stock were placed in as eppendorf tube. The MPs were washed three times with shoot 500 a! of Ix wash buffer by placing the tube on a magnet, inverting the tuhe ISO degrees several times, removing the Ix wash buffer and resuspending the immobilized rmcroparticles in fresh ix wash boffer. After the wash steps, die SHcroparticIes were resuspended in 100 ul of ix wash buffer and 25 of bioiiiiylated capture antibody were added. The mixture was then incubated for 1 hour at room temperature with constant mixing. The micropasticles were washed five times with 500 uL of Ix wash buffer as described above. After the washing steps, the imcroparticles were resaspeaded in 100 uL of assay buffer and stored at 4 C.

[0088] Preparation of Standard and Samples. The standard was dilated with standards diluent to prepare a proper standard carve. Frozen serum and plasma samples need to be filtered using a 96-weil filter plate and centrifuged 10 minutes at room temperature at about 3000 RPM. 100 L of each standard and sample was. pipetted into appropriate wells.

[0089] Capture Target. 100 «1 of MPs (after resuspension to 11 ml in assay buffer) were added to each well. The mixture was incubated on the JitierBug (Boekel,

Feastervilie.;. PA), setting 5, at room temperature for about 2 itrs.

[0090] Post-capture wash and detection. The plate was placed on a magnet and the supernatant was removed after enssring that all microparticles were captured by the magnet. 200 ul of Ix wash buffer were added, and the wash buffer was aspirated. 20 ul detection antibody were added per well (500 ng ml of detection antibody was diluted in assay buffer. The mixture was incubated on the JitterBug, 5 at room temperature far about ί far.

[00 1] Post-detection washes and ekition. The plate was placed on a magnet and washed fou times with wash buffer. After the washes 200 uL of wash baffler were added to die 96- well plate. After the washes the samples were transferred mi© a sew 96-weM plate. ^'The new plate was then placed oil the magnet and a final aspiration of the wash buffer was remove after ensu in mat all aiicropaiticles were captured by She magnet. 10 \iL of elution buffer were then dded and the mixture was incubated am the JitterBug, 5 at room temperature for 10 min. Diuing; incubation, a 384 well plate was prepared by adding 10 μL· of0.5M Tris per well.

[0092] The contents (standard and samples) of the (second) 96- well plate was transferred to previously prepared 384- well plate. The plate was. covered, with pieresabie plate sealer and was ready to be run on the ERENNA Immunoassay System An aJiqsot was pnmped into the analyzer. Individually-labeled antibodies were measured during capillary flow by setting the interrogation volume sach that the emission of only one fluorescent molecule was detected in a defined space following laser excitation. With each signal representing a digital event, this configuration enables extremely higti analytical sensitivities. Total flaorescent signal was determined as a sum of fee

dividual digital, events. Each molecule counted was a positive data point with hundreds to thousands of DMC events/sample. The limit of detection the tMMP-9 assay was deteonined by the mean +3 SD method.

[0093] Using highly sensitive iranianoassays we were able to qua tify me

concentration of a variety of asalytes in serum obtained from OA patients. Importantly, all of the assays had limits- of quantification that wer e lower man lire cooceniration of the analytes measured, in serum. TMs ensured that the measurement of analyte was accurate.

[0094] Overall, the mean plasma MMP-2 levels were 83.3 ± 63.05 ng ml for all OA patient samples. The mean plasma pro-MMP-9 levels were 30.7 ± 32.64 ng mL. The mean plasma total MMP-9 levels were 455.9 ± 290.09 ng mL.. The MMP-2„ pro-MMP-9, and MMP-9 levels for each OA patient sample is shown in Table 9.

[0095] The levels of biomarfcer concentrations for OA patients was compared to the levels of feiomarter concentrations for non-arthritic patients. As shown in Table 3., the level s of MMP-9 in O A patients was higher man the levels seen is healthy patients.

There was little difference in the levels of MMP-2 between the two patient populations. Table 3

Average lasma biGmarker concentrations (ng/ml) of osteoarthritic patients as compared to healthy volunteer control patients.

Data shown as mean ±. standard d lation.

[0096] OA patient samples were men categorized into two groups. (I) high tMMP-9 samples, defined as >375ng ml plasma tMMP-9 (65 samples), and (2) low tMMP-9 samples, defined as <375ng ml plasma tMMP-9 (63 samples). M P-2 patient samples wer also separated into a high/low gronp.

[0097] To evaluate the correlation between biomarker levels and OA progression, we first examined the quantity of bioniarker as compared to the samples with KL scores of L 2 and those with, scores of 3,.4.. Only total MMP-9 positively compared with, the KL score both at baseline

p G..0.15). This relationship is represented in Figure I.

[0098] To farther evaluate th relationship between biomarker levels and OA progression, JSW and JSM was separately examined and compared with biomarker levels. At time Q, or baseline, the mean JSW for the higher tMMP gras samples was 3.04. At time 0, or baseline, the mean JSW for the higher tMMP samples was 3.41. iMMP-9 correlated with radiographic, severity p 0..06i.) at baseline, and wife progressive JSM (r= 0..22S; p<0.015). The results of fee JS W correlation analysis are shown in Figures 2-3. The comparative data (mean values) is depicted in Table 4. MMP-2 or proMMP-9 levels did not correlate wife either KL scoring or JSN as markers for OA progression. Table 4

Higher plasma tMMP-9 is a predictor for increased joint space narao ing

JSN) at 24 months. Values are expressed as mean (SD).

[0099] These findings indicate mat elevated levels of tMMP-9 are a ssociated with, radiographic severity of SKOA. As sash, tMMP-9 concentration is plasma can be rsieasiired as a predictor for increased joint space narrowin (JSN). This study suggests that OA patients who have a tMMP-9 level above a certain threshold {375ng/ml in this study) in. their plasma have as increased risk of progressive joint space narrowing (JSN), a key indicator of OA disease profession.. These foldings show that tMMP-9 is a reliable prognostic hiomarkex tor the progression of OA.

[00100] The measurement of differences in. the biornarker concentrations, either up- or down-regulated, singly or in comb ation, in OA patients at 24 months versus baseline provides opportunities for better («.£., simpler, earlier, faster) disease diagnosis, disease staging, risk classification, disease progression, disease severity and/or identification of therapy responders non-responders.

[00101] Th examples given above are merely illustrative and are not meant to be an exhanstive list of all possible embodiments, applications or modifications of th disclosure. Thas, various modifications and variations of the described methods and systems of me disclosure will be apparent to those skilled in the art without departing from me scope and spirit of the disclosure. Although the disclosure has been described in connectio-i with specific embodiments, it should be understood mat the disclosure as claimed should, not be unduly limited to sod* specific embodiments. Indeed, various modifications of me described modes for carrying out the disclo sure winch are obvious to those skilled in molecular biology, immunology, chemistry, biocbemistt or in the relevant fields are intended to be within the scope of the appended claims.

[00102] Any numerical values recited herein include all values feaa the lower value to the upper value in increments of one unit provided that there is. a separation of at least two units between any lower value and any higher valise. As an example, if it is stated that the concentration of a component or value of a process, variable such as , for example, size, angle size, pressure, time and the like., is, for example, from 1 to 90, specifically fiom 20 to 88, more specifically fioni 30 to 70, it is intended ihat values sucli as 15 to 85, 22 to 68, 43 to 51, 30 to 32, etc. are expressly enumerated is this specification. For values which are less tha one, one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 as appropriate. These are only examples of what is specifically intended and all possible combinations- of numerical values between the lowest value and the highest value enumerated are to he considered to be expressly stated in this application in a similar manner.

[00103] Particular methods, devices, a d materials are described, although any methods and material s similar or equivalent to those describe herein can be vised in the practice or testing of the disclosure. The disclosure of all references and publications cited herein are expressly incorporated by reference in their entireties to the same- extent as if each were incorporated by reference individijally.

η

3ϊ

urn s 2S 28.7 4S.7 19 L 3 Q -I ■45.3 -9 2 1 s 0.6 14

1149 16 26.1 i 32 R δ 0 9 -9.5 -16 S 6 0 5 δ

1141 30.8 63 38.5 1323 3S L 2 I 9 ^■0.7 IS 1 1 0 e.s 15

1142 38 19 24.S SI. S 52 L J © -1 -1 -3S S -I -0.S -25

114 4S.S 61..5 37.7 S.S 43 R 3 -2 e 0 3 s -2 -1 -45

1144 S.2 13 15.S 37.6 5 L 2 1 e δ.3 6 2 1 s 0.6 12

1145 1*3,4 43 5.Ϊ 72.5 26 R 2 3 6 0 -0.6 -2:1

1146 44.6 4S.S 433 136.4 73 R 3 © -1 -5 3 6 -1 e.s 14 nsi 11.2 22 8.7 41.9 41 R I 2 6.7 14

1154 28.-S 52 575 1IS.5 42 S. 0 3 © 0.4 S 0 3 0 6.2 5

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1.4 53 22.6 77.5 26 L 3 0 9 ^■0.3 7 3 6 0 -0.3 lies 41 49 38.8 128.8 72 R 3 -1 •S 3 -IS δ 6 0 -0.1 -3

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«82 33.6 33.5 3Ϊ.5 106.6 36 R i © -i 0.3 1 1 -I -0.4 -13

3IS3 45,6 4S.5 35.9 imo 43 L 3 1 -0.3 -9

11S4 4.8 9 5.6 194 i L 3 © -1 •« 7 -26 3 6 -1 e.s 22

IS 40 .7 71.7 74 R I 1 S 0 6.6 is-

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IIM 31.8 46 44.2 122.® 51 L 3 © -1 8 a I s 0 -0.6 -2δ

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11S7 53.4 67.5 15.δ 135.9 56 L 1 1 & Μ -21 I 1 δ -δ.3 -S

1198 4 12 3.7 19.7 S R 2 0 -I 0.1 3 1 0.1 2

IIS* 33.4 56 75:9 M53 S4 R 3 © -2 0.6 23 5 6 0 6.4 1 im 1.6 2 .2.2 5.8 4 R Ϊ © § -S..1 -2 0 S 0 6.2 5

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Table Btcmarker -aac¾aratiQB m Qsiecnartliii s Pa¾iea¾ Plasma Sam les ( gml

33 OA Patina

amber MMP-.. pi & MP-9 tMMP-0 i i 9 46.8 460 nm 5 13.7 233 li&i 87 10.6 99 nm 180 24.7 395 lies 87 14.7 37

1186 49 36.3 562 nm 198 9,6 29i

1117 58 162 364

1I1S 210 7.2.4 MO

1119 86 30.2 432

ΪΪ22 283 155.4 15S1 inn 227 23.1 503

11.25 41 50.6 S4Q

1126 66 6.4 212

1129 7i 1Q&..9 1275

1134 log S O 22

1135 59 4.9 255

IIM 74 12.6 337

1137 89 6...1 506 ms si 13.3 482

1139 99 37.6 25S nm 76 16.9 297

114Ϊ 6§ 35.5 522

1142 53 5.S 349

1143 75 16.3 36

1144: 122 2.6 198

1145 74 60.S 9 nm 73 12.5 267

1152 5 9.7 215

1154 43 §2.2 109

1156 49 4.5 229

1158 55 4S.7 661 urn 38 37.3 313

!ISI 76 5.0 195

1163 94 60.6 598

I fi 111 9.6 397

1169 232 1 .7 322

1171 254 «7.8 848

1175 46 115.2 1247

1176 I OS 34.6 521

1182 47 1.1.9 259

1.183 62 21.0 333

1184 60 19.7 336

1187 58 9.3 227

1188 59 Ϊ 8.6 275

1189 219 9.8 263 nm 209 1.0.9 275

1192 2 24.2 381

53 2.2.9 342 OA Patina

amber MMP-2 i & MP-? tMMP-4

1197 49 49.4 5SS m& 49 44.7 515

1199 59 36Λ 412

1280 36 10.5 27Q

1281 36 2S2 375

1285 39 20.4 326 nm 39 10.9 «ø

1211 58 163 295

1213 12 6 2S0

1215 40 6.7 ISl

121« 4 43.7 S4S

1217 57 31.2 278

1 18 65 7.3 289

1221 S6 39.7 528

1224 m 35.6 456

1225 54 13.5 328

1227 59 2S.5 41

1238 54 7.4 252

1231 4S 63 221

1234 42 24.0 319

1235 38 7.1 175

1237 39 7.9 235

1238 45 29 J 511

1242 SS 28.6 4Ϊ8

1244 50 9.7 247

1245 56 19.8 347

1247 45 6.3 377

1248 53 10.8 256

12 3 46 108 0 1013

1251 56 52.0 892

12SS 43 ia.9 375

AWG 83,14 3 JS 454. «4 m «3,29 32.76 2WM

Claims

WHAT IS CLAIMED IS;

1. A method for predicting the progression of osteo.a_th.ifes. in a patient coii-g sing determining an amomit of a metailojJFOteinase in a biological sample from ie patient comparing the amount of the meialioprotsinase in the sample to a threshold

concentration, and predicting progression of osteoarthritis when the concentration of the metalloproteinase in the sample is above the threshold concentration.

2. A method for identifying patients at risk for progression of osteoarthritis comprising determining an amount of a mefallo Oteisase in a biological sample fraa the pattest, comparing the amount of tiie nietalloproteinase in tiie sample to a. threshold

concentration, and identifying mat the patient will have a progression of osteoarthritis when the concentration of the rnetalloproteiiiase in the sample is above the threshol concentration.

3. A method for predicting the progression of osteoarthritis in a patient comprising determining an amount of a metaEoproteinase in a biological sample from the pattest, comparing the amoont of tiie me alloproteina&e is the patient sample to a reference sample,, and predicting the profession of osteoarthritis in a patient if the amount of metalloproteinase in the biological sample is above the amount of -the metallopofeiiiase m the reference sample.

4. A method for diagnosing osteoarthritis in a patient comprising determining an amosni of a metaEopretsinase in a biological sample from the patient, comparing the a ou t of a metalloproteiaase in the sample to a threshold concentration representing the concentration in a non-©steoait½itic population, and determining thai the patient lias osteoarthritis when die concentration of a ms taHoproieinase in the sample is above the threshold concentration.

5. A method for detecting osteoarthritis in a patient: comprising detecting an amount of metaEoproteinase in a biological sample from the patient and correlating the amount of metaEoproteinase to osteoailhritis in the patient

6.. The method of any one of claims 1-5 wherein the metailoprotsmase is matrix etallopro einase (MMP-9).

7. A method of deterniining die progression of osteoarthritis in a pattest comprising:

(a) determining as amount of MMP-9 i a. first biological sample from a

pat ent at a first time- point;

(b) determining as amount of MMP-9 in a. second biological sample from a patient at a second time point;

^■(c) comparing the amount of MMP-9 in fir st sample to the amount of MMP-9 n the second sample; and

■(d) determining mat osteoarthritis has progressed in the patie t by et r ining that the amount of ^'MM P-9 in the second sample ½ greater than the amount of MMP-9 in the second sample.

8. ^'The method of any one of claims 6 and 7 wherein the MMP-9 is total MMP-9.

9. The method of any one of claims 6 and wherei die MMP-9 comprises SEQ ID NO:l

10.. The method of any of claims 1-7 wherein the osteoarthritis is symptomatic knee osteoarthritis.

11. The method of any of claims 1-5 and 7, wherein the determining the amount of mefalloproteinas comprises contacting fee biological sampl with an antibody specific for the metalJoproieinase and determining the am unt of specific binding between the antibody and the sielaSoproteinase is me sample.

12. The method of claim 11_., wherein the metaJioproteinase i MMP-9.

13. A method of determining a course of treatment for arthritis in a patient comprising:

(a) determining an amount of MMP-9 in a first biological sample from a

patient at a first time point ^■(b) determining as am unt ©fMMP-9 in a second biological sam le from a patient si a second tmie point;

■(e) comparing the amoun t: of MMF-9 in fcst sample to me som! of MMP-9 in the second, saraple; sand

■(d) determining a course of treatment if the amcnaii of MMP-9 in die second sample is greater t an the anions* of MMP-9 in the second sample.

14. A method of identifying patients that will ha e a worsening of osteoarthritis

comprising:

(a) determining as amount of MMP-9 in a first biological sample from a

patient at a first time posit;

■(b) determining as amount of MMP-9 in a second, biological sample from a patient at a second time point;

■(c) comparing the amount of MMP-9 in fir st sample to the amooat of MMP-9 in the second sample; and

■(d) determining that osteoarthritis will worsen m the patient by determiiimg thai the amoimt of MMP-9 is the second sample is greater than the amount of MMP-9 in the second sample.