WO2005106495A1

WO2005106495A1 - Procollagen ii biomarkers and methods

Info

Publication number: WO2005106495A1
Application number: PCT/IB2005/001152
Authority: WO
Inventors: Mark Allan Abrams; Poonam Pereira Aggarwal; Dawn Rene Dufield; Rodney W. Mathews; Olga Vladimirovna Nemirovskiy; Teresa Sunyer
Original assignee: Pharmacia & Upjohn Company Llc
Priority date: 2004-05-04
Filing date: 2005-04-21
Publication date: 2005-11-10

Abstract

A method of prediction disease progression in a disorder characterized by the uncoupling of collagen type II synthesis and degradation is disclosed. There is further disclosed a method of detecting specific procollagen fragments associated with osteoarthritis. The fragments are derived from procollagen type IIB (PIIBNP). A method for determining the efficacy of an agent or drug intended to decrease or inhibit a collagenase enzyme is disclosed.

Description

PROCOLLAGEN II BIOMARKERS AND METHODS

SEQUENCE LISTING A sequence listing is appended, including a paper copy and a copy in digital media in machine- readable form, which is identical to the paper copy.

BACKGROUND OF THE INVENTION

I. Field of the Invention The present invention relates to biomarkers of arthritis, and more particularly to peptide biomarkers of collagen synthesis and degradation for diagnosing and treating osteoarthritis. The invention relates generally to methods for identifying and quantifying peptides and, more particularly, to methods for identifying and quantifying degradation peptides resulting from enzyme cleavage of collagen, and propeptide fragments resulting from collagen synthesis. The invention also relates to the specific peptides that result from synthesis of collagen type II in humans and animals and the recognition of levels these peptides in biological samples as markers of diseases or physiological conditions characterized by a disequilibrium of synthesis and enzymatic degradation of collagen, such as osteoarthritis and rheumatoid arthritis, and the identification and quantification of the peptides to assess the efficacy of enzyme inhibiting agents and drugs used to treat or control such diseases or physiological conditions.

The invention is further directed to quantifying degradation peptides and synthesis peptides to calculate a ratio thereof, for determining disease progression and disease stage.

II. Background Articular cartilage acts to cushion and support joints in the skeleton. Cartilage tissue features an extensive extracellular matrix. This matrix is synthesized and maintained by a single cell type, the chondrocyte. Major matrix components of cartilage include collagens, particularly type II collagen, and proteoglycans, particularly the large aggregating proteoglycan, aggrecan. Type II collagen imparts mechanical strength to cartilage, while aggrecan provides compressibility and elasticity. Functionally, the fibrous collagen network enmeshes aggrecan. The negative charge conferred upon cartilage by aggrecan through its glycosaminoglycan side chains acts to retain water, while the collagen fiber network resists swelling of the tissue. When collagen type II is synthesized in adult humans, the immature protein (SEQ ID NO. 1) contains three extra domains: a signal sequence (SEQ ID NO. 2), and two propeptides, an NH₂-terminal propeptide (SEQ ID NO. 3) and a carboxy-terminal propeptide (SEQ ID NO. 4). The signal sequence directs the protein to be secreted from the cell, and the propeptides are then cleaved by specific proteases. The resultant^' mature collagen II protein (SEQ ID NO.5) is then capable of being incorporated into the extracellular matrix. Arthritis, particularly osteoarthritis, involves proteolytic cleavage of both collagen and aggrecan. This proteolytic cleavage is carried out by matrix metalloproteases, such as MMP-13 (a collagenase), and disintigrins, such as ADAMTS-4 / ADAMTS-5 (aggrecanases). Both MMP-13 and aggrecanases are known to be upregulated in osteoarthritis. Generally, active and extensive collagen turnover is not considered a prominent feature in healthy adults. Turnover of collagen type II, also known as articular collagen, generally occurs in diseases, such as rheumatoid arthritis, osteoarthritis or other diseases or physiological conditions in which collagen degradation is a factor. The breakdown of collagen type II is believed to be initiated by specific members of the matrix metalloproteinase family of enzymes, the collagenases. Collagens are comprised of three peptide strands wound in a helix formation. For example, collagen type II is comprised of a triple helix of three identical peptide strands referred to as peptide alpha 1, collagen type II. When collagen is degraded or cleaved by a collagenase, the cleavage takes place at a specific intra-helical site leaving the peptides vulnerable to further degradation or cleavage. In any event, the initial cleavage results in the generation of collagen fragments having an end defined by the proteolytic cleavage. For example, collagenase degradation of collagen type II results in a peptide fragment having the C-terminal sequence ending with: GPXGPQG, where X is proline or hydroxyproline. Billinghurst et al described this primary collagenase cleavage site and developed antibodies reactive to both the carboxy-terminal and amino-terminal "neoepitopes" generated by cleavage of native human collagen type II. (J. Clin. Invest. 99:1534-1545 (1997)). Otterness et al have described production of a monoclonal antibody directed against this carboxy-terminal "neoepitope" (Matrix Biology 18: 331-341 (1999)). U.S. Patent No. 6,030,792 to Otterness et. al. discloses antibodies for detecting collagen fragments resulting from collagenase cleavage of type II collagen. Poole et al. U.S. Patent No. 6,132,976 provides a method for evaluating cartilage degradation by immunoassay measurement of type II collagen cleavage. These patents and references describe methods for the detection of collagen type II enzymatic cleavage by detecting the presence of peptide fragments only determine the presence of the target C- terminus portion and immediately adjacent peptide sequence using antibodies specific for these sequence regions. WO 03/021226, the disclosure of which is incorporated herein by reference, describes collagen type II fragment biomarkers of diseases from proteolytic cleavage of collagen type II by metalloproteinase enzymes and methods of identifying and quantifying such peptide fragments. Recently, it has been discovered that in an early stage of osteoarthritis, chondrocytes proliferate, and synthesize more collagen, presumably to compensate for the loss of collagen type II due its degradation by collagenases. A specific collagen, type IIA, was first identified in developing embryos from chondroprogenitor ce'lls in developing skeletal tissues. Type IIA procoUagen (SEQ ID NO. 6) is characterized by a cysteine-rich region in the NH₂ terminal end, termed PIIANP. Normal adult chondrocytes, in contrast, do not express NH₂-terminal type IIA procoUagen, but rather express NH₂- terminal type IIB procoUagen (SEQ ID NO. 3), which lacks the NH₂ cysteine-rich sequence present in PIIANP. Aigner et al. describe a reexpression of type IIA procoUagen in adults suffering from osteoarthritis (Arthritis &^• Rheumatism (1999) Vol 42, No.7, pp. 1443-1450), and Sandell, U.S. Patent No. 5,780,240 describes detection of type IIA procoUagen mRNA or peptide associated with osteoarthritis in adult humans.

Garnero et al. have proposed that a disequilibrium between synthesis of new collagen, characterized by embryonic pro-collagen IIA production and degradation of articular collagen is predictive of osteoarthritis severity and progression. Arthritis & Rheumatism (2002) Vol. 46, No. 10, pp. 2613-2624. In that study, type IIA procoUagen (PIIANP) levels were detected in serum using an ELISA, and C-terminal cross linked telopeptide (CTX-II) were detected in urine using an ELISA, at a baseline and one year time points. A ratio of PIIANP and CTX-II was calculated, and the result was termed an "uncoupling index." Patients diagnosed with osteoarthritis and having relatively low detected levels of PIIANP and high levels of CTX-II exhibited a greater level of disease progression over one year, as determined by radiography, than those patients with either high levels of PIIANP or low levels of CTX-II, although increased levels of CTX-II was determined to be a better predictor of disease progression than relatively high levels of PIIANP. Serum levels of PIIANP, however, are not necessarily correlated to PIIANP levels in synovial fluid. Furthermore, PIIANP has not been identified in urine. Therefore, there is a need for an assay that measures peptides from a consistent biological-fluid or tissue that provides correlation between peptide levels and disease progression.

SUMMARY OF THE INVENTION There is now disclosed a method of predicting disease progression in a disorder characterized by the uncoupling of collagen type II synthesis and degradation. There is further disclosed a method of detecting specific procoUagen fragments associated with osteoarthritis. The present inventors were able to detect PIIBNP fragments in the urine of both normal and osteoarthritis patient urine samples. Accordingly, an embodiment of the present invention is directed to a method of determining the level of type II B procoUagen fragments (PIIBNP, synthesis products) in a biological sample, determining the level of type II collagen degradation fragments (degradation products) in a biological sample, and calculating a ratio of synthesis products to degradation products to determine disease state or predict disease progression. In an embodiment of the present invention, PIIBNP synthesis products and type II collagen degradation products are both collected from urine. The selection of urine as a biological sample provides a less invasive, easier to obtain source of biomarkers than previously described methods. It is believed that obtaining biomarkers from a consistent source will result in improved diagnosis, prognosis, quantification and characterization, since these collagen fragments may be subject to further proteolytic degradation in different portions of the body after their initial cleavage in vivo. In another embodiment of the present invention, specific PIIBNP fragments are characterized and differentiated between healthy adults and adults afflicted with an arthritis condition. In another embodiment of the present invention, the level of type II procoUagen fragments (PIINP, synthesis products) is determined in a biological sample, the level of type II collagen degradation fragments (degradation products) is determined in a biological sample, and a ratio of synthesis products to degradation products is calculated to determine disease state or predict disease progression. In an embodiment of the present invention, PIINP synthesis products and type II collagen degradation products are both collected from urine. The selection of urine as a biological sample provides a less invasive, easier to obtain source of biomarkers than previously described methods. It is believed that obtaining biomarkers from a consistent source will result in improved diagnosis, prognosis, quantification and characterization, since these collagen fragments may be subject to further proteolytic degradation in different portions of the body after their initial cleavage in vivo. In another embodiment of the present invention, specific peptides detected in urine are disclosed. These peptides are QDVRQPGPKGQKGEPGD (SEQ ID NO. 7); QDVRQPGPKGQKGEPGD (SEQ ID

NO. 8); QDVRQPGPKGQKGEPGDIKDIK (SEQ ID NO. 9); QDVRQPGPKGQKGEPGDIKDIVGPKGPPGP (SEQ ID NO. 10); QDVRQPGPKGQKGEPGDIKDIVGPKGPPGPQGPAG (SEQ ID NO. 11); and QDVRQPGPKGQKGEPGDIKDIVGPKGPPGPQGPAGEQGPRG (SEQ ID NO. 12), including post translational modifications and derivatives thereof. The present invention is further directed to a method of determining the efficacy of an agent or drug administered to treat a disease or condition mediated by pathological degradation of type II collagen, particularly osteoarthritis. Another embodiment of the present invention relates to a method of determining the efficacy of an agent or drug intended to decrease or inhibit a collagenase enzyme comprising: obtaining a first biological sample from a subject with a collagen type II degradation mediated disorder; detecting a PIIBNP. in the first biological sample; quantifying the concentration of the PIIBNP in the first biological sample, detecting a collagen type II degradation peptide in the first biological sample; quantifying the concentration of the collagen type II degradation peptide; administering an agent or drug intended to decrease or inhibit a collagenase enzyme; obtaining a second biological sample from the subject; quantifying the concentration of the PIIBNP in the second biological sample, detecting a collagen type II degradation peptide in the second biological sample; quantifying the concentration of the collagen type II degradation peptide; and comparing the concentration of the PIIBNP and the concentration of the collagen type II degradation peptide from the second sample with the concentration of the PIIBNP and the concentration of the collagen type II degradation peptide from the first sample to determine the efficacy of the agent or drug intended to decrease or inhibit a collagenase. Yet another embodiment of the present invention relates to a method of determining an appropriate dose of an agent or drug intended to decrease or inhibit a collagenase enzyme. To this end, the present invention may also be useful in determining an appropriate dose of an agent or drug, tailored to an individual's relative disease state and to an individual's sensitivity to such agent or drug. This embodiment is directed to a method comprising: obtaining a first biological sample from a subject with a collagen type II degradation mediated disorder; detecting a PIINP in the first biological sample; quantifying the concentration of the PIINP in the first biological sample, detecting a collagen type II degradation peptide in the first biological sample; quantifying the concentration of the collagen type II degradation peptide; administering an ^'agent or drug intended to decrease or inhibit a collagenase enzyme in a fixed dose; obtaining a second biological sample from the subject; quantifying the concentration of the PIINP in the second biological sample, detecting a collagen type II degradation peptide in the second biological sample; quantifying the concentration of the collagen type II degradation peptide; and comparing the concentration of the PIINP and the concentration of the collagen type II degradation peptide from the second sample with the concentration of the PIIBNP and the concentration of the collagen type II degradation peptide from the first sample to determine the appropriate dose of the agent or drug intended to decrease or inhibit a collagenase. In addition, prior art biomarkers and methods may be used in combination with the PIINP biomarkers and methods of the present invention. Further advantages of the present invention will be apparent to those of skill in the art, in light of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the performance of a ZCGPKGQKGEPGDIKDI epitope generated antibody column.

FIG 2 shows a generalized process for detecting procoUagen peptides obtained from a biological sample;

FIG 3 is a mass spectrum of a peptide fragment, QDVRQPGPKGQKGEPGDIKD (SEQ ID NO. 9) obtained from the urine of a child;

FIG 4 is a mass spectrum of a peptide fragment QDVRQPGPXGQKGEPGDIKD, with three hexose sugar post-translational modification, where X is hydroxyl-allysine; FIG 5 is a graphic representation comparing the concentration of collagen peptide fragments obtained from twelve osteoarthritis patients to collagen peptide fragments obtained from age-matched healthy control patients;

FIG 6 is a graphic representation comparing the ratio of collagen type II degradation peptides to procoUagen NH₂-terminal synthesis peptides (PIIBNP) between osteoarthritis patients and age-matched healthy control patients; and

FIG 7 is a graphic representation of a correlation between the ratio of collagen type II degradation peptides to procoUagen NH₂-terminal synthesis peptides (PIIBNP) and arthritis score from osteoarthritis patients; and

FIG 8 is a graphic representation of a competition assay of an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is provided to aid those skilled in the art in practicing the present invention. Even so, this detailed description should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery. The contents of each of the references cited herein, including the contents of the references cited within these primary references, are herein incorporated by reference.

a. Definitions

The following definitions may be applied to terms employed in the description of embodiments of the invention. The following definitions supercede any contradictory definitions contained in each individual reference incorporated herein by reference.

Nucleic acids, amino acids, peptides, protective groups, active groups and similar moieties, when abbreviated are abbreviated according to the lUPACIUB (Commission on Biological Nomenclature) or the practice in the fields concerned.

The following are examples. General Abbreviations HPLC High pressure liquid chromatography LC Liquid chromatography MS Mass Spectrometry or Mass spectroscopy MStMS Tandem mass spectrometry MALDI Matrix assisted laser desorption ionization TOF Time-of-flight FAB Fast atom bombardment CF-FAB Continuous flow fast atom bombardment M/Z Mass-to-charge ratio Da Dalton, a unit of molecular mass equal to ¹/ι₂ mass of a ¹²C mw Molecular weight mg milligrams mL milliliters pg picograms M moles, molar nM nanόmoles, nanomolar SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis PCR Polymerase chain reaction Oligo Otig^'onucleotide RT Room temperature, about 22°C CAb Capture antibody DAb Detection antibody Reaαents EDTA Ethylenediamine tetraacetic acid SDS Sodium dodecylsulfate TW-20 Tween-20 NFDM Non-fat dry milk DPBS Dulbecco's phosphate buffered saline Bt Biotinylated HAT Hypoxanthine, aminopterin, thymidine containing media HT Hypoxanthine, thymidine containing media HRP Horseradish peroxidase

Immunoqlobulin-like molecules or chains VH or VH: Variable region of the heavy chain VL or VL: Variable region of the light chain scFv A single chain antibody containing a VL region and a VH region

Nucleic Acids RNA Ribonucleic acid DNA Deoxyribonucleic acid cDNA Compli imentary DNA mRNA Messenger RNA

Nucleic acid bases Purines Pvrimidines A: Adenine T: Thymine G: Guanine C: Cytosine U: Uracil Amino Acids-Single letter codes: Three letter codes: Full names G: Gly: glycine V: Val: valine L: Leu: leucine A: Ala: alanine 1: lie: isoleucine S: Sen serine D: Asp: aspartic acid K: Lys: lysine Hyl hydroxylysine aK allysine aHyl hydroxyallysine R: Arg: arginine H His: histidine F Phe: phenylalanine Y: Tyr: tyrosine T: Thr: threonine C Cys: cysteine M Met: methionine • E: Glu: glutamic acid W: Trp: tryptophan P, Pro: praline O Hyp: hydroxyproline 4Hyp 4-hydroxyproline N Asn: asparagine Q Gin: glutamine X: Xaa unspecified amino acid 2 — linking group

Definitions The term "identification," as used herein, means the characterization of a material, such as a peptide. The characterization may differentiate the material from other materials in a matrix, such as a liquid sample. The term "quantification," as used herein, means a determination of relative concentration or amount of a material, such as a peptide, found in a matrix, such as a liquid sample. The terms "detection" and "detecting," as used herein, means identification, quantification, or both. The term "matrix," as applied to a biological system, means a surrounding substance within which a material, such as a peptide, originates, develops, or is contained. The surrounding substance may be a liquid or a tissue, for example. The term "Liquid Chromatography - Mass Spectrometry (LC/MS)," as used herein, means the process of separation of complex mixtures of non-volatile compounds before introduction into a mass spectrometer. LC/MS is used for compounds that have a high molecular weight or are too heat sensitive to be analyzed by gas chromatography (GC). The most common ionization methods that are interfaced to LC are electrospray ionization (ESI) and Atmospheric Chemical Ionization (APCI) in positive and negative-ion modes. The LC is performed in most cases by RP-HPLC, and the buffer system should not contain involatile salts (such as phosphates). ESI can be used for m/z 50-4000 and is done at low resolving power. The terms "Tandem MS" or "MS/MS," as used herein, refers to a process in which the ion of interest is selected with a first analyzer (MS-1), collided with inert gas atoms in a collision cell, and the fragments generated by the collision are separated by a second analyzer (MS-2). Tandem mass spectrometry is used for structure determination of the peptides of interest. In Ion Trap and Fourier transform experiments, the experiments are carried out in one analyzer, and the various events are separated in time, not in space. The information is used to sequence peptides. In order to increase sample analysis throughput, the use of fast liquid is used for structure determination of molecular ions or fragments. In Tandem MS, chromatography in quantitative bioanalysis based on liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) has become prevalent. The term PIIBNP means procoUagen type IIB NH₂-terminal peptide or peptides, such that the term covers both the singular and plural. The term PIIANP means procoUagen type IIA NH₂-terminal peptide or peptides, such that the term covers both the singular and plural. The term PIINP means either PIIBNP or PIIANP or both. The term "MEROPS," as used herein, means a system of classification of peptidases, and a database of peptidases available on the world wide web at address: http://merops.sanger.ac.uk/. and used in the text Handbook of Proteolytic Enzymes. Barrett. Alan and Rawlings, Neil, ed., Academic Press 1998 (ISBN 0-12-079370-9). The term "MMP," or "matrix metalloproteinase," as used herein, means an enzyme designated clan MB (metzincins), family M10, subfamily A, by the Enzyme Commission, Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), and is a member of the family of zinc coordinating endopeptidases that function to degrade extracellular matrix proteins. MMPs are also known as matrixins. The term "MMP-13," as used herein, means the MMP classified as clan MB, family M10, MEROPS ID M10.013. MMP-13 is also known as collagenase-3. The term "MMP inhibitor," as used herein, means an agent capable of reducing or eliminating the catalytic activity of a matrix metalloproteinase, without regard to specific MMP inhibition. Thus, an MMP inhibitor may reduce or eliminate the catalytic activity of any member of the matrix metalloproteinase family, or all members of the matrix metalloproteinase family. The term "MMP-13. inhibitor," as used herein, means an MMP inhibitor that is capable of reducing or eliminating the catalytic activity of MMP-13, without regard to specificity with respect to other members of the matrix metalloproteinase family. Thus, an MMP-13 inhibitor may selectively inhibit substantially only MMP- 13, but spare other members of the matrix metalloproteinase family, or may alternatively inhibit any number of matrix metalloproteinase enzymes, as long as MMP-13 is inhibited. Those skilled in the art will recognize that with many selective enzyme inhibitors, the particular enzyme inhibiting agent is considered selective at a given dose range, and levels of the enzyme inhibiting agent exceeding that dose range may result in loss of selectivity. The term "agent^'or drug intended to decrease or inhibit a collagenase" means a molecule or collection of molecules that reduces or eliminates the expression, translation or transcription of at least one collagenase, as well as a molecule or collection of molecules that directly inhibits at least one collagenase enzyme. The term "immunoglobulin," or "Ig," as used herein, means a natural or genetically engineered tetrameric protein composed of two light chains of about 23 kD and two heavy chains of about 53-70 kD, depending on the amino acid sequence and degree of glycosylation. Multimers of the tetrameric protein are also formed (IgM and IgA). There are two classes of light chains, kappa (K) and lambda (λ), and several classes of heavy chains gamma (y), mu (μ), alpha (α), delta (δ) and epsilon (ε). There are also subclasses. Each chain, whether a light chain or heavy chain, is made up of two parts. The first part, beginning from the N-terminus of either chain, is called the variable domain. The C-terminal half of the light chain is called the constant region of the light chain and it is the primary determinant whether the light chain is a K or λ type. The constant region of the heavy chain comprise about the C-terminal three- fourths of the heavy chain and determines the class of the immunoglobulin molecule (such as, for example, IgGi or IgM), that is to say, a v heavy chain corresponds to an IgG and a μ heavy chain corresponds to an IgM, for example. V_L and VH : The amino acid sequence of the variable domain of the light chain (VL) and the variable domain of the heavy chain (VH) together determine the binding specificity and the binding (kD) constant of the immunoglobulin molecule. The variable domain comprises about half the length of the light chain and about a quarter of the length of the heavy chain and for both chains, begins at the N- terminus of the chain. The variable regions each contain three (3) hypervariable segments known as the complementarity determining regions or CDRs. CDR and FR: Each variable domain, V or V_H, is comprised of three CDRs: CDR1, CDR2 and CDR3. The intervening sequence segments before, between and after the CDRs are known as framework segments (FR). Each V and VH is comprised of four FR segments FR1 , FR2, FR3 and FR4. V_κ. and V^ : The VL domain is either K or λ, depending on which constant region (C_κ or C_λ) is used during the productive rearrangement of the light chain (VJC_K or VJC_λ). The term "antibodies," as used herein, means specific immunoglobulin molecules produced by B-cells of the immune system in response to challenges by proteins, glycoproteins, virus cells, chemicals coupled to carriers, and other substances. An antibody is simply an immunoglobulin molecule for which its binding partner is known. The substance to which the antibody binds is called an antigen. The binding of such antibodies to its antigen is highly refined and there are a multitude of specificities capable of being generated by changes in amino acid sequence in the variable domains of the heavy and light chains. The term "polyclonal antibodies," as used herein, means that a variety of different antibodies are present which may be directed to different portions of a given antigen. Normal immunization leads to a wide variety of antibodies against the same antigen. Although each B lymphocyte normally produces one immunoglobulin molecule of a defined amino acid sequence, in an immune response, many B lymphocytes are stimulated to make immunoglobulin molecules that react with the antigen, that is to say, antibodies. These different antibodies are characterized by different amino acid sequences in the variable regions of the immunoglobulin molecule which result in differences in the fine specificity and affinity of binding. Such antibodies are called polyclonal antibodies to emphasize the variety of binding specificities and binding constants which arise from the variety of amino acid sequences found in the different immunoglobulin molecules utilized in the immune response. The term "monoclonal antibodies," or "Mab," as used herein, means a plurality of antibodies that are specific to the same portion of an antigen. A B lymphocyte producing a single antibody molecule can be hybridized with an immortal B lymphocyte cell line, such as a myeloma, to derive an antibody producing immortal cell line, that is to say, a hybridoma. The hybrids thus formed are segregated into single genetic strains by selection, dilution, subcloning, and regrowth, and each strain thus represents a single genetic line. It produces a single antibody of a unique sequence. The antibody produced by such a cell line is called "monoclonal antibody" or MAb, referencing its pure genetic parentage and differentiating it from polyclonal antibody, produced from a mixed genetic background, that is to say, multiple B cells. Because a MAb is a pure chemical reagent it gives consistent, uniform results in immune tests. Moreover, because the MAb is produced by an immortal cell line, reagent supply is not limiting. For these reasons, a MAb is generally preferred over polyclonal antibodies for diagnostic purposes. The term "genetically engineered antibody," as used herein, means an artificially created antibody with variable regions similar to those of the naturally occurring antibody, but with altered constant regions. As the binding specificity of an antibody resides in the variable regions of the light and heavy chains, antibodies can be genetically engineered to change or remove the constant regions and, if done properly, it can result in an antibody molecule with different properties and molecular weight, but with the same or very similar antigen binding properties. For example, the V and V (or V_H and V ) genes can be cloned and assembled with an appropriate linker between them. Such a new genetically engineered molecule is called a single chain antibody (abbreviated scFv) and typically has a molecular weight of about 25 to 28 kD, depending on the design of the linker and the addition of other sequences to help in purification, stability, trafficking, detection, and the like. Multimers of the single chain antibody can also be made by appropriate use of linkers in which the order of each VH and VL pair may vary. In addition, some changes in the amino acid sequence of the VL and VH region can be made that retain desirable antigen binding properties. It can be seen that an almost infinite variety of genetically engineered antibodies can be derived from the original antibody sequence which retain binding specificity to the antigen, but which are tailored to fulfill specific characteristics. Other examples of genetically engineered antibodies include, but are not limited to: Fab, F(ab')₂, chi eric antibodies, humanized antibodies, and the like. The term "bispecific antibody, as used herein, means a discreet antibody with two binding sites of different specificity. Normally an IgG antibody has two identical light chains and heavy chains. There are therefore two identical antibody binding sites in the immunoglobulin molecule. By contrast, a bispecific antibody is a single immunoglobulin molecule which has two specificities. It can be made by fusion of two monoclonal antibody producing hybridoma cell lines, where each hybridoma has a different antigen specificity, and selection for a ceil line (a quadroma) that produces an antibody whose composition is a tetramer composed of one light chain and one heavy chain from each hybridoma fusion partner. The antibody produced by the quadroma has only one light and one heavy chain of each parental specificity and has one binding site for each heavy/light chain pair and is bispecific, i.e., it has two binding sites of different specificities. A bispecific antibody can also be made by genetic engineering. It can comprise the V_L linker V_H of one antibody linked through an additional linker to a V_L linker V_H of another antibody molecule. The order of V_L and V_H can be altered, but the end result is a bispecific antibody. The term "epitope," as used herein, means a portion of an antigen to which an antibody specifically binds. Depending on the size, structure and conformation of the antigen, an antibody may bind only to a small part of the entire structure. The part of the antigen molecule to which the antibody binds is called its epitope. Different antibodies may be mapped to different epitopes on the same antigen. The term "neoepitope," as used herein, means an epitope that is revealed after processing of a protein or peptide. The antigen may have an epitope which is hidden so that it cannot bind to a specific antibody. However, a conformational change in the antigen may cause the appearance of the epitope by unfolding or uncovering part of the surface of the molecule. This now allows the antibody to bind to the epitope. In another aspect, the action of an enzyme on the antigen may cause the appearance of a new epitope to which the antibody can bind. For example, after cleavage by a proteolytic enzyme, new N- terminal and new C-terminal sequences are generated. Because the epitope is not observed in the parent molecule and because, after some change in the parent molecule, the epitope is revealed and now can bind antibody, it is called a neoepitope. While the term neoepitope is described in terms of antibody recognition, as used herein the term neoepitope means any such fragment of a protein or peptide, whether or not the fragment is actually determined through immunological or other means, such as, for example, mass spectroscopy. Such a peptide fragment is not found as the native sequence, but is rather formed by enzymatic cleavage of a larger protein, thus forming a new N-terminus, a new C-terminus, or both. The term "biological media," as used herein, means any biological sample, such as a fluid, that might contain the peptide and be of interest to assay by the methods of the present invention. Biological media include, without limitation: blood, synovial fluid, urine, spinal fluid, bronchiolar lavage fluid, lymph, the vitreous humor of the eye, extracts of tissues, tissue culture supernatants, extracts of cartilage, and the like. Biological media need not be limited to human samples, but may also be obtained from a similar variety of animal media in a fashion similar to the examples above. The term "immunoassay," as used herein, means an assay for a substance, such as a complex biological such as a protein, or a small molecule, based on employing the binding properties of an antibody to recognize the substance, which may be a specific molecule or set of homologous molecules. The immunoassay may involve one or more antibodies. Non-limiting examples of applicable immunoassays are provided below. It is noted that the post-translational modifications, 4-hydroxylation of proline and hydroxylation of lysine, appear in varying levels in collagen peptides and peptide fragments in different species. In addition, the conversion of lysine to allysine by lysyl oxidase, and the conversion of hydroxylysine to hydroxyallysine was found in some of the procoUagen peptide fragments. The originally translated peptides themselves, as well as permutations of the post-translational modifications, may be found in urine of subjects and are included among the embodiments of the invention. Identifiable derivatives or modifications of the peptides also can function as markers and are included within the scope of the invention.

Direct Assay In a "direct assay," the antibody binds directly to an antigen such as in a biological specimen (cells, tissues, histological section, and the like) or to antigen adsorbed or chemically coupled to a solid surface. The antibody itself is usually labeled to enable the determination of the amount of antibody bound to the antigen. Alternatively, the antibody (now termed primary antibody) is detected with a secondary labeled antibody that will demonstrate that binding of the primary antibody had occurred.

Competitive Assay An assay based on the binding properties of a single antibody molecule. Typically, a labeled antigen is used to compete with an unknown antigen and the amount of unknown antigen is determined in terms of how much of the labeled antigen is displaced by the unknown antigen. The label may be radioactive, optical, enzymatic, florescent polarizing, florescent quenching, or other label. The antibody may be monospecific or bispecific.

Sandwich Assay This is a double antibody assay in which both antibodies bind to the antigen, forming a trimeric immune complex or sandwich containing the two antibodies with the antigen between them. One antibody is utilized to localize the immune complex to the detection surface or chamber. This antibody is termed the capture antibody. The other antibody bears a label that will allow the immune complex to be detected. It is called the detection antibody. If an immune complex is not formed (no antigen is present), then the capture antibody is unable to bring the detection antibody to the detector. If antigen is present, then an immune complex will form and the capture antibody will be joined with the detection antibody such that the amount of detection antibody in the immune complex is quantitatively related to the amount of antigen present. The assay can be formatted in many ways. For example, the capture antibody can be chemically coupled to a solid surface, or non-specifically adsorbed to a surface, attached via biotinylation to an avidin-like molecule, for example, without limitation, avidin, streptavidin, neutravidin, and the like, streptavidin or avidin-coated surface, coupled to magnetic particles or beads as a means of localizing the immune complex to the measurement device. The detection antibody may be radiolabeled, or it may have a variety of possible enzymatic amplification systems such as horseradish peroxidase (HRP), alkaline phosphatase (AP), urease, and the like, when formatted as an Elisa (Enzyme-linked immune assay). It may have an electrochemical, an optical, a fluorescent or other detection method to determine the amount of detection antibody in the immune complexes. It may immediately be seen that many examples can be derived in which the two antibodies are paired in a sandwich assay using a variety of methods to capture the immune complex in a detection device and a variety of detection systems to measure the amount of immune complex.

Molecular Biology Techniques A skilled artisan could in vitro produce a complete gene coding for the V_H and V_L regions and a completely functional antibody. It can be produced as an immunoglobulin molecule of any given class with constant regions of the heavy chain and light chain added or it can be produced as a scFv with V_H and V joined by a linker with tags added as appropriate. The constructed gene may be engineered by conventional recombinant techniques, for example, to provide a gene insert in a plasmid capable of expression. Thereafter, the plasmids may be expressed in host cells where the host cells may be bacteria such as E. coli or a Bacillus species, yeast cells such as Pichia pastoris or in mammalian cell lines such as, for example and without limitation, Sp2/0, Ag8 or CHO cells.

Preferred Embodiments One embodiment of the invention is directed to the identification and quantification of collagen peptides, particularly in biological samples from humans or animals. The term "biological sample" includes a sample from any body fluid or tissue. The invention includes a method of determining the presence of, and identifying the structure of, cleaved propeptide collagen type IIB fragments, and peptide degradation products of specific collagenase enzyme activity. The present invention can be used to detect cleavage by proteolytic enzymes, such as, for example, matrix metalloproteinases -1, -8, and -13, that results in peptides with C-terminal amino acid sequences having characteristic fragment ions upon collisional activation by tandem mass spectrometry that can be identified by the methods of the present invention. The present invention allows the diagnosis and prognosis of physiological conditions characterized by collagen degradation through the identification and quantification of propeptide collagen type II fragments and collagen degradation peptides discovered to be present in biological samples of subjects exhibiting signs and symptoms of osteoarthritis. This quantification of the peptides can be used for diagnosis or prognosis of diseases such as osteoarthritis, to monitor collagenase enzyme activity in disease or physiological conditions characterized by collagenase activity, and to evaluate drugs or agents used to inhibit collagen degradation, such as matrix metalloproteinase inhibitors, or the levels of active collagenase, particularly those drugs or agents that result in the inhibition of collagen degradation. The identification and quantification of the biomarker peptides can be used in diagnosis and prognosis of diseases or conditions characterized by the ratio of collagen type II propeptide (as a surrogate for collagen synthesis) and collagen type II fragments (as a surrogate for collagen type II degradation).

Purification of Samples It may be desirable to purify a biological sample by selective removal of target procoUagen peptide fragments from the biological sample prior to detection. This is useful to remove other peptide fragments present in the biological sample, which would otherwise create "background noise" in mass spectrometry. The peptides can be separated from the biological matrix components of the sample by an appropriate separation method known to the art. For example, chromatographic or electrophoretic separation can be used in this step. Other appropriate separation or "clean up" methods are contemplated by the invention. In liquid chromatographic separation the eluant containing the peptides of the target mass are introduced to a mass spectrometer through an appropriate liquid chromatography/mass spectrometry interface. Purification may be accomplished, for example, by application of a liquid chromatography column containing antibodies specific to a known region of the propeptide of interest.

Example I - Purification of a Peptide Fragment in a Biological Sample An antibody was raised in chickens to the artificial peptide KLH-ZCGPKGQKGEPGDIKDI (SEQ ID NO. 13) of the NH₂-terminal sequence of PIINP, where "Z" represents a linking group, and "KLH" represents Keyhole Limpet Hemocyanin, using well known techniques. The resulting antibodies were immobilized into columns using epoxide chemistry. Urine samples were applied to the column, and the bound PIINP were eluted into a sample vials. FIG. 1 shows the performance of the antibody column. In a similar manner, an antibody was raised in chickens to the artificial peptide DQAAGGLRQHDAEVDATLCZ-KLH (SEQ ID NO. 14) of the carboxy-terminal propeptide of collagen type II, where "Z" represents a linking group, and "KLH" represents Keyhole Limpet Hemocyanin. While usable, this purified carboxy-terminal peptide did not provide as strong a signal as did the NH₂-terminal

Determination of Molecular Mass Mass spectrometers measure mass-to-charge ratios (m/z) of ions produced from molecules introduced into an ion source. In many ionization methods developed for mass spectrometry, the charge is generally (but not always) unity, so it is often stated that 'mass' is measured with a mass spectrometer, although this is not strictly correct. Several newer ionization techniques routinely produce multiply- charged ions; most notable among these being electrospray ionization (ESI). In the case of peptides, a wide distribution of charges may be recorded for a peptide. Fortunately, these sequentially charged (m/z) states give peaks in the spectrum that can be mathematically processed to derive the original molecular mass of the peptide.

Example II ■ Detection of PIIBNP Fragments in Urine Figure 2 shows the general format used in the detection of PIIBNP from a biological sample. It is to be understood that this is only an exemplary method of detecting PIIBNP, and other analogous techniques could be employed. For example, a single antibody column could be employed, or no purification step may be performed. A sample of urine was injected at a rate of between one and two mL per minute using an Agilent 1100 quaternary pump with mobile phase A: 25 nM ammonium acetate, pH 7 through a series of up to five columns in which antibodies to the peptide sequence ZCGPKGQKGEPGDIKDI (SEQ ID NO. 13) of the NH₂-terminal sequence of PIIBNP was immobilized by either Protein G or epoxide coupling to the resin therein, into an autosampler with a 96 well plate format into 1ml vials. An elution with mobile phase B: 0.1 - 0.5% formic acid or ammonium acetate at pH 4.5 was performed on the column, and the resulting samples were eluted with 300 μL/min. flow rate onto a Betasil C-18 column (ThermoHypersil- Keystone Co., Bellefonte, PA, 5 micron particle size, 100 x 2 mm) with mobile phase composed of with 1% formic acid using a second Agilent 1100 quaternary pump, and collected in a Michrom Bioresources macro peptrap, a C-18 type trap, (Michrom BioResources, Inc., Auburn, CA) that was connected to an HP1100 HPLC system (Agilent, Palo Alto, CA). Waste was expelled by a wash with mobile phase B: 1% formic acid in acetonitrile. The purified samples were analyzed in an API 4000 triple quadrapole mass spectrometer (Applied Biosystems/MDS SCIEX , Foster City, CA 94404, U.S.A.) operated in MRM mode with detection limits between 100 pg/mL - 100 ng/mL. The peptides are fragmented by collisional activation by a neutral gas in a collision cell using collision energies and methods known to the art resulting in collision-induced dissociation of the peptide to create corresponding fragment ions. The spectrum of the fragment ions was analyzed. Propeptide type IIB NH₂-terminal peptide fragments (PIIBNP) were first isolated from a sample of urine from a twelve-year old child. The choice to use a biological sample from a child for detection was made because the rate of synthesis of collagen type II is typically much greater in children than in healthy adults, thereby providing a stronger signal in the mass spectrum. The mass spectrum of the sequence QDVRQPGPKGQKGEPGDIKD (SEQ ID NO. 9) of PIIBNP is shown in FIG. 3.

Post-Translational Modifications Lysine may be converted to allysine (and hydroxylysine may be converted to hydroxyallysine) in vivo by lysyl oxidase by way of the following formula: NH O CH CH i_H" + O ₊ H O ^{LySyl 0XidasB} > CH + H O ₊ NH CH CH CH CH -NH-CH-CO- -NH-CH-CO- Lysiπa Allysine

This post-translational modification was found in several PIIBNP characterized. Such post- translational modifications are intended to be within the scope of the appended claims. For example, SEQ ID NO. 9 was detected in the following modified forms: QDVRQPGP(aK)GQKGEPGDIKD I (1) OH

QDVRQPGP(aK)GQKGEPGDIKD I (2) OGal QDVRQPGP(aK)GQKGEPGDIKD I (3) OGalGlu

QDVRQPGP(aK)GQKGEPGDIKD ) | (4) GalGlu O OGal

QDVRQPGP(aK)GQKGEPGDIKD I I (5) GalGlu O OGalGlu

FIG 4 shows the mass spectrum of example (4), with three hexose sugar modifications. Six other PIIBNP were characterized and quantified in human urine, as follows:

QDVRQPGPKGQKGEPGD (SEQ ID NO. 7); QDVRQPGPKGQKGEPGDIK (SEQ ID NO. 8);

QDVRQPGPKGQKGEPGDIKD (SEQ ID NO. 9); QDVRQPGPKGQKGEPGDIKDIVGPKGPPGP (SEQ ID NO. 10); QDVRQPGPKGQKGEPGDIKDIVGPKGPPGPQGPAG (SEQ ID NO. 11); and

QDVRQPGPKGQKGEPGDIKDIVGPKGPPGPQGPAGEQGPRG (SEQ ID NO. 12). Of these peptides, QDVRQPGPKGQKGEPGD (SEQ ID NO. 7) and QDVRQPGPKGQKGEPGDIKD (SEQ ID NO. 9) were most abundant, in both normal and osteoarthritis patients. Example III - Detection of PIIBNP in Osteoarthritis Patients Urine samples were collected from twelve patients with diagnosed osteoarthritis, and six age- matched, healthy control subjects. LC/MS/MS analysis of the samples was performed as described herein. The samples were further analyzed for detection of collagen type II degradation peptides, as previously described. Patients one through twelve had been previously diagnosed with osteoarthritis, while patients thirteen through eighteen were age-matched, heaithy controls. FIG. 5 graphically depicts the concentration of collagen type II 45 mer degradation peptide present in the samples. The osteoarthritis group had a mean concentration of 6.6 ng/ml collagen type II 45 mer degradation peptide, compared with the control group with a mean concentration of 3.4 ng/ml. When the ratio of collagen type II 45 mer degradation peptide to PIIBNP was calculated, however, an even greater differential between osteoarthritis patients and normal control patients was observed. FIG. 6 graphically depicts the ratio. The osteoarthritis group had a mean ratio of 10, while the control group had a mean ratio of 4.8. Arthritis scores for each of the twelve osteoarthritis patients were assessed, and compared to the ratios for each of the twelve osteoarthritis patients. FIG. 7 graphically shows the results of each patients arthritis score and ratio of collagen type II 45 mer degradation peptide to PIIBNP. As can be seen, there is correlation between arthritis score and collagen type II 45 mer degradation peptide to PIIBNP ratio.

Example IV- ELISA ASSAYS 1. Capture ELISA Assay

A. Selection of a First Monoclonal Antibody

A first monoclonal antibody to be used as a detection antibody is isolated or selected from known, available monoclonal antibodies such that the monoclonal antibody will demonstrate binding to a particular degradation product (a PIIBNP fragment, for example) but no significant binding to intact collagen. The generation and characterization of such a monoclonal antibody is well known in the art. Monoclonal antibodies can be of any class of antibody, for example, IgG, IgA, IgD, IgE, and IgM.

B. Selection of a Second Monoclonal Antibody A second monoclonal antibody to be used as a capture antibody is isolated or selected from known, available monoclonal antibodies such that the monoclonal antibody will demonstrate binding to synthesis products (PIIBNP fragments) but no significant binding to intact collagen. The generation and characterization of such a monoclonal antibody is well known in the art.

C. Sandwich Assay Using a Capture Antibody (CAb) and a Monoclonal Antibody as the Detection Antibody (Dab) for Determining Concentration of a Degradation Product in a Biological Sample.

The second monoclonal antibody of part B, above (the capture antibody, CAb) may be added to Nunc Maxisorp (VWR, Boston, Mass.) 96-well plates or their equivalent, with CAb at 10 μg/mL in 0.05M sodium borate buffer, pH 8.5 using 100 μL/well (except for control wells numbered 4, 5 and 6, see Table 1) and incubated for about 18-48 hours at 4° C. The plate may be washed, for example, for three times with DPBS with 0.05% TW-20 (Sigma), (DPBS/TW-20); 200 μL/well may be used. Wells in the plate are blocked with 1% non-fat dry milk (NFDM) dissolved in DPBS (NFDM DPBS) prepared freshly, that is, held on ice for no more than the day of use, using 100 μL/well incubated for 1 hour at RT. The blocking solution is discarded, the wells rinsed one time with 200 μL of DPBS/TW-20. A peptide, for example corresponding in amino acid sequence to SEQ ID NO 7, is diluted in 0.1% NFDM DPBS to concentrations shown in Table 1. The exemplary peptide has the sequence: Gln-Asp- Val-Arg-Gln-Pro-Gly-Pro-Lys-Gly-Gln-Lys-Gly-Glu-Pro-Gly-Asp, as set forth in the Sequence Listings as SEQ ID NO: 7 and may be synthesized and purified by any custom protein synthesis laboratory, such as Anaspec Inc (San Jose, Calif.), for example. The dilutions of this exemplary peptide of SEQ ID NO: 7, the samples at appropriate dilutions, and the controls are placed into the specified wells of the microtiter plate as shown in Table 1.

TABLE 1

Outline of a microtiter plate and antibody coating scheme Peptide (SEQ ID NO 7) ng/mL

TABLE 2

Additions to the control wells Controls: Biotinylated Anti-biotin antibody CAb peptide DAb HRP-labelled

1 + 2 + 3 + 4 -

The wells are washed three times with 200 μL/well of DPBS TW-20. Biotin-conjugated DAb (Bt-DAb) is added to all peptide of SEQ ID NO: 7 containing wells, all sample wells, and all control wells except 1 ,2, and 5. Bt-DAb (100 μL/well) at 1 μg/mL in 0.1% NFDM DPBS is added to each well and the plate is incubated for 40 min at 37 °C. Optionally, the DAb may be biotinylated using 37 μg of biotin-N-hydroxysuccinamide (available from Pierce Chemical) per mg of DAb for 2 hrs and then dialyzed overnight using a 10 kD cut-off dialysis cassette (available from Pierce Chemical). The wells are washed three times with 200 μL/well of DPBS TW-20. Mouse monoclonal anti-biotin antibody conjugated with HRP (available from Jackson ImmunoResearch) is diluted 1/5000 in 0.1% NFDM DPBS and 100 μL/well is added to all wells and incubated for 30 minutes at RT. The wells are washed three times with 200 μ L/well with DPBS TW-20. 100 μL/well of 1-step Turbo (ready to use 3,3',5,5'-tetramethyl benzidine; available from Pierce Chemical) is added to each well and incubated at RT for approximately 10 minutes. Color development may be stopped with 2N H₂ S0 . The results are read on a spectrophotometer at 450 nm. From the concentrations of a peptide of SEQ. ID NO. 7 and the resulting optical density reading at 450 nm, a standard curve can be constructed. Over the linear portion of the curve, a regression line is used to fit the data. For samples that fall outside of the linear portion, the concentrations can be read off the graph or the samples may be diluted to fall within the standard portion of the curve, or they may be below the limit of detection. The regression between log (nM) and OD450 gives a slope OD450/log(nM) and an intercept OD450. When unknown samples are run, the calibration curve can be used to determine of concentration of PIIBNP fragments from the optical density of the sample. The following calculation can be used to determine the concentration of the degradation fragment in a biological sample: Log(Concentration[nM]) = (Sample OD450 - Intercept) / Slope Thus, the inverse log of the resulting number provides the concentration in nM.

Other appropriate peptides or collagen fragments can be substituted to prepare a standard curve. The units are expressed in terms of molar equivalents of standard. 2. Competitive ELISA Assay This assay is a competitive ELISA using a 96 well format. The ELISA plate is coated overnight at room temperature with chicken IgY antibody against procoUagen II N-terminal propeptide (PIINP) sequence CZGPKGQKGEPGDIKDI (SEQ ID NO. 13). The unbound antibody is removed. A fixed amount of biotynylated PIINP peptide is allowed to compete with standard peptide or unknown samples for 2 hours. Unbound PIINP is removed and biotinylated peptide is detected with Horseradish peroxidase conjugated anti-biotin and developed using Tetramethylbenzidine (TMB) substrate. This assay can be used to detect NPII levels in human, rat, and dog serum, plasma, urine and synovial fluid, since these species share identical homology in the propeptide sequence GPKGQKGEPGDIKDI (SEQ ID NO. 13). SEQ ID NO. 14 is the full-length rat (rattus norvegicus) unprocessed collagen type II alpha 1 sequence. SEQ ID NO. 15 is the procoUagen IIA sequence for dog (Cannis familiaris). SEQ ID NO. 16 is a fragment of the procoUagen IIB sequence for dog (Cannis familiaris). FIG. 8 shows the format of this assay.

3. Competitive Solution Phase Assay Wells of 96-well plates are coated with protein antigen (2 mg/ml in carbonate buffer, 0.13 ml/well), and kept at 4°C overnight. Wells are aspirated and blocked with 2% BSA in TBS (250 ml/well) for 2 hours at room temperature. Using, for example, polypropylene tubes, the antigen is diluted into antibody solution (varying antigen concentration with constant antibody). Optimum concentration of the antibody must be routinely determined. Both antibody and antigen dilutions are conveniently made from stock solutions for which the protein concentrations are determined by absorbance measurement at 280 nm, for example. Typically, an antibody concentration of 0.5 nM and a starting antigen concentration of 10 mM is appropriate. Serial dilutions are made into antibody diluted to the appropriate concentration in 0.1% BSA-TBS. Following a three-hour incubation at room temperature, transfer about 0.1 ml of antigen-antibody solution to duplicate or triplicate wells of antigen-coated 96-well plates, and let the plate sit at room temperature for about thirty minutes. Wells should be washed, about four times, with TBS. Peroxidase-conjugated secondary antibody is added, and incubated for about thirty minutes at room temperature. Wells should again be washed, about four times, with TBS. A chromogenic substrate (such as, for example, o-Phenylenediamine) is then added. The reaction is allowed to develop for about five to ten minutes, and stopped by adding 50 ml of 4N H₂S0₄, for example. The absorbance of the wells is read at an appropriate wavelength, such as, for example, between 450-490 nm. Absorbances are averaged from duplicate wells and Absorbance vs. log [Inhibitor] is plotted.

P. Comparison of Sample Concentration to Known Standard Concentration The concentration calculated from the biological sample is compared with a standard. The standard is preferably provided as adjusted based upon normal function, that is to say, with little synthesis and degradation product concentration. A standard error is taken into account for the approximate standard concentration. Values outside of the standard error are considered to be abnormal.

Example V - Determination of Efficacy of Agents or Drugs that Prevent Cartilage Degradation The present invention may be used to determine the efficacy of agents or drugs administered to treat or prevent a cartilage type II degradation mediated disorder, such as, for example, arthritis. To this end, In a first detection step, PIIBNP fragments, such as SEQ ID. NO. 9, SEQ ID NO. 10, SEQ ID NO. 1 , or SEQ ID NO. 12, or combinations thereof, are detected in a biological sample obtained from an individual exhibiting the signs and symptoms of arthritis, or an individual predisposed to an arthritis condition. Collagen type II degradation fragments may also be detected in a biological sample obtained from the same individual, as previously described. The biological sample may be the same sample, or a different sample. Optionally, both PIIBNP fragments and collagen type II degradation fragments are both obtained from the same individual. In this optional step, the ratio of PIIBNP fragments to collagen type II degradation fragments may be compared, and this ratio may be used to determine the presence or stage of arthritis. An agent or drug intended to decrease or inhibit a collagenase is administered to the individual. A subsequent, second detection step is performed, in which detection of PIIBNP fragments is carried out at a time after administration of the agent or drug intended to decrease or inhibit a collagenase, and optionally, detection of collagen type II degradation fragments is also performed. The presence, and optionally the quantity, of PIIBNP fragments, and optionally the presence and quantity of collagen type II degradation fragments is compared with the results of the first detection step, and the efficacy of the agent or drug administered to decrease or inhibit the collagenase is determined based upon the resulting detection. The following non-limiting list of individual patents and publications describe MMP inhibitors useful in the practice of the present invention: U.S. Patent No. 6,541,489; U.S. Patent No. 6,448,250; WO 00/69821; WO 02/064599; US2002-0156061; US2003-0004172; U.S. published application 2003- 0078276; U.S. published application 2003-0130278; U.S. published application 2002-0193377; U.S. published application 2002-0151558; U.S. published application 2002-0156069; U.S. published application 2002-0151555; U.S. published application 2002-0161000; U.S. published application 2003- 0087924; U.S. published application 2003-0144274; WO 02/064080; WO 02/064568; WO 02/064571; WO 02/064572; WO 02/064578; WO 02/064595; WO 02/064598; WO 03/032999; WO 02/064547; WO 03/076417; WO 03/033478 and WO 04/000811. In particular, the following MMP inhibitors would be useful in the practice of the present invention: N-hydroxy-4-({4-[5-(3,3,4,4,4-pentafluorobutyl)pyrazin-2-yl]phenyl}sulfonyl)tetrahydro-2H-pyran-4- carboxamide hydrochloride; N-hydroxy-4-({4-[5-(4,4,4-trifluorobutyl)pyrazin-2- yl]phenyI}sulfonyl)tetrahydro-2H-pyran-4-carboxamide hydrochloride; N-hydroxy-4-({4-[5-(3,3,3- trifluoropropyl)pyrazin-2-yl]phenyl}sulfonyl)tetrahydro-2H-pyran-4-carboxamide hydrochloride; 1-(2- methoxyethyl)-4-({4-[5-(4,4,4-trifluorobutyl)(2-pyridyl)]phenyl}sulfonyl)piperidine-4-carbohydroxamic acid, dihydrochloride; 4-({4-[5-(2-cyclopropylethyl)pyrazin-2-yl]phenyl}sulfonyl)-N-hydroxytetrahydro- 2H- pyran-4-carboxamide hydrochloride; 4-({4-[5-(3,3-difluorobutyl)pyrazin-2-yl]phenyl}sulfonyl)-N- hydroxytetrahydro-2H-pyran-4-carboxamide hydrochloride; 4-[6-(4-methoxy-benzylcarbamoyl)-4-oxo-4H- pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3-methoxy-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4- d]pyrimidin-3-ylmethyl]-benzoic acid; 4-{4-oxo-6-[(pyridin-3-ylmethyl)-carbamoyl]-4H-pyrido[3,4- d]pyrimidin-3-ylmethyl}-benzoic acid; 4-[6-(4-chloro-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3- ylmethylj-benzoic acid; 4-{4-oxo-6-[(pyridin-4-ylmethyl)-carbamoyl]-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl}- benzoic acid; 4-{6-[(2-methoxy-pyridin-4-ylmethyl)-carbamoyl]-4-oxo-4H-pyrido[3,4-d]pyrimidin-3- ylmethyl}-benzoic acid; 4-[6-(4-methylsulfanyl-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3- ylmethylj-benzoic acid; 4-[6-(4-fluoro-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]- benzoic acid; 4-[6-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d3pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3- chloro-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3-fluoro- benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid;4-[4-oxo-6-(4-trifluoromethyl- benzylcarbamoyl)-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[4-oxo-6-(3-trifluoromethyl- benzylcarbamoyl)-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3,4-difluoro- benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(4-hydroxy-3-methoxy- benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin- 3-ylmethyl]-benzoic acid; 4-[[4-(4-bromophenyl)-4- hydroxy-1-piperidinyl]sulfonyl]- tetrahydro-N- hydroxy-2H-pyran-4- carboxamide: and 4-[6-(4methoxy-benzylcarbamoyl)-4-oxo-4H-pyrido-[3,4-d]pyrimidin-3- ylmethyl]- cyclohexanecarboxyiic acid.

Claims

CLAIMSWhat is claimed is:

1. A method of diagnosing a condition mediated by the pathological degradation of collagen type II in a subject comprising: detecting at least one PIIBNP in a biological sample obtained from said subject; and quantifying the concentration of said PIIBNP from said biological sample.

2. The method of claim 1 wherein said PIIBNP is selected from the group consisting of SEQ ID 9,

SEQ ID 10, SEQ ID 11, and SEQ ID 12.

3. The method of claim 1 wherein a plurality of PIINP are collected.

4. The method of claim 1 further comprising detecting at least one degradation peptide of collagen type II, quantifying the concentration of said degradation peptide of collagen type II, and determining the ratio of said concentration of said degradation peptide of collagen type II to said concentration of said PIIBNP.

5. The method of claim 4 wherein said biological sample is urine.

6. The method of claim 4 wherein said biological sample is collected contemporaneously.

7. The method of claim 6 wherein said biological sample is collected in a single vessel.

8. The method of claim 1 wherein said biological sample obtained from said subject represent a first sample, and second biological sample is collected from said subject at a subsequent time, and at least one PIIBNP in a biological sample is obtained from said subject from said second sample; the concentration of said PIIBNP from said second biological sample is quantified, and said concentration of said PIIBNP from said second sample is compared with the concentration of said first sample.

9. A method of determining the efficacy of an agent or drug intended to decrease or inhibit a collagenase enzyme comprising: obtaining a first biological sample from a subject with a collagen type II degradation mediated disorder; detecting a PIIBNP in said first biological sample; quantifying the concentration of said PIIBNP in said first biological sample, detecting a collagen type II degradation peptide in said first biological sample; quantifying the concentration of said collagen type II degradation peptide; administering an agent or drug intended to decrease or inhibit a collagenase enzyme; obtaining a second biological sample from said subject; quantifying the concentration of said PIIBNP in said second biological sample, detecting a collagen type 11 degradation peptide in said second biological sample; quantifying the concentration of said collagen type II degradation peptide; and comparing the concentration of said PIIBNP and said concentration of said collagen type II degradation peptide from said second sample with the concentration of said PIIBNP and said concentration of said collagen type II degradation peptide from said first sample to determine the efficacy of said agent or drug intended to decrease or inhibit a collagenase.

10. The method of claim 9 wherein said agent or drug intended to decrease or inhibit a collagenase is selected from the group consisting of:

N-hydroxy-4-({4-[5-(3,3,4,4,4-pentafluorobutyl)pyrazin-2-yl]phenyl}suifonyl)tetrahydro-2H-pyran-4- carboxamide hydrochloride; N-hydroxy-4-({4-[5-(4,4,4-trifluorobutyl)pyrazin-2- yl]phenyl}sulfonyl)tetrahydro-2H-pyran-4-carboxamide hydrochloride; N-hydroxy-4-({4-[5-(3,3,3- trifIuoropropyl)pyrazin-2-yl]phenyl}sulfonyl)tetrahydro-2H-pyran-4-carboxamide hydrochloride; 1-(2- methoxyethyl)-4-({4-[5-(4,4,4-trifluorobutyl)(2-pyridyl)]phenyl}sulfonyl)piperidine-4-carbohydroxamic acid, dihydrochloride; 4-({4-[5-(2-cyclopropylethyl)pyrazιn-2-yl]phenyl}sulfonyl)-N-hydroxytetrahydro- 2H- pyran-4-carboxamide hydrochloride; 4-({4-[5-(3,3-difluorobutyl)pyrazin-2-yl]phenyl}sulfonyl)-N- hydroxytetrahydro-2H-pyran-4-carboxamide hydrochloride; 4-[6-(4-methoxy-benzylcarbamoyl)-4-oxo-4H- pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3-methoxy-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4- d]pyrimidin-3-ylmethyl]-benzoic acid; 4-{4-oxo-6-[(pyridin-3-ylmethyl)-carbamoyl]-4H-pyrido[3,4- d]pyrimidin-3-ylmethyl}-benzoic acid; 4-[6-(4-chloro-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3- ylmethyij-benzoic acid; 4-{4-oxo-6-[(pyridin-4-ylmethyl)-carbamoyl]-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl}- benzoic acid; 4-{6-[(2-methoxy-pyridin-4-ylmethyl)-carbamoyl]-4-oxo-4H-pyrido[3,4-d]pyrimidin-3- ylmethyl}-benzoic acid; 4-[6-(4-methylsulfanyl-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3- ylmethylj-benzoic acid; 4-[6-(4-fluoro-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]- benzoic acid; 4-[6-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3- chloro-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3-fluoro- benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-dlpyrimidin-3-ylmethyll-benzoic acid;4-[4-oxo-6-(4-trifluoromethyl- benzylcarbamoyl)-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[4-oxo-6-(3-trifluoromethyl- benzylcarbamoyl)-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3,4-difluoro- benzyIcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(4-hydroxy-3-methoxy- benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin- 3-ylmethyl]-benzoic acid; 4-[[4-(4-bromophenyl)-4- hydroxy-1-piperidinyl]sulfonyl]- tetrahydro-N- hydroxy-2H-pyran-4- carboxamide; and 4-r6-(4methoxv-benzvlcarbamovl)-4-oxo-4H-pvrido-r3.4-dlpyrimidin-3- ylmethyl]- cyclohexanecarboxylic acid.

11. A method of determining the an appropriate dose of an agent or drug intended to decrease or inhibit a collagenase enzyme comprising: obtaining a first biological sample from a subject with a collagen type II degradation mediated disorder; detecting a PIIBNP in said first biological sample; quantifying the concentration of said PIIBNP in said first biological sample, detecting a collagen type II degradation peptide in said first biological sample; quantifying the concentration of said collagen type II degradation peptide; administering an agent or drug intended to decrease or inhibit a collagenase enzyme in a fixed dose; obtaining a second biological sample from said subject; quantifying the concentration of said PIIBNP in said second biological sample, detecting a collagen type II degradation peptide in said second biological sample; quantifying the concentration of said collagen type II degradation peptide; and comparing the concentration of said PIIBNP and said concentration of said collagen type II degradation peptide from said second sample with the concentration of said PIIBNP and said concentration of said collagen type II degradation peptide from said first sample to determine the appropriate dose of said agent or drug intended to decrease or inhibit a collagenase.

12. The method of claim 11 wherein said agent or drug intended to decrease or inhibit a collagenase is selected from the group consisting of:

N-hydroxy-4-({4-[5-(3,3,4,4,4-pentafluorobutyl)pyrazin-2-yl]phenyl}sulfonyl)tetrahydro-2H-pyran-4- carboxamide hydrochloride; N-hydroxy-4-({4-[5-(4,4,4-trifluorobutyl)pyrazin-2- yl]phenyl}sulfonyl)tetrahydro-2H-pyran-4-carboxamide hydrochloride; N-hydroxy-4-({4-[5-(3,3,3- trifluoropropyl)pyrazin-2-yl]phenyl}sulfonyl)tetrahydro-2H-pyran-4-carboxamide hydrochloride; 1 -(2- methoxyethyl)-4-({4-[5-(4,4,4-trifluorobutyl)(2-pyridyl)]phenyl}sulfonyl)piperidine-4-carbohydroxamic acid, dihydrochloride; 4-({4-[5-(2-cyclopropylethyl)pyrazin-2-yl]phenyl}sulfonyl)-N-hydroxytetrahydro- 2H- pyran-4-carboxamide hydrochloride; 4-({4-[5-(3,3-difluorobutyl)pyrazin-2-yl]phenyl}sulfonyl)-N- hydroxytetrahydro-2H-pyran-4-carboxamide hydrochloride; 4-[6-(4-methoxy-benzylcarbamoyl)-4-oxo-4H- pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3-methoxy-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4- d]pyrimidin-3-ylmethyl]-benzoic acid; 4-{4-oxo-6-[(pyridin-3-ylmethyl)-carbamoyl]-4H-pyrido[3,4- d]pyrirriidin-3-ylmethyl}-benzoic acid; 4-[6-(4-chloro-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3- ylmethylj-benzoic acid; 4-{4-oxo-6-[(pyridin-4-ylmethyl)-carbamoyl]-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl}- benzoic acid; 4-{6-[(2-methoxy-pyridin-4-ylmethyl)-carbamoyl]-4-oxo-4H-pyrido[3,4-d]pyrimidin-3- ylmethylj-benzoic acid; 4-[6-(4-methylsulfanyl-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3- ylmethylj-benzoic acid; 4-[6-(4-fluoro-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]- benzoic acid; 4-[6-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3- chloro-benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3-fluoro- benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid;4-[4-oxo-6-(4-trifluoromethyl- benzylcarbamoyl)-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[4-oxo-6-(3-trifluoromethyl- benzylcarbamoyl)-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(3,4-difluoro- benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin-3-ylmethyl]-benzoic acid; 4-[6-(4-hydroxy-3-methoxy- benzylcarbamoyl)-4-oxo-4H-pyrido[3,4-d]pyrimidin- 3-ylmethyl]-benzoic acid; 4-[[4-(4-bromophenyl)-4- hydroxy-1-piperidinyl]sulfonyl]- tetrahydro-N- hydroxy-2H-pyran-4- carboxamide; and 4-[6-(4methoxy-benzylcarbamoyl)-4-oxo-4H-pyrido-[3,4-d]pyrimidin-3- ylmethyl]- cyclohexanecarboxylic acid.

13. A peptide, or post-translationally modified or derivitized peptide selected from the group consisting of:

SEQ ID NO. 9, SEQ ID NO 10; SEQ ID NO. 11 and SEQ ID NO. 12.

14. The use of a peptide, or post-translationally modified or derivitized peptide selected from the group consisting of:

SEQ ID NO. 9, SEQ ID NO 10; SEQ ID NO. 11 and SEQ ID NO. 12, as a biomarker of a collagen type II degradation mediated disorder.