WO2002077639A2 - Osteocalcin fragments assay to monitor bone resorption - Google Patents

Abstract

The amount of osteocalcin-derived fragments in a body fluid is measured by determining binding to said fragments of an immunological binding partner specific for a epitope located at the N or C terminus of a said fragment created upon cleavage of osteocalcin at said terminus during resorption of bone and which is not present in intact osteocalcin. The epitope may be created by cleavage by cathepsin K and may include an isomerised or optically inverted amino acid.

Description

A METHOD OF ASSAYING OSTEOCALCIN FRAGMENTS IN BODY FLUIDS, A

TEST KIT AND MEANS FOR CARRYING OUT THE METHOD AND USE OF THE

METHOD TO MONITOR BONE RESORPTION AND RESPONSE TO

ANTIRESORPTIVE THERAPY

The present invention relates to a method of measuring the amount of osteocalcin fragments in biological fluids. The invention further relates to means, including synthetic peptides, monoclonal and polyclonal antibodies and cell lines for use in carrying out the method of the invention. Still further the invention relates to the use of the above method to assess bone resorption and monitor short-time and long time response to antiresorptive therapy.

Bone is maintained through a continuous remodelling cycle where aged or damaged tissue is replaced by new through the collective action of specialised cells; osteoblasts and osteoclasts. Osteoblasts are the bone forming cells responsible for the synthesis of bone matrix and osteoclasts degrade the bone matrix by releasing protons and proteolytic enzymes.

The extra-cellular bone matrix is composed of hydroxyapatite as well as a variety of different more or less tissue specific proteins. One of these is osteocalcin (OC) a small protein with high affinity for hydroxyapatite. OC is a bone-specific protein consisting of 49 amino acids in a single polypeptide chain and having a calculated molecular weight of 5879 Da (Poser et al. 1980) and makes up approximately 1-2 % of the extracellular bone protein. OC contains up to 3 γ-carboxyglutamic acid (Gla) residues, an amino acid resulting from the vitamin K-dependent post- translational modification of glutamic acid residues (Glu) within the molecule. For this reason OC is also termed bone Gla protein (BGP) . The role of Gla in OC is to enable the protein to bind strongly to hydroxyapatite. OC inhibits hydroxyapatite formation in vitro, its expression is regulated by 1,25 dihydroxyvitamin D₃ (1, 25 (OH) ₂D₃) , and the molecule is believed to be secreted from differentiated osteoblasts. OC has chemotactic properties (Malone et al. 1982, Lucas et al. 1988) and may play a role in the initiation of osteoclast recruitment and bone resorption, however the precise physiological function (s) of OC remain (s) elusive.

A series of data is available on the structure and biosynthesis of OC, but currently only limited data has been published regarding its turnover and degradation (Price 1985, Power et al. 1991). Because OC is partly incorporated into the bone matrix and partly delivered to the circulatory system (Price et al. 1981) circulating osteocalcin is regarded as a specific marker for bone formation. Thus, intact OC is derived from osteoblastic de novo biosynthesis and a large number of studies have established that OC and various OC-derived fragments can be measured in serum or plasma and provide valid surrogate markers of bone formation (Power and Fottrel Crit Rev Clin Lab Sci 1991; 28:287-335).

When bone is resorbed by the osteoclasts, matrix proteins are degraded to smaller peptides that are released to the circulation. These fragments may be subject to further proteolytic processing in the liver and kidneys where some are completely catabolised and re-utilised, whereas others are left more or less unaltered. A part of these fragments may thus survive to be excreted into the urine. Theoretically the formation of proteolytic OC fragments may take place at various stages during the lifetime of the molecule. They may be produced by the action of osteoclasts on the bone matrix or as a result of catalytic degradation of the circulating protein after synthesis by osteoblasts. Although intact OC is not released during bone resorption (Price et al. 1981), some papers have discussed the possibility of the release of OC-related degradation products during bone resorption (Gundberg and Weinstein J Clin Invest 1986; 77:1762-7, Baumgrass et al. 1997, Kurihara et al.1998), and some have focused on the lytic activity of various proteases on OC, (Baumgrass et al. 1997, Novak et al. 1997). In the recent years biological data have been mounting suggesting that the cysteine protease cathepsin K (Tezuka et al. 1994) plays a central role for the matrix solubilisation in the resorption compartment. Thus, cathepsin K is relatively specific to osteoclast (Tezuka et al . 1994, Inaoka et al. 1995, Rantakokko et al. 1996, Drake et al. 1996, Littlewood-Evans et al. 1997), is upregulated by bone resorbing agents (Saneshige et al. 1995, Kakudo et al. 1997), has been detected in the resorption area (Littlewood-Evans et al. 1997, Kamiya et al. 1999), its activity is rate limiting for bone resorption (Gelb et al. 1996, Inui et al. 1997, Dodds et al. 1998, How et al. 1999), and appears to play a key role in the osteoclast mediated lysis of the skeletal collagen (Garnero et al. 1998) . However, at present nothing is known about the action of cathepsin K on OC.

Some experimental evidence for the generation of OC- related fragments during osteoclastic resorption has been provided in vitro by Kurihara and co-workers who studied the release of N-terminal fragments of osteocalcin from osteoclast cell cultures (Kurihara et al. 1998) . These investigators reported that N-terminal OC fragments might be released during osteoclastic bone resorption. However, fragments related to the Mid- and C-terminal parts of the molecule were not mentioned and the observed immunoreactive fragments were not characterised. US patent No. 6,004,765 discloses a process of determination of decarboxylated fragments of osteocalcin in body fluids in order to assess bone fragility and osteoporosis fracture risk. In the context of this patent the expression "decarboxylated" signifies OC having less than the normal number of Gla residues (e.g. molecules having either 0, 1 or 2 Gla residues) . The patent states that the employed antibody should bind to an epitope occurring in the region of amino acids 17-24 and that the binding epitope should not occur in the fully carboxylated form of osteocalcin. The patent does not mention the probable resorptive origin of OC- related fragments and the patent does not contain a characterisation of the molecular nature of OC-fragments immunoreactive in the assay.. PCT application WO99/09058 (Hellman) describes measurement of osteocalcin fragments in urine characterised in that at least one of the glutamic acids in position 17, 21 and 24 of the amino acid sequence is gamma-carboxylated. Furthermore the invention concerns a method for the measurement of the rate of bone turnover (formation and/or resorption) and/or for the investigation of metabolic bone disorders. Although the potential use of OC related fragments to determine bone resorption is mentioned, the patent does not provide any direct evidence to support this statement, thus no data on the short-term effect of anti-resorptive bisphosphonate treatment is provided, and no studies of the release of OC- related fragments from bone-cell cultures are provided to show that the OC-fragments measured in the assay constitute neoepitopes related to bone resorption. In addition, the patent does not mention any of the molecular fragments disclosed in the present invention.

The first reported measurement of urine osteocalcin (Taylor et al. J. Clin. Endocrinol. Metab. 1990; 70: 467-72) is based on a competitive RIA utilizing polyclonal guinea pig anti-human OC antibodies for recognizing the immunoreactive OC fragments (Taylor et al. Metabolism 1988; 37:872-7). The assay (uhOC) is said to be specific for the mid-molecule epitope of human OC molecule according to information obtained from cross-reactivity test with tryptic fragments and synthetic peptides. The authors determine the probable epitope for polyclonal antibody recognition quite widely. It is concluded that the binding site is located in the mid- molecule of the protein and probably involves amino acid 19 and at least a portion of the N-terminal sequence of the 20- 43 tryptic digest fragment prior to amino acid 37. However, a detailed characterisation of the fragments measured by the assay is missing. Furthermore this assay is not suitable for routine measurement of urine because of desalting of urine samples before measurement is inevitable for the proper function of the RIA. In addition, and most importantly the possible resorptive origin of the fragments is not mentioned. With theuhOC assay, multiple immunoreactive OC-fragments have been detected in both serum and urine. However the observed immuno-reactive fragments were not characterised in detail. The correlation between serum and urine samples as measured by the uhOC assay was good (r=0.83, p<0.01) indicating that the assay is detecting osteocalcin originating from the formation process. Besides this, even better correlation was obtained uhOC measured in urine and serum alkaline phosphatase measurements, further indicating that the fragments measured in the uhOC assay reflect the process of bone formation (Taylor et al J Clin Endocrinol Metab 1990; 70:467-72).

Kurihara et al . 1998 describe the measurement of N- terminal OC fragments released as a consequence of bone resorption in vitro. Osteoclasts were cultured on human bone slices for 6 days. The content of N-terminal OC in the culture medium increased in a cell number dependent manner. Incubation of cultured osteoclasts with IL-1 plus IL-6 (cytokines which are known to stimulate osteoclastic activity) slightly increased the concentration of N-terminal OC in the culture medium and conversely incubation with calcitonin (thought to be an inhibitor of osteoclastic resorption) decreased N-terminal OC in the medium. The authors conclude that N-terminal fragments of OC are the major form of osteocalcin released during osteoclastic bone resorption along with small amounts of intact osteocalcin. They suggest that the measurement of N-terminal OC fragments may serve as an index of bone resorption in vitro. However, the paper does not provide any direct evidence to support this statement, and although the authors noted that bone resorption lacunae were found on the surface of the bone slices incubated with osteoclasts, no correlation was made between resorbed area and the content of N-terminal OC in the culture medium to demonstrate that the fragments were indeed related to osteoclastic resorption and not released passively from the bone matrix. In addition, the observed immunoreactive fragments were not characterised. Likewise the epitope recognised by the employed antibodies was not disclosed although it was reported that the antibodies were raised against the 20 N-terminal amino acids of human osteocalcin sequence. Finally the measurements were not carried out on body fluids and the measurement of osteocalcin fragments related to bone resorption in vivo was not mentioned. Thus at present no clear-cut evidence for the release of OC-fragments in bone resorption in vivo has been provided, and no data are available on the possible molecular nature of such fragments. Thus although a number of studies have demonstrated a decreased concentration of OC or OC-related fragments in vivo as a response to long-term anti-resorptive therapy, no one has until now demonstrated responses to short-time bisphosphonate therapy indicative of the resorptive nature of such fragments.

Bone formation markers usually show a lag time of several weeks or months in responding to natural and therapeutically induced changes in bone turnover rates (Delmas PD, Endocrinol Metab Clin North Am 1990;19:1-18), consistent with the concept that formation is coupled to but lags behind resorption at remodelling sites. Most bone resorption markers, in contrast, respond rapidly (within a few days) to intravenous bisphosphonate therapy (Fleish 1995, Eyre 1995) . Thus a critical clinical characteristic of the specificity of bone resorption of all bone turnover markers is their short-term responsiveness in human subjects to antiresorptive therapies, particularly the more advanced generation of bisphosphonates which . are believed to target bone highly selectively (Eyre 1995) . In developing the present invention the bone resorption specificity of OC-related fragments was assessed in vivo in urine and serum by studying their response to short-time bisphosphonate treatment. For this purpose an ELISA was applied measuring an epitope in the mid-region of osteocalcin. This assay is used to demonstrate the presence of OC-fragments related to bone resorption. Applying this assay in conjunction with c romatographic techniques the exact nature of some of these fragments is determined and some of them are shown to reflect bone resorption. One aspect of the present invention provides a method of measuring the amount of osteocalcin-derived fragments in a body fluid comprising contacting a sample of said body fluid with at least one immunological binding partner for said fragments and determining binding of the said immunological binding partner to said fragments, wherein said immunological binding partner is specific for an epitope located at the N or C terminus of a said fragment, which epitope is created upon cleavage of osteocalcin at said terminus during resorption of bone and which is not present in intact osteocalcin.

Said epitope is preferably one created by cleavage of osteocalcin by cathepsin K. Preferably, said fragments are below 2600 Dalton in size. In contrast to the situation in US6004765 it is not necessary that said immunological binding partner should be specific for fully carboxylated or under carboxylated osteocalcin. Preferably, the body fluid is urine, but other body fluids including serum, tear fluid or saliva may in principle be used.

In a preferred assay said fragments are contacted with two immunological binding partners for said fragments in a sandwich assay, said immunological binding partners including a first immunological binding partner which is specific for an epitope located at the N-terminus of a said fragment and which is created upon cleavage of osteocalcin at said terminus in resorption of bone and is not present in intact osteocalcin and a second immunological binding partner which is specific for an epitope located at the C-terminus of a said fragment and which is created upon cleavage of osteocalcin at said terminus in resorption of bone and is not present in intact osteocalcin. In a further aspect, the invention includes an immunological binding partner specific for an epitope present at the N or C terminus of an osteocalcin fragment, which epitope is created upon cleavage of osteocalcin at said terminus during resorption of bone and which is not present in intact osteocalcin. The epitope is preferably one created upon cleavage of osteocalcin by cathepsin K. In a further aspect, the invention includes an assay kit for use in performing a method as described and comprising an immunological binding partner in accordance with the preceding aspect of the invention together with one or more of peptide standards, urine standards, substrate bearing peptides reactive with the immunological binding partner, buffers, antibody reaction stopping solutions, osteocalcin reactive immunological binding partners lacking specificity for isomerised or optically inverted osteocalcin sequences, antibody-enzyme conjugates, enzyme substrates or enyzme reaction indicator substances.

In a still further aspect, the invention includes a method of measuring the amount of osteocalcin derived fragments in a body fluid comprising contacting a sample of said body fluid with at least one immunological binding partner for said fragments and determining binding of the said immunological binding partner to said fragments, wherein said immunological binding partner is specific for an epitope containing an isomerised or optically inverted amino acid. The the amino acid may be aspartic acid or asparagine or glutamic acid or glutamine or γ-carboxylated glutamic acid. The fragments optionally may be larger than 2600 Daltons, but preferably are smaller.

The invention further includes an immunological binding partner specific for an epitope containing an isomerised or optically inverted amino acid in an amino acid sequence of osteocalcin. The invention further includes an assay kit for performing an assay for isomerised or optically inverted osteocalcin fragments, comprising an immunological binding partner as just described in combination with one or more of peptide standards, urine standards, substrate bearing peptides reactive with the immunological binding partner, buffers, antibody reaction stopping solutions, osteocalcin reactive immunological binding partners lacking specificity for isomerised or optically inverted osteocalcin sequences, antibody-enzyme conjugates, enzyme substrates or enzyme reaction indicator substances.

In a further aspect, the invention provides a method of obtaining an indication of the rate of bone resorption in an individual by measuring the amount of certain osteocalcin derived fragments in urine, comprising contacting a sample of urine from said individual with at least one immunological binding partner for said fragments and determining the amount of binding of the said immunological. binding partner to said fragments, wherein said immunological binding partner is such as to bind predominantly to osteocalcin fragments in said sample of not more than 2600 Daltons.

The method according to this and other aspects of the invention may include comparing the measured amounts of the osteocalcin fragments with similar results known to be indicative of a particular bone resorption rate, thereby to determine the rate of bone resorption measured in the assay.

In accordance with this aspect of the invention said binding partner preferably binds to an epitope containing the amino acids EVCE. It is preferred that the population of the bound fragments comprises peptides of one or more of the following sequences : hOC14-24: DPLEPRREVCE (SEQ ID NO: 2) hOC14-31: DPLEPRREVCELNPDCDE (SEQ ID NO: 3) hOC16-36: LEPRREVCELNPDCDELADHI (SEQ ID NO: 4) hOC16-31: LEPRREVCELNPDCDE (SEQ ID NO: 5) hOC17-28: EPRREVCELNPD (SEQ ID NO: 6) hOC20-32: REVCELNPDCDEL (SEQ ID NO: 7) hOC20-31: REVCELNPDCDE (SEQ ID NO: 8) hOC20-29: REVCELNPDC (SEQ ID NO: 9) hOC20-26: REVCELN (SEQ ID NO: 10) hOC21-32: EVCELNPDCDEL (SEQ ID NO: 11) hOC21-31: EVCELNPDCDE (SEQ ID NO: 12) hOC21-29: EVCELNPDC (SEQ ID NO: 13) hOC21-27: EVCELNP (SEQ ID NO: 14) hOC21-26: EVCELN (SEQ ID NO: 15) hOC_Dι24-35: ELNPD_DCDELADH (SEQ ID NO: 16) hOC_D224-35: ELNPDCD_DELADH (SEQ ID NO: 16) hOC_DD24-35: ELNPD_DCD_DELADH (SEQ ID NO: 16) hOC_D30-37: DELAD_DHIG (SEQ ID NO: 17) Preferably, said binding partner binds to an epitope containing or consisting of amino acids EVCELNPDC (SEQ ID NO:13) .

In a further aspect, the invention provides a method of measuring the amount of osteocalcin derived fragments in urine comprising contacting a sample of said urine with at least one immunological binding partner for said fragments and determining the amount of binding of the said immunological binding partner to essentially all osteocalcin fragments in said sample which are reactive therewith, wherein said immunological binding partner is specific for an epitope comprising at the N terminal end of said epitope the amino acid sequence EVCE (SEQ ID NO: 37) . Preferably, said epitope is defined by the amino acid sequence EVCELNPD (SEQ ID NO:18) .

The invention includes cell lines producing monoclonal antibodies constituting immunological binding partners as described above.

Certain aspects of the present invention are based on the discovery of the presence of particular osteocalcin fragments in urine of patients and normal human subjects. The osteocalcin fragments are generated upon osteocalcin degradation and are partially characterised by the presence of osteoclast-related cleavage sites, e.g. by the presence of specific N and C-terminal residues.

Other aspects of the invention are based on measuring osteocalcin-related fragments containing isomerised or optically inverted residues, thus avoiding contributions from bone formation and assuring the specificity to bone resorption. According to the present invention it is found that isomerised and/or optically , inverted fragments of osteocalcin are found in urine, and that the measurements of such fragments reflect bone resorption.

In a preferred embodiment of the method according to the invention, the assaying of osteocalcin fragments in urine is performed by an inhibition ELISA (enzyme linked immunosorbent assay) by metering off a sample of urine and contacting the sample with a synthetic peptide having a sequence derived from osteocalcin, and with a digoxigenin-labelled antibody, which is immunoreactive with the synthetic peptide. The synthetic peptide is immobilised on a solid support. The antibody is one which has been raised against the synthetic peptide.

The combined reagents and sample are incubated, and a peroxidase-conjugated anti-digoxigenin (revealing) antibody is added. After another incubation, a peroxidase substrate solution is added. Following short final incubation, the enzyme reaction is stopped, and the absorbance is measured at 450 nm and compared with a standard curve obtained with standard solutions by the same procedure. Synthetic peptides are used for the preparation of standards. The concentration of synthetic peptide in a stock solution of the relevant synthetic peptide is determined by quantitative amino acid determination. A two-fold dilution is prepared and subsequently used for the construction of the standard curve in the inhibition ELISA.

Preferably, one said assay determines the amount of osteocalcin fragments derived from the degradation of osteocalcin by the requirement that the fragments are spanning i) less than 22 amino acids, and ii) contain the epitope EVCE derived from human osteocalcin (SEQ ID NO:l): 1 10 20 30 40

I I I I I

YLYQWLGAPVPYPDPLEPKREVCELNPDCDELADHIGFQEAYRRFYGPV

Potential sites of isomerisation and optical inversion are underlined in the osteocalcin amino acid sequence given above. The glutamic acids (E) in the positions 17, 21 and 24 may or may not be gamma-carboxylated.

The preferred immunoassay is sensitive to short-time bisphosphonate therapy; these assays should be of interest in monitoring various disease states, particularly of bone metabolism diseases. The assays are thus especially useful in methods for the measurement of the rate of bone resorption and/or for the investigation of bone metabolic disorder and bone-related disease. The assay may take many forms including but not limited to ELISA, RIA or IRMA, procedures for which are too well known to warrant description here.

According to a preferred embodiment of the invention the assay should be specific for a peptide sequence including the sequence EVCE and preferably spanning less than 22 amino acids preferably less than 10 amino acids. It is contemplated that the assay may be constructed in such a way that reactivity in the assay is dependent on the OC fragment being cleaved at a specific residue or at specific residues.

In an alternative aspect of the invention, the assay should be specific for an osteocalcin-derived sequence including at least one isomerised or optically inverted residue. Aspartic acid and asparagine (Asx) and glutamic acid and glutamine (Glx) residues will in some susceptible proteins undergo spontaneous re-arrangement where the normal peptide bond between the Asx and Glx residues and the adjacent residue is transferred from the normal α-carboxyl group to the β-carboxyl group (γ-carboxyl group for Glx residues) of the side chain (Clarke 1987) the isomerisation reaction proceeds via a imide intermediate, which upon spontaneous hydrolysis may result in one of four forms: the normally occurring ocL, the isoform βL, or the two optically inverted forms αD or βD as outlined in the following reaction scheme for aspartic acid/ asparagine. The reaction occurs analogously for Glx containing sequences.

In a recent publication Ritz et al . demonstrated that the content of D-aspartic acid increases with age in human bone (Ritz et al. (1996) Forensic Sci Int; 770:13-26). After purification of osteocalcin from 53 forensic skull bone specimens, the relative content of D-aspartic acid was determined by chiral chromatography. It was found that the relative D-aspartic acid content in osteocalcin was highly correlated to "age at death". The authors concluded that whereas serum osteocalcin originates from de novo synthesis, bone matrix appears to be "aged" and not exchanged during life. They further speculated that only certain components (e.g. collagen) were turned over during the bone remodelling process, whereas others (e.g. osteocalcin) are left over, i.e. not renewed. In WO96/30765 it was disclosed that isomerised or optically inverted fragments of type I collagen provided an improved index of bone resorption.

According to the present invention it is now demonstrated that urinary osteocalcin fragments contain isomerised and optically inverted D-aspartyl residues. Furthermore, it is demonstrated that measurements of these fragments can provide an index of bone resorption. The present invention also provides a method of assay, comprising measuring in a biological fluid the amount of isomerised or optically inverted osteocalcin fragments.

According to this second aspect of the invention said assay is preferably carried out using an immunological binding partner which specifically binds an amino acid sequence comprising isomerised or optically inverted residues. The immunological binding partner should discriminate between the sequence containing isomerised and optically inverted residues and the corresponding native sequence to a degree adequate to provide a useful assay. The cross-reactivity of assay/immunological binding partner according to this aspect of the invention towards the native antigen should be less than 25%, preferably less than 5%.

Preferably, the assay according to the second aspect of the invention determines the amount of at least one *Asx or *Glx containing protein-fragment, wherein *Asx denotes optically inverted or isomerised Asx, and *Glx is optically inverted or isomerised Glx.

The immunological binding partner may be a monoclonal or polyclonal antibody. Suitable immunological binding partners also include fragments of antibodies including but not limited to - Fab, Fab' and F(ab')2 fragments.

The invention also includes cell lines (e.g. hybridomas) that produce monoclonal antibodies immunoreactive with the above-mentioned synthetic peptides. The invention further includes monoclonal antibodies produced by the fused cell hybrids, and those antibodies (as well as binding fragments thereof, e.g. Fab) coupled to a detectable marker. Examples of detectable markers include, but are not limited to, enzymes, chromophores, fluorophores, co-enzymes, enzyme inhibitors, chemiluminescent materials, paramagnetic metals, spin labels and radioisotopes .

The methods of the invention involve quantitating in a biological fluid the concentration of particular osteocalcin fragments derived from osteocalcin degradation. In a representative assay, osteocalcin fragments in the biological fluid and a synthetic peptide immobilized on a solid support are contacted with an immunological binding partner, which is immunoreactive with the synthetic peptide.

The biological fluid may be used as it is, or it may be purified prior to the contacting step. This purification step may be accomplished using a number of standard procedures, including but not limited to, cartridge adsorption and elution, molecular sieve chromatography, dialysis, ion exchange, aluminia chromatograpy, hydroxyapatite chromatography, and combinations thereof.

The invention includes antibodies according to the various aspects of the invention coupled to a detectable marker. Suitable detectable markers include, but are not limited to, enzymes, chromophores, fluorophores, coenzymes, enzyme inhibitors, chemiluminescent materials, paramagnetic materials, spin labels, radio-isotopes, nucleic acid, or nucleic acid analogue sequences. The invention also includes test kits useful for quantifying in a body fluid (or bone cell supernatant) the amount of osteocalcin fragments derived from the degradation of osteocalcin. The kits comprise at least one immunological binding partner, e.g. a monoclonal or polyclonal antibody specific for a peptide derived from the degradation of osteocalcin. If desired, the immunological binding partner of the test kit may be coupled to detectable markers such as the ones described above. Generally speaking, the immunological binding partner is therefore also useful as a diagnostic agent.

It is contemplated that the method may be used for assaying osteocalcin fragments in biological fluids, e.g. for determination of the osteocalcin metabolism. It can also be used during pre-clinical and clinical testing of drugs to assess the impact of these drugs on osteocalcin metabolism and bone resorption.

In a preferred embodiment, the method is based on the competitive binding of osteocalcin in biological fluids (body fluids or cell culture fluids) and of synthetic peptides essentially derived from osteocalcin to immunological binding partners.

In second preferred embodiment, the method is carried out using an immunological binding partner specific for fragment containing an isomerised or optically inverted residue

The method of the present invention may be used for the determination of the degradation of osteocalcin in vivo in mammals, preferably in humans, and in bone cell-cultures.

According to the present invention the identification of resorption-related fragments of osteocalcin is now described. Bone-resorption-driven osteocalcin degradation producing osteocalcin fragments has now been shown in vivo, where Mid- OC concentrations in patients treated with antiresorptive bisphosphonate therapy were markedly decreased in response to short-time bisphosphonate therapy. It has also been shown in vi tro where the release of Mid-OC from total bone cell cultures was highly correlated to a known marker of bone resorption (r²=0.91) and to resorbed area (r²=0.89).

In addition it has been found that urinary fragments of osteocalcin contain isomerised (isoaspartyl) or optically inverted (D-aspartyl) aspartyl residues. The formation of such isomerised and optically inverted residues is generally believed to be associated with ageing of peptides and proteins (Bada et al. 1973, Nature; 245: 308-310, Masters et al 1977, Nature 268:71-73, Geiger and Clarke (1987), J Biol Chem 262:785-794). The presence in such bone peptide fragments of isoaspartyl or D-aspartyl thus provides confirmation that the peptide fragments indeed derive from bone degradation and not some other source such as the degradation of newly formed osteocalcin never incorporated into bone.

For purposes of the present invention, as disclosed and claimed herein, the following terms are defined below:

"Antibody": a monoclonal or .polyclonal antibody or immunoreactive fragment thereof (i.e. capable of binding the same antigenic determinant) , including - but not limited to - Fab, Fab' and F(ab')2 fragments.

Isomerised residues: Amino acid residues within a (poly) peptide where said residues are bound to their adjacent residue through an isopeptide bond (e.g a bond proceeding through the side-chain β or γ-carboxyl group) .

Optically inverted residues: D-amino acid residues. Isomerised (about antigens, peptides, proteins and sequences) : Molecules contain isomerised residues (isopeptide bonds) . Optically inverted (about antigens, peptides, proteins and sequences): Molecules containing D-amino acid residues. Native (about antigens, peptides, proteins and sequences) : Antigens/peptides/proteins and sequences composed of L-amino acid residues linked together by normal peptide bonds. Test kit: A combination of reagents and instructions for use in conducting an assay.

Essentially derived (about structures) : Structures with similar antigenicity, i.e. with an ability, above the level of a non-related peptide, to inhibit the binding of any of the mentioned synthetic peptides to an immunological binding partner immunoreactive with said synthetic peptide.

Biological fluid: Body fluids including urine, blood, serum, plasma saliva, sweat and synovial fluid, as well as fluids derived from cells in culture (e.g. supernatants from bone cell cultures) .

Mid-OC ELISA: Competitive immunoassay based on the reactivity of a monoclonal antibody to the osteocalcin sequence EVCE (SEQ ID NO:37).

Mid-OC: Antigens measured by the Mid-OC ELISA.

Preparation of antibodies

The methods for preparation of both monoclonal and polyclonal antibodies are well known in the art. For example, see Campbell, A. M., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13 (1986) . It is possible to produce antibodies to synthetic peptides by immunization. However, because of the relatively small molecular weight of these compounds it is preferred that the hapten be conjugated to a carrier molecule. Suitable carrier molecules include, but are not limited to, tuberculin, bovine serum albumin, thyroglobulin, ovalbumin, tetanus toxoid, and keyhole limpet hemocyanin. The preferred carrier proteins are tuberculin and thyroglobulin. To present the hapten in its most immunogenic form to the antibody producing cells of the immunized animal a number of alternative coupling protocols can be used. Suitable procedures include, but are not limited to glutaraldehyde, carbodiimide and periodate. Preferred binding agents are glutaraldehyde and carbodiimide. The preparation of antibodies is carried out by conventional techniques including immunization with osteocalcin fragments or synthetic peptides conjugated to a carrier. To improve the immunogenicity it is preferred that the immunogen be mixed with an adjuvant before injection. Examples of adjuvants include, but are not limited to, aluminium hydroxide, Freund' s adjuvant, and immune stimulating complexes (ISCOMs) . ISCOMS can be made according to the method described by Morein B. et al. Nature 308: 457-460 (1984). Either monoclonal or polyclonal antibodies to the hapten carrier molecule can be produced. For the production of monoclonal antibodies it is preferred that mice are immunized. Spleen cells from the immunized mouse are harvested, homogenized, and thereafter fused with cancer cells in the presence of polyethylene glycol to produce a cell hybrid which produces monoclonal antibodies specific for peptide fragments derived from osteocalcin. Suitable cancer cells include, but are not limited to, myeloma, hepatoma, carcinoma, and sarcoma cells. Detailed descriptions of the production of monoclonal antibodies are provided by Goding, J.W., in Monoclonal Antibodies: Principles and Practice, (1986) . A preferred preliminary screening comprises the use of synthetic peptides conjugated to a carrier and coated onto the solid surface of a microtitre plate. For the preparation of polyclonal antibodies, which are reactive with peptide fragments derived from collagen, different animal species can be immunized. Suitable species include, but are limited to, chicken, rabbit and goat, Chicken and rabbit are preferred.

Antibody fragments are prepared by methods known in the art (see Ishikawa, E. Journal of immunoassay 3: 209-327 (1983)).

Conduct of immunoassays

Accordingly by utilization of an immunoassay with the antibodies prepared as above it is possible to assay biological fluids without prior fractionation or hydrolysis.

The specificity for the desired osteocalcin fragments in the biological fluid may be supplied by the antibody in combination with the use of a synthetic peptide analogue

(against which the antibody was raised or in ^' any event with which the antibody is immunochemically reactive) in the assay construction. The immunoassay may be performed using polyclonal or monoclonal antibodies.

The immunoassays themselves may be conducted using any procedure selected from the variety of standard assay protocols generally known in the art. As it is generally understood, the assay is constructed as to rely on the interaction between the specific immunological binding partners. The immunological binding partner may be complexed to a solid support and used as a capture immunological binding partner for the analyte. This protocol may be run in a direct form, wherein the formation of analyte-immunological binding partner complex is detected, e.g. by fluorescent, radioactive or enzymatic label, or it may be run in a competitive format wherein a labelled standard competes with the analyte for the immunological binding partner. The format may also be constructed as an agglutination assay or the complex may be precipitated by addition of a suitable precipitant to the reaction mixture. The specific design of the immunoassay protocol is opened to a wide variety of choice, and the number of clinical assay devices and protocols available in the art is multitudinous. For a variety of such protocols, see US. Patent No. 5,001,225. A homogeneous assay format may be used in which for instance latex particles are conjugated to the peptide and the sample and the particles compete to the bind the antibody. Specific agglutination of the particles by antibody produces a change which is optically detectable as a change in scattering or absorbance and which is inhibited by cross- linking in the sample. The antibodies and revealing reagents for the conduct of an immunoassay using standard detection protocols, for example radioisotope labelling, fluorescent labelling or ELISA, either in a direct or competitive format, may conveniently be supplied as kits which include the necessary components and instructions for the assay. In one embodiment of the invention such a kit includes a microtiter plate coated with a relevant synthetic peptide, standard solutions for preparing a standard curve, a urine control for quality testing of the analytical run, monoclonal murine antibodies reactive with the above-mentioned synthetic peptide, anti-mouse immunoglobulins conjugated to peroxidase, a substrate solution, a stopping solution, a washing buffer and an instruction manual. Since immunoassays can be constructed using antibodies and specific synthetic peptides, the ratios of the corresponding osteocalcin fragment sequences in an appropriate biological fluid can be determined as well as their individual levels and their total. Thus, the assay can be designed to include antibodies that will result in determination of a single peptide sequence, or any desired combination thereof. In addition to the use of the herein specified peptides as indicators of bone resorption, bone metabolic balance is advantageously determined by the substantially simultaneous determination of a marker of the formation of bone in the same or other appropriate biological fluids from the same individual. "Substantially simultaneous" means the same day, preferably within 4 hours. For example such markers include procollagen type I, bone alkaline phosphatase and total alkaline or intact native osteocalcin. The assay of the present invention that provides an index to determination of the metabolic status of bone tissue, which generates osteocalcin-derived peptides when degradation occurs, is useful in a variety of contexts. First, when considering the degradation of osteocalcin, the assays are methods to assess an abnormal condition of a subject by indicating, for example, excessive bone resorption. This may show the presence of an osteoporotic condition or the metastatic progress of malignancy. Other conditions characterized by excessive bone resorption include Paget's disease and hyperparathyroidism. Since the condition of the subject can be monitored continuously, application of these assays can also be used the progress of therapy administered to treat these or other conditions. Further, the assays can be used as a measure of toxicity, since the administration of toxic substances often results in tissue degradation.

Thus the assays may be applied in any situation wherein the metabolic condition of bone can be used as an index of the condition, treatment, or effect of substance directly administered to the subject or to which the subject is exposed in the environment.

In order to assay for osteocalcin fragments produced in vivo by osteoclast protease degradation of osteocalcin in the bone matrix one may employ a monoclonal antibody selected for reactivity with an N- or C-terminal neo-epitope produced from the osteocalcin molecule by such protease action.

One may accordingly subject osteocalcin (either purified from nature or synthetic) to proteolytic degradation with the enzyme cathepsin K, which is the predominant proteolytic enzyme present in the bone resorption compartment of osteoclasts resorbing bone. The peptides so produced may be used as antigen for immunising mice in an otherwise known manner and monoclonal antibodies produced from such an immunisation may be selected for reactivity towards the immunising peptide mixture and lack of reactivity toward intact osteocalcin followed by verification that any antibody so selected has indeed specificity for the presence at the N- or C-terminal of a peptide of a sequence from within osteocalcin.

For producing antibodies to novel C-terminal epitopes, the immunising peptides may be N-terminal conjugated to thyroglobulin. For producing antibodies to N-terminal neo-epitopes it is preferred to use site directed conjugation at the C- terminus, e.g. by SMCC (succinimidyl 4-(N- maleimidomethyl) cyclohexane-1-carboxylate) crosslinking using synthetic peptides prolongated with a cysteine residue at the C-terminus. Synthetic peptides may be used in such immunisations .

The measurement of peptide containing neo-epitopes produced by cleavage of osteocalcin during bone resorption is particularly preferred for use in measurements in body fluids other than urine, e.g. in serum.

For the measurement of short osteocalcin peptides in urine, one may employ antibodies that have no specificity for neo-epitopes and which are reactive with intact osteocalcin. Such methods are described in the following examples. Methods of assay of serum using similar antibodies to those suitable for use in such measurements of small peptides in urine are known. It is especially surprising that applied to measurements of small peptides in urine, such assays produce a valid index of bone resorption, because their use in measurements on serum has been for producing an index of bone formation.

It is not of course essential that all of the peptides taken into the measurement should be less than 2600 Daltons or not more than 12 or 13 amino acids. However, at last 75%, e.g. at least 90%, of the signal in the assay should derive from such short peptides. This can be verified by extensive dialysis of the sample with a membrane having a molecular weight cut off of 2600 Da.

One method of determining the contribution to the assay of different sizes of peptide would be to capture peptides by immunoaffinity chromatography of urine and to separate and quantitate captured and subsequently eluted peptides by HPLC, as exemplified below.

Preferably, the assay does not exclude from the measurement either γ-carboxylated or non-γ-carboxylated fragments. In antibody based assays therefore, antibodies or combinations of antibodies should preferably be used such that there is neither, specificity for γ-carboxylation nor for its absence. The following examples making reference to the attached drawings are intended to illustrate, but not to limit the invention.

The invention will be further described and exemplified in more detail below. Reference is made to the appended drawings in which:

Figure 1 shows a typical standard curve for a Mid-OC ELISA. Figure 2 shows a typical standard curve for a in vitro Mid-OC ELISA.

Figure 3 shows a schematic presentation of osteocalcin showing the epitopes reactive with monoclonal antibodies Mab 504-1 and 539-4.

Amino acid residues are given by their one-letter symbol according to the IUPAC guidelines. Residues are numbered according to the sequence published in GenBank. Glutamic acid residues (E) at positions 17, 21 and 24 may be gamma- carboxylated. Residues at positions 4, 14, 17, 21, 24, 26, 27, 30, 31, 34, 39 and 40 may potentially be isomerised or optically inverted.

Figure 4 shows the correlation between Mid-OC, CrossLaps for culture and resorbed area in bone cell cultures. The concentration of Mid-OC was determined in bone culture supernatants (using the in vitro Mid-OC assay) and correlated to resorbed area (Fig. 4a) and a known marker of bone resorption (CrossLaps for culture), Fig. 4b.

Figure 5 shows HPLC separation of urinary osteocalcin fragments from a patient with Paget's Disease before and after bsphosphonate treatment.

Immunoreactivity profiles of the affinity purified osteocalcin are shown. Eluents were collected in fractions and assayed in the two osteocalcin assays. N-Mid® ELISA ( right panel) , Mid-OC ELISA (left panel) .

Figure 6 shows HPLC separation and purification of urinary osteocalcin fragments from two healthy adult males.

Immunoreactivity profiles of the affinity purified osteocalcin are shown. Eluents were collected in fractions and assayed in the two osteocalcin assays. N-Mid® ELISA (open squares) , Mid-OC ELISA ( filled circles) . Figure 7 shows the sequence of human OC with mapping of urinary fragments and indication of putative cleavage sites.

Figure 8 shows response to 8 days i.v. bisphosphonate treatment in 10 patients with Paget's disease. U-Mid-OC: Mid-OC/Cr in urine, S-Mid-OC: Mid-OC in serum, U-CTx: Urine CrossLaps/Cr, S-CTx: Serum CrossLaps/Cr, U-N- MID-OC: N-Mid/Cr in urine.

Figure 9a and b shows response to 1 and 12 months alendronate (20mg/day) treatment in 9 postmenopausal women. Figure 10 shows measurement of isoaspartyl/D-aspartyl in osteocalcin fragments purified from urine, using the IAMT assay.

Figure 11 shows results obtained in Example 9.

Examples

Example 1 : Mid Osteocalcin ELISA (version optimized for measurement of urine)

Ma terials - All chemicals were of analytical grade from either Sigma (St. Louis, MO) or Merck (Darmstadt, Germany), unless otherwise stated. Streptavidin coated plates were from MicroCoat. Peroxidase-conjugated sheep anti-digoxigenin IgG was from Boehringer Mannheim, (Mannheim, Germany) . Bronidox L5 was from Henkel (Dϋsseldorf, Germany). 3, 3', 5,5'- tetramethylbenzidine (TMB) solution was from Kierkegaard &

Perry, (Gaithersburg, MD) . Buffers used were as follows;

Assay buffer: 1.5 mM KH₂P0₄, 8.5 mM Na₂HP0₄-2H₂0, 2.7 mM KCl,

137 mM NaCl, 1 % (w/v) bovine serum albumin (BSA) , 0.1 %

(w/v) Tween-20, 0.36 % (w/v) Bronidox L5, pH 7.0. Mid Osteocalcin ELISA Procedure - The Mid Osteocalcin ELISA is an immunoassay developed for the measurement of fragments related to the Mid-region of osteocalcin. The assay (moreover denoted Mid-OC ELISA) is a competitive ELISA employing a monoclonal antibody (MAb 504-1) directed against residues 21-29 of human osteocalcin and is optimized for measurement in urine. The assay was performed as follows

(all incubations are carried out at 20 °C on a mixing apparatus (300 rpm) ) . Streptavidin coated microtiter plates

(MicroCoat) were pre-incubated for 30 min with biotinylated synthetic human osteocalcin 20 ng/ml, 100 μl/well. Twenty μl of either standards, diluted controls or unknown samples were pipetted into appropriate wells in the microtiter plate, followed by 150 μl of digoxigenin-labeled Mab 504-1 diluted in assay buffer. The wells were covered with sealing tape and incubated for one hour. Wells were emptied and washed five times using the washing solution. One hundred μl of peroxidase conjugated anti-digoxigenin IgG (diluted in a protein stabilized buffer) was added to all wells, which were incubated for one hour. After another washing step 100 μl of TMB solution was added to each well and the plate was incubated in darkness. The enzyme reaction was stopped after 15 minutes by adding 100 μl of 0.18 mol/L H₂S0 per well and the absorbance measured at 450 nm (with 650 nm as the reference wavelength) using a microtitre plate reader (E_max Easy Microtitre Reader; Molecular Devices, Menlo Park, CA) . Finally a standard curve was constructed on the basis of six standards and the concentration of antigen in each unknown sample was determined by interpolation on this curve. The immunoassay was calibrated using synthetic human OC (in concentrations from 0-2096 ng/ml) , the concentration of which had been determined by amino acid analysis. Amino acid analysis was performed after acid hydrolysis by ion exchange chromatography combined with post column derivatization as described (Barkholdt and Jensen 1989) . Figure 1 shows a typical standard curve for the Mid-OC ELISA. Example 2 : In-vitro Mid Osteocalcin ELISA (version optimized for measurement of bone cell-cultures)

The In Vitro Mid Osteocalcin ELISA is a competitive ELISA employing a monoclonal antibody (MAb 504-1) directed against residues 21-29 of human osteocalcin and is optimized for measurement in bone cell-cultures. The assay is performed as the standard Mid-OC assay described as in Example 1. The only deviation is that the concentration of biotinylated synthetic human osteocalcin (used for coating of plates) is reduced to a concentration of 5 ng/ml, 100 μl/well, to enhance the sensitivity of the assay. Correspondingly the concentration of the primary antibody solution is increased to obtain a suitable absorbance. Likewise the standard curve is changed to cover the range from 0- 257.2 ng/ml. Figure 2 shows a typical standard curve for the in-vitro Mid-OC ELISA.

Example 3 : Characterization of Antibody Specificity

The specificity of the antibodies (Mab 504-1, Mab 539-4) employed in the Mid-OC and N-Mid™ OC ELISA immunoassays was clarified by epitope-mapping using Pepscan™ analysis.

Polypeptides of 14 amino acids, shifted by two amino acids and spanning residues 3 to 34 of human osteocalcin were synthesized by spot synthesis using Whatman 5-iO paper (Maidstone, U.K.), using the previously described procedures

(Frank et al., Kramer et al . 1994). Peptides were automatically prepared using a spot synthesis method (Abimed,

Langenfeld FRG) and covalently attached to the cellulose support by their C-termini. The cellulose membrane containing peptides was incubated with the monoclonal antibodies.

Incubation and visualization of the bound antibodies was performed using conventional immuno-blotting techniques as described (Kramer et al. 1994) .

The results of the Pepscan™ analysis showed that the monoclonal antibody MAb 539-4 recognizes a seven amino acid epitope VPYPQPL^10"16. The MAb 504-1 specifically recognized a 9 amino acid epitope EVCELNPDC^21"29 (SEQ ID NO: 13) spanning both cysteine residues (Fig. 3) . The detected epitopes are in good agreement with specificity data obtained by inhibition ELISA (Rosenquist et al.1995).

Example 4 : Measurement of OC in Mixed Bone Cell Culture Supernatants

To determine whether Mid-OC fragments are generated as neo-epitopes by osteoclasts, mixed bone cell culture supernatants were measured using the in-vitro Mid-OC ELISA and correlated to resorbed area. For comparison the content of collagen type I related telopeptide fragments was assessed in the culture medium using the CrossLaps for culture ELISA (a known bone-resorption assay) . CrossLaps™ for culture ELISA- The CrossLaps™ for culture ELISA is a commercially available sandwich type assay employing two monoclonal antibodies (Mab F1102 and Mab F12) both directed against the collagen type I specific sequence EKAHDGGR containing a isoaspartyl bond between the aspartic acid and glycine residue. The assay is optimized for measurement in bone culture supernatants where it provides a valid index of bone resorption. The assay was performed as recommended by the manufacturer (Osteometer BioTech A/S) . the in-vitro Mid-OC ELISA was manufactured and performed as described in example 2.

Procedure - Unfractionated populations of bone cells were prepared as described by Tezuka et al. (Tezuka et al. 1992) with minor modifications. Briefly, long bones from 10- day old rabbits were minced in α-modified essential medium (α-MEM) (Life Technologies, Paisley, Scotland) and agitated for 30 s. with a vortex mixer. After sedimentation of bone fragments for 1.5 min. the supernatant was harvested and washed twice (700 rpm for 2 min.). Cells were resuspended in α-MEM supplemented with 2% (v/v) fetal calf serum (FCS) . The mixed bone cells were seeded unto slices of bovine femur cortical bone (diameter 6 mm, thickness 0.2 mm) in 96 well plates. After a settling period of 90 min., non-adherent cells were removed by replacing media. The cells were cultured in α-MEM, 2% FCS in the presence or absence of different inhibitors of bone resorption for 3 days at 37°C, 5% C0₂. After culture the conditioned media was harvested for measurement of Mid-OC and CTx assays. The bone slices were stained for resorption pits with Mayer's Hematoxylin. Resorbed area was measured using C.A.S.T-GRID SYSTEM. The net concentration in the conditioned medium was determined after subtraction of the contributions from i) the serum in the culture medium, ii) the isolated bone cells and iii) the spontaneous release of antigen from the substratum.

Statistical analysis - Correlation between biochemical markers and resorbed area were assessed by least square regression analysis. Results - There was a highly significant correlation between the Mid-OC concentration in the culture medium and resorbed area (r²= 0.89, p<0.0001), Figure 4a. The concentration of Mid-OC in the culture medium was likewise highly correlated to the concentration of the CrossLaps for culture resorption marker (r²= 0.89, p<0.0001), Figure 4b..

These results demonstrate that osteocalcin fragments reactive in the Mid-OC ELISA are released as a consequence of osteoclastic bone resorption and reflect bone resorption (osteoclastic activity) .

Example 5 : Characteriza tion of Urinary Mid-OC Fragments and Analysis of their Response to Short-time Bisphosphonate Therapy

Materials : All chemicals were of analytical grade from either Sigma (St. Louis, MO) or Merck (Darmstadt, Germany), unless otherwise stated. Acetonitrile was from Rathburn

(Walkerburn, Scotland) , and triflouroacetic acid (TFA) was from Applied Biosystems (Foster City, CA) . The reverse phase C18 HPLC column used for chromatographic separation of urinary OC-fragments was from Vydac (Hesperia, ^' CA) , cat. No. 218TP54, 250 mm x 4.6 mm I.D., 5 mm particle size. CNBr activated Sepharose 4B was from Amersham-Pharmacia BioTech, (Uppsala, Sweden) . Buffers used were as follows; Immunoaffinity buffer: 1.5 mM KH2P04,_ 8.5 mM Na2HP04.2H20, 2.7 mM KCl, 137 mM NaCl, 0.1 % (w/v) Tween-20, 0.36 % (w/v) Bronidox L5, pH 7.0

Immunoaffinity Chromatography of Urinary Osteocalcin - OC-related fragments were affinity purified from three different urine samples: two urine samples from a patient with Paget's disease, (i) before and ( ii) after 3 days bisphosphonate treatment, and ( Hi) a five liter sample (pooled urine from two healthy adult males 35 and 37 years of age) . The creatinine concentration of the former Pagetic sample (i) was adjusted to match the latter (ii) by addition of immunoaffinity-buffer. All urine samples were subsequently diluted 1+1 in the same buffer before being applied to the affinity column. The monoclonal antibody Mab 504-1 was covalently linked to CNBr-activated Sepharose 4B (8 mg antibody/ml gel) according to the manufactures instructions (Amersham-Pharmacia BioTech) . 20 ml gel was packed in a 16 mm (i.d.) x 100 mm column. 30 mL CNBr-activated Sepharose 4B coupled with a non-sense antibody was used in a pre-column.

The affinity column and pre-column were equilibrated at 4°C with 50 ml immunoaffinity buffer. Urine (diluted in IA- buffer) was passed onto the column. The pre-column was removed and the affinity column was washed with 100 ml IA- buffer and subsequently eluted with 20 ml 1% TFA. The eluted material was frozen directly and lyophilized. All chromatographic step were performed at 4°C with a flow-rate of 1 ml/min. In order to prevent cross-contamination between runs, a new affinity column was used for each sample.

Eluents were desalted on a Sep-Pak column as previously described (Fledelius et al. 1997) . The three batches of immunoaffinity purified OC were subsequently separated by reversed phase HPLC as described below. HPLC profiles of the two batches immunoaffinity purified OC from a Pagetic patient before (i) and after ( ii) bisphosphonate treatment were compared, to identify possible resorption specific fragments. The last large batch of immunoaffinity purified OC ( iii) was used for molecular characterization of the purified fragments. Thus selected immuno-reactive HPLC fractions from this batch were subsequently analyzed by N-terminal sequencing and mass-spectrometry as described below. Separation and Purification of Urinary OC by Reverse- phase HPLC - OC fragments were separated using reverse-phase HPLC on a Vydac Cl8-column. Chromatography was performed at room temperature employing a 2.5-50% acetonitrile gradient containing 0.1% TFA over 75 minutes at a flow-rate of 1 ml/min. The HPLC effluent was collected in 500 μl fractions, lyophilized, reconstituted in assay buffer and measured in the N-Mid™ OC and Mid-OC assays. N-Mid™ Osteocalcin ELISA -The N-Mid™ OC ELISA is a commercially available sandwich type assay employing two monoclonal antibodies Mab 504-1 and 539-4 directed against the Mid- and N-terminal region of OC respectively. The assay is optimized for measurement in serum where it provides a valid index of bone formation (Rosenquist et al. 1995). The assay is manufactured and performed as recommended by the manufacturer (Osteometer BioTech A/S) and as described in previous publications (Rosenquist et al. 1995) . The Mid-OC ELISA was manufactured and performed as described in example 1.

N-terminal Sequencing - N-terminal sequencing was performed on a 494A protein sequencer with an on-line 120A analyzer (Applied Biosystems) and chemicals recommended by the manufacturer.

Mass Spectrometry - Mass spectrometry was done using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) in a .Reflex III spectrometer

(Bruker Analytical Systems Inc., Billerica, MA, USA). Lyophilized material was re-dissolved in 20 μl of 30% acetonitrile (v/v) containing 0.15 % TFA (v/v) . A 2 μl aliquot was mixed with 2 μl of saturated α-cyano-4- hydroxycinnaic acid as matrix in the same solvent. 0.7 μl of this mixture was spotted onto the target plate. Samples were evaporated and analyzed in reflector and linear mode at 22.5 kV. The spectra were averaged from 50-100 laser-beam shots and calibrated externally with proteins of known masses.

Resul ts - All material reactive in the N-Mid™ OC and Mid-OC assays was retained on the affinity column as judged by immunoassay analysis (data not shown) . The immuno-affinity purified urinary OC fragments were subjected to reverse phase HPLC and immunoassay measurement of the eluted fractions demonstrated that most were sensitive to short time bisphosphonate treatment, Figure 5. In order to characterize the molecular identity of fragments, another batch of OC fragments were purified from a large quantity of urine, and separated by R.P. HPLC, Figure 6. The analytical results for some of the purified molecules are compiled in table II and Figure 7.

Example 6: Short Time Response of Urinary Mid-OC to Bisphosphona te

Matched urine and serum samples from patients with Paget's disease undergoing bisphosphonate therapy were measured in the Mid-OC and N-Mid OC ELISAs to assess the resorption specificity of these markers. Urine and Serum Samples - Matched urine and serum samples were collected from 10 patients with Paget's disease

(women and men) at baseline and after 8 days of bisphosphonate treatment. At the, entry of the study

(baseline) none of the patients were receiving any drugs known to influence bone metabolism. Informed consent was obtained from all participants according to the Helsinki Declaration of 1975, as revised in 1983. As is usual for urinary test, marker values were expressed relative to urinary creatinine (Cr) . Materials - The immunoassays CrossLaps™ ELISA, Serum CrossLaps™ one-step ELISA, and N-Mid™ osteocalcin ELISA were from Osteometer Biotech A/S, Herlev Denmark. The N-Mid™ OC ELISA is a commercially available sandwich type assay employing two monoclonal antibodies Mab 504-1 and 539-4 directed against the Mid- and N-terminal region of OC respectively. The assay is optimized for measurement in serum where it provides a valid index of bone formation (Rosenquist et al. 1995) . The assay is manufactured and performed as recommended by the manufacturer (Osteometer BioTech A/S) and as described in previous publications (Rosenquist et al. 1995) . The Mid-OC ELISA was manufactured and performed as described in example 1. Sta tistical Analysis - To assess longitudinal changes, the values were calculated for each person and expressed as the percentage of the initial values. Wilcoxon' s ranked sum test was used to assess differences between measurements before and after treatment with bisphosphonate and differences in response between the various markers. For all tests p<0.05 was considered significant.

Results - After eight days of treatment with bisphosphonate the Mid-OC/creatinine values decreased by 53±34% (mean ± SD) in urine. In contrast serum values of this marker remained unaltered. Figure 8 shows the response in excretion rates of Mid-OC, N-Mid OC and CrossLaps (CTx) in serum and urine after 8 days of anti-resorptive bisphosphonate therapy. These results demonstrate that urinary osteocalcin fragments reactive in the Mid-OC ELISA are responsive to short-time antiresorptive therapy. Thus urinary Mid-OC primarily or exclusively reflects bone resorption.

Example 7: Long Time Response to Bisphosphonate

The response of Mid-OC to long-term anti-resorptive treatment was investigated in 9 postmenopausal women receiving bisphosphonate therapy (oral alendronate, 20 mg/day plus a daily 500 mg calcium supplement) . At the entry of the study (baseline) none of the patients were receiving any drugs known to influence bone metabolism. Informed consent was obtained from all participants according to the Helsinki Declaration of 1975, as revised in 1983. Second morning void urine samples were collected after an overnight fast at entry of the study and after 1 and 12 months of therapy. Urine samples were subsequently measured in the Mid-OC ELISA (according to the procedures described in example 1) and in a known marker of bone resorption; CrossLaps (CTx) ELISA. As is usual for urinary tests, marker values were expressed relative to urinary creatinine (Cr) .

Statistical Analysis - To assess longitudinal changes, the values were calculated for each person and expressed as the percentage of the initial values. Wilcoxon' s ranked sum test was used to assess differences between measurements before and after treatment with bisphosphonate and differences in response between the various markers. For all tests p<0.05 was considered significant.

Results - Figure 9a and 9b shows the response in urinary excretion rates of Mid-OC/Cr and CrossLaps/Cr after 1 and 12 months of anti-resorptive bisphosphonate therapy. The Mid- OC/Cr values were significantly decreased (p<0.01) as a response to anti-resorptive therapy.. Thus on a group basis Mid-OC/Cr was decreases to approximately 30 and 20% of baseline values at 1 and 12 months respectively. For comparison the CrossLaps (CTx) /Cr values were decreased to approximately 20 and 10% of baseline values. The two marker were fairly correlated r²=0.40.

These results demonstrate that urinary osteocalcin fragments reactive in the Mid-OC ELISA are responsive to long-time anti-resorptive therapy.

Example 8: Detection of isoaspartyl/D-aspartyl in osteocalcin fragments purified from -urine. Fragments derived from osteocalcin were immunoaffinity purified and separated using R.P. HPLC as described above (example 5) . Eluents from HPLC were freeze-dried and reconstituted in phosphate buffered saline and analyzed for the presence of isomerised (isoaspartyl) or optically inverted Asx residues by an enzyme assay with the enzyme L- isoaspartate (D-aspartate) methyltransferase (IAMT) . Briefly described this assay is based on detection of isomerised or optically inverted residues by labeling with radioactive (tritiated) methionine by the IAMT enzyme. The assay is carried out as follows: In 600 μl eppendorf tubes the following reagents are added: 15 μl bovine red blood cell lysate containing the IAMT activity (prepared according to Murray and Clarke 1984) , 10 μl assay buffer (0.25 M NaH₂P0₄/NaOH, pH 7.0), 15 μl sample (or calibrator made up of synthetic isomerised peptide solutions of known concentration) and 10 μl SAM tracer (prepared as follows: 3 ml "cold" SAM is added to 26.1 ml freshly prepared 10 mM HC1. To 20 ml of this solution 100 μl "hot SAM" (Amersham TRA 236, 1000 μmol/1) is added, and the solution is stored in 1 ml aliquots at -18°C) . After whirly mixing the vials are incubated for 60 ±1 min. at 37 °C on a water bath. The reaction is stopped by addition of 50 μl quenching solution (0.2 M NaOH, 1% w/v SDS) followed by mixing. 75 μl of this solution is spotted onto a filter-paper (0.75 x 5.5 cm, pre- folded in "accordion-pleats") . The filter paper is placed in a 6 ml scintillation, tubes containing 2.5 ml Ecoscint H scintillation fluid (submersed approximately 1.5 cm in the tube) . The tubes are left at room temperature for approximately 18 hours (overnight) in order to allow radioactive methanol to diffuse into the scintillation fluid. The filter strips are removed and the vials are counted in a β-counter with the following stop conditions: 900 sec, or a maximum of 6400 CPM. The concentrations of unknown samples are calculated from the standard curve prepared from the measurements of the calibrators made up of synthetic isopeptides of known concentrations. These measurements demonstrated that some of the affinity purified osteocalcin fragments contain isomerised and/or optically inverted residues (Figure 10) . Here, using several independent lines of evidence we have shown urinary Mid-OC fragments (as defined as fragments being reactive in the Mid-OC ELISA) predominantly reflect bone resorption.

We have shown in vitro through the study of bone cells in culture that Mid-OC fragments are released from bone during osteoclastic resorption. The release of Mid-OC to the culture medium is highly correlated to a known marker of bone resorption and to the extent of pit-formation. Furthermore, urinary Mid-OC responds rapidly and markedly to anti- resorptive bisphosphonate therapy further emphasizing that an assessment of urinary Mid-OC fragments provides an index of bone resorption. This is the first description of osteocalcin fragments being generated in vivo as a result of resorptive

(osteoclastic) activity. The study likewise contains the first thorough molecular characterization of such resorption related fragments.

Surprisingly, it appears that most urinary OC-fragments measured by the Mid-OC assay reflect bone resorption. This is contrary to serum were most OC (fragmented or intact) appears to be un-responsive to short time bisphosphonate therapy. An explanation for the bone-resorption specificity of OC in urine may be that some specific post-translational modifications (i.e. cross-linking, glycosylations etc.) protect OC fragments derived from resorption against renal and hepatic degradation, securing their survival into urine. Conversely, fragments derived from bone formation may lack such modifications and may consequently be unprotected against proteolysis resulting in complete metabolization of such fragments.

The Mid-OC assay is able to measure both newly synthesized (intact) OC as well as fragments generated by osteoclastic resorption. Although OC fragments of resorptive origin are undoubtedly present in serum, the large background stemming from synthesis of bone probably masks them; therefore the Mid-OC assays response to bisphosphonate treatment is not detectable when measured in serum. The assessment of urine Mid-OC appears to provide an index of the rate at which bone is resorbed. In contrast, the related fragments in serum provide an index of bone formation.

In summary, we have shown that urinary fragments derived from the Mid-part of OC reflect bone resorption. The Mid-OC assay shows promise as a convenient non-invasive index of bone resorption.

All cited patents, patent applications and literature are incorporated by reference in their entirety. In case of conflict, however, the present disclosure controls.

The invention has been described above with reference to specific embodiments. It will be apparent to those skilled in the art, however, that many additions, deletions and modifications are possible without departing from the spirit of the invention as claimed above. ' Example 9: D-osteocalcin fragments in urine inhibit binding of monoclonal D-osteocalcin antibodies to immobilized antigen .

Materials - All chemicals were of analytical grade from either Sigma (St. Louis, MO) or Merck (Darmstadt, Germany), unless otherwise stated. Triflouroacetic acid. Methanol. Acetonitrile was from Rathburn. PBS buffer. C18 Sep-Pak Cartridge was from Waters (Milford, USA) . Streptavidin coated plates were from MicroCoat. Horse radish peroxidase conjugated anti-IgG antibody was from Jackson ImmunoResearch

Laboratories (West Grove, USA) . Bronidox L5 was from Henkel

(Dϋsseldorf, Germany). 3, 3' , 5, 5' -tetramethylbenzidine (TMB) solution was from Kierkegaard & Perry, (Gaithersburg, MD) .

Buffers used were as follows; Assay buffer: 1.5 mM KHP0, 8.5 mM Na₂HP0₄'2H₂0, 2.7 mM KCl, 137 mM NaCl, 1 % (w/v) bovine serum albumin (BSA) , 0.1 % (w/v) Tween-20, 0.36 % (w/v) Bronidox L5, pH 7.0.

D-Osteocalcin antibodies - D-osteocalcin antibodies were raised in mice by immunisation with the synthetic peptide ELNPD_DCD_DELADH (aa 24-35 in osteocalcin) . Three monoclonal antibody producing cell lines were generated by methods well known in the art, MAb 7004A3B8, MAb 7004A3B5 and MAb 7002D7G7. The antibodies are specific towards osteocalcin peptides containing optically inverted amino acids (D- osteocalcin) and did not recognize the L-osteocalcin peptides (not shown) . A competitive immunoassay was developed to verify that D-osteocalcin fragments could be measured in urine by means of an immunoassay.

Urine samples from healthy individuals were pooled and desalted by the following procedure. Fifty mL heat treated urine was precipitated with 450 μl triflouroacetic acid (TFA) for 30 min at room temperature. The mixture was filtered. A C18 Sep-Pak Cartridge was activated with 3 x 10 mL 80% methanol and equilibrated with 3x10 L 0.1% TFA. The filtrate was added to the column, and the column was washed with 3 x 0.1% TFA. The bound material was eluted with 2 x 10 ml 40% acetonitrile containing 0.1% TFA. The desalted urine eluate was freeze-dried and resuspended in 10 mL PBS.

The assay was performed as follows (all incubations are carried out at 20°C on a mixing apparatus (300 rpm) ) . Streptavidin coated microtiter plates (MicroCoat) were pre- incubated for 45 min with biotinylated synthetic peptide with the following sequence ELNPD_DCD_DELADHG 10 ng/ml, 100 μl/well. Wells were washed five times using the washing solution. Fifty μl of diluted urine samples (undiluted, 1:2, 1:4, 1:8, 1:16 and 1:32) were pipetted into appropriate wells in the microtiter plate, followed by 50 μl of either MAb 7004A3B8, MAb 7004A3B5 or MAb 7002D7G7 diluted 1:400 in assay buffer to the appropriate wells. The wells were covered with sealing tape and incubated for one hour. Wells were emptied and washed five times using the washing solution. One hundred μl of horse radish peroxidase conjugated anti-IgG antibody

(diluted in a protein stabilized buffer) was added to all wells, which were incubated for one hour. After another washing step 100 μl of TMB solution was added to each well and the plate was incubated in darkness. The enzyme reaction was stopped after 15 minutes by adding 100 μl of 0.18 mol/L H₂S0₄ per well and the absorbance measured at 450 nm (with 650 nm as the reference wavelength) using a microtitre plate reader (E_maχ Easy Microtitre Reader; Molecular Devices, Menlo Park, CA) . The absorbance obtained for the antibodies were ploted with decreasing dilution of the urine samples (figure 11) . This clearly shows that urine contains D-osteocalcin fragments, which can be detected in a competitive immunoassay.

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Table 1 : Correlation Between Resorbed Area, in vitro Mid-OC and CrossLaps for Culture in Bone Cell Cultures

The concentration of OC was determined using the in vitro Mid-OC ELISA. Correlations were determined using linear least squares regression.

Table II: Predicted and Determined Mass of Molecular Species in the Various Peaks Purified by R.P. HPLC.

Peak Peptide as Predict Mass Probable Identity Comment No. deduced from ed Found N-terminal Exact sequencing Mass + H+

EVXELNPDXD 1308.46 1307.27 EVCBLNPDCDE (SEQ ID 7Anu.de (SEQ ID NO: 12) NO:19) formation at C- terminus?

(-D

1419.56 1419.55 REVZELNPDZDE (SEQ ID NO: 8)

1463.56 1463.38 REVZBLNPDZDE (SEQ ID NO:21)

1507.56 1507.37 RBVZBLNPDZDE (SEQ ID NO: 22)

1576.64 1576.57 REVZBLNPDZDEL (SEQ ID NO: 23)

862.40 861.03 RE CELN (SEQ ID NO:10)

2510.13 2510.10 LEPRREVCBLNPDCDELADHI (SEQ ID NO:24)

1430.63 1430.69 DPLEPRRBVCB (SEQ ID NO:25)

3009.35 3009.27 LGAPVPYPDPLEPRRBVZBLN PDZDE (SEQ ID NO: 26)

EVXELNPD (SEQ 847.35 847.29 EVCBLNP (SEQ ID ID NO:20) NO:27) (major 891.35 891.30 BVCBLNP (SEQ ID sequence) NO:28)

907.35 907.27 BVCBLNP (SEQ ID Sulfenic NO:28) acid from C (+16)

935.26 935.29 BVCBLNP (SEQ ID disodium NO:28) (+44)

Table II continued.

Peaks are not absolutely pure but contain various molecular species some of which are created by post- translational modifications of the same peptide (γ- carboxylation etc. ) .

Unless otherwise stated masses are calculated assuming hydrogen as N-terminal group and a free acid C-terminally.

B = Gammacarboxyglutamic acid (Gla)

X = Cysteine (potential disulfide bridge)

Z = Cysteine (disulfide bridge)

Claims

73CLAIMS

1. A method of measuring the amount of osteocalcin-derived fragments in a body fluid comprising contacting a sample of said body fluid with at least one immunological binding partner for said fragments and determining binding of the said immunological binding partner to said fragments, wherein said immunological binding partner is specific for an epitope located at the N or C terminus of a said fragment, which epitope is created upon cleavage of osteocalcin at said terminus during resorption of bone and which is not present in intact osteocalcin.

2. A method as claimed in claim 1, wherein said epitope is created by cleavage of osteocalcin by cathepsin K.

3. A method as claimed in claim 1 pr claim 2, wherein said fragments are below 2600 Dalton in size.

4. A method as claimed in any preceding claim, wherein said immunological binding partner does not depend on the carboxylation state of osteocalcin fragments for binding.

5. A method as claimed in any preceding claim, wherein the body fluid is urine.

6. A method as claimed in claim 5, wherein said fragments are contacted with two immunological binding partners for said fragments in a sandwich assay, said immunological binding partners including a first immunological binding partner which is specific for an 74

epitope located at the N-terminus of a said fragment and which is created upon cleavage of osteocalcin at said ^• terminus during resorption of bone and is not present in intact osteocalcin and a second immunological binding partner which is specific for an epitope located at the C-terminus of a said fragment and which is created upon cleavage of osteocalcin at said terminus during resorption of bone and is not present in intact osteocalcin.

7. An immunological binding partner specific for an epitope present at the N or C terminus of an osteocalcin fragment, which epitope is created upon cleavage of osteocalcin at said terminus during resorption of bone and which is not present in intact osteocalcin.

8. An immunological binding partner as claimed in claim 7, wherein said epitope is one created upon cleavage of osteocalcin by cathepsin K.

9. An assay kit for use in performing a method as claimed in claim 1 and comprising an immunological binding partner as claimed in claim 7 or 8 together with one or more of peptide standards, urine standards, substrate bearing peptides reactive with the immunological binding partner, buffers, antibody reaction stopping solutions, osteocalcin reactive immunological binding partners lacking specificity for isomerised or optically inverted osteocalcin sequences, antibody-enzyme conjugates, enzyme substrates or enyz e reaction indicator substances . 75

10. A method of measuring the amount of osteocalcin derived fragments in a body fluid comprising contacting a sample of said body fluid with at least one immunological binding partner for said fragments and determining binding of the said immunological binding partner to said fragments, wherein said immunological binding partner is specific for an epitope containing an isomerised or optically inverted amino acid.

11. A method as claimed in claim 10, wherein the amino acid is aspartic acid or asparagine or glutamic acid or glutamine or γ-carboxylated glutamic acid.

12. An immunological binding partner specific for an epitope containing an isomerised or optically inverted amino acid in an amino acid sequence of osteocalcin.

13. An assay kit for performing an .assay for isomerised or optically inverted osteocalcin fragments, comprising an immunological binding partner as claimed in claim 11 in combination with one or more of peptide standards, urine standards, substrate bearing peptides reactive with the immunological binding partner, buffers, antibody reaction stopping solutions, osteocalcin reactive immunological binding partners lacking specificity for isomerised or optically inverted osteocalcin sequences, antibody-enzyme conjugates, enzyme substrates or enyzme reaction indicator substances.

14. A method of obtaining an indication of the rate of bone resorption in an individual by measuring the amount of certain osteocalcin derived fragments in urine, 76

comprising contacting a sample of urine from said individual with at least one immunological binding partner for said fragments and determining the amount of binding of the said immunological binding partner to said fragments, wherein said immunological binding partner is such as to bind predominantly to osteocalcin fragments in said sample of not more than 2600 Daltons.

15. A method as claimed in claim 14, wherein the population of the bound fragments comprises peptides of one or more of the following sequences:

hOC14-24 DPLEPRREVCE (SEQ ID NO: 2) hOC14-31 DPLEPRREVCELNPDCDE (SEQ ID NO: 3) hOC16-36 LEPRREVCELNPDCDELADHI (SEQ ID NO: 4) hOC16-31 LEPRREVCELNPDCDE (SEQ ID NO: 5) hOC17-28 EPRREVCELNPD (SEQ ID NO: 6) hOC20-32 REVCELNPDCDEL (SEQ ID NO: 7) hOC20-31 REVCELNPDCDE (SEQ ID NO: 8) hOC20-29 REVCELNPDC (SEQ ID NO: 9) hOC20-26 REVCELN (SEQ ID NO: 10) hOC21-32 EVCELNPDCDEL (SEQ ID NO: 11) hOC21-31 EVCELNPDCDE (SEQ ID NO: 12) hOC21-29 EVCELNPDC (SEQ ID NO: 13) hOC21-27 EVCELNP (SEQ ID NO: 14) hOC21-26 EVCELN (SEQ ID NO: 15)

16. A method as claimed in claim 14 or 15, wherein said binding partner binds to an epitope containing the amino acids EVCE (SEQ ID NO:37).

17. A method as claimed in any of the claims 14 to 16, wherein said binding partner binds to an epitope 77

containing one or more isomerised and/or optically inverted amino acids.

18. A method as claimed in claim 14 or 17, wherein the population of the bound fragments comprises peptides of one or more of the following sequences: hOC_Di24-35 ELNPD_DCDELADH (SEQ ID NO: 16) hOC_D224-35 ELNPDCD_DELADH (SEQ ID NO: 16) hOC_DD24-35 ELNPD_DCD_DELADH (SEQ ID NO: 16) hOC_D30-37: DELAD_DHIG (SEQ ID NO: 17)

19. A method as claimed in any one of claims 14 to 16, wherein said binding partner binds to an epitope containing or consisting of amino acids EVCELNPDC (SEQ ID NO:13).

20. A method of measuring the amount of osteocalcin derived fragments in urine comprising contacting a sample of said urine with at least one immunological binding partner for said fragments and determining the amount of binding of the said immunological binding partner to essentially all osteocalcin fragments in said sample which are reactive therewith, wherein said immunological binding partner is specific for an epitope comprising at the N terminal end of said epitope the amino acid sequence EVCE (SEQ ID NO: 37).

21. A method as claimed in claim 18 wherein said epitope is defined by the amino acid sequence EVCELNPD (SEQ ID NO:18) . 78

22. A cell line producing a monoclonal antibody constituting an immunological binding partner as claimed in claim 7, claim 8 , or claim 12.