WO2007107002A1

WO2007107002A1 - Markers for the diagnosis, monitoring and prognosis of osteoporosis and for assessing the risk for osteoporosis and for osteoporotic fractures

Info

Publication number: WO2007107002A1
Application number: PCT/CA2007/000457
Authority: WO
Inventors: Sean Alexander Fitzgerald Peel; Yaoming Gu
Original assignee: Osteopharm Inc.
Priority date: 2006-03-22
Filing date: 2007-03-22
Publication date: 2007-09-27
Also published as: WO2007107003A1

Abstract

Use of biomarkers in the diagnosis, monitoring and prognosis of osteoporosis and assessing the risk for osteoporosis and for osteoporotic fractures. The biomarkers include CGRP, include CGRPα and CGRPβ, and can further include a chemokine such as CXCL1, CXCL4, CXCL6 and/or CXCL8.

Description

MARKERS FOR THE DIAGNOSIS, MONITORING AND PROGNOSIS OF OSTEOPOROSIS AND FOR ASSESSING THE RISK FOR OSTEOPOROSIS AND

FOR OSTEOPOROTIC FRACTURES

FIELD OF THE INVENTION

This invention relates to the use of biomarkers in the diagnosis, monitoring and prognosis of osteoporosis and assessing the risk for osteoporosis and for osteoporotic fractures.

BACKGROUND

Osteoporosis has been defined as "a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture" (Anonymous. Consensus Development Conference: diagnosis, prophylaxis, and treatment of osteoporosis. Am J Med. 1993;94:646-650). Osteoporosis has been further classified as primary (osteoporosis in the absence of known conditions that cause bone loss) and secondary osteoporosis (osteoporosis caused by another condition or use of a drug). Primary osteoporosis has also been subdivided into postmenopausal (also called type I) and senile (type II) osteoporosis. Broadly, osteoporosis relates to any condition which results in significant loss of bone mass and/or quality which results in a significant reduction in mechanical strength and increased risk of fracture.

Osteoporosis is a major health problem worldwide. It has been estimated that more than one third of adult women will experience a fracture due to osteoporosis. There were approximately 3 million osteoporotic fractures worldwide in 2005, and this is expected to increase dramatically as the world population continues to age.

Fractures due to osteoporosis are a principal cause of pain, disability and death. Osteoporotic hip and vertebral fractures result in 10-20% excess mortality within a year. The health cost of treating osteoporotic fractures in Europe is estimated to be €4.8 billion and over $12 billion in the US, and may reach US$240 billion by 2040. Consequently, osteoporosis diagnosis and prevention is considered a priority of health care in many countries.

Osteoporotic bone loss is a gradual process that is typically asymptomatic until fractures occur. Even after osteoporotic fractures have occurred, fewer than one third of patients are appropriately diagnosed. This has led the US Preventative Services Task Force to recommend universal screening of all women over 65. This population numbers over 70 million in Europe and the United States and 270 million worldwide.

The World Health Organization has classified individuals as osteoporotic based on their bone mineral density (BMD) scores (WHO Study Group 1994). An individual's BMD, measured by densitometry and expressed in g/cm2, is compared with a "normal value." The normal value is the mean BMD of sex-matched young adults at their peak bone mass, sometimes referred to as the "young adult mean." When compared with the normal value, a patient's BMD can be expressed in terms of the number of standard deviations (SD) from the normal value. A convenient way to express this is a T-score. Based on this scoring system the WHO classifies individuals as being normal (T score >-1.0) osteopenic (-1.0>T score>-2.5) and osteoporotic (-2.5> T score). WHO also classified individuals as having severe or established osteoporosis if their T score was less than -2.5 and they had at least one fragility fracture.

Although low BMD correlates with an increased risk for fracture, it is estimated that as high as 82% of osteoporotic fractures occur in women with a BMD score above that used to diagnose osteoporosis (T score <-2.5).

Another concern with the use of BMD is the minimum change in BMD that can be detected. Currently the DEXA scanner, which is considered the "gold standard" for measuring BMD, requires changes of between 3.1 to 6.2% in BMD to be able to detect the change. However, when DEXA units from different manufacturers are used to measure BMD of a single patient, results may differ by as much as 10 to 15%. Even manufacturer intersystem difference after primary calibration is approximately 2%. Thus when trying to determine whether individuals are losing BMD if different machines are used very large changes in BMD must have occurred before bone loss can be definitely identified. As many of the current osteoporosis therapies result in increases in BMD of 6 to 7% after eighteen months it is recognized that BMD can only be useful in determining whether therapy is efficacious after years of treatment have occurred.

Further, since BMD is only a useful diagnostic tool once there has been significant bone loss, BMD is not a good indicator for initiating therapy to prevent the initial bone loss. Another concern with using BMD to diagnose osteoporosis is that the cost and availability of bone scanners limits their use. Consequently in the US less than 9% of women who meet the criteria for a BMD scans had received one in 2000. Biomarker assays are cheaper and more convenient than BMD scans. Further, if appropriate markers are used they offer the possibility of identifying those who are at greater risk for osteoporotic bone loss prior to that loss occurring, providing the opportunity to initiate preventative measures, such as life style changes or therapeutic interventions, other than having to restore lost bone later.

Current FDA approved bone biomarker assays are approved for use as aids in the diagnosis of osteoporosis and for monitoring the effectiveness of therapy. All of the approved biomarkers are based on the measurement of markers for bone turnover, and fall into two categories: markers of bone formation (e.g. osteocalcin, bone alkaline phosphatase, procollagen I N and C telopeptides), or markers of bone resorption (e.g. hydroxyproline, pyridinoline, deoxypridinoline, N-telopeptide, C-telopeptide, and tartrate resistant acid phosphatase). However their diagnostic accuracy is poor (typically one or both of sensitivity and/or specificity are <65%).

The calcitonin gene related peptides (CGRP) are 37 amino acid peptides which are expressed in the central and peripheral nervous systems and act as neurotransmitters. There are two CGRPs: CGRP alpha (CGRPa) and CGRP beta (CGRPb). CGRPa is a product of the CT/CGRP gene. Alternate splicing of the primary RNA product of this gene can produce calcitonin or CGRPa. CGRPb is derived from a second gene, thought to exist due to gene duplication of the CT/CGRP gene. Human CGRPa and b differ by 3 amino acids.

CGRP is widely expressed in many organs and tissues including the heart, blood vessels, pituitary, thyroid, lung, and gastrointestinal tract, and it possesses a broad range of biological effects including neuromodulation, vasodilation, cardiac contractility, angiogenesis and bone growth. The peptide is released from motor neurons at the neuromuscular junction and sensory neurons of spinal cord. The source of serum CGRP is believed to be from the thyroid in old age and from perivascular nerves at all ages.

Both major cell types in bone, osteoblasts and osteoclasts, express receptors for CGRP. CGRPa has been demonstrated to stimulate osteogenesis in vitro, while CGRPb is reported to have no effect. Transgenic mice overexpressing osteoblast-derived CGRPa have a skeletal phenotype consistent with anabolic effects on bone. CGRPa has also been demonstrated to inhibit bone resorption in vitro and in vivo and systemic administration of CGRPa partially reverses the bone loss in rats caused by ovariectomy. CGRP appears to exist in several immunological forms in serum. Various reports have indicated that CGRP levels decline in postmenopausal women or following orchidectomy in rats, and, at least partially, are restored with hormone replacement therapy, or testosterone supplementation respectively. Patients who have experienced trauma resulting in bone fracture show elevated CGRP levels immediately post fracture, which begin to decline within 24 hours.

A single study has reported that plasma CGRP was elevated in Chinese women who reported back pain and were subsequently shown to have an osteoporotic BMD score (T<-2.5; n = 38) compared to those who reported back pain and had normal BMD scores (T>-1.0; n = 12)(CGRP 37±7 vs. 29±6ng/ml PO.05). (Lin J., Lu C, Gao L. Study on the level of plasma calcitonin gene-related peptide and adrenomedulin in subjects with primary osteoporosis. Zhonghua Yi Xue Za Zhi 2001 ; 81 :841-3). This led the authors to conclude that further studies should be carried out to determine the value of CGRP as an osteoporosis diagnostic. However they also reported that there were no differences between women with osteopenic BMD (T = -1.0 to -2.5; n = 25; CGRP = 38±7ng/ml) and women with osteoporotic BMD scores. Further, when the mean CGRP concentration is calculated for the 2 groups with the non-osteoporotic BMD scores combined the result is 35 ng/ml compared to 37±7 ng/ml for the osteoporotic group, suggesting that CGRP could not distinguish those with non- osteoporotic BMDs from those with osteoporotic BMDs.

A need thus exists for a robust and reproducible biomarkers assay for osteoporosis which possesses improved accuracy compared to current biomarkers, and desirably with the sensitivity and specificity required for diagnosis of the disease and ability to evaluate the risk of osteoporotic fracture.

SUMMARY OF THE INVENTION

According to this invention an assay is provided based on the surprising and unexpected finding that the CGRPs and/or specific CXC chemokines are useful biomarkers for the diagnosis of osteoporosis.

One objective of this invention is to provide improved biomarker assays, which can be used to diagnose a predisposition to osteoporosis, the presence of the disease and also to monitor the progression of the disease. According to one aspect of this invention a method of diagnosing osteoporosis is provided which comprises the following steps:

a) taking a biological sample from an individual;

b) measuring the level of one or both CGRPs and/or at least one CXC chemokine; and

c) diagnosing osteoporosis or the risk of osteoporosis or risk of osteoporotic fracture by comparing the measured level(s) to known level(s) associated with osteoporosis or the risk of osteoporosis or osteoporotic fracture.

In one embodiment a method is provided for determining whether an individual has or is at risk of having osteoporosis and/or at risk of having osteoporotic fractures, comprising the steps of (a) taking a sample, preferably whole blood, plasma or serum from a patient, (b) measuring the levels of at least one calcitonin gene related peptide (CGRP) in the sample, and (c) diagnosing osteoporosis or the risk of osteoporosis or risk of osteoporotic fracture by comparing the measured level(s) to known level(s) associated with osteoporosis or the risk of osteoporosis or osteoporotic fracture. The sample may of course be processed, as appropriate, prior to step (b), so the measuring step can thus be of the CGRP in a specimen comprising whole blood, plasma or serum, or in a fluid, or a specimen comprising a fraction of whole blood, fraction of plasma, or fraction of serum.

DETAILED DESCRIPTION OF INVENTION

The present invention provides methods and means for the diagnosis and prognosis of osteoporosis and for assessing the risk for osteoporosis and for osteoporotic fractures in individuals.

The present invention is particularly useful for physicians or others who do not have ready or have no access to bone mineral density testing, by providing a simple method for screening patients to determine their risk for osteoporosis and/or osteoporotic fractures.

The present invention provides simple methods to determine the risk for osteoporosis and/or osteoporotic fractures prior to significant bone loss.

According to the present invention osteoporosis relates to all forms of osteoporosis. Diagnosis according to the present invention includes determination, confirmation, sub- classification and prediction of osteoporosis.

Prognosis according to the present invention relates to the monitoring of osteoporosis. Monitoring relates to keeping track of an already diagnosed osteoporosis or complication, e.g. to analyze the progression of the disease or condition, or influence of treatment on progression of the disease or condition.

Risk stratification according to the present invention relates to the use of clinical risk factors to allow a physician to classify an individual into various categories of risk for developing osteoporosis or osteoporotic fractures e.g. low, moderate or high.

The term "individual" according to the present invention relates to a healthy individual, an apparently healthy individual or an individual with osteoporosis.

The present invention allows diagnosing whether an individual is at risk of having osteoporosis or of having an osteoporotic fracture.

The invention takes advantage of certain biomarkers. Usually a biomarker is a protein, peptide, vitamin, lipid or other biochemical entity and may also include minerals or nucleic acids, which is elevated, decreased or altered in the presence or absence of a condition, disease or complication. The level of a suitable biomarker can indicate the presence, absence or relative risk of the condition, disease or complication and thus allow for assessment of disease risk, diagnosis, or prognosis.

The biomarkers according to the present invention comprise intact calcitonin gene related peptides, GCRP alpha (CGRPa) and CGRP beta (CGRPb) their fragments or variants and intact chemokines of the CXC sub-family and their fragments or variants. The preferred CGRP would be CGRPb. The preferred CXC chemokines would be CXCLl, CXCL4, CXCL6, and CXCL8.

The term "fragments" in the present invention relates to individual components derived from the intact molecule. These may be proteolytic degradation products.

The term "variants" in the present invention relates to molecules which are substantially similar to the said biomarkers. In particular a variant may be an isoform or an allele which shows amino acid exchanges compared to the amino acid sequence of the most prevalent isoforms in the human population.

The method may also comprise the step of taking a sample, e.g. a body fluid or tissue sample from the patient. The body fluid can be but is not restricted to whole blood, plasma, serum, urine or saliva. Preferably the sample is blood, plasma or serum.

A person skilled in the art will be familiar with different methods of measuring the level of a biomarker in a sample. The term "level" relates to the amount or concentration of the analyte to be measured.

The term "measuring" according to the present invention relates to determining the amount or concentration of the biomarker preferably semi-quantitatively or quantitatively. Measuring can be done directly or indirectly. Direct measurement methods include the use of high performance liquid chromatography (HPLC), near or mid infra-red spectroscopy or mass spectroscopy. Indirect measurement includes measuring cellular responses, bound ligands, labels, or enzymatic reaction products. Measurement can be done according to any method known in the art.

A preferred method of measurement is an immunoassay. The term "immunoassay" in the present invention relates to a test that uses the binding of antibodies to antigens to identify and measure certain substances. Immunoassay refers to all immunological methods of measuring an analyte including radioimmunoassay (RIA), enzyme immunoassays (EIA) e.g. enzyme linked immunosorbant assay (ELISA), western blot and immunoprecipitation.

In a preferred embodiment of the invention the method for measuring the biomarker of interest comprises the steps of (a) contacting the analyte with a specific binding ligand (b) (optionally) removing non-bound analyte, and (c) measuring the amount of the bound ligand.

Preferably the binding ligand in (a) should be specific for the analyte to be measured. "Specific" in the present invention means that the ligand does not "cross-react" with another substance present in the sample to be investigated. The term "cross-react" in the current invention means the degree in which the other (interfering) substances are shown to generate positive test results. Preferably other structurally similar substances should have a percent cross reactivity of <35%, more preferably <20%, and most preferably <10%. The invention further relates to a method of producing immunoassays as defined above. Methods of producing such immunoassays are generally known.

In another preferred embodiment a multiplexed assay will be used where multiple markers will be assayed simultaneously. The term "multiplexed" refers to the measurement of more than one analyte, or more than one fragment or variant of the same analyte simultaneously in a single small volume of the sample.

Such an assay can be performed using an array. Said array will be capable of quantitating at least two biomarkers. According to the present invention the term "array" refers to a solid- phase or gel-like carrier upon which at least 2 binding ligands are attached or bound in one-, two-, or three dimensions. Such arrays (including protein chips, antibody arrays and the like) are generally known to a person skilled in the art and are typically generated on glass slides, or membranes such as those based on nitrocellulose or nylon.

In another preferred embodiment the multiplexed assay will utilize a microsphere-based flow cytometric assay (Carson, R.T.; Vignali, D.A., "Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay" J. Immunol. Methods 1999; 41). In such an assay the carrier, e.g. a microbead or microsphere, is present in suspension. Each different binding ligand is attached to a different microbead type.

The invention further relates to a method of producing multiplexed assays as defined above. Methods of producing such multiplexed assays are generally known.

The person skilled in the art is able to determine the known levels of markers which are associated with the presence or risk of suffering from osteoporosis or osteoporosis related fractures. Such levels can be determined by well known methods as described in Examples 1, 2, 3, and 4. For example the measured levels in a population of patients with osteoporosis can be compared to a population of patients without osteoporosis. A reference level can be determined above or below which a diagnosis is made or risk stratification is made. Evaluating the levels in further patients, e.g., in cohort studies, can help refine the reference levels and distinguish between different grades of severity and status of disease progression.

The reference levels given in the examples may serve only as a first guideline to diagnose the risk of an individual. The person skilled in the art will be able to determine other reference levels. The value of the reference level may also depend on the desired sensitivity and specificity of the diagnosis. This level can be determined empirically or through the use of statistical methods. A preferred method is by using the Receiver Operating Characteristic (ROC) curve analysis to optimize the diagnostic accuracy.

EXAMPLES

Example 1

Serum Samples

118 individual human serum samples were obtained from Bioreclamation (East Meadow, NY). A single pooled set of samples was obtained for use as a normalization standard. All samples were from donors who were fasted overnight. The samples were stored at -80⁰C until use. The specifications for each group were:

Young Normal (YNMU

Thirty healthy Caucasian females taking no medications aged 30-45.

Postmenopausal (PMP)

Fifty healthy Caucasian females aged 55+ who were postmenopausal (minimum 1 year since menses) and were not on hormone therapy.

Osteoporotic (OSP)

Thirty Caucasian females, diagnosed osteoporotic based on a BMD T-score at any site of <2.5. A further 8 Caucasian women who had been diagnosed osteoporotic but had BMD scores >-2.5 were also analyzed.

Pooled.

Samples pooled from 10 healthy Caucasian female donors aged 20 to 35.

Diagnostic Assays

Commercial assays were used for determining serum levels of CGRP alpha (Bachem Catalog No. S-1198) and CGRP beta (Bachem Catalog No. S- 1200) in each of the samples. For comparison, levels of the markers for bone resorption (Serum CrossLaps™, Nordic Catalog No. 4CRL 4000) and bone formation (NMID Osteocalcin, Nordic Catalog No. 3OSC 400) were also determined for the same samples. All assays were performed according to manufacturer's published procedures.

Data Analyses

As several samples had analytes below the limit of detection, these were given a nominal value of zero in the appropriate test. This resulted in many of the data sets not being normally distributed.

To minimize the effect of age a subset of 40 donors from the postmenopausal (n=20) and osteoporotic groups (n=20) were selected that were aged between 59 and 70.

All data was analyzed using the Sigma Stat 3.2 (Systat Software Inc., Richmond CA). Normally distributed data was analyzed by 1 way ANOVA followed by Holm-Sidak post hoc analysis to identify significantly different factors. ANOVA on Ranks was used for non- normally distributed data with the Dunn's post hoc test. A P value of 0.05 was considered significant in these analyses.

Four measures of diagnostic accuracy were determined. These were sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).

These are defined as:

Sensitivity Proportion of people with disease who have a positive test result

Specificity Proportion of people without disease who have a negative test result

PPV Proportion of people with a positive test who have the target disorder

NPV Proportion of people with a negative test who are free of the target disorder

Sensitivity, specificity, PPV and NPV are calculated by the following formulae:

Sensitivity = a/a+c, Specificity =d/b+d, PPV = a/a+b, and NPV = d/c+d; where a is the number of true positives and c is the number of false negatives among the osteoporotic samples, and b is the number of false positives and d is the number of true negatives among the non-osteoporotic samples.

Results

Bone formation and resorption markers

Osteocalcin was significantly higher in young normal compared to postmenopausal or osteoporotic samples. However there were no differences between postmenopausal and osteoporotic samples. CrossLaps was significantly lower in young normal compared to postmenopausal or osteoporotic samples. However there were no differences between postmenopausal and osteoporotic samples. The results are summarized in Tables 1 and 2.

CGRP biomarkers

Of the 118 samples assayed for CGRPa, 52 had undetectable levels. When considered by sample type the distribution was young normal 21/30 (70% of YNML), postmenopausal 11/49 (22.4% of PMP), osteoporotic 20/39 (51.3% of OSP). By comparison only 1 of the 118 samples had undetectable levels of CGRPb.

CGRP levels in young normal post menopausal and osteoporotic samples

Serum CGRPa and b levels were significantly increased within the postmenopausal group compared to both the young normal and the osteoporotic samples (Table 3).

CGRP levels in age range matched samples

Neither CGRPa nor b were correlated with age. When the subsets of osteoporotic and postmenopausal samples that lay within the same age range (59 to 70) were compared CGRPa and CGRPb were still significantly elevated in the postmenopausal non-osteoporotic samples compared the osteoporotic samples (P<0.01)(Table 3). Table 1 Summary of Results for Age, Osteocalcin, CrossLaps and T-scores

Young Normal Postmenopausal Osteoporotic

Age 36.6 ± 4.3 57.9 ± 3.7 a 70.8 ± 6.1 a,b

Osteocalcin (ng/ml) 6.217 ± 2.427 3.527 ± 3.896 a 3.801 ± 3.225 a

CrossLaps (ng/ml) 0.010 ± 0.042 0.135 ± 0.204 a 0.128 ± 0.282 a

T-score (all sites) ND (not done) ND -2.4 ± 0. 7

Results are presented as mean ± SD

a significantly different from young normal (P<0.05)

b significantly different from postmenopausal (P<0.05)

Table 2 Osteocalcin, CrossLaps and T-score in age matched samples

Postmenopausal Osteoporotic

Osteocalcin 2.87 ± 3.53 3.22 ± 3.39

CrossLaps 0.15 ± 0.20 0.10 ± 0.23

T-score ND -2.4 ± 0.6

Results are presented as mean ± SD

Table 3 Summary of Results for CGRPa & b

Young Normal Postmenopausal Osteoporotic

CGRPa (ng/ml) 0.065 ± 0.159 3.274 ± 8.360 a 1.377 ± 3.710 b

CGRPb (ng/ml) 0.308 ± 0.611 2.877 ± 4.619 a 0.544 ± 0.979 b

Age Matched

CGRPa (ng/ml) 1.94 ± 3.74 0.91 ± 3.05 b

CGRPb (ng/ml) 3.57 ± 5.04 0.44 ± 0.82 b

Results are presented as mean ± SD

a significantly different from young normal (P<0.05)

b significantly different from postmenopausal (P<0.05)

Diagnostic accuracy of CGRPa & b in differentiating between osteoporotic and non- osteoporotic groups

As CGRP has been reported to stimulate bone formation and inhibit resorption and it appears to be lower in women with osteoporosis than in undiagnosed postmenopausal women it is possible that elevated CGRP levels may protect women from osteoporosis. Further, it may be possible to identify women at risk for osteoporosis prior to significant bone loss as those who have lower CGRP levels than age matched controls.

To determine the diagnostic accuracy of using CGRP levels to diagnose osteoporosis, samples were ranked by concentration of CGRP and a concentration below which a diagnosis of osteoporosis would be made was chosen based on each CGRP. As the young normal women were shown to have low levels of CGRPs, osteoporotic samples were compared to postmenopausal women only, (n = 38 osteoporotic and n = 50 postmenopausal). A similar analysis was performed using only samples within the age range 59 to 70 (n = 20 for each condition). A truth table was then created and the diagnostic accuracy calculated, for both all postmenopausal and all osteoporotic, or just the age matched samples. Using a level of CGRPa less than 0.16ng/ml for a diagnosis of osteoporosis, CGRPa had a sensitivity and specificity of 68% (both) for the whole group and of 80% and 65% when considering the age matched samples. Using 0.25ng/ml of CGRPb for a diagnosis the values of sensitivity and specificity were 80% and 72% for the entire set, and 80% and 74% for the age matched sub set.

The diagnostic accuracy of a CGRPa and a CGRPb assay for the diagnosis of osteoporosis in the age range matched samples is shown in Tables 4 & 5.

Table 4 Diagnostic accuracy of CGRPa for osteoporosis

Estimate 95% CI

Sensitivity 80% [58.4 to 91.9]

Specificity 65.0% [43.3 to 81.9]

Positive Predictive Value 69.6% [49.1 to 84.4]

Negative Predictive Value 76.5% [52.7 to 90.4]

These results are based only on the postmenopausal and osteoporotic samples in the same age range. Samples were diagnosed as osteoporotic if they had CGRPa levels of 0.16ng/ml or less.

Table 5 Diagnostic accuracy of CGRPb for osteoporosis

Estimate 95% CI

Sensitivity 80.0% [58.4 to 91.9]

Specificity 73.7% [51.2 to 88.2]

Positive Predictive Value 76.2% [54.9 to 89.4]

Negative Predictive Value 77.8% [54.8 to 91.0]

These results are based only on the postmenopausal and osteoporotic samples in the same age range (n = 20 for both). Samples were diagnosed as osteoporotic if they had CGRPb levels of 0.25ng/ml or less.

Claims

1. A method of diagnosing and/or monitoring osteoporosis in an individual which comprises the following steps; a) taking a biological sample from the individual; b) measuring the level of at least one CGRP in said sample; and c) comparing the level(s) in said sample with the level in a control biological sample.

2. The method of claim 1 wherein the control biological sample is from an individual known not to be afflicted by osteoporosis or from the same individual at different times.

3. A method of claim 1 or 2, which comprises the following steps: a) contacting the biological sample with an antibody specific for each CGRP, said antibody being labeled with a detectable substance; b) measuring the detectable substance to quantitate each CGRP in the sample; and c) comparing the quantitated CGRP levels to levels for a control sample.

4. A method of claim 3 which further comprises: in step a), further contacting the sample with a second antibody specific for the CGRP which is immobilized; following step a) separating the first antibody from the second antibody to provide a first antibody phase and second antibody phase; and in step b), measuring the detectable substrate in either the first or second antibody phase, thereby quantitating the level of CGRP in the biological sample.

5. A method of claim 4, wherein in step a) the first and second antibodies are contacted simultaneously or sequentially with the biological sample.

6. A method of diagnosing an individual's osteoporosis risk or assessing the risk of osteoporotic fracture comprising the steps of examining the concentration profile in a biological sample of the individual of at least one CGRP and correlating the profile with osteoporosis diagnosis.

7. A method of claim 6, wherein the examining step includes exposing the sample to an antibody of each of the at least one CGRP to determine the level of the CGRP in the sample and the correlating step includes comparing the level of the CGRP in the sample with the level of the CGRP in a control population.

8. A method of claim 7 wherein the control population is comprised of individuals each diagnosed as not having osteoporosis, and optionally, wherein the control population is matched to the age of the individual.

9. A method of claim 7 wherein the control population is comprised of individuals each diagnosed as having osteoporosis, and optionally, wherein the control population is matched to the age of the individual.

10. A method of diagnosing osteoporosis, or assessing the risk of osteoporotic fracture in a postmenopausal female individual comprising the steps of examining the concentration profile in a biological sample of the patient of a CGRP and correlating the profile with osteoporosis diagnosis.

11. A method of claim 10, wherein the correlating step includes comparing the level of the CGRP in the sample with the level of CGRP in a control population of non-osteoporotic postmenopausal females in which a positive diagnosis is indicated by a non-elevated level of CGRP in the individual relative to the control population.

12. A method of claim 10 or 11, wherein the correlating step includes comparing the level of the CGRP in the sample with the level of CGRP in a positive control population of osteoporotic postmenopausal females in which a negative diagnosis is indicated by an elevated level of CGRP in the patient relative to the positive control population.

13. A method as claimed in any one of the preceding claims in which the biological sample comprises a whole blood sample, a plasma sample, or a serum sample.

14. A method according to any preceding claim wherein the CGRP is CGRP/3.

15. A method according to any one of the preceding claims which further comprises measuring the level of at least one chemokine.

16. A method of claim 15, wherein the chemokine is selected from the group consisting of CXCLl, CXCL4, CXCL6 and CXCL8.

17. A method of any one of the preceding claims, further comprising correlating the level of a bone turnover marker(s) selected from the group of osteocalcin, alkaline phosphatase, bone sialoprotein, pro-collagen I N-telopeptide, pro-collagen I C-telopeptide, tartrate resistant acid phosphatase (TRAP), TRAP 5b, deoxypryidiniline, pyridinoline, Cathepsin K, and combinations thereof in said sample with the level of the marker(s) in a control population.

18. A method according to any preceding claim, wherein the individual is female, optionally a postmenopausal female.

19. A kit for differentiating between an osteoporotic and a non-osteoporotic condition in an individual comprising: a) a device for obtaining a sample of a biological fluid from the individual; and b) a test for determining the level of a chemokine in said sample relative to the level in a control population.

20. A device for differentiating between an osteoporotic and a non-osteoporotic condition in an individual comprising: a) a capture antibody bound to a support, said capture antibody being specific for a CGRP; and b) a reporter antibody, said reporter antibody being specific for said CGRP and having an indicator attached thereto such that the level of the chemokine in said sample relative to the level in a control population can be determined.

21. A method of claim 19 or 20 wherein the fluid comprises a blood sample of the individual, or comprises a whole blood sample, a plasma sample, or a serum sample.