WO2006119545A1

WO2006119545A1 - Bone fracture risk assay based on c-reactive protein levels

Info

Publication number: WO2006119545A1
Application number: PCT/AU2006/000592
Authority: WO
Inventors: Hans Schneider; Geoff Nicholson
Original assignee: Bayside Health; The University Of Melbourne
Priority date: 2005-05-06
Filing date: 2006-05-08
Publication date: 2006-11-16

Abstract

The present invention provides a sensitive assay for measuring the risk of a bone fracture in an individual based on the level of CRP in the individual. The invention also provides kits and methods for diagnosing the risk of future bone fracture and conditions that predispose to fracture including osteoporosis and osteopenia.

Description

BONE FRACTURE RISK ASSAY BASED ON C-REACTIVE PROTEIN LEVELS

1 BONE FRACTURE ASSAY

The present invention relates to a method of analysing a risk of bone fracture. The invention also provides kits and methods for diagnosing the risk of future bone fracture and conditions that predispose to fracture including osteoporosis and osteopenia.

BACKGROUND

Osteoporosis and associated bone disorders and consequences such as bone fractures are a major health care problem mostly in western societies particularly among the aged. Fractures due to poor bone strength can lead to much pain and suffering as well as consequent morbidity and mortality.

Medications are available to patients to reduce the incidence of fractures. However, these medications are generally given at a stage where a patient already shows signs of a high risk to bone fracture or they have had previous fractures. The medication then becomes a preventative to further fractures.

Tests that are currently used to diagnose bone conditions involve the measurement of bone mineral density (BMD) by radiological means and the measurement of bone resorption and bone formation markers in blood or urine. While these tests are helpful, they are not clearly identifying a population at highest risk who should be treated preventively. They are also not accurate enough to identify those at risk at an early stage.

Accordingly, the present invention provides an assay to predict the risk of bone fracture. The assay may provide an insight into the condition of the bone and of bone disorders such as osteoporosis or osteopenia which predispose the bone to fractures.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a method of diagnosing a risk of bone fracture in an individual, said method including determining a level of C-reactive protein (CRP) or an associated compound in a biological sample from the individual; wherein the level of the CRP or associated compound is indicative of the risk of bone fracture and analysing the risk based on the level of the CRP or associated compound relative to a control sample.

The present invention provides a sensitive assay for measuring the risk of a bone fracture in an individual based on the level of CRP in the individual. As an indicator of a risk of bone fracture, this may also be indicative of a bone disorder. The risk of the bone fracturing provides an insight into the condition of the bone and hence may signal a disorder associated with bone strength.

A high risk of bone fracture often indicates a bone disorder. It is preferred that the bone disorder is selected from the group including osteoporosis, osteopenia, abnormal bone formation, and abnormal bone resorption.

The level of CRP may be measured in the blood, serum or plasma. However, any measure of the CRP from an individual may be used as an indicator of a risk of bone fracture. This measure will be used to assess the likelihood of a fracture in the bones of the individual, generally based on normalised values of CRP in patients of various degrees of risk of bone fracture.

The invention also provides kits that may be used to diagnose the risk of bone fracture by measuring the level of CRP in an individual from which an assessment can be made of the risk of bone fracture.

FIGURES

Figure 1 shows a survival plot (Kaplan Meier) showing the probability of remaining fracture-free for the highest CRP quartile (quartile 4) compared with quartiles 1 -3.

Figure 2 shows a relative risk of fracture in postmenopausal women in the highest quartile compared with women in the other 3 quartiles, adjusted for BMD at the spine (a) and hip (b). DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a sensitive assay for measuring the risk of a bone fracture in an individual based on the level of CRP in the individual. CRP has previously been used as a predicator of future risk of heart attacks and sudden death and ischemic heart disease. The assays are standardized to an international standard. However, the compound has not been applied to act as an indicator of a risk of bone fracture that may be indicative of a bone disorder. This measurement provides an insight into the condition of the bone and hence may signal a disorder associated with bone strength.

Measurement of CRP as an indicator of bone condition and risk of bone fracture is provided in the present invention. However, CRP may also be measured indirectly via an associated compound which can also act as an indicator of CRP. Accordingly it is within the scope of the present invention to use compounds that are indicative of CRP to measure CRP indirectly which provides a prediction of the risk of bone fracture.

A high risk of bone fracture often indicates a bone disorder. It is preferred that the bone disorder is selected from the group including osteoporosis, osteopenia, abnormal bone formation, and abnormal bone resorption. Preferably, the bone disorder is osteoporosis or osteopenia. However, instances of increased risk of bone fracture is generally seen in populations with an increased risk of osteoporosis and/or osteopenia. Furthermore, the present assay is sufficiently sensitive to determine a risk of bone fracture independent of Bone Mineral Density (BMD) determinations.

The determination of CRP may be conducted by any method available to the skilled addressee. Standard tests are presently available. The use of quantitative immunoassays may be employed. However, the present invention preferably utilises a highly sensitive CRP assay. Highly sensitive CRP assays utilised in routine laboratories are capable of measuring CRP concentrations to below 0.1 -0.2 mg/L. The techniques utilised are mainly nephelometry and immunoturbidimetry. In these assays antibodies to CRP are bound to small latex particles. These form complexes with CRP present in the analysed sample. The number and size of the particles will cause increased light scattering (nephelometry). The light adsorption (light loss by scattering on the surface of a particle) is proportional to the diameter of the particle and also depends on the wavelength. Improvements in the production of the latex particles used, optimisation of the software integrating the data and optimal choice of antibodies allow higher sensitivity than the standard CRP assays. The Roche CRP assay is particularly useful for determining CRP in serum and plasma and for use in the present invention to determine CRP.

It is required that a level of CRP is determined and this may equate as an absolute determination of the concentration of CRP or relative levels of CRP may be determined and compared against other samples determined in a similar manner. A change in the CRP relative levels will allow for an assessment of the risk of bone fracture. Changes in the levels can be used to assess and monitor the progression of the risk of bone fracture and hence the progression of a bone disorder such as osteoporosis or osteopenia.

A standard may be developed which provides standard levels of CRP in patients who are not at risk of the bone fracture to which an unknown sample can be compared. Preferably the standard is collated using a patient having little or no risk of bone fracture. In the present invention a high level of CRP is generally indicative of an increased risk of bone fracture. Applicants have conducted studies to determine levels of CRP in patients with osteoporosis (Example 1 ). Over the population of patients sampled, the CRP levels varied over a range in which the Applicants divided into four quarters. The top quarter represented those at highest risk of bone fracture. In that Example levels greater than 4.9mg/ml for instance may be indicative of a high risk of bone fracture. This level is merely relative to the sample population collected. However, the example merely provides an illustration that high CRP levels can correlate to high risk of bone fracture.

Patients with lower CRP concentrations (< 1 mg/L) are less at risk of future fracture compared with patients with higher CRP concentrations. However, as herein described, relative measurements of CRP taken from one time period to another may also provide an indication of an increased or stabilised risk of bone fracture in a patient.

Consideration of bone deficits such as bone mineral density (BMD) may also contribute to diagnosis of a risk of bone fracture. Increased bone deficits such as BMD along with increased CRP levels contribute to increased fracture risk and may indicate an increased risk of bone disorders or the need to treat.

CRP may be determined from any biological sample from which CRP can be analysed and can be compared against a control or standardized level. Preferably, the CRP is determined in blood, serum, or plasma.

The assay is useful to predict a risk of bone fracture in any individual. Generally, those at high risk or who have shown previous fracture or are prone to osteoporosis would find this assay useful. In particular, the test is relevant to the aged and post menopausal women.

The method of analysis of the risk of bone fracture of the present invention may encompass the diagnosis of the bone disorder or bone condition or the monitoring of the progress of the bone disorder or condition. CRP may be used as an indicator of the progression of the disorder or condition. The method may be used to monitor an increasing risk of bone fracture when the level of CRP present in the individual increases over time to reflect an increased risk of bone fracture. This may also indicate an increased risk of bone disorders such as osteoporosis or osteopenia.

The present invention also provides a kit for diagnosing a risk of bone fracture said kit including a reagent for determining CRP in a sample. Such reagents may include antibodies to CRP, and other reagents used to detect binding of the CRP present in the sample to the antibody latex particles coated with CRP antibody and other detection means to determine the presence of a complex comprising the CRP and the antibody to CRP. Methods are available to the skilled addressee to determine the presence of a complex.

The kit will also include solutions such as buffers to perform the analysis and assist in the reaction that detects the presence of CRP. Other components present may include control samples to determine the relative risk of bone fracture. For instance, the kit may include samples of CRP that are indicative of little or no risk of bone fracture. Similarly, the kit may include samples of CRP that are indicative of varying degrees of risk of bone fracture.

Preferably, the kit also includes instructions as are necessary for carrying out the test in the kit and also may include reference material to determine and compare the CRP level to make an assessment of the risk of bone fracture.

Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

Examples of the procedures used in the present will be now more fully described. This should be understood however that the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES Example 1 : Correlation of serum - CRP with increased risk of bone fracture.

Women from an osteoporosis study were analysed. 744 post menopausal women from the study were randomly selected and their CRP levels were determined. Fractures were identified amongst the women and correlated with their CRP levels.

CRP was measured using a CRP assay distributed by Roche Diagnostics. Serum samples taken from the women were analysed. Multivariate cox proportioned hazards regression was used to determine the association between CRP and fracture.

135 fractures were sustained during 4008 person-years of follow-up. After adjusting for potential confounders, women with CRP in the highest quartile (>4.9 mg/L) had a 1 .6-fold (95%CI 1 .1 -2.4) greater risk of fracture than the lower pooled quartiles, independent of Bone Mineral Density (BMD). Table 1 provides the results of the study.

Table 1 : Distribution of characteristics for the whole group (n=744) and for quartiles 1 -3 (combined, n=558) compared with quartile 4 (n=186). Data are shown as number (%) unless otherwise indicated.

Both increased CRP and increased bone deficits such as osteoporosis or osteopenia contributed to increased fracture risk. The probability of remaining fracture free in the highest quartile is less over time (Figure 1 ). Using CRP quartiles 1 -3 and normal hip BMD (T-score>-1 .0) as the referent group, women in the highest CRP quartile and with low BMD (T-score<-2.5) had the highest adjusted fracture risk (RR=9.1 ; 95%CI 3.6-23). (See Table 2 below).

Table 2: RR (95%CI) for fracture associated with serum CRP. The RRs are determined for the comparison between the highest quartile with the lowest 3 combined as the referent group.

Abbreviations: RR relative risk, Cl confidence interval, CRP C-reactive protein, BMD bone mineral density.

^*Adjusted for age, weight, smoking (ever), glucocorticoid use, site-specific BMD (as shown in table).

Elevating CRP from quartiles 1 -3 into the highest quartile was equivalent to increasing the fracture risk normally associated with osteopenia to that of osteoporosis, and for normal BMD to that of osteopenia (see Figure 2). A similar pattern was observed for BMD measured at the spine. Therefore circulating CRP is an independent predictor of fracture risk in postmenopausal women. These results may implicate systemic inflammation as a factor in the pathophysiology of osteoporosis. Furthermore they establish high sensitivity CRP (hsCRP) measurement as a potential new marker of the future risk of fracture.

Example 2: High sensitivity C-reactive protein and fracture risk in elderly women. (a) Method

An age-stratified sample of women (99% Caucasian, 77.1% participation) was randomly recruited from electoral rolls for the Geelong Osteoporosis Study. The study region is characteristic of Australia in age distribution and socioeconomic indicators. This analysis includes 444 of the potential 522 eligible women aged >65 years (exclusions: hormone therapy, glucocorticoid use, no serum). Except for a smaller proportion using calcium and vitamin D supplements (P=0.01 ), participants were comparable to those excluded for characteristics in Table 3 (P>0.05, data not shown). Table 3: Subject characteristics at baseline (n = 444). Data are shown as number (%) unless otherwise indicated.

BMI body mass index CTx C-telopeptide

BSAP bone-specific alkaline phosphate SD standard deviation IQR interquartile range

Baseline hsCRP levels were measured by the Roche/Hitachi Tina-quant (Latex) assay (Roche Diagnostics, Australia) and imprecision is < 10% at 0.15 mg/L. Serum C-telopeptide (CTx) and bone-specific alkaline phosphatase (BSAP) were measured as markers of bone resorption and formation, respectively (β- Crosslaps and Bone-ALP, Roche). Bone mineral density (BMD) was measured by LUNAR DPX-L and analyzed using Lunar DPX-L software version 1.31 (LUNAR Corporation, Madison, Wis, USA). Incident fractures were identified prospectively using a computerized keyword search of all radiological reports from the three medical imaging centers serving the region. This comprehensive method of ascertaining fractures from all causes was validated prior to the commencement of the study. Baseline assessments were performed 1994-7 and subjects were followed prospectively until sustaining a fracture, death or migration from the study region, or until the end of 2002. Relative risk (RR) for fracture was determined from Cox proportional hazards models, predicting 'time to fracture' and controlling for BMD, prevalent fracture (at least one self- reported low trauma fracture since age 50 years), age, anthropometry, lifestyle , medication use and co-morbidity. Values for hsCRP (log transformed, In- hsCRP) and BMD were expressed as standard deviation (SD) units. Linear regression techniques were used to determine associations between In-hsCRP and In-CTx, In-BSAP and BMD. The study was approved by the ethics committee and written consent was obtained.

(b) Results

The hsCRP distribution was positively skewed, median 2.44 mg/mL (Table 3).

Few women (26/444) had hsCRP values >10 mg/L. During the study period 56 women died, 13 left the region and 102 sustained a fracture. Ninety-six incident fracture cases were included in the analysis: 32 spine, 21 hip, 9 humerus, 8 ribs, 7 wrist, 7 radius/ulna, 3 pelvis and 1 1 other. One pathological and 5 minor (finger, face, patella) fractures were excluded. The median follow-up period was 5.5 years per subject (interquartile range, IQR, 3.8-6.2), giving 2,208 person-years of follow-up.

Unadjusted RR for fracture increased by 23% for each SD increase in In-hsCRP (RR = 1 .23; 95%CI 1 .01 -1 .51 ). The age-standardized absolute risk for fracture during the study period increased from 16.3% (95%CI 6.8-25.8) for In-hsCRP <- 1 SD (ie < 0.96 mg/L) to 28.9% (95%CI 17.7-40.1 ) for In-hsCRP >+1 SD (ie > 6.35 mg/L). Models developed sequentially by adjusting for potential confounders consistently included significant contributions from In-hsCRP, prevalent fracture and BMD, irrespective of site for BMD measurement (Table 4). Table 4: Relative risk for fracture (95% confidence interval) associated with increases in standard deviation units of In-hsCRP, decreases in standard deviation units of BMD, and prevalent fracture. Separate models were developed for BMD measured at the spine, proximal femur sub- regions, whole body and distal forearm sub-regions.

Thus, for each SD increase in In-hsCRP there was an independent 24-30% increase in fracture risk, depending on site-specific BMD used in the model. Fracture risk was independently increased 52-79% for each SD decrease in BMD, and 52-73% by previous fracture. Further adjustment for bone turnover markers, lifestyle factors, diet, medication use and co-morbidity had only modest effects on the point estimate and failed to explain the observed increased risk for fracture.

There were no significant correlations between In-hsCRP and In-CTx or In- BSAP. Trends for age- and weight-adjusted In-hsCRP to be positively associated with In-hsCTx and negatively with In-BSAP (regression coefficient ± SEM, 0.038 ± 0.032, P = 0.2, and -0.049 ± 0.032, P = 0.1 , respectively) were not significant. No association was detected between In-hsCRP and BMD.

This report shows that increased serum hsCRP concentration is associated with increased fracture risk. The 23% increase in unadjusted fracture risk associated with each SD increase in In-hsCRP was not explained by differences in BMD, prevalent fracture, markers of bone turnover, diet, lifestyle, medications or co-morbidity.

Finally, it is understood that various other modifications and/or alterations may be made without departing from the spirit of the present as outlined herein.

Claims

CLAIMS:

1 . A method of diagnosing a risk of bone fracture in an individual, said method including determining a level of C-reactive protein (CRP) or associated compound in a biological sample from an individual wherein the level of CRP or associated compound is an indication of the risk of bone fracture and analysing the risk based on the level of the CRP or associated compound relative to a control sample.

2. A method according to claim 1 wherein the level of the CRP is an indication of a risk of bone fracture predisposed by a bone disorder or condition.

3. A method according to claim 1 or 2 wherein a high level of CRP compared to a normal CRP level is an indication of a high risk of bone fracture predisposed by a bone disorder or condition.

4. A method according to any one of claims 1 to 3 wherein an increased CRP level over time is an indication of an increased risk of bone fracture predisposed by a bone disorder or condition.

5. A method according to any one of claims 1 to 3 wherein a decreased CRP level over time is an indication of a decreased risk of bone fracture predisposed by a bone disorder or condition.

6. A method according to any one of claims 1 to 5 further including determining bone mineral density (BMD).

7. A method according to claim 5 wherein decreased BMD compared to a normal BMD and an increased level of CRP is an indication of an increased risk of bone fracture.

8. A method according to any one of claims 1 to 7 wherein the CRP is the determined in a biological sample selected from the group including blood, serum, and plasma.

9. A method according to any one of claims 1 to 8 wherein the CRP is determined in the serum.

10. A method according to any one of claims 2 to 9 wherein the bone disorder and/or bone condition is selected from the group including osteoporosis, osteopenia, abnormal bone resorption, or abnormal bone formation.

1 1 . A method according to any one of claims 2 to 10 wherein the bone disorder or condition is osteoporosis.

12. A method according to any one of claims 2 to 10 wherein the bone disorder or condition is osteopenia.

13. A method according to any one of claims 1 to 12 wherein the CRP is measured using nephelometry or immunoturbidimetry.

14. Use of CRP or an associated compound for the diagnosis of a risk of bone fracture.

15. Use of CRP or an associated compound for the diagnosis of a risk of bone fracture for the prevention or treatment of a bone disorder or condition.

16. Use according to claim 14 or 15 wherein the bone disorder or bone condition is selected from the group including osteoporosis, osteopenia, abnormal bone resorption, or abnormal bone formation.

17. A method according to any one of claims 14 to 16 wherein the bone disorder or condition is osteoporosis.

18. A method according to any one of claims 14 to 16 wherein the bone disorder or condition is osteopenia.

19. A kit when used in a method of diagnosing a risk of bone fracture in an individual according to any one of claims 1 to 13, said kit comprising a means to determine CRP in a biological sample and a reference to analyse the risk of bone fracture.

20. A method of measuring progression of a bone disorder or condition, said method comprising determining a risk of bone fracture in an individual by determining a level of C-reactive protein (CRP) or associated compound in a biological sample from an individual wherein the level of CRP or associated compound is an indication of the risk of bone fracture and analysing the risk based on the level of the CRP or associated compound relative to a control sample.

21 . A method according to claim 20 wherein the bone disorder or bone condition is selected from the group including osteoporosis, osteopenia, abnormal bone resorption, or abnormal bone formation.

22. A method according to claim 1 substantially as hereinbefore described.

23. A use according to claim 14 substantially as hereinbefore described.