European Journal of Clinical Pharmacology

, Volume 62, Issue 10, pp 781–792

An update on biomarkers of bone turnover and their utility in biomedical research and clinical practice

  • D. J. Leeming
  • P. Alexandersen
  • M. A. Karsdal
  • P. Qvist
  • S. Schaller
  • L. B. Tankó
Review Article

DOI: 10.1007/s00228-006-0174-3

Cite this article as:
Leeming, D.J., Alexandersen, P., Karsdal, M.A. et al. Eur J Clin Pharmacol (2006) 62: 781. doi:10.1007/s00228-006-0174-3

Abstract:

Background:

Maintenance of the structural and functional integrity of the skeleton is a critical function of a continuous remodeling driven by highly associated processes of bone resorption and synthetic activities driven by osteoclasts and osteoblasts, respectively. Acceleration of bone turnover, accompanied with a disruption of the coupling between these cellular activities, plays an established role in the pathogenesis of metabolic bone diseases, such as osteoporosis. During the past decades, major efforts have been dedicated to the development and clinical assessment of biochemical markers that can reflect the rate of bone turnover. Numerous studies have provided evidence that serum levels or urinary excretion of these biomarkers correlate with the rate of bone loss and fracture risk, proving them as useful tools for improving identification of high-risk patients.

Objective:

The aim of the present review is to give an update on biomarkers of bone turnover and give an overview of their applications in epidemiological and clinical research.

Discussion:

Special attention is given to their utility in clinical trials testing the efficacy of drugs for the treatment of osteoporosis and how they supplement bone mass measurements. Recent evidence suggests that biochemical markers may provide information on bone age that may have indirectly relates to bone quality; the latter is receiving increasing attention. A more targeted use of biomarkers could further optimize identification of high-risk patients, the process of drug discovery, and monitoring of the efficacy of osteoporosis treatment in clinical settings.

Keywords

Bone resorption markers Bone formation markers Bone regulatory proteins Bone mineral density Fracture risk Treatment Monitoring Postmenopausal women 

Introduction

Osteoporosis and the related fragility fractures remain major epidemiological burdens of postmenopausal women. Currently, the prevalence of osteoporosis in industrialized countries is estimated to be 40% in women in their sixties, and 70% in women in their eighties [25]. Elderly men also lose bone with aging and, due to increasing longevity of the elderly, more and more men reach the state of osteoporosis and have increased risk of fragility fractures. The estimated lifetime risk of fractures in men over 50 years of age is estimated to be 17% [78]. The impact of osteoporotic fractures on morbidity and mortality is greater in elderly men than in women [3], emphasizing the need to give a bi-gender focused consideration to the clinical management of osteoporosis.

A central component of the pathogenesis of osteoporosis is an imbalance between the function of two key players of bone turnover, namely osteoclasts and osteoblasts. Estrogen deficiency arising after the menopause leads to acceleration of bone turnover, the rate of bone resorption exceeding the rate of bone formation. This leads to a net negative calcium balance and consequent demineralization of bone [54].

The rate of bone resorption and formation can be estimated by immunoassays measuring the serum concentration or urinary excretion of different target molecules specific to these cellular processes [27]. Over the past decade, a wide array of such immunoassays has been launched for in vitro, ex vivo, and in vivo investigations. Their systematic validation led to the recognition of their utility for assisting biomedical research, targeting the better understanding of the pathogenesis of osteoporosis, the improvement of clinical diagnostic, and the evaluation of novel treatment modalities.

The aim of this review is to provide an up-to-date summary of recent developments in the field of bone markers and to discuss their utility in assisting in prediction of fracture, drug development, and monitoring of treatment efficacy.

Cellular and structural elements of bone

Bone is composed of type I collagen fibres, crystals of hydroxyapatite [3Ca3(PO4)2]·(OH)2], and ground substance. Based on structural differences, bone can be subdivided into cortical and trabecular compartments. Cortical bone (the outer layer) is composed of a thick and dense layer of calcified tissue, whereas trabecular bone (the central part) is composed of thin trabeculae forming a robust, though slightly flexible, framework [5]. Bone homeostasis is critical for maintenance of bone strength and endurance. During the remodeling process of bone, osteoclasts and osteoblasts interconnect in the Basic Multicellular Units (BMU), to degrade old bone and replace it with the exact same amount of new bone. In premenopausal women, the activity of osteoblasts and osteoclasts are balanced so that the net result parallel resorption and formation is zero [63]. However, after the menopause, coupling between bone resorption and formation becomes partially disrupted, i.e. the rate of resorption increases more pronouncedly than the rate of bone formation, which in turn leads to a negative calcium balance and consequent bone loss. Continuous bone loss leads to low bone mass and micro-architectural deterioration of bone tissue that enhances bone fragility and leads to an increased risk of fractures [24].

Biochemical markers of bone turnover

Markers of bone turnover can be stratified regarding their origination from the BMU Fig. 2:
  1. a.

    Collagenous bone resorption markers: Measures of collagen type I degradation products released during osteoclastic resorption of bone.

     
  2. b.

    Osteoclast regulatory proteins markers: Proteins that either regulates the differentiation and proliferation of osteoclast precursors into mature osteoclasts or are involved in the coupling between osteoblasts and osteoclasts.

     
  3. c.

    Bone formation markers: Measures of enzymatic activity of osteoblasts, bone proteins and fragments of pro-collagens released during bone formation.

     

Collagenous bone resorption markers

The most common markers of bone resorption measure peptide fragments deriving from collagen type I, such as CTX-I, NTx, ICTP, and pyridinolines. Collagenous markers reflect the rate of bone resorption, but may also provide information on the composition and thereby quality of bone [12]. The location of the collagen derived resorption fragments are denoted in Fig. 1.
Fig. 1

Structure of type I pro-collagen and mature type I collagen. Location of the collagen epitopes are indicated by arrows

CTX is an 8-amino acid fragment from the C-telopeptide of type I collagen (Fig. 1). CTX is generated by cathepsin K activity and the rate of its release from bone is a useful reflection of the resorbing activity of osteoclasts [9]. The CTX epitope contains an aspartyl-glycine motif (DG) that is prone to spontaneous isomerization. In other words, EKAHD(α)GGR epitopes are released during degradation of newly synthesized type I collagen, whereas EKAHD(β)GGR epitopes are released from matured collagen type I. It has been established that the α/β ratio is a useful measure of the age of bone tissue; the lower the ratio, the older the bone tissue [36]. Resorption rate of newly synthesized collagen type I can be assessed by specific immunoassays targeting the detection of αCTX in urine samples [23]. Degradation rate of matured, isomerized, collagen can be estimated by another specific assay targeting βCTX in both urine and serum samples. The intra- and inter-assay variations (CV%) for these assays are <9% [23, 88].

NTX is an 8 amino acid epitope (JYDGKGVG; Fig. 1) derived from the N-telopeptide of type I collagen [48]. This fragment is cleaved by cathepsin K and the rate of its release is also a useful measure of bone resorption. NTX can be measured in serum and urine by a specific immunoassay. The intra- and inter-assay variations are 6.1% and 4%, respectively [48].

ICTP measures a relatively large hydrophobic phenylalanine-rich pyridinolines cross-link of the two α-1 chain in the C-terminal telopeptides of matured collagen type I [86]. The ICTP epitope neighbours CTX (Fig. 1) but it is released as a result of MMP activity [42]. Cathepsin K activity eradicates the ICTP epitope. ICTP can be measured in serum and plasma. The inter- and intra-assay variations of the assay were reported to be between 3 and 8% [86].

PYR (deoxypyridinoline and pyridinolines) are found in mature type I collagen and involved in the formation of cross-links between adjacent collagen polypeptides. Pyridinium cross-links do not appear to be metabolised. Because these cross-links are formed during the late stage of fibril formation, measurements of these are considered as an index for degradation of mature collagen [38]. Pyridinolines may be detected in both serum and urine by specific immunoassays. The intra- and inter-assay variation for one DPD assay has been reported to be 4–8% and 3–5%, respectively [69].

Osteoclast regulatory proteins

The transition of osteoclast precursors to mature osteoclasts that are capable of resorbing bone is tightly regulated by osteoclast regulatory proteins Fig. 2. The OPG/RANKL/RANK cytokine system is essential for osteoclast biology. A number of studies point out that alteration of this system is involved in the pathogenesis of metabolic bone diseases. Estrogen deficiency is accompanied by increases in RANKL and decreases in OPG levels leading to increased number and lifespan of mature osteoclasts [52]. Accordingly, resorption will increase and bone loss becomes evident with time. Consequently, the OPG to RANKL ratio is of great interest and may be a more powerful indicator of osteoclast activity than separate evaluation of the two proteins.
Fig. 2

Schematic presentation of type I collagenous, pro-collagenous non-collagenous and osteoclast regulatory proteins released during the activity of a BMU. Markers released during osteoclastogenesis (red), bone resorption (blue) and bone formation (black) are indicated by arrows

Markers of osteoclastogenesis are relatively new players that are not fully characterized regarding their potentials in biomedical research and clinical trials of osteoporosis treatment. However, preliminary observations suggest that they can be helpful in the elucidation of the mechanism of action and efficacy of novel drugs acting on osteoclast function. Markers of osteoclastogenesis include RANKL and OPG, whereas markers of osteoclast number include TRAcP and Cat K.

Markers of osteoclastogenesis

RANKL (receptor activator of nuclear factor NF-κB ligand) is a member of the tumor necrosis factor (TNF) family and is produced by bone-forming osteoblasts and activated T lymphocytes. It activates its specific receptor RANK on osteoclasts precursors, thereby promoting cellular maturation in the presence of macrophage colony stimulation factor (MCS-F). It is the main mediator of osteoclast activation, differentiation, and survival [59, 62, 100]. RANKL expression is inversely correlated with serum levels of 17β-estradiol and positively correlated with bone resorption markers [52] and may therefore be considered as a measure of osteoclast activity. As an example the intra-and inter-assay variation for one total RANKL kit (Immundiagnostik, Bensheim, DE) is 2.2% and 8.2%, respectively.

Osteoprotegerin (OPG) is a soluble decoy-receptor, which is produced in different tissues, e.g., bone, liver, stomach, intestine, and lung. Osteoblasts secrete OPG that binds to RANKL and thereby inhibit the regulatory effect of RANKL on osteoclast activation and proliferation [51, 99]. OPG production is positively correlated with estrogen levels [60]. Intra- and inter-assay variation for an available OPG assay (Immundiagnostik, Bensheim, Germany) is <10%.

Markers of osteoclast number

TRAcP (tartrate-resistant acid phosphatase) is a glycoprotein produced in mature osteoclasts, activated macrophages and dendritic cells. It is active as a phosphatase and as a generator of reactive oxygen species [76]. The polypeptide chain of TRAcP is cleaved by proteases into two isoforms 5a and 5b, which activate phosphatase activity [76]. The isoform TRAcP 5b is derived from osteoclasts and has been proposed to reflect osteoclast number rather than bone resorption [2, 17]. It is known that TRAcP 5b increases with age in healthy women and after the menopause [15], hence TRAcP 5b may be useful in clinical trials evaluating novel treatments of osteoporosis. TRAcP 5b can be measured in serum samples. The intra- and inter-assay variation of two selected TRAcP 5b assays were 2.1–7.9% and 4.9–13%, respectively [74].

Cathepsin K is a member of the cysteine protease family. This enzyme plays a critical role in osteoclastic degradation of collagen type I. Dissolution of the inorganic phase of bone in the resorption lacunae at low pH is a prerequisite for the degradation of the organic phase, which is mainly mediated by cathepsin K [57]. The enzyme is secreted into the lacunae, where it cleaves both helicoidal and telopeptide regions of the collagen molecules. Cathepsin K is an abundantly synthesized by mature resorbing osteoclasts [53]. This marker can be measured in serum samples. The intra-and inter-assay variations range from 4% to 8% [53].

Bone formation markers

Formation of bone is most often evaluated using the following biomarkers: bone specific alkaline phosphatase, osteocalcin, or PICP/PINP.

Enzyme activity markers

Bone specific alkaline phosphatase (BSAP)

Approximately half of serum alkaline phosphatase activity comes from the bone-specific isoenzyme [89]. BSAP concentrations demonstrate a linear relationship with osteoblast and osteoblastic precursor activity. During the immediate post-proliferative period (12–18 days), the bone extracellular matrix endures a succession of modifications rendering competence for mineralization. In cultures that progress into the mineralization stage, all cells become alkaline phosphatase (AP) positive immunohistochemically, indicating that AP is involved in the mineralization of bone. Serum concentration of BSAP may be assessed indirectly after precipitation with lectin. The intra-assay and inter-assay CVs are <4% and <10%, respectively [89].

Bone protein markers

Osteocalcin (OC, former Gla-protein) is synthesized and secreted by osteoblasts and constitutes the major non-collagenous protein of bone matrix [102]. The physiological role of osteocalcin is related to its high affinity to calcium, thereby changing the osteocalcin polypeptide (a 49 amino-acid residues) into a compact α-helical confirmation in which the glutamic acid residues stimulate the absorption to hydroxyapatite within bone matrix [102]. In both preclinical and clinical studies, circulating OC has been shown to correlate with histomorphometric measurements [33, 55, 91]. Using specific immunoassays, osteocalcin can be quantified in plasma or serum samples. Intra- and inter assay for osteocalcin assays have been reported to be 5.7–6.4% and 5.9–6.1% [88], <8% and <15% [72], <2.3% and 2.5% [79], respectively.

Pro-collagen markers

PICP/PINP represent the C- and N-terminal pro-peptides of type I collagen, respectively. These pro-peptides are trimeric, globular, peptides that are enzymatically released from newly synthesized pro-collagen prior to its incorporation into the extracellular matrix. Circulating levels of PINP have also been demonstrated to correlate directly with histomorphometric indices of bone formation [35]. PICP and PINP can be detected in serum/plasma by specific assays. The intra- and inter-assay variation for a PINP ELISA was 4.6–5.3% and 2.9–4.9% [77], respectively. For a PICP ELISA, these parameters were 5–7% and 5–7% [69].

Variation of biochemical markers

Collagenous markers of bone resorption have been reported to exhibit marked biological circadian variation with a nadir during the day and in the afternoon with a peak during the night and early morning hours [80, 94]. Thus, the increased bone resorption occurring at night is counterbalanced by an equally large inhibition of bone resorption during the day (the ‘area-under-the-curve’ being zero over a 24-h period in premenopausal women). Circadian variation is independent of gender, age, menopausal status (although the baseline level of bone degradation in postmenopausal women is higher than before the menopause), mobility, vision, and pituitary hormone secretion [95]. Recently, the major component of the circadian variation in resorption markers has been attributed to the effects of food intake, probably involving endogenous secretion of glucagons-like peptide 2 (GLP-2) [50]. The cause of circadian variation remains unknown, but it is speculated that serum calcium homeostasis is crucial for the nature of this phenomenon [50]. Consequently, in order to obtain valid estimates of bone resorption markers, blood and urine sampling must be performed in the fasting state of individuals and within relatively narrow time frames (0800–1000 hours). Circadian variability of approximately 10–20% has been found for serum OC and serum BSAP, and approximately 40% for the urinary resorption marker NTx and 60–66% for serum CTX [21].

Another source of variation for biochemical markers is the long-term variability found in an individual over days and months. Intra-individual short-term (3 days) and long-term (2 months) variation for urinary NTx has recently been found to be 13.1% and 15.6%, respectively. The corresponding numbers for serum NTx were 6.3% and 7.5%, respectively [21].

Factors that might affect these variations include dietary habits, smoking, exercise, and medication. Bisphosphonate treatment such as alendronate reduces the circadian variability by about 50% in magnitude [21]. Compared with this, the impact of the aforementioned lifestyle factors is modest, yet their contribution should still be taken into consideration when applying biochemical markers for estimating efficacy. It is important to emphasize that there is still no accepted WHO criterion of “high” bone turnover.

The use of biochemical markers for risk prediction

In this section we will discuss biochemical markers in relation to:
  1. a.

    BMD and bone loss

     
  2. b.

    Fracture risk

     

BMD and bone loss

With the exception of a few [6, 58], most clinical investigations support the existence of an inverse correlation between bone turnover markers and BMD. Several prospective studies have shown significant correlation between levels of bone turnover markers and rates of bone loss assessed by serial BMD or BMC measurements at different skeletal sites over 1–13 years [98], indicating that bone turnover markers may provide additional information to BMD measurements. Whereas, in premenopausal women, bone turnover rates account only for 0–10% of the variation in bone mass, this percentage increases up to 52% in elderly women [38]. After the menopause, the inverse association of BMD with biochemical markers of bone turnover becomes stronger with advancing age [28], and stronger for resorption markers than for formation markers. In groups of untreated postmenopausal women, investigators found a significant correlation between baseline measures of bone turnover markers and the subsequent rate of bone loss at the hip or wrist [6, 68], but apparently not at the lumbar spine [98]. Explanation of this latter finding rests in methodological limitations of DEXA scanning in the elderly. After the age of 65 years, presence of progressive vascular calcification in the lumbar aorta and degenerative changes of the lumbar spine interfere with objective measurement of BMD. Monitoring over time reveals increases rather than decreases as is otherwise seen at peripheral skeletal sites [97]. A prospective study has shown that increased levels of biochemical markers could identify a subgroup of subjects who were ‘rapid bone losers’ (i.e. >3% loss in BMD per year) in the subsequent 2–12 years [4]. However, biochemical markers of bone turnover alone are not suitable for estimating BMD, bone loss, or fracture risk in an individual subject, although they might be useful as supplements to BMD measurements. The conflicting opinions regarding the ability of bone turnover markers to predict bone loss is mainly focused on the lumbar spine and to some extent prediction of hip BMD in individuals [6] and the predictive value of a single measurement of these markers [58]. We believe that bone turnover markers are not optimal in predicting bone loss in elderly patients, due to the above-mentioned reasons in the aorta, and that serial measurements of markers should be performed in order to predict bone loss.

In a 4-year prospective study of 305 women from the OFELY cohort (mean age 64 years), bone resorption (NTx, CTX) and formation markers (OC, PINP) were evaluated [40]. Baseline values of bone turnover markers were highly correlated with the rate of BMD loss in the forearm (r=−0.19 to −0.30, p<0.001), independent of age. In early postmenopausal women (years since menopause<5 years) with the highest rate of bone loss, the correlation coefficient increased to 0.53. Another prospective study examined the ability of formation markers (OC, BSAP) and resorption markers (NTx, PYR, D-PYR CTX) to predict hip bone loss in 295 elderly women (age >67 years) [6]. Increased levels of all four resorption markers were significantly associated with fast rates of bone loss at the total hip, although not at the femoral neck. Women with OC levels above the median value were also associated with increased rate of bone loss, whereas BSAP did not seem to provide information on the rate of hip bone loss. In a recent 5-year follow-up study, including 429 pre-and postmenopausal women by Lofman et al., it was found that formation markers (OC, ALP) and resorption markers (hydroxyproline, calcium) at baseline correlated significantly with BMD at 5 years at group level [65] indicating that biochemical markers of bone turnover provides information about future bone loss.

In a study of 105 male individuals, 65 osteoporotic men and 40 controls, levels of estradiol, the sex hormone-binding globulin (SHBG), bone formation (OC, BSAP) and bone resorption (ICTP, CTX) were determined [66]. There was no correlation between estradiol and spinal BMD, and only weak correlation to femoral neck BMD. However, SHBG was significantly increased in the osteoporotic individuals compared to controls (p<0.01) and negatively correlated to BMD at the femoral neck (r=−0.37, p<0.01). SHBG also correlated to sCTX (r=0.37, p<0.01), but none of the other bone markers. In another population of 283 healthy, ambulatory men <70 years of age, bone formation (OC, BSAP) and bone resorption (sCTX, uCTX, Dpd) were negatively associated to BMD, all significantly at the proximal femur and distal forearm [47]. Serum CTX was highly significant correlated at all sites measured by BMD (p<0.001). The same inversely relation between BMD and bone turnover markers (NTx, OC) has also been shown previously by Krall et al. in 1997 in 272 elderly healthy men aged 65–87 years [61]. Here, the men in the lowest quartile of NTx or OC were associated with 11% higher femoral neck BDM as compared to men in the highest quartile.

Fracture risk

The clinical complications of osteoporosis are fragility fractures. BMD is a widely used estimate of future fracture risk, but around 33–50% of patients with fragility fractures have BMD values above the diagnostics threshold of osteoporosis (T-score less than −2.5 SD) [71, 93]. Increasing number of studies [11, 38, 39, 44, 67] point out that fracture risk is also related to the level of bone turnover reflected by a single or a combination of biomarkers. Woo and colleagues were one of the first groups to investigate biochemical markers as predictors of osteoporotic fractures in elderly subjects [103]. They studied the ability of hydroxyproline to predict fractures in 283 elderly Chinese subjects aged ≥60 years and concluded that increased levels of this marker may be used as a predictive measure. A limitation in this study was a limited number of fractured subjects (n=7). Later, Garnero et al. (1996) [38] evaluated markers of bone resorption for prediction of risk for hip fracture in elderly women participating in the EPIDOS study. A total of 7,598 healthy women >75 years old participated, of which 126 sustained hip fractures during the 22-month follow-up period. Urinary NTx, CTX and free D-PYR levels at baseline were compared between subjects with or without fractures at follow-up. Increased levels of bone resorption markers predicted increased incidence of hip fracture, independently of initial bone mass. Women with high CTX or high free D-PYR levels had a 4.8 or 4.1-fold increased risk of a hip fracture, respectively. Another study by Garnero et al. in 1998 [39] pointed out that combining urinary CTX measurements with history of prevalent fractures performs as well as hip BMD measurements when estimating the risk of future hip fractures in elderly women. Another group investigated the utility of urinary CTX, serum OC, and BSAP for long-term prediction of vertebral fracture in 603 postmenopausal osteoporotic women. Baseline values of bone turnover markers correlated inversely and significantly with baseline and follow-up (i.e. 36 months later) measures of spine BMD [11]. It was observed that two sequential measurements of serum OC and urine CTX performed at 3-month intervals in combination with BMD measurements could help identify women with the highest risk to present new vertebral deformities. Women in the lowest quartile of a 3-month change in OC had a 69% decreased risk for a vertebral fracture in the subsequent 36-month period compared to those in the highest quartile.

In the OFELY study, BMD and biochemical markers of bone turnover (OC, BSAP and CTX) were assessed in 671 postmenopausal women. During the 9.1-year follow-up period, a total of 158 incident fractures were recorded in 116 women [96]; 48% of fractures were seen in osteopenic women, 44% in osteoporotic women and 8% in women with normal BMD. In the osteopenic women, low BMD was associated with increased fracture risk with an age-adjusted hazard ratio of 2.5. In addition to BMD, age, prior fractures, and bone turnover markers were also independently associated with an increased risk of fractures. In the whole group of osteopenic women, there was a 5.3-fold increased risk of sustaining a fracture if low BMD combined with a prior fracture or a BSAP level corresponding to the highest quartile. The 10-year probability of fracture was 26% if at least one of the three predictors was present, whereas it was only 6% in women without any of the three predictors.

The ability of urinary OC, serum OC, and TRAcP 5b to predict fracture was assessed in 1,040 elderly women [44] of whom 178 women sustained at least one osteoporotic fracture. Both urinary and serum OC were significantly increased in women with a fracture of any type or with vertebral fracture only compared with women without fracture. TRAcP5b also was able to predict the occurrence of a fracture of any type.

Until now only a few studies have assessed the utility of biomarkers for risk evaluation in men. In a case-control cohort study, Meier and colleagues [67] followed 151 elderly men for 6.3 years, 50 men with incident low-trauma fractures and 100 without fractures. In this analysis, S-ICTP was independently associated with fracture incidence; subjects in the highest quartile of S-ICTP had a 2.8-fold increased risk of fracture compared to those within the lowest quartile.

Collectively, serum or urinary levels of bone turnover markers are independent predictors of fracture risk [16, 33, 38, 44]. However, it has not yet been demonstrated whether biochemical markers can really sum up the information on all determinants of the fracture risk. Therefore, the current recommendation for assessment of fracture risk is combined measurements of BMD and biochemical markers [67].

Utility of biomarkers in biomedical research

In this section, we wish to emphasize the potentials and relative advantages of biochemical markers for establishing optimal doses of novel drug candidates in Phase II studies. We provide illustrative data on how these efficacy parameters behaved in Phase II trials that contributed significantly to the approval and marketing of numerous drugs. Finally, we revisit the utility of biochemical markers for improving patient compliance to long-term use of antiresorptive agents.
  1. a.

    Biochemical markers for drug development

     
  2. b.

    Biochemical markers for monitoring treatment efficacy

     

Biochemical markers for drug development

The diagnosis of osteoporosis is inherently linked to low bone mass, and thus monitoring of changes in BMD during intervention is the main endpoint of the efficacy of drugs targeting the treatment of osteoporosis. Ideal dose of a given agent is established in Phase II trials. It is to be emphasized that annual changes in BMD are relatively small both when regarding the spontaneous loss of bone mass in the placebo treated group as well as in the group receiving active medical interventions (typically a few % change). Moreover, BMD measurements have an imprecision of 1–2% when using repetitive measurements [34, 43, 49, 56]. Furthermore, despite BMD being important as a surrogate marker of drug efficacy, the access to DXA can be limited. Consequently, much attention has been given to the search for more simple yet useful surrogate markers over the last two decades. The utility of biochemical markers as a powerful tool for fracture risk prediction has recently been emphasized by the observation that increases in BMD during treatment can provide only partial explanation of the reduction in fracture risk [30].

In clear contrast to the imaging techniques, changes in biochemical markers of bone resorption in serum or urine are markedly larger compared with the imprecision of the assays. As an example, changes observed within the first 3 weeks of treatment with bisphosphonates include decreases in serum CTX in the magnitude of 75% with an average short-term intra-individual coefficient of variation (CV) of 7.9% [22]. As the biochemical markers of bone resorption have a low ‘noise-to-signal’ ratio combined with rapid changes in the biochemical marker in response to treatment, use of biochemical markers may provide critical information about the relative effect of an antiresorptive or anabolic treatment that may be used for optimal dosing of various anti-osteoporotic drugs.

Previous studies have indicated that 3- to 6-month changes in the biochemical markers correlate with the change of BMD over 2 years [45, 46, 81, 82]. Accordingly, biomarker based assessment of various doses of drug candidates could considerably improve Phase II development and decrease costs. Biochemical markers can furthermore assist the elucidation of the relative effects of medical interventions on bone formation and resorption, which is useful when distinguishing between antiresorptive and anabolic properties of drug candidates.

Biochemical markers for monitoring treatment efficacy

The ultimate aim of treatment is prevention of fractures, both vertebral and non-vertebral (hip).

Current treatment of osteoporosis includes several drugs targeted towards inhibition of bone loss by decreasing osteoclast activity. Some of these drugs have in clinical trials been demonstrated to reduce the risk of fracture by approximately 40–50% [90] and the effect is usually seen with changed biomarker concentrations within 3–6 months of treatment and increase in BMD after 24 months. Antiresorptive drugs include hormone replacement treatment (HRT), selective estrogen receptor modulators (SERMs), tibolone, bisphosphonates, calcitonin and strontium renalate. Another compound PTH stimulates bone resorption but may also stimulate bone formation when dosed by intermittently injections. In this section, we shortly summarize some of the largest phase III studies conducted for these drug (Table 1) [8, 10, 13, 18, 20, 31, 32, 64, 70, 75, 83].
Table 1

Summary of phase III studies conducted for some of the most utilized drugs for treatment of osteoporosis

Study

Treatment

Duration (years)

Participants

ΔResorption markersa

ΔFormation markersa

ΔBMD (%)a

Fracture reduction

Type

Age (years)

Number

WHI/Women’s HOPE [13, 64]

0.625 mg CEE+2.5 mg MPA

5.6/2

Healthy PM women

63/52

16,608/695

−49.2% (NTx)

−35.8% (OC)

Hip +3.7%

Hip −33%

Spine +3.46%

Vertebral −35%

T1 vs T0

T1 vs T0

T2 vs T0

 

BONE study [31]

Ibandronate 2.5 mg/day

3

Osteoporotic PM women

70

2,946

−65,3% (CTX)−68,3% (NTx)

−35.8% (OC)

Hip +3.4%

Hip −50%

Spine +6.5%

Vertebral −62%

T3 vs T0

T3 vs T0

T3 vs T0

 

FIT Study [8, 10]

Alendronate 5 mg/day

3

PM women with low BMD

71/63

2,027/202

−65% (NTx)

−50% (BSAP)

Hip +4.7%

Hip −50%

Spine +6.2%

1 vertebral −50%

>1 vertebral −90%

T3 vs T0

T3 vs T0

T3 vs TPl

 

VERT study [83]

Risendronate 5 mg/day

3

Osteoporotic PM women

72

1,226

−33% (Dpyr)

−37% (BSAP)

Fem. Neck +2%

Vertebral −49%

Spine +6.5%

Non-vertebral −33%

T6mo vs T0

T6mo vs TPl

T3 vs T0

 

SOTI study [70]

Strontium ranelate 2 g/day

3

Osteoporotic PM women

69

1,649

−12.2% (CTX)

+8.1% (BSAP)

Hip +8.6%

 

Spine +6.8%

Vertebral −41%

T3mo vs Pl3mo

T3mo vs Pl3mo

T3 vs T0

 

FPT Study [18, 75]

PTH (1–34) 20 or 40 μg/day

2

Osteoporotic PM women

70–72

1,637

+42% (NTx)

+10% (BSAP)

Hip +2.6%

 

Spine +9.7%

1 vertebral −65%

>1 vertebral −69%

Non-vertebral −40%

T6mo vs T0

T6mo vs T0

T2 vs TPl

 

PROOF study [20, 32]

Calcitonin 100–400 IU/day

5

Osteoporotic PM women

68–69

1,255

−12–30% (CTX)

−9% (BSAP)

Spine +1–1.5%

Vertebral −33%

Non-vertebral −18–36%

T1 vs T0

T1 vs Tpl

T5 vs T0

 

BSAP: bone specific alkaline phosphatase, OC: osteocalcin, Dpyr: deoxypyridine, CTX and NTX: C and N-telopeptide of collagen type I. aPartly estimated from graphs

T0, intial value; Tpl, placebo values; T3mo, T6mo, value at 3 and 6 months; T1, T2, T3, value at 1, 2 and 3 years

From these larges studies, we see changes in bone turnover markers are soon as 3 months after drug administration, and see a negative correlation between most treatments and markers. Bone markers assist here in determining the efficacy of the drug in question, which is valuable information for a drug development company and not least for patients. Several studies show that bone turnover markers show correlation to BMD changes following drug administration [1, 14, 26, 73, 84, 85, 87, 101]. These studies all show decrease in bone turnover markers following treatment in populations represented from around the world (Scandinavia, Northern Europe, Japan, North America, Thailand, Australia, etc.). Special attention should be paid to the meta-analysis by Crane et al. [26]. Data on spine BMD and five bone turnover markers (OC, BSAP, uNTx, uCTX, sCTx,) from 85 studies using bisphosphonate treatments were analyzed. Spearman correlations were computed to assess the strength of the associations between markers and BMD changes. Baseline BMD at 6 and 12 months were compared to marker changes at 1, 3 and 6 months and revealed modest to strong associations (r=0.63–0.90). In particular, the association was strong for changes at 3 and 6 months in BSAP, OC and NTx, and at 1, 2 and 6 months for uCTX and sCTX. Interestingly, the strongest correlation was found for the 1-month assessment of sCTX (r=0.90).

Compliance

Compliance is an important issue of long-term therapy of chronic diseases. Trivial inconveniences could impair compliance, especially when the medication is given to treat chronic asymptomatic diseases such as osteoporosis. Strict daily dosing of a drug might cause problems for some patients and may obstruct their compliance, which in turn hampers the long-term efficacy of medication. Although a trivial and most cost-effective way of monitoring compliance is by asking the patients, this may not be a completely objective measure. Very useful information can be added by serial measures of bone markers for monitoring patients. In a study of 200 healthy postmenopausal women with an average age of 63.1 years, serial measurements of serum CTX were performed in patients receiving different dosing regimes of ibandronate mimicking different compliances to the oral treatment [99]. The results illustrated that when patients were monitored by serial measurements of CTX, important information could be obtained. The biomarker measurements can not only inform the physician about the efficacy of the treatment, but can also be used to confront the patient regarding her or his achievements, or the need of more rigorous compliance to ensure maximal benefits. The low sensitivity of BMD measurements is not able to provide such early feedback. This advantage of serial measurements of bone markers over the initial 3-month period is also emphasized by the fact that there is strong correlation between drug-induced responses in bone markers at month 3 and subsequent BMD changes at 24–36 months [7, 19, 29, 37, 41, 46, 81]. While the potentials of biomarkers to serve such purposes seems rationalized by the aforementioned studies, the concept needs practical verification by prospective studies.

Summary

In the present review, we attempted to give an updated list of biomarkers including three recently established (Cat K, OPG, RANKL) and eight already well-established (CTX, ICTP, NTx, PYR, BSAP, OC, PINP/PICP, TRAcP) ones. Biomarkers have become key players in bone-related biomedical research. The strengths of biochemical markers lies within their dynamics that makes it possible to document early responses to interventions, which correlate well with subsequent changes in bone mass and fracture risk. The development of medical drugs for the treatment of osteoporosis is an expensive process, which at present time demands the participation of hundreds (phase II) or thousands (Phase III) of patients in trials run for at least 2 years. Biochemical markers are widely used in in vivo, ex vivo and in vitro experiments [92].

The application of biochemical bone markers should be based on careful consideration as to which metabolic events are in need of assessment. The markers reviewed here are all available in test formats having acceptable technical performance, and the selection of any specific marker should therefore be based on a clear understanding of the metabolic events leading to the generation of the analyte. In clinical and epidemiological studies of osteoporosis and other metabolic bone diseases, bone resorption and bone formation is often assessed on the basis of measurements of serum samples, as the marker levels here do not have to be corrected for creatinine. Serum CTX-I in combination with either PINP or OC seems a reasonable choice as they reflect the degradation and synthesis of matrix molecules that are very abundant in the skeleton. Consequently, these markers have been used in numerous studies, some of which have been referenced here. Further studies are needed to gain more experience with what advantages we can gain by combining collagenous resorption markers with the different non-collagenous osteoclast markers that provide insights into changes of osteoclast number under different pathophysiological processes or during treatment with antiresorptive or anabolic drugs.

However, it is still an ongoing debate, whether biomarkers combined with BMD measurements can be primary end-points of drug evaluation. If the answer to this question is yes, we will be able to lower the costs of drug-development, and provide patients with less expensive medications for the prevention and treatment of osteoporosis.

Conflict of interest

Diana J Leeming, Morten A. Karsdal and Per Qvist are employed by Nordic Bioscience A/S, a company engaged in the development and marketing of bone and cartilage markers.

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • D. J. Leeming
    • 1
  • P. Alexandersen
    • 2
  • M. A. Karsdal
    • 3
  • P. Qvist
    • 1
  • S. Schaller
    • 3
  • L. B. Tankó
    • 2
  1. 1.Nordic Bioscience Diagnostics A/SHerlevDenmark
  2. 2.Center for Clinical and Basic Research A/SBallerupDenmark
  3. 3.Pharmos Bioscience A/SHerlevDenmark

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