The anti-tumor agent sagopilone shows antiresorptive effects both in vitro and in vivo.

UNLABELLED: Sagopilone, a fully synthetic epothilone and very potent anti-tumor agent, has proved to be efficient in inhibiting bone destruction and tumor burden in a mouse model of breast cancer bone metastasis. In addition to its antiproliferative effects, this study shows direct effects of sagopilone on bone resorption and osteoclast activity. INTRODUCTION: Sagopilone, a novel fully synthetic third-generation epothilone, has proved to be efficient in inhibiting bone destruction and tumor burden in a mouse model of breast cancer bone metastasis. The aim of this study was to investigate whether the effect was primarily due to sagopilone's antiproliferative effect and consequent inhibition of tumor cell growth, or if sagopilone exerts direct effects on bone resorption and osteoclast activity. METHODS: Sagopilone was studied and compared to paclitaxel in vitro in human osteoclast differentiation and activity cultures. For studying the potential of sagopilone for inhibiting bone resorption in vivo, a mouse model of ovariectomy (ovx)-induced osteoporosis was utilized. RESULTS: Sagopilone inhibited osteoclast differentiation and activity more efficiently than paclitaxel and showed less cytotoxicity. Whereas sagopilone showed inhibitory effects on human osteoclast differentiation and activity already at 5 and 15 nM, respectively, paclitaxel started to show effects only at 20 and 100 nM concentrations, respectively. Sagopilone treatment increased BMD In the mouse ovx model even though a non-optimized dose was used which is effective in tumor-bearing mice. CONCLUSION: This is the first study to evaluate sagopilone's effects on bone resorption in non-cancerous situation. The evidence that sagopilone is beneficial for bone will strengthen the status of sagopilone as an anti-cancer compound compared to other microtubule stabilizing agents.

Strong prediction of fractures among older adults by the ratio of carboxylated to total serum osteocalcin.

We examined serum total osteocalcin (TOC), carboxylated osteocalcin (COC), and their ratio (COC/TOC) by one-step two-site immunofluorescent assays in 87% (n = 792) of all home-dwelling persons of 70 years or older living in a defined area in northern Finland. Other baseline subject-related risk factors of fractures were assessed by postal questionnaires, interviews, clinical examinations, and tests. During a 5-year follow-up period, all falls and fractures (n = 106) were recorded by regular phone calls and by examining all the medical records yearly. Serum TOC and COC concentrations increased with advancing age and were higher in women than in men, but corresponding differences were not found in the case of COC/TOC. The adjusted relative risk of fracture was elevated in association with low (< or =-1 SD from the mean) COC; hazard ratio (HR, 95% CI) 2.00 (1.20-3.36) and low COC/TOC; HR 5.32 (3.26-8.68), the relative risk being highest in the population older than 80 years; and HR 7.02 (2.42-20.39). The predictive value of low COC/TOC lasted 3 years. The multivariable-adjusted relative risk of hip fracture (n = 26) in regard to low COC/TOC ratio was 3.49 (1.12-10.86), as compared with the persons who did not suffer hip fractures. Our results suggest that serum COC concentrations and, more strongly, COC/TOC, predict the occurrence of fractures in older community-dwelling adults. The risk of fracture associated with low COC/TOC equals the hip fracture risk previously verified for concomitant high serum undercarboxylated OC concentrations and low bone mineral density.

Development and evaluation of three immunofluorometric assays that measure different forms of osteocalcin in serum.

BACKGROUND: Circulating human osteocalcin (hOC) has been used as a marker of bone formation. Our aim was to validate three immunofluorometric assays (IFMAs), measuring different forms of hOC. METHODS: The two-site IFMAs were based on previously characterized monoclonal antibodies. Assay 2 recognized intact hOC, assays 4 and 9 measured the NH(2)-terminal mid-fragment and the intact hOC. In addition, assay 9 required hOC to be gamma-carboxylated. RESULTS: A 76-79% increase of serum immunoreactive hOC was found in the postmenopausal group compared with the premenopausal group with all IFMAs. With EDTA-plasma samples, the observed increases were lower (49-65%). The hOC concentration in the postmenopausal group receiving hormone replacement therapy was 42-44% lower than that in the postmenopausal control group in both serum and EDTA-plasma samples. The depressed carboxylation in warfarin-treated patients was accompanied by lower results in assay 9. The ratio of assay 9 to assay 4 totally discriminated the warfarin-treated patients from the controls. Assay 9 showed the smallest decreases in measured hOC after storage of serum or plasma for 4 weeks at 4 degrees C, followed by assay 4 and assay 2. Results from the last assay were <17% of their initial values after 4 weeks of storage. No diurnal variation was observed with assay 9 as opposed to the two other IFMAs. CONCLUSION: The three assays with their distinct specificity profiles (intact vs fragmented and carboxylated vs decarboxylated hOC) may provide valuable tools for investigating the significance of different hOC forms in various bone-related diseases.

The proportion of carboxylated to total or intact osteocalcin in serum discriminates warfarin-treated patients from control subjects.

We assessed the serum concentration of gamma-carboxylated osteocalcin (OC), total OC, and full-length OC in a clinical setting of 37 patients on continuous warfarin treatment (international normalized ratio 2.0-3.8). A comparison was done with the results from 30 untreated age-matched controls. Four monoclonal antibodies, previously generated and characterized as to their ability to recognize different human OC forms and fragments, were used in three two-site immunofluorometric assays. The warfarin-treated patients had significantly lower levels of carboxylated OC 4.9 +/- 3.8 (+/- 1 SD) ng/ml compared with the controls 13.1 +/- 9.7 (p < 0.0001). There was no difference in the levels of total OC or full-length OC between the two groups of patients. A strong correlation was found between the serum concentration of carboxylated OC and total OC, both for the warfarin-treated patients (r = 0.98) and for the controls (r = 0.99). There was a distinct cut-off level at 0.80, in the quotient carboxylated OC/total OC, at which all warfarin-treated patients fell below and all controls above this level. Hence, the concentration or ratio of serum gamma-carboxylated OC in clinical settings such as warfarin-treated patients could be measured using two-site immunoassays.

Demonstration of the predominant urine osteocalcin fragments detectable by two-site immunoassays.

We have isolated and characterized human osteocalcin (OC) fragments from pubertal urine. The fragments were isolated by immunoaffinity chromatography based on monoclonal antibody 6F9 and further purified by reverse phase chromatography. The major isolated forms, which were detectable with two-site immunofluorometric assays for serum OC, span residues 6-30 and 7-30 as determined by mass spectrometry and N-terminal amino acid sequencing. Full-length OC was not detectable in the supernatant fraction of urine but could be extracted with guanidinium hydrochloride from the sediment of urine samples. Urine samples from subjects with different menopausal status were measured by two different two-site assays. Urine OC (uOC) concentrations were 12- to 16-fold higher in the pubertal group than in the adult group. Also, the uOC concentration in a postmenopausal group was significantly higher than in a premenopausal group. The difference was 125% and 75% (values for p < 0.0001), respectively, when measured with the two assays. uOC concentrations in postmenopausal subjects on hormone replacement therapy were indistinguishable from the premenopausal subjects. The fact that uOC can be measured by a noncompetetive two-site assay design offers improved analytical sensitivity. Urine as the sample matrix is also especially interesting because the predominant markers of bone resorption, collagen type I peptides or cross-links, are performed on urine samples. Our results from the technical validation of two-site assays for uOC and from applying these to human pubertal and pre- and postmenopausal samples calls for more extensive clinical validation.

Two-site immunoassays for osteoclastic tartrate-resistant acid phosphatase based on characterization of six monoclonal antibodies.

Tartrate-resistant acid phosphatase (TRAP), an enzyme expressed in bone-resorbing osteoclasts, is secreted into the circulation during bone resorption. We used six monoclonal antibodies (MAbs) to optimize direct two-site fluoroimmunoassays for determining serum TRAP concentrations. Four of the MABs, 1F1, 2H1, 4E6, and 5C1, were raised against recombinant human TRAP, and the other two, O1A and J1B, against human bone TRAP. 2H1, J1B, and O1A appeared to be highly specific for TRAP. 1F1 and 4E6 were poor in recognizing bone TRAP and were not useful in the assay. 5C1, while having a good affinity for the bone enzyme, was not specific. Serum TRAP is relatively stable, because 7 days of storage of serum samples at 4 degreesC and -20 degreesC or five thawing-freezing cycles, did not change the TRAP concentration detected using the two-site assays. All studied assays detected an increase in serum TRAP concentrations of postmenopausal women compared with premenopausal women, the difference being highest with MAB pairs 2H1-5C1 and O1A-J1B. These results suggest that serum TRAP may be a useful bone resorption marker, and the MAB pairs 2H1-5C1 and O1A-J1B may be useful in determining the bone resorption rate.

A dual-label immunofluorometric assay for human osteocalcin.

Circulating human osteocalcin (hOC) has been shown to be comprised of two main forms: the intact 1-49 form and the proteolytic N-terminal midfragment (N-mid) spanning amino acid residues 1-43 or 1-44. We used three monoclonal antibodies (MAbs) raised against hOC and bovine osteocalcin in developing a dual-label assay for the simultaneous measurement of the proportions of the intact and N-mid forms in serum samples. The assay is based on time-resolved fluorescence utilizing differently labeled trace MAbs. Biotinylated MAb 2H9 is used as a capture antibody for both the intact hOC and the N-mid. Tracer MAb 6F9 labeled with a Europium (III)-chelate binds to the intact the N-mid and the intact hOC, whereas tracer MAb 3G8 labeled with a Terbium (III)-chelate binds to the intact hOC only. The simultaneous binding of the antibodies was tested by comparing full-length hOC purified from human bone and hOC shortened from the C terminus by four amino acid residues with carboxypeptidase Y. Serum hOC measurements with the dual-label assay were in agreement with the corresponding single-label assays (r = 0.96 for intact + N-mid assay and r = 0.81 for intact assays, n = 91). The lower correlation between the intact assays was attributable to proteolytic susceptibility of the intact form due to one additional freezing and thawing cycle in carrying out the dual-label assay. As measured with the dual-label assay, the levels (mean +/- SD) of serum intact + N-mid OC were 6.2 +/- 2.1 ng/ml in the premenopausal group (n = 44), 13.9 +/- 4.9 ng/ml in the postmenopausal group without hormone replacement therapy (HRT; n = 13), and 7.5 +/- 3.4 ng/ml in the postmenopausal group with HRT (n = 13). The levels of intact hOC in the same groups were 4.8 +/- 1.4 ng/ml, 9.8 +/- 2.9 ng/ml, and 5.3 +/- 2.1 ng/ml, respectively. Whether the main forms of OC or their relative proportions in serum can be used for predicting bone diseases or for monitoring the progression and management of diseases awaits further investigations.

Characterization of serum tartrate-resistant acid phosphatase and development of a direct two-site immunoassay.

Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP) to the circulation, where the amount of TRAP is expected to correlate with the bone resorption rate. We have developed two monoclonal antibodies, O1A and J1B, using purified human bone TRAP as antigen. The antibodies recognized different epitopes, allowing us to develop a two-site fluoroimmunoassay. The immunoreactivity in fresh serum specimens was less than 10% of the concentrations measured from the same specimens after 24 h of storage at 4 degrees C, or after addition of 5 mM EDTA or EGTA to them. When fresh serum was gel filtrated using Sephacryl S-200 column, all of the enzyme eluted in the void volume as a complex with a molecular weight of more than 250 kDa. If the serum was treated with EDTA before the gel filtration, the complex was destroyed and the enzyme eluted in fractions corresponding to a molecular weight of 30 kDa, the size of monomeric purified human bone TRAP. The immunoassay was used to measure TRAP concentrations from serum samples that had been stored at 4 degrees C for 24 h. According to the assay, premenopausal women had 13.1 +/- 3.1, postmenopausal women 17.6 +/- 4.2, and children 32.6 +/- 12.2 microg TRAP/l of serum. We conclude that TRAP circulates in the serum as part of a complex, which also contains Ca2+, and that TRAP-immunoassay is a potentially useful method for determining bone resorption rates, as long as the complex is destroyed before the assay.

Purification and characterization of recombinant osteocalcin fusion protein expressed in Escherichia coli.

Human osteocalcin (hOC) is a 49-amino-acid peptide produced mainly by bone osteoblasts. The amount of hOC in the circulation reflects the status of bone metabolism and it is used to monitor various bone-related diseases. The aim of this study was to produce recombinant human osteocalcin (rhOC) in Escherichia coli and use it for designing new osteocalcin fluorescence immunoassays. Recombinant DNA technology was used to fuse synthetic hOC coding sequences to an affinity handle system based on glutathione S-transferase (GST) gene. GST-rhOC fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The affinity-purified fusion protein was cleaved with activated protease factor X releasing the rhOC portion. The structure of rhOC was confirmed by mass spectrometry and amino acid sequencing. The fusion protein and its proteolytic cleavage product proved to be immunoreactive as shown by Western blotting analysis and by a new osteocalcin immunoassay based on time-resolved fluorescence. When osteocalcin was tested for its ability to bind to hydroxyapatite, there were no differences between the recombinant forms and native human osteocalcin purified from bone, suggesting that the Gla residues might be important only in oriented high-affinity binding.

Epitope mapping of nine monoclonal antibodies against osteocalcin: combinations into two-site assays affect both assay specificity and sample stability.

Nine monoclonal antibodies (Mabs) were raised against human recombinant osteocalcin fusion protein (rGST-hOC) or bovine osteocalcin (bOC) and selected to develop two-site immunoassays for human osteocalcin (hOC). The detection system was based on the time-resolved measurement of the fluorescence of europium chelates conjugated to the tracer Mabs. Based on the ability of the Mabs to recognize different forms of hOC (carboxypeptidase Y-digested, alkylated hOC, thermally decarboxylated hOC, recombinant forms of hOC, and tryptic peptides derived from hOC) and the information obtained from combinations of the Mabs in two-site assays, an epitope map was created. The epitope map was useful in understanding the behavior of the two-site combinations of the Mabs with serum samples. The two-site combinations could be divided into subgroups detecting either full-length hOC or full length+large NH2-terminal fragment as stimulated by the carboxypeptidase Y-digested form of hOC (it lacks four COOH-terminal residues), which with intact specific assays showed cross-reactivities ranging from 7 to 14% when compared with full-length hOC. In addition, differences were observed in the ability of the combinations to detect thermally decarboxylated hOC (lacks gamma-carboxylation at residues 17, 21, and 24) with cross-reactivities ranging from 8 to 85% when compared to gamma-carboxylated hOC. The analysis of human serum samples showed considerable differences in the concentration and stability of serum OC. This was attributed as the varying ability of the Mabs to detect different proteolytic fragments derived from hOC and/or differences in the degree of gamma-carboxylation of hOC. The in vitro generation of the large NH2-terminal fragment during incubation of the serum samples at room temperature (RT) and during prolonged storage at -20 degrees C in an undercooled state was detectable as loss of immunoreactivity (ranging from -42 +/- 17 to -50 +/- 15% in 16 h at RT, n = 3) with Mab combinations detecting only full-length hOC. Two-site combinations detecting full-length+large NH2-terminal fragment showed no loss of immunoreactivity after incubation of the serum samples at RT for 16 h. With all assays, an increase of serum OC ranging from 16 to 38% was found in postmenopausal samples (n = 24) when compared with premenopausal samples (n = 17), but the degree of statistical significance varied from not significant to p < 0.01.