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Bisphosphonate pharmacokinetics may affect individual responses. Skeletal retention of pamidronate infused monthly to patients with bone metastases was highly variable (12-98%) and did not diminish with time, showing the capacity of the skeleton to retain large amounts of bisphosphonate. Relationships between skeletal retention of pamidronate and rate of bone resorption are complex and depend on previous treatment and the total amount of retained bisphosphonate. Introduction: Bisphosphonates (BPs) given intravenously every 3-4 weeks are effective in the management of metastatic bone disease from breast cancer, but responses among patients vary, and it is not known whether current dose and dose intervals are appropriate for an individual patient. An influence of pharmacokinetics of BPs on antiresorptive action may contribute to this variation in response. To test this hypothesis, we determined the skeletal retention of intravenous pamidronate and its association to the rate of bone resorption in patients with bone metastases from breast cancer. Materials and Methods: In a cross-sectional study, 24-h urinary excretion of pamidronate and the biochemical marker of bone resorption N-terminal telopeptide of type 1 collagen and serum alkaline phosphatase were measured in 40 patients with bone metastases from breast cancer at the beginning, after 3-6 months, and after 1 year of treatment with intravenous pamidronate 90 mg every 3-4 weeks. Results and Conclusions: Skeletal retention (dose - amount excreted into urine) 2 days after infusion varied between 12% and 98% (mean, 62%) of the administered dose, but there were no differences in retention between patients receiving pamidronate for the first time or after 3-6 months or after 1 year of treatment. Retention of pamidronate was related to the prevalent rate of bone turnover in previously untreated patients, whereas no such relationship was found in previously treated patients. Rate of bone resorption after treatment seemed to be related to the amount of pamidronate retained. During 1 year of treatment, retention of pamidronate remained constant, indicating no saturation of skeletal binding sites with treatment. The variability in retention among individual patients can be attributed to the number of available binding sites. This is related, however, to bone turnover only before the start of treatment. The apparent relationships between skeletal retention and antiresorptive effect could have implications for the design of optimal therapeutic regimens with BPs in patients with bone metastases from breast cancer.

In a recent multinational, double-blind, placebo-controlled, randomized, phase III study (BONE: IBandronate Osteoporosis Vertebral Fracture trial in North America and Europe), oral daily ibandronate (2.5 mg) significantly and substantially reduced the risk of new vertebral fractures by 62% relative to placebo after 3 years of treatment. The objective of the present study was to retrospectively analyze data from the BONE study to examine the efficacy of oral ibandronate in preventing incident vertebral fractures of greater severity. This analysis was conducted on the placebo and oral daily ibandronate (2.5 mg) arms of the BONE study, comprising a total of 1964 women (aged 55-80 years, >or=5 years postmenopause) with osteoporosis. Vertebral fractures on annual lateral radiographs of the spine were graded as mild, moderate, or severe, using criteria derived from an established semiquantitative technique. The findings demonstrate that in addition to being effective in significantly reducing the risk of new vertebral fractures of all severities, oral daily ibandronate has a pronounced effect on the more severe, most clinically relevant, vertebral fractures: a significant and sustained reduction of 59% in the relative risk of combined new moderate and severe vertebral fractures was observed at years 1 (P = 0.0164), 2 (P = 0.0004), and 3 (P < 0.0001).

We report the case of a severe symptomatic hypophosphatemic osteomalacia in a 66-year-old patient with hormone-refractory prostate cancer metastatic to the skeleton. A follow-up of 2 years from diagnosis to development of hormone refractoriness and death allowed us to study the natural history of this uncommon disturbance of mineral homeostasis in this common malignancy. Relevant to the difficult management of the late stages of prostate cancer is the failure of hypophosphatemia to respond to conventional therapeutic approaches and the favorable outcome of antitumor therapy suggesting that this group of patients, although having a poor prognosis, could still benefit from aggressive second line therapy. In this malignancy in which metastases have a predilection for bone, failure to recognize osteomalacia can only result in significantly increasing the burden of skeletal complications.

Bisphosphonates (BP) inhibit osteoclast-mediated bone resorption, and have been reported to decrease the rate of cartilage degradation. The anti-resorptive effect of BP is determined by the amount of BP retained by the skeleton. In rheumatoid arthritis (RA) the uptake is not confined only to the skeleton, but BP is also retained in joints, which could have implications for dose regimens. We investigated the whole body retention (WBR) of pamidronate and its relationship to bone resorption and cartilage degradation in patients with active RA.Twenty-six patients received placebo, 45 mg, or 90 mg intravenous pamidronate. Serum and urine samples were collected before and for 12 days after drug administration. Rate of bone resorption was assessed by the biochemical markers: serum carboxy terminal cross-linked telopeptide of type I collagen, urinary carboxy terminal cross-linked telopeptide of type I collagen normalized to creatinine and urinary amino-terminal telopeptide of type I collagen normalized to creatinine; and rate of cartilage degradation by urinary carboxy terminal telopeptide of type II collagen normalized to creatinine. WBR was derived from urinary excretion of pamidronate data.Pamidronate induced a rapid and sustained decrease in the level of biochemical markers of bone resorption and cartilage degradation. The mean WBR of pamidronate was 69% of the administered dose, and showed a remarkably wide range (41-96%). The decrease in rate of bone resorption, but also rate of cartilage degradation appeared to be related to the WBR of pamidronate.This is the first study in which the effect of BP treatment has been studied in relation to the amount of BP retained by the body in patients with active RA. The total amount of BP retained by the body shows a remarkably wide range and is comparable with literature on patients with osteoporosis. The apparent relationships between the amount of BP retained by the body and the effect could have implications for therapeutic regimens in patients with RA.

Objective To estimate the occurrence of familial Paget's disease of bone in The Netherlands, to examine the prevalence of mutations of the sequestosome 1 gene (SQSTM1) in identified families, and to assess potential genotype–phenotype associations. Methods We performed a case–control study of patients with Paget's disease and a mutation analysis of the SQSTM1 gene of index patients with familial disease and of the relatives of those with a mutation. Serum alkaline phosphatase (AP) activity was assessed, and bone scintigraphy was performed. Results Five percent of patients had at least 1 first-degree relative with the disease, compared with 0.5% of the controls (relative risk 10; 95% confidence interval 1.3–75.6). In 38.9% of patients with familial disease, heterozygous mutations in the SQSTM1 gene were identified. These were the previously described P392L mutation, which was present in 22.2% of patients, and 3 new mutations, S399P, G425R, M404T, 9 of which were present in 3 different families. All mutations were located in the ubiquitin-associated domain of the gene. There was a relationship between serum AP activity, as a marker of the disease, and the presence or absence of the G425R and P392L mutations, the subject's age, and the presence of Paget's disease. Conclusion Our data provide further evidence of a causal role of SQSTM1 gene mutations in the pathogenesis of Paget's disease and allow the design of a strategy based on measurements of serum AP activity and age for investigating asymptomatic relatives of patients with familial Paget's disease of bone.

Treatment with alendronate, a potent and specific inhibitor of bone resorption, is known to significantly reduce fracture risk among women with postmenopausal osteoporosis. The purpose of this meta-analysis was to assess the consistency of the effect of alendronate in reducing the risk of hip fracture among different studies and populations. Data from completed, randomized, treatment studies were pooled in a meta-analysis. The duration of the studies ranged from 1–4.5 years. The dose of alendronate ranged from 5–20 mg/day, with over 95% of patients receiving either 5 or 10 mg/day during the trials. In patients with a T-score of less than or equal to −2.0, or with a vertebral fracture, the effect on hip fracture risk consistently favored patients receiving alendronate therapy, with an overall reduction in risk of hip fracture of 45% [95% confidence interval (CI) 16% to 64%, P=0.007]. For patients who met the criteria of osteoporosis, as defined by the World Health Organization (WHO), the overall risk reduction was 55% (95% CI 29% to 72%, P=0.0008). In both analyses we performed a sensitivity analysis by removing one study at a time. The strength of the evidence was not dependent on any one study. We conclude that therapy with alendronate is associated with significant and clinically important reductions in the incidence of hip fracture in women with postmenopausal osteoporosis. The overall reduction is consistent among different patient populations.

UNLABELLED: Serum ALP may be a good indicator of Paget's disease in epidemiologic studies. Subjects with raised and normal ALP from a population cohort were matched (1 in 6, total 548), and radiographs were taken. ALP was an excellent marker of the disease (RR, 10.9), but the majority of those affected had normal ALP. INTRODUCTION: Evidence from radiographic surveys of limited skeletal sites has shown that Paget's disease of bone (PDB) is common in the elderly and has a distinct geographic variation. There is no information, however, about the relation of serum alkaline phosphatase (ALP) activity, a marker of the disease, and its prevalence in the population. MATERIALS AND METHODS: We analyzed data from a well-defined Dutch population cohort (the Rotterdam study) with the following specific aims: (1) to assess the relationship between serum ALP activity and prevalence of radiographically diagnosed PDB, (2) to estimate the overall prevalence of the disease in the Netherlands, and (3) to assess the appearance of the disease with time. Using a nested case-control design, subjects with an increased serum ALP and normal serum liver enzymes were matched for gender and age (1 to 6) with subjects with normal serum ALP activity. Radiographs of the thoracic and lumbar spine, pelvis, proximal femurs, knees, wrists, and hands were taken. RESULTS AND CONCLUSIONS: PDB was diagnosed in 20.5% of subjects with elevated serum ALP activity and in 2.3% in those with normal serum ALP activity, increasing with age in both groups. The relative risk (RR) for PDB in the presence of raised serum ALP activity was 10.9 (95% CI, 4.8, 24.9). The estimated prevalence of PDB in the population was 3.6%, and the large majority (about 86%) had normal serum ALP activity, contrasting findings in bone clinics where the opposite is the case. Finally, in subjects with normal and raised serum ALP activity but no PDB at baseline, radiographs taken 6-9 years later showed no evidence of the disease. This study demonstrated that serum ALP activity is a sensitive marker of PDB in men and women >55 years of age, but the majority of those affected have normal serum ALP activity.

Bisphosphonates are the treatment of choice of Paget's disease of bone. For optimal patient care determinants of the induction and duration of remission of the disease after bisphosphonate therapy must be defined. We addressed these issues in a longitudinal study of 157 patients with biochemically active disease (serum alkaline phosphatase activity >120 U/L) treated with the bisphosphonate olpadronate and followed for a median period of 37 months (range 3-162, mean 46 ± 30). Two different total doses of olpadronate were used: an effective dose (40 mg intravenously given over 5 or 10 consecutive days) and a high dose consisting of the effective dose followed by oral olpadronate 200 mg/day for 15 days. Treatment induced biochemical remission, defined as normalization of serum alkaline phosphatase activity, in 89.2% of the patients. There were no differences between the two treatment regimens. The only independent determinants of induction of remission were baseline serum alkaline phosphatase activity and number of affected bones. In contrast, duration of remission depended on the dose of olpadronate given (high versus effective dose, RR of relapse 0.49, 95% CI 0.27-0.89). Additional independent determinants of relapse were nadir value of serum alkaline phosphatase activity after treatment, number of previous therapies, and number of affected bones. Pain scores decreased with therapy in 88% of patients with pain complaints. Pain scores were significantly related to the probability of relapse (RR1.54, 95% CI 1.04-2.27). In this long-term study of a large cohort of patients with Paget's disease we confirmed the efficacy of olpadronate therapy. In addition, we identified and quantified determinants of the response to bisphosphonate that can help in improving the management of patients with Paget's disease with bisphosphonates.

Bisphosphonates are the treatment of choice of Paget's disease, but variable responses have been reported, and despite the availability of potent bisphosphonates, biochemical remission is not achieved in a substantial number of patients. This may, in part at least, be because of the influence of pharmacokinetics of bisphosphonates on their pharmacodynamics. That is the response of bone turnover to treatment. To address this issue, we examined the pharmacokinetics and pharmacodynamics of the bisphosphonate olpadronate given intravenously to 75 patients with Paget's disease, using a specific assay for olpadronate concentrations in serum and urine. The skeletal uptake of olpadronate varied greatly among patients and ranged between 10% and 90% of the administered dose. The two major determinants of skeletal uptake were renal function and prevalent rate of bone turnover. Serum and urinary data were well described by a physiology-based four-compartment pharmacokinetic model that takes into account the distribution of the bisphosphonate in the bone and its subsequent elimination. Bone turnover was suppressed to well within the normal range in virtually all patients. This, together with the absence of resolution of effect during 1 year, does not allow the construction of an adequate integrated pharmacokinetic/pharmacodynamic model. However, the pharmacokinetic model, described for the first time in Paget's disease, can accurately simulate the amount of bisphosphonate delivered to the skeleton with different dose regimens as well as the amount still present in bone after 1 year. Such approaches can lead to improved patient care and individualization of treatment of Paget's disease with bisphosphonates.

The diagnosis of osteoporosis is made from the measurement of BMD. DXA at the hip is the appropriate diagnostic site. Current clinical guidelines follow the principle that BMD measurements are indicated in individuals with risk factors for fracture and that treatment is recommended in those with a BMD below a critical value. In some countries reimbursement for the costs of treatment depend upon such thresholds for BMD. In Europe the critical value corresponds to a T-score of-2.5 SD, whereas in the USA less stringent criteria are used. It is evident, however, that fracture risk at any given T-score varies markedly according to age and other risk factors. This has led to the view that interventions should be targeted to those at high risk, irrespective of a fixed BMD threshold. In this sense, BMD is utlized as a risk assessment, since in many instances intervention thresholds will be less stringent than the diagnostic threshold. Thus, intervention thresholds need to differ from diagnostic thresholds and be based on fracture probabilities. A 10-year fracture probability appears to be an appropriate time frame. There are a number of problems to be overcome in the development of assessment guidelines. They need to take account of not only the risk of hip fracture but also that of other fractures which contribute significantly to morbidity, particularly in younger individuals. A promising approach is to weight fracture probabilities according to the disutility incurred compared with hip fracture probability. Account also needs to be taken of the large geographic variation in fracture probabilities worldwide. A further challenge for the future will be to identify risk factors that predict fracture with high validity in different regions of the world and their independent contributions, so that models of risk prediction can be constructed and ultimately validated in independent cohorts.

Invasion of the mineralized matrix by endothelial cells and osteoclasts is a key event in endochondral bone formation. To examine the putative role of osteoclast activity in the angiogenic process, we used two in vivo models of suppressed bone resorption: mice treated with the bisphosphonate clodronate and in osteoclast-deficient, osteopetrotic mice. Angiogenesis was assessed in caudal vertebrae of these neonatal mice. This model enables us to study the interaction between osteoclasts and endothelial cells during endochondral bone formation. In control conditions, sinusoid-like structures were detected in the vicinity of tartrate resistance acid phosphatase positive (TRAcP+) osteoclasts. Treatment with clodronate completely abolished osteoclastic bone resorption, whereas angiogenesis remained unaffected. In line with these observations, in the osteopetrotic mouse mutants c-fos knockout mice and op/op mice, capillaries invaded the calcified cartilage in the absence of osteoclasts. In conclusion, our data strongly suggest that during endochondral bone formation, vascular invasion can occur in the absence of osteo(chondro)clastic resorption. In addition, bisphosphonates show no apparent effect on angiogenesis in this in vivo model. These findings may have important clinical implications in the management of skeletal disorders such as metastatic bone disease, in which both osteoclastic bone resorption and angiogenesis contribute to tumor growth. On the other hand, our results confirm that bisphosphonates can be used safely in the treatment of disorders that affect the growing skeleton, such as in juvenile osteoporosis.

Background: Bisphosphonates are used extensively in the management of skeletal disorders including osteoporosis. Intermittent i.v. regimens with bisphosphonates are being explored as alternatives to oral regimens. The design of therapeutic regimens with bisphosphonates is mainly based on pharmacodynamic (PD) information. A combined pharmacokinetic (PK) and PD model of bisphosphonate action can help in the design of more effective therapeutic strategies. Aim: The objective of this study was to hypothesise on a PK/PD model for intermittent i.v. pamidronate in patients with osteoporosis. Methods: Serum and urine pamidronate PKs were studied in nine patients with osteoporosis treated with pamidronate (15 mg/day via i.v. infusion) on five consecutive days. Long-term renal excretion of pamidronate was extrapolated from literature data on alendronate. The PD response (urinary hydroxyproline) was assessed in 13 patients with osteoporosis, who were given the same regimen every 3 months for 1 year. Pooled serum and urine data were fitted into a physiology-based compartment model. In order to develop a mechanistically based PK and PD model, three different Emax models were examined. Results: A physiology-based three-compartment model was able to describe pamidronate PKs with a central compartment representing the serum, a second compartment representing the bone surface and a third compartment representing deep bone. The typical effect of 1 year of 3-monthly i.v. therapy was described adequately using an Emax model, in which the effect depended on both the amount of pamidronate attached to bone (second compartment) and on the amount of pamidronate buried into bone (third compartment). Conclusions: A combined PK/PD model for pamidronate is described which incorporates the specific pharmacology of bisphosphonates. The model may be used to describe long-term effects of an i.v. pamidronate regimen in osteoporotic patients.

Osteoblasts and adipocytes arise from a common progenitor cell in bone marrow. Whether estrogen directly regulates the progenitor cells differentiating into osteoblasts or adipocytes remains unknown. Using a mouse clonal cell line KS483 cultured in charcoal-stripped fetal bovine serum (FBS), we showed that 17beta-estradiol (E2) stimulates the differentiation of progenitor cells into osteoblasts and concurrently inhibits adipocyte formation in an estrogen receptor (ER)-dependent way. E2 increased alkaline phosphate (ALP) activity and nodule formation and stimulated messenger RNA (mRNA) expression of core-binding factor alpha-1 (Cbfa1), parathyroid hormone/parathyroid hormone-related protein receptors (PTH/PTHrP-Rs), and osteocalcin. In contrast, E2 decreased adipocyte numbers and down-regulated mRNA expression of peroxisome proliferator-activated receptor-gamma (PPARgamma)2, adipocyte protein 2 (aP2), and lipoprotein lipase (LPL). Furthermore, the reciprocal control of osteoblast and adipocyte differentiation by E2 was observed also in the presence of the adipogenic mixture of isobutylmethylxanthine, dexamethasone, and insulin. Immunohistochemical staining showed that ERalpha and ERbeta were present in osteoblasts and adipocytes. A new mouse splice variant ERbeta2 was identified, which differed in two amino acid residues from the rat isoform. E2 down-regulated mRNA expression of ERalpha, ERbeta1, and ERbeta2. The effects of E2 are not restricted to the KS483 cell line because similar results were obtained in mouse bone marrow cell cultures. Our results indicate that estrogen, in addition to stimulation of osteogenesis, inhibits adipogenesis, which might explain the clinical observations that estrogen-deficiency leads to an increase in adipocytes.

Nitrogen-containing bisphosphonates (NBps) are taken up by osteoclasts and inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway. There is evidence, however, that cells other than mature osteoclasts, like osteoclast precursors and osteoblasts, are also involved in the action of Bps on bone resorption in vitro. To examine this issue further, we developed a new in vitro model, which allows the study of the effects of additives on early osteoclast precursors. In this model, osteogenic cells are essential for osteoclastogenesis. The model consists of 15-day-old fetal mouse metatarsals. At time of explantation, these bone rudiments do not yet contain a mineralized matrix or osteoclasts; only early osteoclast precursors are present in the perichondrium. During culture and after the addition of Nabeta-glycerolphosphate, the bones form a mineralized matrix that is consequently resorbed by osteoclasts that develop from their precursors. Short treatment of these explants with Bps, before the formation of a mineralized matrix, resulted in a subsequent dose-dependent inhibition of bone resorption. The relative potencies of eight Bps to suppress resorption were comparable with those observed after the addition of Bps after the formation of a mineralized matrix, the natural target of Bps. In addition, the effects of the NBp olpadronate, but not of clodronate, on osteoclastic resorption, could be partly reversed by geranylgeraniol. Results indicate that Bps can suppress osteoclastic resorption in vitro by a direct action on very early osteoclast precursors at the bone surface, and not by affecting the osteoclastogenic capacity of osteogenic cells. Moreover, the mechanism of action of the NBp olpadronate, but not clodronate, on early tartrate-resistant acid phosphatase-negative osteoclast precursors involves inhibition of protein geranylgeranylation, indicating a molecular mechanism similar to that established for mature osteoclasts.

Spinal cord compression (SCC) is a devastating complication of metastatic cancer. We investigated the potential beneficial effect of two palliative therapies--strontium-89 (Metastron) and the nitrogen-containing bisphosphonate olpadronate--on the incidence of SCC in hormone-refractory prostate cancer (HRPC) metastatic to the skeleton. We retrospectively studied 415 patients with histologically proven prostate cancer who underwent bone scintigraphy at the time of diagnosis and were followed up at the Leiden University Medical Center between 1990 and 1999. Medical or surgical castration was undertaken in 172 patients with evidence for skeletal metastases. Within 2 years, 147 of these patients (85%) developed HRPC associated with severe progressive bone pain. Palliative treatment was given to 131 patients in the form of local radiotherapy ( n=10), 89Sr ( n=46) or intravenous olpadronate ( n=66), with ( n=57) or without ( n=9) maintenance oral olpadronate. Nine patients received both 89Sr and olpadronate at various intervals. Sixteen patients who did not receive any of these treatments were used as historical controls. There was no significant difference in baseline characteristics between treatment modalities. The incidence of SCC was 17% in the whole group, and highest in controls receiving no palliation (50%). None of the patients treated with local radiotherapy, only 4% of patients receiving 89Sr and 21% of patients given olpadronate developed this complication. Our findings suggest a significant reduction in SCC in patients with symptomatic HRPC metastatic to the skeleton who receive palliative therapies. Local radiotherapy completely prevents the incidence of SCC, 89Sr leads to an important decrease in this complication and olpadronate induces a significant, albeit smaller decrease in the incidence of SCC. The use of these agents opens new avenues in the difficult management of patients with advanced prostate cancer who are most at risk of developing SCC.

The efficacy and safety of oral pamidronate was examined in a double-blind, placebo-controlled trial in women and men with established osteoporosis. Seventy-eight postmenopausal women and 23 men with at least one prevalent vertebral fracture were randomized separately to 150 mg/day of pamidronate or placebo for 3 years followed by 150 mg/day of pamidronate for an additional 2 years. In addition, all patients received 400 U/day of cholecalciferol and 500 mg/day of elemental calcium. Pamidronate increased significantly bone mineral density of the lumbar spine (LS-BMD) and of the femoral neck (FN-BMD). The total increase in BMD of the spine after 5 years of treatment was 14.3%. Lateral spine radiographs were obtained at baseline and after 3 years of treatment. Fractures of previously normal vertebrae occurred in 15 of 45 patients treated with placebo (33.3%) and in 5 of 46 patients treated with pamidronate (11%). The relative risk was 0.33 (95% CI, 0.14-0.77). Treatment was well tolerated and there was no difference in gastrointestinal toxicity between pamidronate and placebo-treated patients. One hundred fifty milligrams daily of pamidronate is an effective and safe treatment of women and men with established osteoporosis.

Background Denosumab (DMAb) is an approved therapy for the treatment of postmenopausal women with osteoporosis at increased risk for fracture. The effects of DMAb treatment for up to 10 years are being evaluated in the 3-year FREEDOM study and its 7-year extension. Objectives To report the 5-year results of the FREEDOM extension study, representing up to 8 years of continued DMAb treatment. Methods During the extension, all women received 60 mg of DMAb every 6 months and daily calcium and vitamin D. In this analysis, women in the long-term group received 8 years of DMAb (3 years in FREEDOM and 5 years in the extension); women in the cross-over group received 5 years of DMAb (3 years of placebo in FREEDOM and 5 years of DMAb in the extension). Results Of the women who entered the extension, 66% completed the 5th year. With 8 years of DMAb treatment in the long-term group, mean bone mineral density (BMD) continued to increase from FREEDOM baseline for cumulative gains of 18.4% at the lumbar spine (LS) and 8.3% at the total hip (TH) (all p p Conclusions DMAb treatment for up to 8 years was associated with continued increases in BMD, persistent reduction of bone turnover, and low fracture incidence. The benefit/risk profile for DMAb remains favorable. Disclosure of Interest C. Roux Grant/research support: Bongrain, Lilly, MSD, Consultant for: Amgen, MSD, Novartis, S. Papapoulos Consultant for: Amgen, Axsome, Gador, GSK, Merck, Speakers bureau: Amgen, Eli Lilly, GSK, Merck, Novartis, K. Lippuner Consultant for: Amgen, Eli Lilly, MSD, Takeda, UCB, C. Lin Shareholder of: Amgen Inc., Employee of: Amgen Inc., D. Kendler Grant/research support: Amgen, Astalis, Eli Lilly, Novartis, Pfizer, Consultant for: Amgen, Eli Lilly, Merck, Pfizer, Warner Chilcott, Speakers bureau: Amgen, Eli Lilly, Pfizer, E. M. Lewiecki Grant/research support: Amgen, Lilly, Merck, Consultant for: Agnovos, Amgen, Lilly, Merck, Radius Health, M. Brandi Grant/research support: Abiogen, Alexion, Amgen, Bruno, Eli Lilly, Farmaceutici, MSD, Servier, Shire, Stroder, Consultant for: Alexion, Servier, Speakers bureau: NPS, E. Czerwinski Grant/research support: Amgen Inc., Merck Serono, Servier, E. Franek Grant/research support: Amgen, Consultant for: Novartis, Speakers bureau: Amgen, MSD, Novartis, Servier, Teva, P. Lakatos Consultant for: Amgen, Lilly, Servier, Speakers bureau: Amgen, Lilly, Merck, Roche, Servier, Teva, C. Mautalen Consultant for: Merck, Servier, S. Minisola Speakers bureau: Abiogen, Amgen, Bruno Farmaceutici, Eli Lilly, Merck Sharp & Dohme, J.-Y. Reginster Grant/research support: Amgen, Boehringer, Bristol Myers Squibb, Chiltern, Danone, Galapagos, GlaxoSmithKline, Lilly, Merck Sharp & Dohme, Novartis, Organon, Pfizer, Roche, Rottapharm, Servier, Teva, Therabel, Theramex, Consultant for: Amgen, Asahi Kasei, GlaxoSmithKline, IBSA-Genevrier, Lilly, Merckle, Negma, Novartis, NPS, Nycomed-Takeda, Roche, Servier, Theramex, UCB, Wyeth, S. Jensen: None declared, N. Daizadeh Shareholder of: Amgen Inc., Employee of: Amgen Inc., A. Wang Shareholder of: Amgen Inc., Employee of: Amgen Inc., M. Gavin Shareholder of: Amgen Inc., Employee of: Amgen Inc., R. Wagman Shareholder of: Amgen Inc., Employee of: Amgen Inc., H. Bone Grant/research support: Amgen, Merck, Novartis, NPS, Consultant for: Amgen, Merck, Novartis, Noven, Sucampo, Tarsa, Speakers bureau: Amgen DOI 10.1136/annrheumdis-2014-eular.1119

Background Prolia has marketing authorization in the EU, US, Canada, Japan, and over 40 countries or administrative districts worldwide for the treatment of postmenopausal women with osteoporosis at high/increased risk for fracture. Since product registration, new data have provided further information about efficacy and safety. In the FREEDOM extension trial, 8 years of continued denosumab (Prolia) therapy was associated with continued increases in bone mineral density and maintenance of low fracture incidence. Rates of adverse events did not increase over time.1 In addition, a rigorous pharmacovigilance program has been established to complement safety data collection from clinical trials. Spontaneous adverse event reports, while often with insufficient information, are the cornerstone of safety surveillance programs and help detect rare and serious adverse drug reactions (ADR). Objectives We characterize post-marketing (PM) experience for 4 ADRs with Prolia: atypical femoral fracture (AFF), osteonecrosis of the jaw (ONJ), severe symptomatic hypocalcemia (SSH), and anaphylaxis. Methods The Amgen PM database undergoes continual assessment of adverse events reported from health care providers, patients, and other sources. AFF and ONJ cases were assessed and adjudicated by independent committees. SSH and anaphylaxis prompted further assessment by Amgen Global Safety because causality due to Prolia could not be excluded. Results As of September 2013, estimated exposure with Prolia was 1,252,566 patient-years. Four PM reports have been adjudicated as consistent with the ASBMR definition for AFF.2 All patients had prior bisphosphonate (BP) use. Two subjects had healing and two did not have follow-up information. For ONJ, 32 PM reports were adjudicated as consistent with the AAOMS definition.3 Risk factors included ≥1: glucocorticoids, chemotherapy, prior BP use, older age, and invasive dental procedures. One-third of reports indicated resolution, 1/3 were ongoing, and the remainder were unknown. Eight reports of SSH included symptoms of seizures and/or tetany; nearly all (7 of 8) had chronic kidney disease, a risk factor for hypocalcemia; most SSH events occurred within 30 days of Prolia administration and responded to IV/PO calcium/vitamin D. For anaphylaxis, 5 reports included hypotension, dyspnea, throat tightness, facial and upper airway edema, pruritus, and/or urticaria. Most events occurred within 1 day of the first Prolia dose; emergency room treatments included antihistamines and IV/PO steroids with no fatal outcomes. Conclusions These PM events with Prolia have not shown any unexpected findings; the benefit/risk profile for Prolia remains favorable. Ongoing safety surveillance will continue in the clinical trial program and pharmacovigilance activities. References Papapoulos et al., JBMR 2013. Shane et al., JBMR 2010. Position Paper, AAOMS 2009. Disclosure of Interest M. Geller Shareholder of: Amgen Inc., Employee of: Amgen Inc., R. Wagman Shareholder of: Amgen Inc., Employee of: Amgen Inc., P. Ho Shareholder of: Amgen Inc., Employee of: Amgen Inc., S. Siddhanti Shareholder of: Amgen Inc., Employee of: Amgen Inc., C. Stehman-Breen Shareholder of: Amgen Inc., Employee of: Amgen Inc., N. Watts Shareholder of: OsteoDynamics, Grant/research support: Merck, NPS, Consultant for: AbbVie, Amarin, Amgen, Bristol-Meyers Squibb, Corcept, Endo, Imagepace, Janssen, Lilly, Merck, Novartis, Noven, Novo Nordisk, Pfizer/Wyeth, Quark, Radius, sanofi-aventis, S. Papapoulos Consultant for: Amgen, Axsome, Gador, GSK, Merck, Speakers bureau: Amgen, Eli Lilly, GSK, Merck, Novartis DOI 10.1136/annrheumdis-2014-eular.1170

Preterm neonates frequently develop nephrocalcinosis (NC). However, the cause has not yet been elucidated. This study focuses on the effects of urine from preterm neonates on crystallization kinetics. Urine samples were collected and renal ultrasound examinations of preterm neonates (gestational age32 weeks) were performed during the first weeks of life, at term, and ages 6, 12, and 24 months. The effect of urine on crystallization was determined using a seeded crystal growth system, which measures the square root of solubility product ( radicalLc), percentage of growth inhibition (GI), and agglomeration inhibition ([tm]) of calcium oxalate crystals. Data for preterm neonates in the first weeks of life (n = 19) were compared with those for full-term neonates (n = 17) and healthy adults. Moreover, the correlation between [tm] and urinary (U)citrate level was studied. Mean radicalLc (0.27 +/- 0.1 versus 0.36 +/- 0.08 mmol/L) and mean [tm] (81 +/- 32 versus 143 +/- 97 minutes) were lower and mean Ucalcium-creatinine (2.20 +/- 1.74 versus 0.46 +/- 0.73 mol/mol) and Uoxalate-creatinine ratios (0.39 +/- 0.21 versus 0.16 +/- 0.09 mol/mol) were greater in preterm neonates in the first weeks of life compared with full-term neonates (p0.05). Furthermore, [tm] was less than the lower limit for healthy adults for all but one preterm neonate; [tm] increased and Ucalcium-creatinine and Uoxalate-creatinine ratios decreased with age (p0.005). There was a correlation between [tm] and citrate excretion (coefficient of 38; P0.001). Patients with and without NC at term did not differ statistically in mean radicalLc, percentage of GI, or [tm]. In conclusion, urine from preterm neonates in the first weeks of life is highly supersaturated and has a defective ability to inhibit calcium oxalate crystal agglomeration. This ability improves with age and is citrate mediated. We suggest that both the high level of supersaturation and defective ability to inhibit calcium oxalate crystal agglomeration contribute to the high incidence of NC.

Interactions between specific cell-surface molecules, which include the urokinase receptor (uPAR) and integrins, are crucial to processes of tumor invasion and metastasis. Here we demonstrate that uPAR and beta1-integrins may cluster at distinct sites at the cell surface of metastatic MDA-MB-231 breast cancer cells and form functional complexes. Attachment assays performed in the presence of a synthetic peptide (p25), which interferes with the formation of uPAR-integrin complexes, reveal that uPAR is able to regulate the adhesive function of integrins in breast cancer cells. On dissociation of the uPAR-integrin complexes by p25, tumor cell attachment to the extracellular matrix was either decreased (vitronectin) or increased (fibronectin). Moreover, the tumor cells display remarkable morphological changes when cultured on fibronectin in the continuous presence of p25, leading to increased cell spreading and attachment. In marked contrast to control conditions, increased cellular adhesion to fibronectin after p25 treatment was entirely beta1-integrin-mediated. The role of uPAR-integrin complexes in tumor progression was studied in an in vivo bone xenograft model. Stably transfected MDA-MB-231 cells that overexpress p25 showed a significant reduction in tumor progression in bone (Por = 0.0001 versus mock-control). In line with these observations, continuous administration of p25 (25 microg/mouse/day, osmotic minipumps) for 28 days resulted in significantly reduced tumor progression of MDA-MB-231 cells in bone (Por = 0.005) when compared to scrambled control peptide. In conclusion, our data demonstrate that uPAR can act as an adhesion receptor in breast cancer and is capable of regulating integrin function. Our findings strongly suggest that adhesive and proteolytic events are tightly associated in metastatic breast cancer cells and that functional integrin-uPAR complexes are involved in tumor progression in vivo.

Bone remodelling accounts for the continuous renewal of the adult skeleton, and its disturbances constitute the pathophysiological basis of most skeletal disorders. Recently identified bone signalling pathways explain the action of known therapeutic agents and provide targets for new developments in the management of bone diseases.

Interleukin-17 (IL-17) is a proinflammatory cytokine produced exclusively by activated memory T cells and has recently been found to stimulate osteoclastic resorption. Like other proinflammatory cytokines, IL-17 may affect osteoclastic bone resorption indirectly via osteoblasts, possibly by mechanisms previously reported for chondrocytes that respond in very similarly to osteoblasts. As in chondrocytes, but only in combination with tumor necrosis factor-alpha (TNF-alpha), IL-17 induced nitric oxide (NO) production in osteoblastic cells and fetal mouse metatarsals by a nuclear factor-kappaB (NF-kappaB)-dependent mechanism. This effect was associated with elevated mRNA levels of the NF-kappaB isoforms RelA and p50. In fetal mouse metatarsals, IL-17 stimulated osteoclastic bone resorption only in combination with TNF-alpha. The pathway by which the cytokine combination exerts this effect was examined using inhibitors of NO synthesis and NF-kappaB activation. Although both inhibitors used abolished NO production, they did not prevent the stimulatory effect of the cytokine combination on osteoclastic resorption. In contrast, the inhibitors slightly increased osteoclastic resorption, suggesting a suppressive rather than stimulatory effect of NO on cytokine-induced bone resorption. In addition, we showed that IL-17 + TNF-alpha stimulated osteoclastic resorption independent of NF-kappaB signaling. To further examine the pathway by which osteoclastic resorption was stimulated, we used osteoprotegerin, a specific inhibitor of the receptor activator of NF-kappaB (RANK)/receptor activator of the NF-kappaB ligand (RANKL) pathway. Osteoprotegerin partially inhibited IL-17 + TNF-alpha-stimulated osteoclastic resorption only at the high concentration of 1000 ng/mL, whereas it completely blocked parathyroid hormone-related peptide-stimulated resorption at 300 ng/mL. In conclusion, IL-17 stimulated NO production by an NF-kappaB-dependent pathway in osteoblastic cells and fetal mouse metatarsals only in combination with TNF-alpha. Neither NO production nor NF-kappaB signaling, and only partly the RANK/RANKL pathway, were involved in the stimulatory effect of the cytokine combination on osteoblastic bone resorption in these long bones, suggesting the existence of other pathways by which osteoclastic resorption can be stimulated.

Fetal mouse metatarsals are well-known models to study cartilage differentiation and osteoclastic resorption. We show here the outgrowth of PECAM-1 positive tubelike structures from the bone rudiments. This feature can be used to study angiogenesis in vitro. The area of outgrowth significantly increased with culture time, as shown by computerized image analysis of PECAM-1 positive tubelike structures. Treatment with recombinant human vascular endothelial growth factor (rhVEGF-A) stimulated the formation of tubelike structures. Treatment of explants with the angiogenesis inhibitor endostatin, the chemokine IP-10, and the thalidomide derivative phatolyl glutamic acid (PG-acid) resulted in an inhibition of the formation of PECAM-1 positive tubelike structures of 48.8% (+/- 4%), 50.2% (+/- 12%), and 80.8% (+/- 3%), respectively. Outgrowth of tubelike structures was partly dependent on endogenous VEGF-A because treatment with anti-mVEGF-A and truncated VEGF receptor 1 (soluble fms-like tyrosine kinase 1, sFIt1) strongly inhibited the formation of tubelike structures 74% (+/- 4%) and 38% (+/- 5%), respectively. Neither onset of tube formation nor total area of tubelike structures were changed when metatarsals were cultured on a fibrin gel or collagen type I gel. Tube formation required activation of matrix metalloproteinases because treatment of the bones with an inhibitor of matrix metalloproteinases completely inhibited migration and tube formation, whereas treatment with an inhibitor of plasmin had no effect. In conclusion, we describe a new in vitro model to study angiogenesis that can be used to test the angiogenic or antiangiogenic potential of novel test compounds that also combines the multicellularity of in vivo assays with the accessibility and flexibility of in vitro assays.

Publisher Summary Bisphosphonates are synthetic compounds that have high affinity for calcium-containing crystals, concentrate preferentially in the skeleton, and affect bone surface related processes. In this chapter, pharmacological issues relevant to their application to the management of postmenopausal osteoporosis are discussed. When given daily, biphosphonates reduce the risk of vertebral fractures. More importantly, alendronate and risedronate are currently the only treatments shown in prospective, controlled studies to decrease the incidence of non vertebral fractures, including those of the hip, in vitamin D- and calcium-replete women with postmenopausal osteoporosis. The issue of dose and mode of bisphosphonate administration was addressed in animal studies. Rats were treated with daily subcutaneous injections of pamidronate and changes in bone resorption and in calcium balance were followed. Treatment suppressed bone resorption dose dependently and increased calcium retention significantly. More importantly, with all doses used, suppression of resorption reached a plateau that was also dose-dependent, but did not decrease further despite the continuous administration of the drugs. These results suggested for the first time that it may be possible to suppress bone resorption mildly and to induce significant increases in calcium balance with low-dose bisphosphonate given daily. Moreover, they demonstrated that the daily administration of the bisphosphonate is not necessarily accompanied by progressive suppression of bone resorption and thus the accumulation of the bisphosphonate in the skeleton is not associated with a cumulative effect on bone metabolism. This pattern of response has now been repeatedly shown in humans treated for prolonged periods with oral bisphosphonates given daily.

Endochondral bone formation is regulated by systemically and locally acting growth factors. A role for vascular endothelial growth factor (VEGF) in this process has recently been proposed, because inactivation of VEGF inhibits endochondral bone formation via inhibition of angiogenesis. Despite the known effect of VEGF as specific endothelial growth factor, its effects on osteoblast differentiation have not been studied. We, therefore, examined the expression of VEGF-A, -B, -C, and -D and their receptors in a model of osteoblast differentiation using the mouse preosteoblast-like cell line KS483. Early in differentiation, KS483 cells express low levels VEGF-A, -B, and -D messenger RNA, whereas during mineralization, KS483 cells express high levels. In addition, expression of the VEGF receptors, VEGFR1, VEGFR2, and VEGF165R/neuropilin, coincided with expression of their ligands, being maximally expressed during mineralization. VEGF-A production during osteoblast differentiation was stimulated by insulin-like growth factor I that enhances osteoblast differentiation and was inhibited by PTH-related peptide that inhibits osteoblast differentiation. Furthermore, continuous treatment of KS483 cells with recombinant human VEGF-A stimulated nodule formation. Although treatment of KS483 cells with soluble FLT1, an agent that blocks binding of VEGF-A and -B to VEGFR1, did not inhibit nodule formation, this observation does not exclude involvement of VEGFR2 in the regulation of osteoblast differentiation. As it is known that VEGF-A, -C, and -D can act through activation of VEGFR2, other isoforms might compensate for VEGF-A loss. The expression pattern of VEGFs and their receptors shown here suggests that VEGFs play an important role in the regulation of bone remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblast differentiation.

Rank (receptor activator of nuclear factor-\kappaB), encoded by the TNFRSF11A gene, is one of the most important proteins in osteoclastogenesis and bone remodeling. Mutations in TNFRSF11A have been reported to cause Paget's disease of bone (PDB)-like diseases (i.e. familial expansile osteolysis, expansile skeletal hyperphosphatasia and early onset PDB) and an osteoclast-poor form of osteopetrosis. Yet, the role of the TNFRSF11A gene in classical PDB has not been investigated in detail. We have conducted an association study in 196 Belgian sporadic PDB patients (83 females and 112 males) and 212 controls (86 females and 126 males). Based on HapMap, 27 tagSNPs and 5 multimarker tests (MMTs) were selected in and around TNFRSF11A. In addition, we have included 1 non-synonymous SNP (H141Y) which is not present in HapMap. Genotyping was carried out by KASPar technique (KBioscience, UK), TaqMan assay and direct sequencing. Statistical analysis indicates that 13 SNPs and 2 MMTs are significantly associated (P-values between 0.037 and 3.17×10−4), the majority of them due to an association in the female subcohort. Six SNPs and 1 MMT withstand the Bonferroni correction (P

Bisphosphonates (Bps), inhibitors of osteoclastic bone resorption, are used in the treatment of skeletal disorders. Recent evidence indicated that farnesyl pyrophosphate (FPP) synthase and/or isopentenyl pyrophosphate (IPP) isomerase is the intracellular target(s) of bisphosphonate action. To examine which enzyme is specifically affected, we determined the effect of different Bps on incorporation of [14C]mevalonate (MVA), [14C]IPP, and [14C]dimethylallyl pyrophosphate (DMAPP) into polyisoprenyl pyrophosphates in a homogenate of bovine brain. HPLC analysis revealed that the three intermediates were incorporated into FPP and geranylgeranyl pyrophosphate (GGPP). In contrast to clodronate, the nitrogen-containing Bps (NBps), alendronate, risedronate, olpadronate, and ibandronate, completely blocked FPP and GGPP formation and induced in incubations with [14C]MVA a 3- to 5-fold increase in incorporation of label into IPP and/or DMAPP. Using a method that could distinguish DMAPP from IPP on basis of their difference in stability in acid, we found that none of the NBps affected the conversion of [14C]IPP into DMAPP, catalyzed by IPP isomerase, excluding this enzyme as target of NBp action. On the basis of these and our previous findings, we conclude that none of the enzymes up- or downstream of FPP synthase are affected by NBps, and FPP synthase is, therefore, the exclusive molecular target of NBp action. Chemicals/CAS: Carbon Radioisotopes; Carbon-Carbon Double Bond Isomerases, EC 5.3.3.-; Dimethylallyltranstransferase, EC 2.5.1.1; Diphosphonates; Enzyme Inhibitors; indolepropanol phosphate, 40716-80-1; Indoles; isopentenyldiphosphate delta-isomerase, EC 5.3.3.2; Mevalonic Acid, 150-97-0

Blomstrand osteochondrodysplasia (BOCD) is a rare lethal skeletal dysplasia characterized by accelerated endochondral and intramembranous ossification. Comparison of the characteristics of BOCD with type I PTH/PTH-related peptide (PTHrP) receptor-ablated mice reveals striking similarities that are most prominent in the growth plate. In both cases, the growth plate is reduced in size due to a strongly diminished zone of resting cartilage and the near absence of columnar arrangement of proliferating chondrocytes. This overall similarity suggested that an inactivating mutation of the PTH/PTHrP receptor might be the underlying genetic defect causing BOCD. Indeed, inactivating mutations of the PTH/PTHrP receptor have been recently identified in two cases of BOCD.We describe here a novel inactivating mutation in the PTH/PTHrP receptor. Sequence analysis of all coding exons of the type I PTH/PTHrP receptor gene and complementary DNA of a case with BOCD identified a homozygous point mutation in exon EL2 in which one nucleotide (G at position 1122) was absent. The mutation was inherited from both parents, supporting the autosomal recessive nature of the disease. The missense mutation resulted in a shift in the open reading frame, leading to a truncated protein that completely diverged from the wild-type sequence after amino acid 364. The mutant receptor, therefore, lacked transmembrane domains 5, 6, and 7; the connecting intra- and extracellular loops; and the cytoplasmic tail. Functional analysis of the mutant receptor in COS-7 cells and of dermal fibroblasts obtained from the case proved that the mutation was indeed inactivating. Neither the transiently transfected COS-7 cells nor the dermal fibroblasts responded to a challenge with PTH or PTHrP with a rise in intracellular cAMP levels, in sharp contrast to control cells. Our results provide further evidence that BOCD is caused by inactivating mutations of the type I PTH/PTHrP receptor and underscore the importance of this receptor in mammalian skeletal development

Interleukin-17 (IL-17) is a recently cloned cytokine that is exclusively produced by activated T cells, but its receptor has been found on several cells and tissues. Like other proinflammatory cytokines produced by activated T cells, IL-17 may affect osteoclastic resorption and thereby mediate bone destruction accompanying some inflammatory diseases. In the present study, we investigated whether osteogenic cells possess the receptor for IL-17 (IL-17R) and whether IL-17 affects osteoclastic resorption. We found that IL-17R mRNA is expressed both in mouse MC3T3-E1 osteoblastic cells and fetal mouse long bones, suggesting that osteogenic cells may be responsive to IL-17. In fetal mouse long bones, IL-17 had no effect on basal and IL-1beta-stimulated osteoclastic bone resorption, but when given together with tumor necrosis factor-alpha (TNF-alpha) it increased bone resorption dose dependently in serum-free conditions. In addition, IL-17 increased TNF-alpha-induced IL-1alpha, IL-1beta, and IL-6 mRNA expression in fetal mouse metatarsals and IL-1alpha and IL-6 mRNA expression in MC3T3-E1 cells. In conclusion, IL-17R mRNA was expressed by mouse osteoblastic cells and fetal mouse long bones, and IL-17 in combination with TNF-alpha, but not IL-1beta, increased osteoclastic resorption in vitro. IL-17 may therefore affect bone metabolism in pathological conditions characterized by the presence of activated T cells and TNF-alpha production such as rheumatoid arthritis and loosening of bone implants.

Bisphosphonates, synthetic compounds used in the treatment of skeletal disorders, suppress osteoclast-mediated bone resorption by a yet unidentified mechanism. Previous studies showed that some bisphosphonates can inhibit enzymes of the mevalonate pathway, and nitrogen-containing bisphosphonates inhibit protein prenylation in mouse macrophages. In the present study, we examined the involvement of the mevalonate pathway in basal and bisphosphonate-inhibited osteoclastic resorption in fetal mouse long bone explants, an experimental model representative of the in vivo action of bisphosphonates. Mevastatin inhibited bone resorption at concentrations similar to those of the potent bisphosphonate ibandronate. This effect could be totally reversed by the addition of mevalnate and geranylgeraniol but not farnesol. The first two intermediates but not the latter could also stimulate basal bone resorption. The inhibitory effect of ibandronate on bone resorption could be totally reversed by the addition of geranylgeraniol and to a small extent only by mevalonate and farnesol, indicating that the bisphosphonate acts at a level of the mevalonate pathway different from that of mevastatin. Histologic sections of ibandronate-treated bone explants showed further rescue of functioning osteoclasts during concomitant treatment with geranylgeraniol. Finally, the reversibility of bisphosphonate inhibited osteoclastic resorption by geranylgeraniol was also demonstrated for the potent nitrogen-containing bisphosphonates alendronate, olpadronate, and risedronate but not for the non-nitrogen-containing bisphosphonates clodronate and etidronate. These studies demonstrate that protein geranylgeranylation but not farnesylation is important for osteoclast-mediated bone resorption and that nitrogen-containing bisphosphonates exert their antiresorptive action probably by affecting enzymes of the mevalonate pathway involved in the generation of geranylgeranyl pyrophosphate.

This chapter focuses on pharmacological issues relevant to the clinical application of bisphosphonates and to their use in the management of osteoporosis. Geminal bisphosphonates are synthetic compounds; they are analogs of natural pyrophosphosphate in which the oxygen has been replaced by a carbon. During early experiments bisphosphonates were shown to inhibit bone resorption, an observation that initiated their development as treatments for skeletal disorders. The effect of bisphosphonates on bone resorption is determined primarily by the structure of the R 2 side chain of the molecule, which is also referred to as the “bioactive moeity.” The mechanism of antiresorptive action of bisphosphonates has been a subject of intensive research for many years, but their molecular mechanism of action remained unknown until recently. Bisphosphonates share a number of pharmacological properties: low intestinal absorption, rapid elimination from the circulation, selective uptake by the skeleton, lack of circulating metabolites, renal excretion, and long skeletal retention. All bisphosphonates, independently of mode or route of administration, have been shown to significantly increase bone mineral density, predominantly of trabecular bone.

To define structure-activity relationships for bisphosphonate activity, we examined the bone binding and antiresorptive properties of heterocycle-containing analogs of risedronate, a pyridylbisphosphonate, in cultures of mouse fetal bone explants. Our studies indicated that hydroxybisphosphonates with the nitrogen molecule in the pyridyl ring were very potent inhibitors of osteoclastic resorption. Changing the place of the nitrogen in the ring structure of risedronate or its methylation did not significantly alter antiresorptive potency in relation to risedronate. Extension of the R2 chain, however, reduced efficacy. In binding experiments, we found that all heterocyclic bisphosphonates with a hydroxyl group in R1 had comparable affinity for bone mineral and inhibited calcium incorporation into bone explants to a similar extent. The affinity of a risedronate analog without R1 was markedly reduced. We also examined the properties of a risedronate analog (NE-10790) belonging to the group of phosphonocarboxylates in which one of the phosphonate groups is substituted by a carboxyl group. NE-10790 had strongly reduced binding affinity, but still retained some antiresorptive activity. Interestingly, the continuous presence of NE-10790 in cultures of fetal mouse metacarpal bones increased its antiresorptive efficacy by about 40-fold compared with 24 h preincubation, whereas, under the same conditions, the potency of high-affinity hydroxybisphosphonates did not change or only slightly increased. This may be explained by the differences in pharmacokinetic behavior between compounds of high and of low affinity for bone mineral. These data show that, as with alkylbisphosphonates, heterocycle-containing bisphosphonates with a nitrogen functionality in the R2 chain are potent antiresorptive agents and a hydroxyl substitution in the R1 chain confers high affinity for bone mineral, probably due to tridentate configuration. The group of phosphonocarboxylates, with strongly reduced bone affinity, provides an interesting therapeutic option.

To the Editor: Bisphosphonates are widely used in the management of osteoporosis. They are cleared rapidly from the circulation, with about half the administered dose taken up by the skeleton and t...

Replacing the hydroxyl group in the bone-binding site of three clinically useful bisphosphonates (etidronate, pamidronate, and olpadronate) by an amino group resulted in great differences in their antiresorptive potencies in vitro. In the present study, this is also shown in vivo in mice treated with the six bisphosphonates at doses of up to 16 microM/kg/day for 12 days. Because binding to bone mineral is nearly the same for all tested bisphosphonates, these findings suggest that the aminosubstitution affects the cellular action of the bisphosphonates. This was tested in the cellular slime mould Dictyostelium discoideum in which cellular effects of bisphosphonates can be examined independently of binding to bone mineral. Etidronate and its aminosubstituted analog were equipotent in inhibiting amebal growth, while pamidronate was somewhat more potent than its analog. Whereas olpadronate was a potent inhibitor of axenic growth of Dictyostelium amebae, the aminosubstitution reduced its potency drastically (IC50 12 microM and 700 microM, respectively). The similarities between the inhibitory effects of the bisphosphonates tested on bone resorption in vitro and in vivo and on the growth of Dictyostelium amebae confirm that the differences in antiresorptive potencies found reflect differences in cellular effects and suggest that bisphosphonates may bind to more than one intracellular target.

Osteoporosis in children and adolescents is relatively uncommon and usually secondary to identifiable causal factors. There are no generally accepted therapies for patients with no treatable underlying cause of disease. Any treatment of young patients with bone-acting compounds should be not only effective but also devoid of adverse effects on bone growth and remodeling. For many years we have been studying the effects of bisphosphonates-an effective treatment of postmenopausal osteoporosis-on the growing skeleton. We review here our experience in the treatment of young patients with osteoporosis with special emphasis on issues of skeletal safety and effectiveness, and we discuss the available literature data. We studied 12 patients aged between 10.7 and 17.2 years with symptomatic osteoporosis and multiple fractures treated with the bisphosphonates pamidronate or olpadronate for periods between 2 and 8 years continuously. Linear growth continued normally on treatment; there was even a catch-up growth in prepubertal patients, and there was no excessive suppression of bone remodeling, assessed biochemically. Bone biopsies obtained at various stages during treatment showed bone of normal lamellar structure without mineralization defects. There was an increase in calcium balance, already evident within 10 days, the level of which was maintained for at least 3 years of treatment. This was associated with progressive increases in bone mineral density along a different slope from that of healthy peers as well as correction of vertebral deformities on X-rays in patients given bisphosphonates before puberty. Treatment was well tolerated and clinical improvement was remarkable. Our studies, supported by literature data, strongly suggest that bisphosphonate therapy can be beneficial to young patients with osteoporosis for whom no other options are currently available, and justify planning controlled studies in more common conditions for which no treatment is currently available, such as osteogenesis imperfecta.

Hypercalcemia is a well known complication of renal cell carcinoma (RCC). As RCCs can produce IL-6, and IL-6 may stimulate bone resorption and cause mild hypercalcemia, we examined whether IL-6 is involved in renal cancer-associated hypercalcemia in vivo. Three human renal cell carcinoma tumor lines (RC-8, RC-9, and NC-65) growing in nude mice were studied. Tumors were implanted sc, and parameters of bone metabolism and serum human IL-6 levels were determined in relation to tumor volume (TV). All three tumor lines secreted human IL-6, although in different quantities. The maximum level of IL-6 in RC-8 was 434 pg/ml (TV, 200 mm3), that in RC-9 was 81 pg/ml (TV, 1800 mm3), and that in NC-65 was 2368 pg/ml (TV, 1800 mm3). Hypercalcemia developed in RC-8 and RC-9 tumor-bearing animals, but not in NC-65-bearing animals. The hypercalcemia in both RC-8 and RC-9 tumor lines was associated with elevated levels of PTH-related peptide (PTHrP) and loss of trabecular bone volume. Serum calcium and phosphate concentrations showed an almost linear relationship with plasma PTHrP independently of the tumor line and serum IL-6 levels. No hypercalcemia occurred in the NC-65 animals, which had the highest levels of IL-6, but no detectable plasma PTHrP and PTHrP messenger RNA expression in the tumor. Administration of neutralizing antibodies to IL-6 to RC-8 animals normalized serum calcium concentrations and PTHrP values and induced a significant inhibition of tumor growth. No such effect on tumor growth of anti-IL-6 was seen in the other two tumor lines. The normalization of serum calcium in RC-8 mice is most likely attributed to the growth-inhibiting effect of anti-IL-6 on RC-8 tumor. We conclude that IL-6 secreted by RCC does not contribute directly to hypercalcemia, but may enhance hypercalcemia by stimulating the tumor growth of a subpopulation of PTHrP-secreting carcinomas.

Bisphosphonates suppress bone resorption by inhibiting the activity of mature osteoclasts as well as the formation of osteoclasts from bone marrow-derived precursor cells. It is not yet known at which level of osteoclast development this latter action is exerted and whether this is due to a systemic effect of circulating bisphosphonate or to an action at the bone surface, given the property of these compounds to concentrate specifically at active bone sites. We addressed these questions in an ex vivo study in mice. The animals were treated with the bisphosphonate alendronate for various periods, and the central compartment of the bone marrow was isolated and cultured together with osteoclast-free fetal mouse bone explants. In this way the capacity of bone marrow cells, exposed previously to alendronate in vivo, to generate osteoclasts and induce resorption in vitro was examined. Alendronate (0.25 mg/kg, subcutaneously) given to mice for periods up to 4 weeks suppressed bone resorption, as expected, but did not affect the capacity of bone marrow cells to develop into mature osteoclasts and resorb the calcified matrix of bone explants. In addition, using monoclonal antibodies specific for different macrophage-granulocyte precursor subsets, we found that alendronate treatment did not affect the pattern of antigen expression of bone marrow cells, which is in line with the lack of an effect of the bisphosphonate on the ability of bone marrow cells to induce resorption in vitro. In contrast, in control experiments, lipopolysaccharide (0.1 mg) treatment induced marked changes in the pattern of antigen expression of bone marrow cells. In conclusion, our studies demonstrate that the inhibition of bone resorption by alendronate does not involve alteration of the osteoclastogenic potential of osteoclast progenitors (precursors) from the central bone marrow compartment. Moreover, this treatment did not alter the expression of markers specific for monocyte-macrophage precursors as well as mature macrophages. These results suggest that the effects of alendronate are exerted at the bone surface at late osteoclast precursors (and mature osteoclasts).

Paget's disease of bone is a focal disorder of bone remodelling due to abnormally increased osteoclast-mediated bone resorption. It rarely presents before the age of 35 years and its prevalence increases with age affecting 2–5% of the population above 50 years, making Paget's disease the most common skeletal disorder after osteoporosis. Its aetiology is not known but available evidence favours an infection of genetically predisposed individuals with a paramyxovirus. Affected bones change in shape, size and direction causing considerable morbidity but the majority of patients are asymptomatic. In Paget's disease increased bone resorption is tightly linked to increased bone formation which are reflected in the proportional increases in biochemical indices of bone turnover. Because the primary abnormality lies in the osteoclasts, inhibitors of bone resorption are used for its therapy and bisphosphonates are currently the treatment of choice. These decrease bone turnover effectively and their effect lasts after stopping treatment. With available bisphosphonates clinical and biochemical, long-lasting, remissions can be obtained in the majority of patients. Symptomatic disease and preparation for orthopaedic surgery are no longer the only indication for treatment, but asymptomatic patients with localizations at sites likely to induce complications should be considered candidates for bisphosphonate therapy.

Withdrawal of gender steroids in both women and men is associated with an increase in bone turnover with bone resorption exceeding bone formation leading to bone loss. To further investigate this process, the osteoclastogenic potential of mouse bone marrow cells was assessed at different time-points after ovariectomy (ovx) or orchidectomy (orx). Cocultures of osteoclast-free fetal mouse long bones together with bone marrow from ovariectomized or orchidectomized mice indicated that the withdrawal of gender steroids in female and male mice induces a transient increase in osteoclastogenesis. The osteoclastogenic potential of the bone marrow cells was increased 7 days after ovx or orx. However, osteoclastic resorption was not increased at 3 days after surgery and had normalized 30 days after either ovx or orx. These results suggest that the withdrawal of gender steroids induces a transient increase in osteoclastogenesis in mice of both genders, which is associated with the early phase of rapid bone loss.

Background Denosumab (DMAb) is approved for the treatment of postmenopausal women with osteoporosis at increased risk for fracture. A favorable risk/benefit profile was demonstrated in the pivotal, 3-year FREEDOM trial.1 The open-label, active-treatment FREEDOM extension study is evaluating the long-term efficacy and safety of DMAb for up to 10 years. Objectives Report results from the first 3 years of the extension, representing up to 6 years of DMAb treatment. Methods In the extension, each woman is scheduled to receive 60 mg DMAb every 6 months and supplemental calcium and vitamin D daily. For women given placebo during FREEDOM, the data here reflect 3 years of DMAb exposure (cross-over group). For women given DMAb during FREEDOM, the data reflect 6 years of DMAb exposure (long-term group). Results There were 5928 women eligible for the extension. Of these, 4550 (77%) enrolled (2207 cross-over; 2343 long-term). In the first 3 years of DMAb treatment during the extension, the cross-over group had significant gains in bone mineral density (BMD) at the lumbar spine (9.4%) and total hip (4.8%), comparable to those observed in the long-term DMAb group during the first 3 years of FREEDOM (lumbar spine, 10.1%; total hip, 5.7%). In the long-term group, further significant increases in BMD occurred for cumulative 6-year gains of 15.2% at the lumbar spine and 7.5% at the total hip. After the 1st (cross-over) or 7th (long-term) DMAb dose, sCTX was rapidly and similarly reduced with the characteristic attenuation at the end of the dosing period. In the cross-over group, yearly incidences of nonvertebral, new vertebral, clinical vertebral, and clinical fractures were lower than those in the FREEDOM placebo group. Fracture incidence remained low in the long-term group. Incidences of adverse events (AEs) and serious AEs did not increase over time with DMAb. Two subjects in each group had AEs adjudicated to ONJ. Both cases in the cross-over group healed without further complications; one subject continues to receive DMAb. One case in the long-term group healed, while the other continues to be followed. There were no atypical femur fractures. Conclusions DMAb treatment for 3 years in the cross-over group reproduced FREEDOM observations. DMAb treatment for 6 years (long-term group) continued to significantly increase BMD, maintained reduced bone turnover, and remained well tolerated. Fracture incidence remained low. References Cummings et al. N Engl J Med. 2009;361:756 Disclosure of Interest R. Chapurlat Grant/Research support from: Pfizer, Amgen, Servier, Consultant for: Amgen, Servier, Novartis, Roche, Lilly, S. Papapoulos Consultant for: Amgen, Eli Lilly, GSK, Merck, Novartis, J. Brown Grant/Research support from: Abbott, Amgen, Bristol-Myers Squibb, Eli Lilly, Merck, Novartis, Pfizer, Roche, sanofi-aventis, Servier, Warner Chilcott, Consultant for: Amgen, Eli Lilly, Merck, Novartis, sanofi-aventis, Warner Chilcott, Speakers Bureau: Amgen, Eli Lilly, Novartis, N. Franchimont Shareholder of: Amgen, Employee of: Amgen, M. Brandi Consultant for: Amgen, Servier, MSD, Roche, Glaxo, Eli Lilly, Nycomed, NPS, Pfizer, E. Czerwinski Grant/Research support from: Amgen, AstraZeneca, Danone Research, Eli Lilly, Merck Serono, Merck Sharp & Dohme, Novartis, Pfizer, Roche, Servier, M.-A. Krieg: None Declared, Z. Man Grant/Research support from: Merck, Amgen, Lilly, Consultant for: Novartis, Merck, Sanofi aventis, D. Mellstrom: None Declared, S. Radominski Grant/Research support from: BMS, Pfizer, Amgen, Roche, Novartis, Speakers Bureau: Aventis, Lilly, Novartis, J.-Y. Reginster Grant/Research support from: BMS, Merck Sharp & Dohme, Rottapharm, Teva, Lilly, Novartis, Roche, GSK, Amgen, Servier, Consultant for: Servier, Novartis, Negma, Lilly, Wyeth, Amgen, GSK, Roche, Merckle, Nycomed, NPS, Theramex, UCB, H. Resch Consultant for: MSD, Lilly, Amgen, Sankyo, Roche, Servier, J. Roman Grant/Research support from: Roche, MSD, Consultant for: Actelion, Pfizer, C. Roux Grant/Research support from: Amgen, Novartis, Consultant for: Amgen, Novartis, MSD, N. Daizadeh Shareholder of: Amgen, Employee of: Amgen, M. Geller Shareholder of: Amgen, Employee of: Amgen, S. Smith Shareholder of: Amgen, Employee of: Amgen, R. Wagman Shareholder of: Amgen, Employee of: Amgen, S. Cummings Consultant for: Amgen, H. Bone Grant/Research support from: Merck, Amgen, Nordic Bioscience, Novartis, Takeda, Tarsa, Consultant for: Merck, Amgen, Takeda, Tarsa, Speakers Bureau: Amgen, Pfizer.

The purpose of this study was to examine the role of the R1 moiety of bisphosphonates in binding to bone mineral and for antiresorptive action. For this, the R1 chain of three clinically useful hydroxybisphosphonates (etidronate, pamidronate, and olpadronate) was substituted with an amino group. The effects of the aminosubstituted bisphosphonates were compared with those of their hydroxy counterparts in a crystal growth assay and in fetal mouse long bone cultures which are representative of bisphosphonate actions in vivo. It was found that all three aminosubstituted compounds and their hydroxy analogs bound with similar affinity to bone mineral and inhibited the growth of calcium oxalate crystals to the same extent. Surprisingly, the antiresorptive effect of olpadronate was totally abolished by the amino substitution of the hydroxyl group while that of pamidronate was reduced by about six-fold and that of etidronate did not change. These studies demonstrate the involvement of the entire bisphosphonate molecule in the cellular mechanism of antiresorptive action. In addition, the aminosubstituted analog of olpadronate, which lacks any antiresorptive action but retains all other properties of olpadronate, provides an excellent tool for the study of specific cellular effects involved in bisphosphonate action.

Bisphosphonates are used with increasing frequency in the management of skeletal complications in patients with breast cancer. In this paper, we have investigated whether bisphosphonates, besides their known beneficial effects on tumor-associated osteoclastic resorption, are capable of inhibiting breast cancer cell adhesion to bone matrix. For that we used two in vitro models for bone matrix (cortical bone slices and cryostat sections of trabecular bone from neonatal mouse tails). Four bone matrix-bound nitrogen-containing bisphosphonates (pamidronate, olpadronate, alendronate, and ibandronate) inhibited adhesion and spreading of breast cancer cells to bone dose-dependently, whereas etidronate and clodronate had little or no effect. Strikingly, the relative order of potency of the bisphosphonates in inhibiting the adhesion of cancer cells to cortical and trabecular bone corresponded to their relative antiresorptive potencies in vivo as well as their ranking in in vitro bone resorption assays with predictive value for their clinical efficacy. It appears that nitrogen-containing bisphosphonates alter selectively the adhesive properties of the extracellular bone matrix preventing the attachment of breast cancer cells to it. Besides the beneficial effects of bisphosphonates on tumor-induced osteoclastic resorption, the previously unrecognized effect presented in this paper makes these agents suitable for earlier pharmacologic intervention in patients with breast cancer at risk of developing bone metastases.

Bisphosphonates are used with increasing frequency in the treatment of patients with osteoporosis. Continuous administration of low doses of nitrogen-containing bisphosphonates by mouth is the preferred mode of therapy. The skeletal half-life of bisphosphonates is long, however, and little is known about their long term effects on skeletal metabolism. We examined the changes in biochemical parameters of bone turnover [serum alkaline phosphatase and urinary hydroxyproline (OHP)], in bone mineral density, and in fracture frequency after discontinuation of long term (mean, 6.5 yr, range, 5-9 yr) therapy with oral pamidronate (150 mg/day) in 30 patients with osteoporosis and vertebral fractures. Serum alkaline phosphatase and urinary OHP were significantly lower at the end of long term treatment (90% and 72% of basal values, respectively). Serum alkaline phosphatase had increased to basal values within 6 months of stopping treatment, whereas OHP increased significantly to a maximum average of 92% of pretreatment values. There was no change in the every 6-month bone mineral density measurements of the lumbar spine and the femoral neck during the 2 yr after stopping treatment. Spine fracture index, calculated by the method of Raymakers and co-workers, was 0.83 +/- 0.12 before treatment, 0.85 +/- 0.12 at the end of treatment, and 0.85 +/- 0.13 2 yr after stopping treatment (nonsignificant). There was also no significant change in the rate of new vertebral fractures on or up to 2 yr after stopping treatment (48.5 of 1000 and 46.5 of 1000 patient yr, respectively). Our data demonstrate that the sustained suppression of bone turnover induced by long term treatment with pamidronate is readily reversible on stopping treatment. The beneficial effect of this treatment regimen on the skeleton, however, appears to be maintained for at least 2 yr after discontinuation of treatment.

The mechanism of bone loss following cessation of ovarian function is still unclear. Several studies have shown an increase in bone turnover following natural or surgical menopause which could be prevented by estrogen administration. However, a direct effect of estrogen on osteoclast-mediated bone resorption has been difficult to demonstrate in vitro. Recent evidence suggested that estrogen withdrawal stimulates the production of bone resorbing cytokines, (e.g., interleukin-6, IL-6), which regulate osteoclast formation in the bone marrow microenvironment. We studied the effects of 17beta-estradiol on osteoclastic resorption, measured as 45calcium release, in vitro using cultures of fetal mouse long bone explants in which different stages of osteoclast development and activity are represented. 17beta-estradiol (10(-12)-10(-8) M) had no effect on basal or parathyroid hormone (PTH)-stimulated resorption of bone explants in which mature osteoclasts (radii/ulnae) or osteoclast precursors/progenitors (metacarpals) are present. 17beta-estradiol, however, inhibited significantly the PTH-stimulated resorption of osteoclast-free metacarpals cultured together with mouse fetal liver as a source of early osteoclast progenitors; basal resorption was also not inhibited in this system. In ex vivo studies we further examined the effects of culturing bone marrow cells from ovariectomized (OVX) or sham-operated mice as an osteoclastic source together with osteoclast-free metacarpals on 45calcium release and bone histology. Cocultures of the bone marrow cells from OVX mice with osteoclast-free metacarpals increased significantly the osteoclast formation and subsequent osteoclastic resorption compared with control cocultures. This increase in resorption was prevented by either treatment of the OVX animals with estrogen for 1 week starting immediately after OVX or injection of the OVX animals with an IL-6 neutralizing antibody. We conclude that estrogens suppress the increased osteoclastic resorption induced by PTH or OVX through an effect on hematopoietic progenitor cells of the osteoclast lineage. Furthermore our data suggest that IL-6 is involved in the increase in osteoclastic resorption following OVX.

Bisphosphonates, synthetic compounds that are taken up preferentially by the skeleton and that suppress bone resorption, are currently used in the management of patients with osteoporosis. Long-term uninterrupted administration of low oral doses is the preferred mode of treatment in current clinical trials with newer bisphosphonates. These compounds have, however, a long residence time in the skeleton, and there is no information about their long-term effects on blood calcium homeostasis. We examined the effect of long-term therapy with oral bisphosphonate on blood calcium homeostasis following an acute hypocalcemic stimulus. Twenty patients with vertebral osteoporosis (10 untreated controls and 10 treated with oral pamidronate, 150 mg/day for at least 5 yr) were given intravenous infusions of sodium EDTA, and the concentrations of calcium and PTH in blood were followed for 24 h. Serum calcium concentrations decreased similarly in both groups (maximum decrease 0.21 mmol/L and 0.22 mmol/L, respectively). The recovery of serum calcium concentrations was identical in both groups, and all patients had normal concentrations at 24 h. Plasma PTH increased to a peak of 17.3 +/- 2.5 pmol/L in the control group and to 17.0 +/- 3.1 pmol/L in the pamidronate-treated patients. During the whole study period, there was no difference in either the peak PTH response or in the recovery of plasma PTH values between the two groups. However, when only PTH responses between 60 min and 24 h were examined, there were differences between the two groups. Plasma PTH values, although strictly within the normal range, were significantly higher in the pamidronate-treated patients (P = 0.001). There were no differences in the calcemic responses during this period. Further, there were no detectable changes in immunoreactive PTH-related protein in either group after the EDTA infusions. In conclusion, our study showed that longterm therapy with oral pamidronate does not affect the calcemic response to an acute hypocalcemic stimulus in patients with osteoporosis.

The plasminogen activator (PA)/plasmin system is thought to be involved in processes such as tumor invasion and wound healing, during which epithelial and mesenchyznal cells come close together. However, information on regulation of the PA/plasmin system during epithelial-mesenchymal interactions is scarce. Therefore, we examined the in vitro modulation of the production and activity of the components of the PA/plasmin system in squamous carcinoma cells (SCC-4) and normal human keratinocytes in relation to cell density and the presence or absence of fibroblasts (3T3 cells). There was an inverse relation between cell density and mRNA expression for urokinase-type plasminogen activator (u-PA) and u-PA receptor in both SCC-4 cells and keratinocytes. In addition, such a relation was found for plasminogen activator inhibitor types 1 (PAI-1) and 2 (PAI-2) In SCC-4 monocultures, but not in keratinocyte monocultures. In contrast to monocultures, variation of cell density did not affect the mRNA expression of the components of the PA/plasmin system in co- cultures of SCC-4 cells or keratinocytes with 3T3 cells. However, the relative expression of mRNAs in co-cultures was clearly different from that in mono- cultures, especially at low cell density. For most of the components of the PA/plasmin system, a decrease in mRNA expression and u-PA receptor protein was observed at most cell densities, whereas for PAI-1 only in keratinocytes a marked increase was documented. Zymography of supernatants revealed that the levels of both free u-PA and PA-PAI were increased in SCC-4/3T3 co-cultures, whereas in keratinocyte/3T3 co-cultures, only levels of the PA-PAI complex were increased, while the amount of free u-PA activity decreased, This occurred despite the increased u-PA immunoreactivity and was probably caused by the markedly elevated levels of immunoreactive PAI-1. The results of the present study reveal that the production and synthesis of various components of the PA/plasmin system in keratinocytes of SCC-4 cells depend on the density of epithelial cells and are modulated by fibroblasts, probably through a direct cell-cell or cell-matrix contact. Fibroblast-induced modulations are similar in keratinocytes and SCC-4 cells except for the regulation of PAI-1, which is markedly enhanced only in keratinocytes. This suggests that the modulation of PA activity in the direct microenvironment may be different under physiologic and pathologic conditions.

We investigated the structural requirements for the binding of bisphosphonates to bone mineral and the relation between their affinity for bone and their effects on bone resorption in vitro. For this we used fetal mouse long bones in culture and bisphosphonates with variable R1 and R2 structures. In addition, we studied the effect of structural differences in the incorporation of calcium into bone. We found that bisphosphonates containing a hydroxyl group in the R1 position have the highest affinity for bone mineral. This was related to their capacity to inhibit the incorporation of calcium into long bones but not to their antiresorptive potency. The latter was primarily determined by R2. Furthermore, the effect of bisphosphonates on calcification, but not on resorption of bone explants, was mainly determined by the mode of addition. The continuous presence of bisphosphonate during culture inhibited calcification even at very low concentrations, but short incubation of the bones with relatively high concentrations had no effect. This is probably a result of differences in the availability of the compound to the process of calcification. Because, in vivo, the more potent bisphosphonates inhibit resorption without adversely affecting mineralization of the skeleton and they disappear rapidly from the circulation after administration, we suggest that cultures of bone explants incubated with bisphosphonates for short times rather than cultures in which the drugs are continuously present provide more accurate information about the in vivo effect of these compounds on both resorption and calcification.