Incidence of fractures of the femur, including subtrochanteric, up to 8 years since initiation of oral bisphosphonate therapy: a register-based cohort study using the US MarketScan claims databases.

UNLABELLED: In a cohort study of users of bisphosphonates, we evaluated the incidence of fragility fractures at all sites on the femur following for up to 8 years of therapy with alendronate or risedronate. We did not find evidence for a reversal of fracture protection with long-term use of bisphosphonates. INTRODUCTION: Few studies have acquired adequate data with prolonged follow-up on bisphosphonate users in the general population to evaluate their long-term effects on the risk of hip fractures including those in the subtrochanteric region. METHODS: This cohort study utilizes a large USA database (January 1, 2000 to June 30, 2009). We compared patients with higher versus lower degrees of compliance [medication possession ratio, MPR <1/3 (the reference), 1/3-<2/3, or ≥ 2/3]. Radiographic adjudication of fracture site and features were not performed. Hazard ratios (HR) for fracture were estimated using time-dependent Cox models. Restricted cubic splines (RCS) were used to plot HRs for fracture against duration of therapy. RESULTS: There were 3,655 incident cases of femoral fracture (764 subtrochanteric/shaft, 2,769 hip) identified during 917,741 person-years of follow-up (median = 3 years) on 287,099 patients (267,374 were women) from the date when they initiated oral bisphosphonate therapy. The corresponding HRs (95% confidence interval, CI) for overall femoral fractures associated with each additional year of therapy were 0.93 (0.86-1.01) within 5 years, and 0.89 (0.77-1.03) beyond 5 years for risedronate and 0.86 (0.81-0.91) and 0.95 (0.84-1.07) for alendronate, respectively. The corresponding estimates for subtrochanteric/shaft fractures were 1.05 (0.87-1.26) and 0.89 (0.60-1.33) for risedronate and 0.99 (0.92-1.05) and 1.05 (0.92-1.20) for alendronate, respectively. The HRs (95% CI) for overall femoral fractures associated with each additional year of alendronate or risedronate therapy within 5 and beyond 5 years were not significantly different. CONCLUSION: Our study showed persistence of overall hip fracture protection with long-term use of alendronate or risedronate.

Risedronate in adults with osteogenesis imperfecta type I: increased bone mineral density and decreased bone turnover, but high fracture rate persists.

UNLABELLED: Bisphosphonates can increase bone mineral density (BMD) in children with osteogenesis imperfecta (OI). In this study of adults with OI type I, risedronate increased BMD at lumbar spine (but not total hip) and decreased bone turnover. However, the fracture rate in these patients remained high. INTRODUCTION: Intravenous bisphosphonates given to children with OI can increase BMD and reduce fracture incidence. Oral and/or intravenous bisphosphonates may have similar effects in adults with OI. We completed an observational study of the effect of risedronate in adults with OI type I. METHODS: Thirty-two adults (mean age, 39 years) with OI type I were treated with risedronate (total dose, 35 mg weekly) for 24 months. Primary outcome measures were BMD changes at lumbar spine (LS) and total hip (TH). Secondary outcome measures were fracture incidence, bone pain, and change in bone turnover markers (serum procollagen type I aminopropeptide (P1NP) and bone ALP). A meta-analysis of published studies of oral bisphosphonates in adults and children with OI was performed. RESULTS: Twenty-seven participants (ten males and seventeen females) completed the study. BMD increased at LS by 3.9% (0.815 vs. 0.846 g/cm(2), p = 0.007; mean Z-score, -1.93 vs. -1.58, p = 0.002), with no significant change at TH. P1NP fell by 37% (p = 0.00041), with no significant change in bone ALP (p = 0.15). Bone pain did not change significantly (p = 0.6). Fracture incidence remained high, with 25 clinical fractures and 10 major fractures in fourteen participants (0.18 major fractures per person per year), with historical data of 0.12 fractures per person per year. The meta-analysis did not demonstrate a significant difference in fracture incidence in patients with OI treated with oral bisphosphonates. CONCLUSIONS: Risedronate in adults with OI type I results in modest but significant increases in BMD at LS, and decreased bone turnover. However, this may be insufficient to make a clinically significant difference to fracture incidence.

Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy.

In human breast carcinomas, overexpression of the macrophage colony-stimulating factor (CSF-1) and its receptor (CSF-1R) correlates with poor prognosis. To establish if there is a causal relationship between CSF-1 and breast cancer progression, we crossed a transgenic mouse susceptible to mammary cancer with mice containing a recessive null mutation in the CSF-1 gene (Csf1(op)) and followed tumor progression in wild-type and null mutant mice. The absence of CSF-1 affects neither the incidence nor the growth of the primary tumors but delayed their development to invasive, metastatic carcinomas. Transgenic expression of CSF-1 in the mammary epithelium of both Csf1(op)/Csf1(op) and wild-type tumor-prone mice led to an acceleration to the late stages of carcinoma and to a significant increase in pulmonary metastasis. This was associated with an enhanced infiltration of macrophages into the primary tumor. These studies demonstrate that the growth of mammary tumors and the development to malignancy are separate processes and that CSF-1 selectively promotes the latter process. CSF-1 may promote metastatic potential by regulating the infiltration and function of tumor-associated macrophages as, at the tumor site, CSF-1R expression was restricted to macrophages. Our data suggest that agents directed at CSF-1/CSF-1R activity could have important therapeutic effects.

Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture and in vivo.

Two diphosphonates containing the P-C-P bond, Cl(2)C(PO(3)HNa)(2), and H(2)C(PO(3)HNa)(2) retard the rate of dissolution of apatite crystals in vitro. They inhibit bone resorption induced by parathyroid extract in mouse calvaria in tissue culture and in thyroparathyroidectomized rats in vivo.

Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vivo.

Two diphosphonates containing the P-C-P bond, CH(3)C(OH)(PO(3)HNa)(2) and H(2)C(PO(3)HNa)(2), inhibit the crystallization of calcium phosphate in vitro and prevent aortic calcification of rats given large amounts of vitamin D(3). The diphosphonates therefore have effects similar to those described for compounds containing the P-O-P bond but are active when administered orally.

Regulation of bim in glucocorticoid-mediated osteoblast apoptosis.

Osteoblasts undergo apoptosis both in vitro and in vivo in response to high dose glucocorticoid (GC) treatment. However, the molecular mechanisms remain elusive, hindering the prevention and treatment of this side-effect. Apoptosis was induced by dexamethasone (Dex) in murine MBA-15.4 osteoblasts within 24-48 h of treatment. We found dose- and time-dependent upregulation of Bim protein, a pro-apoptotic Bcl-2 family member, with highest levels at 24-48 h for 1 microM Dex. This was also observed in primary human bone marrow stromal cells. Bim is subjected to stringent transcriptional and post-translational regulation in osteoblasts. Bim mRNA was upregulated in response to 1 microM Dex; both cycloheximide and the GC receptor antagonist, RU486, prevented Dex-induction of Bim protein, indicating transcriptional regulation involving the GC receptor. The proteasome inhibitor, MG132, potently increased Bim protein levels. Bim was also upregulated in osteoblasts undergoing apoptosis in response to serum deprivation and matrix detachment. Gene silencing experiments show that short interference RNA (siRNA) specific for Bim or the downstream effector Bax both reduced apoptosis induced by Dex in osteoblastic cells. These findings suggest that Bim is a novel regulator of osteoblast apoptosis and may be a therapeutic target.

Agonists of TRAIL death receptors induce myeloma cell apoptosis that is not prevented by cells of the bone marrow microenvironment.

The growth and survival of myeloma cells is critically regulated by cells of the bone marrow microenvironment, including osteoblasts. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of myeloma cell apoptosis, however, this antimyeloma activity is inhibited by osteoprotegerin (OPG) released from osteoblasts. Therefore, we hypothesized that specific agonists of TRAIL death receptors would not be inhibited by OPG released from osteoblasts and thus may represent a novel therapeutic approach in multiple myeloma. In the present study, TRAIL-induced apoptosis was demonstrated to be mediated through both DR4 and DR5. Specific agonist antibodies to DR4 or DR5 dose-dependently induced myeloma cell apoptosis, which was not prevented by OPG or by medium conditioned by osteoblasts. Co-culture of myeloma cells with osteoblasts protected against TRAIL-induced apoptosis of myeloma cells, and this protective effect was due to OPG. In contrast, the co-culture of myeloma cells with osteoblasts had no protective effect on apoptosis induced by specific agonists of DR4 or DR5. TRAIL has been proposed as a potential antitumour therapy, but within the bone marrow microenvironment OPG may interfere with the action of TRAIL. Specific agonists of TRAIL death receptors would not be subject to this inhibition and thus may provide an alternative specific antimyeloma therapy.

Stimulation of the proliferation of human bone cells in vitro by human monocyte products with interleukin-1 activity.

The process of induction of bone formation, which follows bone resorption during normal and pathological bone turnover, is well documented. However, the mechanisms responsible for this process are unclear. Mononuclear phagocytes present at the sites of bone remodeling could play a role in this "coupling" of bone formation to bone resorption. This study was designed to investigate such a possibility. By measuring both the increase in [3H]thymidine incorporation and in cell number, we found that human monocytes in culture released factors capable of stimulating the proliferation of osteoblast-like cells derived from human bone. Rapidly dividing cells exhibited a greater response to interleukin 1 (IL-1) than confluent cells. The factors are similar to IL-1 in that they exhibited the same molecular weight and isoelectric point, were present in fractions that contained IL-1 activity after gel filtration chromatography and isoelectric focusing, and showed similar dose-response characteristics. Proliferation was more marked when prostaglandin production by the cells, which was also stimulated by these factors, was inhibited by indomethacin. A factor produced by monocytes that affects osteoblast activity may be important in the coupling of osteoclast and osteoblast actions.

Constitutive and inducible expression of HLA class II determinants by human osteoblast-like cells in vitro.

Activated immune cells release cytokines which modulate the activity of bone cells in vitro. Expression of major histocompatibility complex (HLA in humans) class II determinants on bone surface cells may be important in local immune cell activation. In this study, expression of HLA-DR and DQ by cultured human bone cells (HBC) derived from normal trabecular bone surfaces was assessed by fluorescence-activated cell sorter (FACS) analysis and immunoperoxidase techniques using monoclonal antibodies. A subset of HBC (10-30%) expressed DR constitutively while 5-15% displayed DQ during long-term culture. HBC lacked a number of monocyte and lymphocyte markers. In addition, both DR+ and DR- HBC (FACS separated) produced osteocalcin stimulated by 1,25-dihydroxyvitamin D2 (1,25(OH)2D3). This suggests that both phenotypes belong to the osteoblast lineage. The number of DR+ HBC was increased by interferon-gamma (IFN gamma; 40-95% DR+ cells) whereas DQ+ HBC remained unchanged or was slightly increased (5-20% DQ+ cells). Moreover, 1,25(OH)2D3 enhanced IFN gamma-induced DR expression and at high concentration (10(-7) M) augmented DR expression by itself. Other major osteotropic factors, parathyroid hormone, interleukin 1, and calcitonin, did not affect HBC DR expression. The findings suggest that HBC may participate in activation of the immune system and that some osteotropic factors may regulate this function.

Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow.

Bisphosphonates inhibit bone resorption in vivo and in vitro by unknown mechanisms. The effect of bisphosphonates on the formation of osteoclasts from their mononuclear hematopoietic precursors was investigated using human long-term marrow cultures in which multinucleated cells form that express most of the known features of the osteoclast phenotype (e.g., bone resorption, tartrate-resistant acid phosphatase, calcitonin responsiveness, and reactivity with specific MAbs). The five bisphosphonates that were tested strongly inhibited 1,25-dihydroxyvitamin D3-stimulated formation of these cells with the same relative potencies as they inhibit bone resorption in vivo. Two representative compounds (3-amino-1-hydroxypropylidene-1,1-bisphosphonate and dichloromethylene bisphosphonate) failed to inhibit the proliferation of precursors of the osteoclast-like cells. However, these compounds decreased the proportion of mononuclear and multinucleated cells expressing an osteoclast antigen, thus suggesting a degree of specificity for cells of the osteoclast lineage. We conclude that bisphosphonates are potent inhibitors of osteoclast-like cell formation in long-term human marrow cultures, and that this may be related to their ability to inhibit bone resorption in vivo.

Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and other disorders of bone.

An isotope dilution method, using (32)P-labeled pyrophosphate, has been developed for the measurement of inorganic pyrophosphate (PP(1)) in human plasma. The specificity of the method was better than 90% as assessed by elution patterns during ion-exchange chromatography, by paper chromatography, and by incubation with inorganic pyrophosphatase. The 99% confidence limits for a single estimation of plasma PP(1) was +/-13%. There were no differences in plasma PP(1) between men and women, but the values in young people (0-15 yr) were slightly higher than in older people. The mean concentration (+/-SE) of PP(1) in the plasma of 73 men and women was 3.50 +/-0.11 mumoles/liter (0.217 +/-0.007 mug P/ml) and the normal range (99% limits) was 1.19-5.65 mumoles/liter (0.074-0.350 mug P/ml). It has been suggested that PP(1) may be important in calcium metabolism because PP(1) can prevent the precipitation of calcium phosphates in vitro and in vivo, and can slow the rates at which hydroxyapatite crystals grow and dissolve. Plasma PP(1) was therefore measured in several disorders of bone. Normal values were found in osteogenesis imperfecta, osteopetrosis, "acute" osteoporosis, and primary hyperparathyroidism. Plasma PP(1) was invariably raised in hypophosphatasia. The excess of PP(1) in plasma might be the cause of the defective mineralization in hypophosphatasia and the function of alkaline phosphatase in bone may be to act as a pyrophosphatase at sites of calcium deposition.

Individual somatic H1 subtypes are dispensable for mouse development even in mice lacking the H1(0) replacement subtype.

H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H1(0), the most divergent subtype, develop normally. Examination of chromatin in H1(0-/-) mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H1(0) to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H1(0) (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434-6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H1(0-/-) mice, each of the three H1 knockout mouse lines was bred with H1(0) knockout mice to generate H1c/H1(0), H1d/H1(0), or H1e/H1(0) double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.

Disruption of the Cockayne syndrome B gene impairs spontaneous tumorigenesis in cancer-predisposed Ink4a/ARF knockout mice.

Cells isolated from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair, which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS group A (CSA) or group B (CSB) do not exhibit an increased spontaneous or UV-induced cancer rate. In order to investigate the effect of CSB deficiency on spontaneous carcinogenesis, we crossed CSB(-/-) mice with cancer-prone mice lacking the p16(Ink4a)/p19(ARF) tumor suppressor locus. CSB(-/-) mice are sensitive to UV-induced skin cancer but show no increased rate of spontaneous cancer. CSB(-/-) Ink4a/ARF(-/-) mice developed 60% fewer tumors than Ink4a/ARF(-/-) animals and demonstrated a longer tumor-free latency time (260 versus 150 days). Moreover, CSB(-/-) Ink4a/ARF(-/-) mouse embryo fibroblasts (MEFs) exhibited a lower colony formation rate after low-density seeding, a lower rate of H-Ras-induced transformation, slower proliferation, and a lower mRNA synthesis rate than Ink4a/ARF(-/-) MEFs. CSB(-/-) Ink4a/ARF(-/-) MEFs were also more sensitive to UV-induced p53 induction and UV-induced apoptosis than were Ink4a/ARF(-/-) MEFs. In order to investigate whether the apparent antineoplastic effect of CSB gene disruption was caused by sensitization to genotoxin-induced (p53-mediated) apoptosis or by p53-independent sequelae, we also generated p53(-/-) and CSB(-/-) p53(-/-) MEFs. The CSB(-/-) p53(-/-) MEFs demonstrated lower colony formation efficiency, a lower proliferation rate, a lower mRNA synthesis rate, and a higher rate of UV-induced cell death than p53(-/-) MEFs. Collectively, these results indicate that the antineoplastic effect of CSB gene disruption is at least partially p53 independent; it may result from impaired transcription or from apoptosis secondary to environmental or endogenous DNA damage.

Ibandronate: pharmacology and preclinical studies.

Over the past three decades, changes to the chemical structures of the bisphosphonates have resulted in progressive improvements in their antiresorptive potencies. Ibandronate is a potent, nitrogen-containing bisphosphonate that possesses a tertiary nitrogen group on its R2 side chain and a hydroxyl group on its R1 side chain, which together confer one of the highest antiresorptive potencies of all bisphosphonates. In common with other nitrogen-containing bisphosphonates, ibandronate is a strong inhibitor of farnesyl pyrophosphate synthase, which probably accounts for its major effects on osteoclast activity. In addition, it binds strongly to hydroxyapatite. The pharmacological efficacy and safety of various continuous and intermittent regimens of ibandronate have been extensively investigated in experimental models of osteoporosis in several animal species, including rats, dogs, and monkeys. In ovariectomized (OVX) rats, intermittent (dosing interval 2, 4, and 6 weeks) and continuous ibandronate regimens provided equivalent results per total dose irrespective of the dosing regimen. Similar results were obtained in OVX dogs and monkeys. High doses of ibandronate many times those used therapeutically were well tolerated and did not impair bone quality or mineralization in rats. Moreover, bone mass, architecture, and strength were maintained or improved, and bone healing was not adversely affected in animal models, regardless of whether ibandronate was administered intermittently or daily. The findings from all these studies demonstrate the efficacy and safety of intermittent ibandronate regimens and support the development of such regimens for the clinical management of postmenopausal osteoporosis.

Novel insights into actions of bisphosphonates on bone: differences in interactions with hydroxyapatite.

Bisphosphonates are now the most widely used drugs for diseases associated with increased bone resorption, such as osteoporosis. Although bisphosphonates act directly on osteoclasts, and interfere with specific biochemical processes such as protein prenylation, their ability to adsorb to bone mineral also contributes to their potency and duration of action. The aim of the present study was to compare the binding affinities for hydroxyapatite (HAP) of 6 bisphosphonates currently used clinically and to determine the effects of these bisphosphonates on other mineral surface properties including zeta potential and interfacial tension. Affinity constants (K(L)) for the adsorption of bisphosphonates were calculated from kinetic studies on HAP crystal growth using a constant composition method at 37 degrees C and at physiological ionic strength (0.15 M). Under conditions likely to simulate bisphosphonate binding onto bone, there were significant differences in K(L) among the bisphosphonates for HAP growth (pH 7.4) with a rank order of zoledronate > alendronate > ibandronate > risedronate > etidronate > clodronate. The measurements of zeta potential show that the crystal surface is modified by the adsorption of bisphosphonates in a manner best explained by molecular charges related to the protonation of their side-chain moieties, with risedronate showing substantial differences from alendronate, ibandronate, and zoledronate. The studies of the solid/liquid interfacial properties show additional differences among the bisphosphonates that may influence their mechanisms for binding and inhibiting crystal growth and dissolution. The observed differences in kinetic binding affinities, HAP zeta potentials, and interfacial tension are likely to contribute to the biological properties of the various bisphosphonates. In particular, these binding properties may contribute to differences in uptake and persistence in bone and the reversibility of effects. These properties, therefore, have potential clinical implications that may be important in understanding differences among potent bisphosphonates, such as the apparently more prolonged duration of action of alendronate and zoledronate compared with the more readily reversible effects of etidronate and risedronate.

The bisphosphonate incadronate (YM175) causes apoptosis of human myeloma cells in vitro by inhibiting the mevalonate pathway.

It has recently been suggested that bisphosphonates may have direct antitumor effects in vivo, in addition to their therapeutic antiresorptive properties. Bisphosphonates can inhibit proliferation and cause apoptosis in human myeloma cells in vitro. In macrophages, bisphosphonate-induced apoptosis was recently found to be a result of inhibition of the mevalonate (MVA) pathway. The aim of this study was to determine whether bisphosphonates also affect human myeloma cells in vitro by inhibiting the MVA pathway. Incadronate and mevastatin (a known inhibitor of the MVA pathway) caused apoptosis in JJN-3 myeloma cells and inhibited cell proliferation. Geranylgeraniol and farnesol prevented incadronate-induced apoptosis and had a partial effect on cell cycle arrest. MVA and geranylgeraniol prevented mevastatin-induced apoptosis and inhibition of proliferation and completely prevented the effect of mevastatin on the cell cycle. These observations demonstrate that incadronate-induced apoptosis in human myeloma cells in vitro is the result of inhibition of the MVA pathway.

Loss of anti-mitotic effects of Bcl-2 with retention of anti-apoptotic activity during tumor progression in a mouse model.

Bcl-2 is an anti-apoptotic and anti-proliferative protein over-expressed in several different human cancers including breast. Gain of Bcl-2 function in mammary epithelial cells was superimposed on the WAP-TAg transgenic mouse model of breast cancer progression to determine its effect on epithelial cell survival and proliferation at three key stages in oncogenesis: the initial proliferative process, hyperplasia, and cancer. During the initial proliferative process, Bcl-2 strongly inhibited both apoptosis and mitotic activity. However as tumorigenesis progressed to hyperplasia and adenocarcinoma, the inhibitory effects on mitotic activity were lost. In contrast, anti-apoptotic activity persisted in both hyperplasias and adenocarcinomas. These results demonstrate that the inhibitory effect of Bcl-2 on epithelial cell proliferation and apoptosis can separate during cancer progression. In this model, retention of anti-apoptotic activity with loss of anti-proliferative action resulted in earlier tumor presentation.

Identification of ecto-nucleoside triphosphate pyrophosphatase in human articular chondrocytes in monolayer culture.

In cultured monolayers of human articular chondrocytes we have observed an enzyme activity which catalyzes the extracellular conversion of ATP to AMP and PPi. The enzyme was active at very low concentrations of ATP (microM) and exhibited optimal activity at concentrations of ATP of approx. 100 microM. The enzyme was active in intact cells as judged by measurement of the release of the cytoplasmic marker enzyme lactate dehydrogenase. No increase in production of PPi from ATP was observed on mechanically disrupting the cells and no activity was shed into the medium by intact cells. Activity was stable between days 4 and 8 after subculturing the cells and was not affected by the timing of the final medium change prior to assay. Activity was also observed with other nucleoside triphosphates (GTP, CTP and UTP). We suggest that this activity is attributable to ecto-nucleoside triphosphate pyrophosphatase. This observation may be important in relation to the pathogenesis of the human disease of chondrocalcinosis in which crystals of calcium pyrophosphate dihydrate deposit in articular cartilage.

Evidence that ecto-nucleoside-triphosphate pyrophosphatase serves in the generation of extracellular inorganic pyrophosphate in human bone and articular cartilage.

Extracellular inorganic pyrophosphate (PPi) is important in the regulation of mineralisation of bone, and in the pathogenesis of chondrocalcinosis, an arthritic disease in which calcium pyrophosphate dihydrate crystals form in articular cartilage. Nucleoside-triphosphate pyrophosphatase, which catalyses the formation of PPi, was previously observed at the surface of human articular chondrocytes in culture. A similar enzyme has been identified in osteoblast-like human bone cells in culture, and is active towards purine and pyrimidine nucleoside triphosphates. The enzyme has high affinity for ATP and is located on the cell surface, and thus could serve in the generation of extracellular PPi. Moreover, no other mechanism for the catabolism of small amounts of exogenous ATP is present in human bone cells. Further evidence for ecto-nucleoside-triphosphate pyrophosphatase serving in the generation of extracellular PPi in articular cartilage and bone was obtained by studying the ability of alternative substrates (which do not yield PPi) to inhibit generation of PPi from ATP. In both articular chondrocytes and bone cells, the enzyme exhibited an apparent preference for ATP over dinucleotide and phosphodiester substrates. Some potential inhibitors of the enzyme activity were also studied in both cell types. ADP moderately inhibited the activity but two bisphosphonate drugs were only slightly inhibitory.