R.G.G. Russell

Bisphosphonates induce apoptosis in human myeloma cell lines: a novel anti-tumour activity.

Bisphosphonates are in widespread use to prevent bone resorption in a number of metabolic and tumour‐induced bone diseases including multiple myeloma. Recent reports suggest that bisphosphonate treatment may be associated with an increase in patient survival, raising the possibility that these compounds may have a direct effect on the tumour cells. We have investigated whether the bisphosphonates clodronate, pamidronate and YM175 can directly affect the human myeloma cell lines U266‐B1, JJN‐3 and HS‐Sultan in vitro. The effect of bisphosphonate treatment on cell number and cell cycle progression was examined using flow cytometry. The ability of bisphosphonates to induce apoptosis in human myeloma cell lines was determined on the basis of changes in nuclear morphology and of DNA fragmentation. Pamidronate and the more potent bisphosphonate, YM175, significantly decreased cell number (P < 0.001) in JJN‐3 and HS‐Sultan cells. YM175 also caused cells to arrest in the S‐phase of the cell cycle in the JJN‐3 cell line. Both pamidronate and YM175 also caused an increase in the proportion of cells with altered nuclear morphology (P < 0.05) and fragmented DNA, characteristic of apoptosis, in both JJN‐3 and HS‐Sultan cells. In contrast, clodronate had little effect on cell number and did not cause apoptosis at the concentrations examined. These data raise the possibility that some bisphosphonates could have direct anti‐tumour effects on human myeloma cells in vivo.

Molecular mechanisms of action of bisphosphonates.

BPs can be grouped into two general classes according to their chemical structure and the molecular mechanism by which they inhibit osteoclast-mediated bone resorption. The simple BPs can be metabolically incorporated into non-hydrolysable analogues of ATP that accumulate intracellularly in osteoclasts, causing osteoclast cell death by apoptosis. By contrast, the more potent N-BPs inhibit FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids that are required for the prenylation of small GTPase signalling proteins necessary for osteoclast function. Inhibition of FPP synthase in cells other than osteoclasts also appears to account for the adverse effects of N-BPs in vivo (including the acute phase reaction) and for the anti-tumour effects of N-BPs in vitro.

Bisphosphonates: pharmacology, mechanisms of action and clinical uses.

The bisphosphonates represent a novel class of drugs, the use of which have opened up major new approaches to the therapy of bone diseases. It has, however, taken over 30 years since the discovery of their profound effects on calcium metabolism for them to become well established as clinically successful antiresorptive agents. There are several useful recent reviews of the chemistry, pharmacology and clinical applications of bisphosphonates [1–11]. Several bisphosphonates, including etidronate, clodronate, tiludronate, pamidronate, alendronate, risedronate and ibandronate, have been registered for various clinical applications in various countries. Although it is different for each compound, these include use as agents for bone scanning and as drugs for the treatment of Paget’s disease, hypercalcemia of malignancy, bone metastases and osteoporosis. Those most used in cancer are pamidronate given parenterally, or clodronate given orally. In osteoporosis the major drugs are etidronate and alendronate, which are approved therapies in many countries. Bisphosphonates used as inhibitors of bone resorption all contain two phosphonate groups attached to a single carbon atom, to give a ‘P–C–P’ structure. The bisphosphonates are therefore stable analogs of naturally occurring pyrophosphate compounds, which now helps to explain their intracellular as well as their extracellular modes of action. The mechanism of action of bisphosphonates was originally ascribed to their ability to adsorb strongly to hydroxyapatite crystals, and to inhibit their growth and dissolution [12,13]. It has gradually become clear that this explanation is insufficient to account for all their effects, and cellular actions are involved, particularly with the more potent compounds. The Early Development of Bisphosphonates

Prevention of Bone Loss with Risedronate in Glucocorticoid-Treated Rheumatoid Arthritis Patients

Abstract: The aim of the study was to assess risedronate’s effect on bone mineral density in postmenopausal women with rheumatoid arthritis receiving glucocorticoids. We carried out a two center, 2 year, double-masked, placebo-controlled trial with a third year of nontreatment follow-up. We studied 120 women requiring long-term glucocorticoid therapy at >2.5 mg/day prednisolone randomized to treatment with daily placebo; daily 2.5 mg risedronate; or cyclical 15 mg risedronate (2 out of 12 weeks). At 97 weeks, bone mineral density was maintained at the lumbar spine (+1.4%) and trochanter (+0.4%) in the daily 2.5 mg risedronate group, while significant bone loss occurred in the placebo group (–1.6%, p= 0.03; and 4.0%, p<0.005, respectively). At the femoral neck, there was a nonsignificant bone loss in the daily 2.5 mg risedronate group (–1.0%) while in the placebo group bone mass decreased significantly (–3.6%, p<0.001). The difference between placebo and daily 2.5 mg risedronate groups was significant at the lumbar spine (p= 0.009) and trochanter (p= 0.02) but did not reach statistical significance at the femoral neck. Although not significantly different from placebo at the lumbar spine, the overall effect of the cyclical regimen was similar to that of the daily 2.5 mg risedronate regimen. Treatment withdrawal led to bone loss in the risedronate groups that was significant at the lumbar spine. A similar number of patients experienced adverse events (including upper gastrointestinal events) across treatment groups and risedronate was generally well tolerated. Thus risedronate preserves bone mass in postmenopausal women with rheumatoid arthritis receiving glucocorticoids while patients receiving a placebo have significant bone loss.

Structure-activity relationships among the nitrogen containing bisphosphonates in clinical use and other analogues: time-dependent inhibition of human farnesyl pyrophosphate synthase.

The nitrogen-containing bisphosphonates (N-BPs) are the main drugs currently used to treat diseases characterized by excessive bone resorption. The major molecular target of N-BPs is farnesylpyrophosphate synthase. N-BPs inhibit the enzyme by a mechanism that involves time dependent isomerization of the enzyme. We investigated features of N-BPs that confer maximal slow and tight-binding by quantifying the initial and final K(i)s and calculating the isomerization constant K(isom) for many N-BPs. Disruption of the phosphonate-carbon-phosphonate backbone resulted in loss of potency and reduced K(isom). The lack of a hydroxyl group on the geminal carbon also reduced K(isom). The position of the nitrogen in the side chain was crucial to both K(i) and K(isom). A correlation of K(isom) and also final K(i) with previously published in vivo potency reveals that the isomerization constant ( R = -0.77, p < 0.0001) and the final inhibition of FPPS by N-BPs ( R = 0.74, p < 0.0001) are closely linked to antiresorptive efficacy.

The effects of recombinant human interleukin-1β on cellular proliferation and the production of prostaglandin E2, plasminogen activator, osteocalcin and alkaline phosphatase by osteoblast-like cells derived from human bone

There is mounting evidence implicating cytokines such as interleukin-1 in the local regulation of bone homeostasis. In this report we show that recombinant human interleukin-1 beta (rhIL-1 beta) influences several activities of osteoblast-like cells derived from human trabecular bone explants in vitro. rhIL-1 beta stimulated cellular proliferation and the synthesis of prostaglandin E2 and plasminogen activator activity in the cultured human osteoblast-like cells in a dose-dependent manner. However, the induction of osteocalcin synthesis and alkaline phosphatase activity in response to 1,25(OH)2D3, two characteristics of the osteoblast phenotype, were antagonized by rhIL-1 beta over a similar dose range. This study adds further support to the potential role of interleukin-1 in the physiological and pathological modulation of bone cell metabolism.

Regulation of osteogenic differentiation of human bone marrow stromal cells: Interaction between transforming growth factor-β and 1,25(OH)2 Vitamin D3 in vitro

Abstract. Bone marrow stromal cells are believed to play a major role in bone formation as a major source of osteoprogenitor cells, however, very little is known about how the osteogenic differentiation of these cells is regulated by systemic hormones and local growth factors. We examined the effects of TGF-β and its interaction with 1,25(OH)2 Vitamin D3 [1,25(OH)2D3] on the differentiation and proliferation of human bone marrow stromal cells (hBMSC) in secondary cultures. Alkaline phosphatase (ALP) activity was inhibited by TGF-β (0.1–10 ng/ml) and increased by 1,25(OH)2D3 (50 nM), however, co-treatment of TGF-β and 1,25(OH)2D3 synergistically enhanced ALP activity with maximal stimulation occurring at about 8 days after treatment. This synergistic effect was independent of proliferation because, in contrast to TGF-β alone, combined treatment with TGF-β and 1,25(OH)2D3 had no effect on hBMSC proliferation. As no synergistic effect was seen with combinations of 1,25(OH)2D3 and other osteotrophic growth factors, including BMP-2, IGF-I, and basic fibroblast growth factor (bFGF), it would seem likely that the synergistic interaction is specific for TGF-β. The increased ALP activity was due to an enhancement of 1,25(OH)2D3-induced ALP activity by TGF-β, rather than vice versa. In contrast, TGF-β inhibited 1,25(OH)2D3-induced osteocalcin production. Taken together, these results indicate that TGF-β and 1,25(OH)2D3 act synergistically to stimulate the recruitment of BMSC to the osteoblast lineage. This interaction may play an important role in bone remodeling.

The use of dichloromethylene diphosphonate for the management of hypercalcaemia in multiple myeloma

Summary. We have assessed the effects of the diphosphonate, dichloromethylene diphosphonate (Cl2MDP), in 19 patients with hypercalcaemia and increased bone resorption due to myeloma. Cl2MDP (800–3200 mg daily by mouth or 300 mg daily by intravenous infusion) decreased plasma calcium and biochemical indices of increased bone resorption in 16 of 19 patients. This effect persisted for the duration of treatment (up to 14 weeks). Prolonged treatment was associated with a progressive rise in serum alkaline phosphatase and only a transient fall in hydroxyproline suggesting the stimulation of bone repair. Since myeloma is associated with significant morbidity and mortality due to progressive bone loss, these results suggest that long‐term treatment of myeloma with Cl2MDP is worthy of further study.

Calcium and orthophosphate deposits in vitro do not imply osteoblast-mediated mineralization: mineralization by betaglycerophosphate in the absence of osteoblasts.

It has been shown in several laboratories that addition of beta-glycerophosphate (beta GP), a substrate for alkaline phosphatase (AP), to cultured osteoblast-like cells induces deposition of orthophosphate (Pi) and Ca within seven days. Even though this effect is regarded as an in vitro model of bone mineralization, it is not known whether it is specific for osteoblasts. We have, therefore, studied the amounts of Pi and Ca deposited after seven days with 10 mM beta GP in culture wells containing confluent cultures of osteoblast-like cells (OB) derived from human trabecular bone explants, human skin fibroblasts (SF), or culture medium alone (MED). Ox liver AP at an activity considerably greater than the endogenous AP activity of the cells, but comparable with that of other osteoblast models, was added to ensure a similar rate of Pi generation from beta GP in all wells. beta GP was converted quantitatively to Pi within seven days, leading to a nonphysiological 10-fold increase in the Pi concentration in the culture medium. After thorough rinsing on day seven, the OB and SF wells contained deposits of Pi and Ca, but the amounts were comparable for the two cell types. Smaller, but significant, amounts of Pi and Ca were also detectable even in rinsed MED wells. This suggests that the detection of such deposits in beta GP experiments cannot necessarily be interpreted as a specific property of osteoblast cultures in vitro, and may simply reflect the presence of AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of halogenated bisphosphonates by the cellular slime mould dictyostelium discoideum

Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonates was taken up by the amoebae and incorporated into the corresponding beta, gamma-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.