Donald J. Watts

Overview of bisphosphonates

Michael J. Rogers, Ph.D. B Donald J. Watts, Ph.D. isphosphonates (BPs) were first studied over 30 years ago as stable analogues of a naturally occurring inorganic pyrophosphate (PPi), R. Graham G. Russell, M.D., Ph.D. which were shown to be able to inhibit the precipitation of calcium phosphate in vitro and biologic calcification in vivo. A key step oc1 Department of Human Metabolism and Clinical curred when it was shown that they also inhibited bone resorption Biochemistry, University of Sheffield Medical induced by a wide variety of agents and had profound effects on School, Sheffield, United Kingdom. calcium metabolism in vivo. 2 Department of Molecular Biology and BiotechThe first clinical uses of BPs followed soon after in the late 1960s nology, University of Sheffield, Sheffield, United and included their use as agents for bone scanning, based on their Kingdom. ability to adsorb to bone mineral, for which they remain outstandingly useful. Concurrently they were shown to be strikingly effective in clinical disorders associated with increased bone resorption, initially in Paget’s disease of bone, then in hypercalcemia of malignancy, myeloma, and bone metastases, and much later in osteoporosis. As a result, several BPs (e.g., etidronate, clodronate, pamidronate, alendronate, and tiludronate) now are licensed as drugs for various indications, and more should follow (risedronate, ibandronate, zoledronate, etc.). Because of the current emphasis on osteoporosis, their value in patients with Paget’s disease and in those with malignancy runs the risk of being undervalued. The mode of action of BPs originally was ascribed to their ability to adsorb strongly to hydroxyapatite crystals, and to inhibit their growth and dissolution, but it has gradually become clear that this is insufficient to account for all their effects. The ability to make BPs with minimal inhibitory effects on bone mineralization but increased relative potency on bone resorption was achieved many years ago. Remarkable progress has been made in increasing their potency as inhibitors of bone resorption by factors of 100–10,000 fold. One key feature appears to be the insertion of a nitrogen atom at critical positions in the side chain. These may be in alkyl side chains, (e.g., butyl-NH2) as in alendronate, or in the highly potent -N substituted derivatives of aminohydroxypropylidene bisphosphonate (APD) (pamidronate) (e.g., dimethyl APD, or methylpentenyl APD [ibandronate]). Comparable high potencies are obtained with heterocyclic ring Presented at the Skeletal Complications of Macompounds containing nitrogen (e.g., with imidazole, pyridinyl [e.g., lignancy Symposium, Bethesda, Maryland, April as in risedronate] or picolyl groups). These compounds are remark19–20, 1997. ably potent, and are able to suppress bone resorption in experimental animals at doses of õ 1 mg per day. Address for reprints: Graham Russell, Department of Human Metabolism and Clinical BioA simple hypothesis for the effects of BPs on bone resorption is chemistry, University of Sheffield Medical that the compounds are selectively concentrated in bone, where they School, Beech Hill Road, Sheffield, S10, 2RX, interfere with the action of osteoclasts by as yet poorly defined mechaUnited Kingdom. nisms. It is likely that BPs are internalized by osteoclasts and interfere Received June 3, 1997; accepted June 16, 1997. with specific biochemical processes. Thus BPs interfere with osteo-

The Intracellular Target for the Antiresorptive Aminobisphosphonate Drugs in Dictyostelium discoideum Is the Enzyme Farnesyl Diphosphate Synthase

Aminobisphosphonate (aBP) drugs inhibit osteoclast‐mediated bone resorption and also growth of amoebas of Dictyostelium discoideum apparently by interaction with the same intracellular target. Identification of the target in Dictyostelium therefore could also identify the target in osteoclasts. The aBPs (100 μM alendronate and 30 μM YM‐175) inhibited conversion of [14C]mevalonate into sterols by cultures of Dictyostelium amoebas. One of three enzymes (isopentenyl diphosphate [IDP] isomerase, farnesyl diphosphate [FDP] synthase, and squalene synthase) appeared to be the target for this inhibition because conversion of [14C]IDP into squalene, the immediate precursor for sterol biosynthesis, was inhibited in extracts of wild‐type amoebas by alendronate (IC50 = 75 nM) or risedronate (IC50 = 30 nM) whereas, when the extract had been prepared from amoebas of strains selected for having partial resistance to the growth‐inhibitory effects of alendronate (strain MR102) or risedronate (strain RB101), the values of IC50 were increased to 700 nM for alendronate (MR102 extract) or 130 nM for risedronate (RB101 extract). Neither IDP isomerase nor squalene synthase was inhibited significantly by alendronate or risedronate but both of these aBP drugs, and all others tested, inhibited FDP synthase. Determination of the nucleotide sequences of complementary DNAs (cDNAs) encoding FDP synthase in the wild‐type and aBP‐resistant strains of Dictyostelium indicated that there had been no changes in the amino acid sequence of the enzyme in the mutant strains. However, both mutant strains overproduce FDP synthase. It is concluded that FDP synthase is the intracellular target for the aBP drugs. (J Bone Miner Res 2000;15:971–981)

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.

Inhibition of growth of Dictyostelium discoideum amoebae by bisphosphonate drugs is dependent on cellular uptake

AbstractPurpose. The aim of the study was to determine whether bisphosphonates are internalised by Dictyosteliumamoebae and whether cellular uptake is required for their growth-inhibitory effects. Bisphosphonates inhibit growth of amoebae of the slime mould Dictyostelium discoideum, by mechanisms that appear to be similar to those that cause inhibition of osteoclastic bone resorption. Methods. Cell-free extracts prepared from amoebae that had been incubated with bisphosphonates were analysed by 3lP-n.m.r. spectroscopy or ion-exchange f.p.l.c., to identify the presence of bisphosphonates or bisphosphonate metabolites respectively. The growth-inhibitory effect of bisphosphonates towards Dictyostelium amoebae was also examined under conditions in which pinocytosis was inhibited. Results. All of the bisphosphonates studied were internalised by Dictyostelium amoebae, probably by fluid-phase pinocytosis, and could be detected in cell-free extracts. Amoebae that were prevented from internalising bisphosphonates by pinocytosis were markedly resistant to the growth-inhibitory effects of these compounds. In addition, bisphosphonates encapsulated within liposomes were more potent growth inhibitors of Dictyostelium owing to enhanced intracellular delivery of bisphosphonates. Conclusions. All bisphosphonates inhibit Dictyostelium growth by intracellular mechanisms following internalisation of bisphosphonates by fluid-phase pinocytosis. It is therefore likely that bisphosphonates also affect osteoclasts by interacting with intracellular, rather than extracellular, processes.

Differential Effects of Aminosubstituted Analogs of Hydroxy Bisphosphonates on the Growth of Dictyostelium discoideum

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 μM/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 μM and 700 μM, 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.

Farnesyl diphosphate synthase, the target for nitrogen-containing bisphosphonate drugs, is a peroxisomal enzyme in the model system Dictyostelium discoideum

NBP (nitrogen-containing bisphosphonate) drugs protect against excessive osteoclast-mediated bone resorption. After binding to bone mineral, they are taken up selectively by the osteoclasts and inhibit the essential enzyme FDPS (farnesyl diphosphate synthase). NBPs inhibit also growth of amoebae of Dictyostelium discoideum in which their target is again FDPS. A fusion protein between FDPS and GFP (green fluorescent protein) was found, in D. discoideum, to localize to peroxisomes and to confer resistance to the NBP alendronate. GFP was also directed to peroxisomes by a fragment of FDPS comprising amino acids 1–22. This contains a sequence of nine amino acids that closely resembles the nonapeptide PTS2 (peroxisomal targeting signal type 2): there is only a single amino acid mismatch between the two sequences. Mutation analysis confirmed that the atypical PTS2 directs FDPS into peroxisomes. Furthermore, expression of the D. discoideum FDPS–GFP fusion protein in strains of Saccharomyces cerevisiae defective in peroxisomal protein import demonstrated that import of FDPS into peroxisomes was blocked in a strain lacking the PTS2-dependent import pathway. The peroxisomal location of FDPS in D. discoideum indicates that NBPs have to cross the peroxisomal membrane before they can bind to their target.

The Synthesis and Evaluation of Quaternary Pyridinium Bisphosphonates as Potent Anti-Resorptives

Abstract Several novel quaternary pyridinium bisphosphonates have been synthesised and their efficacy as potential anti-resorptive bone agents have been tested in Dictyostelium discoideum. This assay has been shown to accurately reflect the potency of a bisphosphonic acid as an anti-resorptive compound. All the quaternary bisphosphonates are very potent growth inhibitors but results indicate that the more potent compounds are those containing hydrophobic, bulky groups.