The anti-resorptive drug clodronate is metabolised to a non-hydrolysable ATP analogue by mammalian cells in vitro
Frith JC., Graham R., Russell G., Michael Blackburn G., Watts DJ., Rogers MJ.
Bisphosphonates (BPs) are stable analogues of pyrophosphate that contain P-C-P bonds. Clodronate (dichloromethylenebisphosphonate) is widely used in the treatment of bone diseases characterised by excessive bone résorption such as Paget's disease, tumour-induced osteolysis and hypercalcaemia of malignancy. The mechanisms of action of BPs have not been identified but may involve a toxic effect on bone-resorbing osteociast cells due to induction of apoptosis. It is generally considered that BPs are not metabolised. We have found that mammalian cells in vitro (human MG63 osteosarcoma cells and mouse J774 macrophage-like cells) can metabolise clodronate to a non-hydrolysable ATP analogue, β,γ-dichloromethylene ATP. This metabolite could be detected in deproteinised cell extracts prepared from J774 and MG63 cells that had been incubated for 48 hours with 250μM or 750μM clodronate respectively. These concentrations significantly inhibited cell proliferation. An additional peak of absorbance at 254nm that was not present in control extracts could be detected after anion-exchange f.p.l.c. analysis of extracts from clodronate-treated cells. This peak, corresponding to the metabolite of clodronate, co-eluted with synthetic β,β-dichloromethylene ATP. This is the first report that clodronate can be metabolised by mammalian cells. Similarly, 1007mu;M methylenebisphosphonate (MBP) was metabolised into β,γ-methylene ATP and also a methylene analogue of Ap4A, diadenosine 5′5‴-P1,P4-(p2/p 3-methylene tetraphosphate). We have obtained similar results with amoebae of the eukaryotic slime mould Dictyostelium discoidemn, the growth of which is inhibited by BPs. In Didyostelium, the metabolites of clodronate and MBP appear to be produced by a back-reaction catalysed by certain aminoacyl-tRNA synthetases. The toxic and anti-proliferative effects of clodronate on osteoclasts and other cells may therefore be due to accumulation of a non-hydrolysable ATP analogue or to inhibition of aminoacyl-tRNA synthetase enzymes.