INHIBITION OF BONE RESORPTION

Abstract: A method of inhibiting bone resorption in vivo is provided. The method comprises administering a therapeutically effective amount of a composition containing the perchlorate (ClO4<->) ion. A composition possessing inhibitory properties towards bone resorption is also provided, the composition containing the perchlorate (ClO4<->) ion. The invention also provides a method for use in the treatment of diseases or conditions characterised by bone resorption, e.g. osteoporosis, comprising administering a pharmaceutical composition containing, as active ingredient, the perchlorate (ClO4<->) ion and a method of preventing and/or treating inflammatory osteolysis which comprises administering a therapeutically effective amount of the perchlorate (ClO4<->) ion.

Description: INHIBITION OF BONE RESORPTION This invention is concerned with the inhibition of bone resorption, and relates inter alia to methods of effecting such inhibition, and to agents and compositions which possess such inhibitory properties. Background of the Invention Agents that inhibit bone resorption have potential clinical applications for management of conditions such as Paget's disease, malignancy-associated hypercalcaemias and osteoporosis; these conditions may be associated with a primary pathological osteoclastic resorption of existing bone. However, available therapeutic options are currently limited. Diphosphonates are thought to offer the most effective and long-lasting treatment in relieving the hypercalcaemia of malignancy. They may act through binding hydroxyapatite in areas of increased bone turnover, their direct cytotoxic effects, or by competition with acid phosphatase action on pyrophosphate, but some (e.g. etidronate may also have long term inhibitory effects on bone mineralization. Calcitonin has been used to treat Paget's disease. It may act through an acute inhibition of osteoclast activity, followed by a more delayed effect on osteoclast recruitment from bone marrow. However, its action is transient owing to its rapid destruction in vivo, and receptor downregulation on the osteoclast. The availability of new methods for osteoclast isolation and culture, and for the assessment of their function, may allow the effects of further compounds that influence osteoclast function to be more readily characterized. Summary of the Invention We have found that the perchlorate ion (CIO^-) has inhibitory effects on bone resorption. Accordingly, in one aspect this invention provides a method of inhibiting bone resorption in vivo, which comprises administering to a subject a therapeutically effective amount of a composition containing the perchlorate (CIO4 "" ) ion. In another aspect, the invention provides a composition possessing inhibitory properties towards bone resorption, characterised in that the composition contains the perchlorate (CIO^-) ion. According to a third aspect of this invention, there is provided, for use in the treatment of diseases or conditions characterised by bone resorption, e.g. osteoporosis, a pharmaceutical composition containing, as active ingredient, the perchlorate (CIO4 "" ) ion. According to a fourth aspect of the invention, there is provided a method of preventing and/or treating inflammatory osteolysis. which comprises administering a therapeutically effective amount of the perchlorate (C10 4 -) ion. Perchlorate modifies the activation of a range of cell types. As it does not traverse lipid bilayers, one would expect it to act at the cell surface, rather than at a site within the cell. It modifies Ca channel gating in the plasma membrane and may thereby modulate excitation secretion coupling in pancreatic beta cells. In skeletal muscle, Ca 2+ influx across the external membrane develops over too prolonged a timecourse to explain the build up of cytosolic free [Ca 2+ ] (Aimers & Palade, 1981; Sanchez & Stefani, 1978). Rather, calcium is mobilized from intracellular stores through a process that does snot involve extracellular Ca 2+ entry (see e.g. Luttgau & Spiecker, 1979). This process is thought to be regulated through configurational changes in voltage sensors located in transverse tubular membrane (for a review see Huang, 1988). Perchlorate also potentiates tension development (Gomolla et al, 1983), and Antipyrylazo III absorbance signals monitoring intracellular [Ca Λ ] in striated muscle (Csernoch et al, 1987) in response to surface membrane depolarization. It may act through a selective modification of particular components of the intramembrane charge movement, that may in turn reflect configurational changes in voltage sensors regulating Ca 2+ release (Huang, 1986; 1987). In skinned muscle fibers perchlorate additionally could trigger the release of calcium directly, again probably through an influence at a tubular site (Fill & Best, 1990). Finally, these actions appeared selective to the activation of cellular Ca 2+ mobilization: perchlorate did not influence other processes such as sodium or potassium channel activation with a similar overall dependence upon membrane potential (Gomolla et al, 1983). We have demonstrated, through a variety of independent experimental investigations, that perchlorate is a potent inhibitor of osteoclastic resorption of bone and that it may possibly act though an influence on cell motility properties. The effects of perchlorate anion (CIO^-) on osteoclastic bone resorption were investigated using a number of independent in vitro and in vivo procedures. Its intravenous infusion significantly reduced plasma [Ca 2+ ] in young (50 g) Wistar rats, without influencing plasma [ g 2+ ] or [albumin] . A maximal effect was attained around 20 minutes after administration, at a dose of 600 μMol/rat. Scanning electronmicroscopic image studies suggested that perchlorate (1 to 10 mM) reduced both the total area of cortical bone resorbed by freshly disaggregated rat osteoclasts, and the number of osteoclastic excavations, to an extent dependent upon dose. Lower concentrations were ineffective. Adding 5 mM [Ca ] produced similar effects; these were potentiated by low (1 mM) doses of perchlorate. In contrast, thiocyanate was ineffective at concentrations (10 mM) that would have produced comparable lyotropic effects as perchlorate. Perchlorate (10 mM) elicited a transient six-fold elevation of cytosolic free [Ca 2+ ], as measured using indo-1 dual emission microspectrofluorimetry, in isolated cultured osteoclasts. Time-lapse video recordings indicated that this was followed by marked osteoclastic retraction (by up to 70% of control cell area). However, perchlorate did not alter morphometric measures of pseudopodial motility, margin ruffling, granule movement and cell migration. Nor did it influence supernatant concentrations of tartrate-resistant (osteoclastic) acid phosphatase in cultures of resorbing osteoclasts. These findings indicate that perchlorate is a potent inhibitor of osteoclast function, acting possibly through an influence on intracellular [Ca ] and in turn upon the degree of cell retraction. Detailed description of evaluative techniques Assay of hypocalcaemic effects in vivo When assessing the influence of perchlorate administration in vivo, young male Wistar rats (weight 48.9 ± 1.2 g: means ± S.E. of the mean; Charles Rivers, Margate, Kent, U.K.), were fasted overnight prior to the experiments. At a set time after injection of test agent, dissolved in Ringer solution, the animals were anaesthetized with ether, and bled via the dorsal aorta. Serum was separated and [Ca 2+ ] and albumin concentrations were measured on samples using a Technicon RA 1000 autoanalyser. Serum [Mg ] was measured manually using the Merck kit 3338. For the assessment of dose responsiveness, groups of rats (n = 5 rats per group) were injected intravenously, via the tail vein, with a range of doses of sodium perchlorate (B.D.H., Poole, Dorset, U.K.). The animals were then anaesthetized and bled a fixed time, 30 minutes, after injection. When following the timecourse of the drug response, groups of rats (n = 4 rats per group) were also injected intravenously with sodium perchlorate. They were then anaesthetized and bled at timed intervals between 15 and 105 minutes. Osteoclast isolation and culture For in vitro studies, osteoclasts were isolated for culture from femurs and tibias, under sterile condition of newborn Wistar rats killed by decapitation. Adheren soft tissues were removed and the bones cut across thei epiphyses in N-2-hydroxyethylpiperazine-N'-2- ethanesulphonic acid (HEPES)-buffered Medium 199 supplemented with heat-inactivated fetal calf serum (FCS 10% v/v; Gibco, U.K.), benzyl penicillin (Glaxo, Middlesex, U.K.; 100 mIU/1) and streptomycin (Glaxo; lOOmg/l: SP). Osteoclasts were mechanically disaggregated by curetting the bones of each rat with a scalpel blade into 1 ml of medium 199 and agitating the suspension with a pipette. Larger fragments were allowe to settle for 10 seconds, before the supernatant was dropped onto appropriate substrate (bone slices, plastic Petri dishes or glass coverslips; see below). Bone resorption assays The osteoclast-bone resorption assay employed was based on the ability of isolated osteoclasts to resorb devitalized cortical bone in vitro. Unlike other isolated cell systems, which depend either on measuremen of a secretory product or on quantification of a cytochemical change, this approach entirely relies on direct visualization and quantification of the activity of the responding cell, the osteoclast. This method als enables the direct assessment of the responsiveness to added agents in vitro of osteoclasts that are taken away from the complex microenvironment of bone. Assays were performed on postmortem human bone from patients without evidence of bone disease. This was cleaned and the cortex cut longitudinally with a low speed saw (Buehler, Illinois, USA). The pieces of bone were then cut into 3 mι ώ slices of thickness 0.1 mm, cleaned by sonication in sterile distilled water, and stored dry at room temperature. The cells were then dropped onto the slice placed in wells of Sterilin 100 x 18 mm multiwell dishes. The cells were allowed to sediment and attach to the substrate over 30 minutes at 37°C. The slices were then washed in Minimal Essential Medium (MEM) containing fetal calf serum (10%), glutamine, penicillin, streptomycin (as described above) and sodium bicarbonate (0.85 g/1), and placed in separate wells containing 2 ml of the same medium. Following incubation for 18 hours at 37°C in 10% humidified C0 2 , with or without added test substance, the culture medium was sampled and the slices were fixed with 10% v/v glutaraldehyde, and stained with toluidine blue. The latter enabled the assessment of numbers of multinucleate osteoclasts and mononuclear cells. The latter were assessed in two representative experiments on ten randomly selected fields per bone slice, through an eyepiece graticule. Additionally, in all experiments, 5 to 6 bone slices were incubated with parathyroid hormone (0.1 iu/ml). Those cultures then showing an increased greater than twofold over basal bone resorption were discarded. For scanning electronmicroscopy, the slices were then bleached by immersion in sodium hypochlorite solution (10% v/v) and dehydrated in aqueous alcohol (80% v/v) The bone slices were finally sputter coated with gold and examined under a scanning electron microscope (Cambridge 360, Cambridge Instruments, Bar Hill, Cambs., U.K.). The area of bone resorbed and the number of osteoclastic excavations was assessed by tracing the outline of each excavation into a digitizer linked to an IBM-AT-controlled image processor (Sight Systems, Newbury, Berks., U.K.). When performing a statistical evaluation of the osteoclast-bone resorption assay, each response, whether the total area of resorption on each bone slice (or the numbers of osteoclastic excavations on each bone slice) was transformed by taking its square root or square root with the addition of a constant factor, respectively, to give response metameters. Such a transformation yielded normal, Gaussian, frequency distributions of the data points which would otherwise have been skewed, and led to more stable variances over a wider dosage range. The effect on such response metameters of treatment, compared to control, and the relationship of response metameters to the logarithm of the dose of the administered substance, was assessed using one-way analyses of variance, estimates of ED^ Q and relative potencies (where necessary) were calculated from dose-response curves using logistic regression analysis, and in certain instances where stated in the Results, prohibit analysis. Measurements of acid phosphatase Total acid phosphatase activity was measured by incubating samples (usually 0.15 ml) of medium exposed to osteoclast, cultures either on bone or plastic, with 4- nitrophenyl phosphate (final concentration 10 mM) in citrate buffer (pH 5.0; 100 mM) at 37°C for 30 in in a total volume of 0.45 ml. The reaction was stopped and the coloured quinoid form of the 4-nitrophenate ion was developed by the addition of 0.2 ml sodium hydroxide (0.5 mM) before absorptiometry at 405 nm. Catalytic activity concentrations (μmol substrate hydrolysed per minute (U) per litre sample) were calculated from the absorbance coefficient of 4-nitrophenate (e=1.85 1 mMol -1 mm -1 ). In order to measure tartrate-resistant acid phosphatase, dextrorotatary tartaric acid was added to the incubation medium to achieve a final reagent concentration of 150 mM. Measurements of intracellular [Ca 2+ ] in isolated osteoclasts (21) Intracellular free calcium concentrations, [Ca 2+ ] were measured in single isolated osteoclasts by a dual emission microspectrofluorimetric method using indo-1. This is a dual emission dye, whose emission spectrum is affected by changes in [Ca ] . Preliminary cuvette studies of indo-1 (1 μM) using a Perkin-Elmer LS-5 spectrofluorimeter, indicated an excitation wavelength of 340 nm in the spectra between 400 to 500 nm, obtained at 20°C. As the [Ca ] was progressively increased from 100 nM to 1000 nM, the peak emission decreased and shifted to smaller wavelengths. Thus, by determining the position of the emission spectrum, it was possible to derive the [Ca ] in the environment of the dye.. This was done by measuring the intensity of emission at two wavelengths, 405 nm and 470-490 nm, and calculating the ratio of intensities at these wavelengths. Thus, using ratiometri methods, gave measurements of [Ca 9+] which were not greatly influenced by changes in dye concentration or cell volume. We constructed a dual emission microspectrofluorimeter from an inverted phase-contact microscope fitted with epifluorescence using a 100 W Xenon lamp source (Diaphot, Nikon, U.K., Telford, U.K.). The cells, settled on glass coverslips (20 mm diameter, grade thickness) were incubated with 1 μg/ml indo 1-AM i balanced salt solution without bovine serum albumin or serum for 10 minutes. Balanced salt solution (BSS) consisted of 140 mM NaCl, 5 mM KC1, 1,5 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Na 2 HP0 4 , 5 mM NaHC0 3 , 10 mM glucose. 1 g/1 bovine serum albumin (BSA), and 10 mM HEPES, and buffere to pH 7.4. Dye-loaded osteoclasts were then transferred to a perspex bath (1 ml) mounted on a heated stage on th microspectrofluorimeter. Solutions were pre-war ed to 37°C and passed through the chamber at the rate of 1.8 ml/min. The dye-loaded cells were excited at 340 nm and the fluorescence signal from the stage directed to the sideport to which was attached a variable aperture, a shutter, and a beam splitter containing a dichroic mirro (455nm). Transmitted and reflected light through the dichroic mirror was filtered at 470-490 nm and 405 nm respectively and the intensities recorded by separate photomultiplier tubes (PM28b, Thorn EMI, U.K.). Single photon currents in each tube were converted to TTL pulse and counted by a dual photon counter (Newcastle Photometric systems, Newcastle-upon-Tyne, U.K.). Photo counts per second in each channel were recorded in a BBC microcomputer and the ratio of intensities (at 405nm/470-490nm) calculated and displayed. The dye signals were calibrated by perfusing the experimental chamber with solutions of 1 to 20 μM Indo 1 (Molecular Probes, U.S.A.), in a solution containing 150 mM KC1, 10 mM hepes, and 10 mM EGTA. Following determinations of the optimal dye concentration, which was found to be 10 μM, calibration curves were constructed by perfusing the chamber with dye-containing solutions of known concentrations of free ionized calcium. Measures of cell spread area and cell motility ( 22, 23 ) Osteoclasts were settled in a tissue culture dish (35 mm; Cellcut, Sterilin, Middlesex, U.K.), and were incubated at 37°C for 20 min to allow cell sedimentation and attachment. The wells were then washed with Medium 199, and 2 ml of the same medium was placed in each well. The dishes were placed in eh incubation chamber of an inverted phase-contrast microscope (Diaphot, Nikon U.K. Ltd.), linked via a CCD camera to a 480 hour-mode time lapse video recorder (Mitsubishi, Japan). The taped video records could be played back for subsequent analysis at 120 times the recording speed. The effect of added reagent on cell form was followed over intervals of 70 min. The composite video signal was fed into a 256- grey level imaging system (Sight Systems, Newbury, Berks., U.K.). Digitizing grey images were captured into the computer memory at real time intervals of 2 min. This was followed by redigitization of the cell boundary using a Microsoft mouse (Microsoft, U.S.A.), an edge detection facility and a binary overlay. The position of each image was standardized by ensuring that a pair of reference etch marks previously made in the examined field were superimposable in successive digital images. each cell outline was traced independently by different observers; this procedure resulted in a coefficient of variation of <12% with 10 independent readings. All responses were analysed by a one-way analysis of variance, and differences were considered significant where p < 0.01. Viability of osteoclasts were confirmed through the comparison of osteoclast numbers on control slices with those on treated slices. Furthermore, cell numbers on bone slices counted prior to incubation with test reagent were compared with those obtained following an 18 hour incubation with perchlorate. Recovery of osteoclasts after treatment by test reagents was assessed by comparing bone resorption on treated slices at 18 hours with those obtained after incubating for 36 hours. RESULTS The results obtained by the techniques described above will now be described with reference to the accompanying drawings, in which: Figure 1 shows plasma [Ca 2+ ] measurements (mean ± S.E. of the mean; 5 rats) made 30 min after administration of different doses of perchlorate; Figure 2 shows changes in plasma [Ca 2+ ], [Mg 2+ ] and [albumin] (mean ± S.E.; 4 rats) against time after administration of 300 μMol perchlorate per rat (**:p<0.01; *: p<0.05 on testing against control); Figure 3 illustrates the effects of perchlorate and/or calcium chloride on bone resorption; and Figure 4 illustrates the effect of 15mM perchlorate ion on osteoclast surface area. Perchlorate mimicked the hypocalcemic effects of human calcitonin, in that it depressed serum calcium, but not magnesium or albumin levels in vivo. Fig. 1 displays serum [Ca 2+ ] (means ± S.E. of the mean) 30 minutes after administration of different doses of perchlorate in vivo (5 rats in each group). Perchlorate thus causes a significant (p < 0.01) dose-dependent hypocalcemic response. This gave an ED^ Q of 450 μmol per rat using probit analysis. When the response to infused perchlorate was followed over time, plasma calcium concentrations were significantly depressed 5, 10 and 20 minutes following administration, when compared to control results from infusions of equivalent volumes of normal saline. In contrast, perchlorate little influenced serum [Mg2+] or [albumin] . The effect reached a maximum 20 minutes after presentation, and decayed towards normal plasma calcium levels after this (Fig. 2). The fall in plasma [Ca 2+ ] might most simply arise from diminished osteoclastic activity reducing Ca 2+ flux bone to plasma, although concentrations from renal Ca 9+ excretion would not be excluded. However the in vitro findings that follow do suggest a primary action at the osteoclast level. Effects on osteoclastic bone resorption in vitro The above basis in vivo test justified further analysis of the effect of perchlorate on osteoclast bone resorption in vitro. Osteoclasts were identified as a sparse population of large multinucleate cells of complex morphology, with dorsal microvilli over a central area of unopposed cell surface. Most osteoclasts demonstrated at least over a part of their periphery, lobulated and folded pseudopodia, often in one direction. Elsewhere, the cell margin showed fine filopodia of irregular length and morphology. Osteoclasts were scattered across the bone substrate, separated by large areas of intervening cell-free bone surface. The number of cells per bone slice ranged from 0 to 15 with a mean cell number (± S.E. of the mean) of 6.2 ± 0.9; 85% of the slices had 5 to 7 cells. Incubation of the slices with 15 mM perchlorate for 18 hours did not result in any significant change in cell numbers as compared with control counts at the outset. Nor was there any evidence of morphological changes that would suggest a toxic response. This suggests that cells remained viable in the presence of perchlorate, at least at the concentrations studied here, and over the periods entailed by the present experiments. Examination of bone slices incubated in the absence of perchlorate suggested the presence of characteristic areas of excavation attributable to bone resorption. The morphological features of such excavations were similar in perchlorate-treated and control bone slices. Thus, in both cases, the bone surface showed regular saw-markings interspersed with osteocyte lacunae and occasional vascular channels. Osteocyte lacunae appeared as depressions with finely fibrillated walls, perforated with canaliculi. Vascular channels were generally exposed longitudinally for a considerable distance, before they were found to disappear as tunnels beneath the bone surface. Between the osteocyte lacunae and vascular channels, the bone had a highly predictable structure. Osteoclastic excavations were never found on slices without added osteoclasts, and ' were quite unlike either osteocytic lacunae or vascular channels. They had a fibrillary base, and usually, a well defined margin. They were often found in the proximity of, and often half covered by, an osteoclast. Such excavations were not found in association with any type of bone cells. The excavated surface consisted of a network of anastomising and branching bundles of fibrils either running parallel or exposed transversely to the bone slice. In untreated slices, the areas of excavation covered a wide range of approximately 268 to 106780 μm 2 . Addition of human parathyroid hormone (0.1 iu/ml) did not cause any increase of bone resorption compared to findings in control slices in the absence of perchlorate. Figure 3 shows the degree of bone resorption achieved in several cases. Perchlorate at a concentration of 1 mM (plot B) did not affect bone resorption compared to the control (plot A). However, a concentration of 10 mM markedly depressed the total area of bone resorbed per slice (see plot C) as well as the number of osteoclastic excavations. There was no change in the mean size of individual excavations in the presence of perchlorate. Similarly, increasing extracellular [Ca Λ 9+] from ImM to 5 mM significantly reduced bone resorption (by 60% - see plot D). A non- inhibitory concentration of 1 mM perchlorate potentiated the effect of 5 mM to result in a near complete inhibition of resorption (see plot E). In contrast, the lyotropic ion thiocyanate (SCN ~ ) did not inhibit bone resorption by osteoclasts at comparable concentrations (10 mM). Effects on acid phosphatase release Exposure of resorbing osteoclasts to perchlorate did not inhibit the release of tartrate-resistant (osteoclastic) acid phosphatase at the concentrations tested (1, 10 and 15 mM). Whereas an extracellular [Ca ] of 5mM also failed to inhibit enzyme release, one of 20 mM [Ca ] produced a significant inhibition. In addition, neither compound altered the levels of tartrate-labile (lysozomal) acid phosphatase released into the culture supernatant. The latter estimations provide an indication of the extent of cell lysis. Effects on intracellular [Ca 2+ ] in isolated osteoclasts Perchlorate influenced intracellular [Ca 2+ ] in single osteoclasts at those applied concentrations where it had significant effect on bone resorption. Thus, a five minute exposure to 1 mM perchlorate, whether in extracellular solutions containing 1.5 mM [Ca ], or in calcium-free solutions, failed to affect intracellular [Ca 2+ ] . In contrast, higher perchlorate concentrations (10 mM or above) caused a transient elevation of intracellular [Ca 2+ ] lasting approximately four seconds. Resting intercellular [Ca 9+] levels were approximately 100 nM, and perchlorate elicited peak intracellular [Ca ] responses in the region of 700 nM. Effects on cell spread area and motility of single osteoclasts Osteoclast spread area and motility in response to perchlorate were measured in experiments conducted over 70 minutes. An empirical morphometric analysis was performed through the comparison of successive traces of cell outlines obtained at the image capture intervals employed. First, cell-spread area, A(t) at each time t, was obtained. It was expressed as a percentage of control area A(0) (means ± S.E. of the mean), obtained prior to addition of perchlorate. This yielded a measure of the overall degree of cell retraction. Exposure of osteoclasts to 15 mM perchlorate led to a marked reduction of cell spread area top approximately 40% of the original cell area (as shown at time zero) (Fig. 5). This response had a time course similar to that resulting from an elevated extracellular [Ca 2+ ] . For example, the estimated half-time of the decay in response with an applied extracellular [Ca 9"+] of 20 mM was 9 minutes; applications of 15 mM perchlorate gave 10.5 min. When perchlorate was washed away, the cells recovered with a longer timecourse. Second, a tracing of each cell obtained at time t bounding the set of pixels {A(t)> was overlaid on the previous outline obtained at time (t-Δt). Comparison of the two traces would give two sets {Δp} and {Δr> of points that would not fall in both sets (A(t)} and {A(t-Δt)}. The degree of cell retraction {Δr} would be reflected in points enclosed within the set {A(t-Δt)}, but excluded from (A(t)}; conversely, cell protrusion {Δp} would correspond to the set {Δp} falling within {A(t)} but not {A(t-Δt)}. The observed pseudopodial protrusion and retraction, and cell movement, indicated that these parameters were also not altered by exposure to C10 4 ~. The lack of effect of perchlorate on cell motility was confirmed by observing time-lapse video recording of individual cells. The cells remained viable and healthy throughout the experiments, and there was no evidence of cytotoxicity up to 15 mM perchlorate. This study investigated the effect of perchlorate ions on osteoclast function. It employed four independent experimental approaches: (i) In vivo studies on the effect of perchlorate infusion on plasma [Ca 2 —] in young rats, with a high bone turnover. (ii) In vitro studies on bone resorption. (iii) Biophysical studies of cytosolic free [Ca 9+], and of spreading and motility in single osteoclasts, and (iv) biochemical assays of acid phosphatase release. All these studies separately indicate that perchlorate is a potent inhibitor of bone resorption. Earlier reports of the effects of perchlorate on other cell types include not only instances of cell activation dependent upon an influx of extracellular calcium, as may occur in pancreatic beta cells, but also in situations where intracellular calcium stores, rather than the extracellular ion, play the major part. Thus, in skeletal muscle, direct mechanical or chemical coupling between voltage sensors in the tubular membrane, and calcium channels in sarcoplasmic reticular membrane has been suggested (Fleischer et al, 1985; Leung et al, 1987). This interaction could occur at 'triad' complexes where the two membrane systems are in close proximity though remaining electrically distinct (Franzini- Armstrong, 1970; Schneider and Chandler, 1973), and can proceed even in Ca - free solutions. Nevertheless, the voltage sensor in the tubular system, in common with calcium channels, binds to dihydropyridines (Rios & Brum, 1987; Curtis & Caterall, 1984). In addition, extracellular calcium does modulate contractile activation (Luttgau & Spiecker, 1979). Recent findings (Zaidi et al, 1989) suggest that osteoclasts may not require voltage-dependent calcium current activation to modify resorptive activity. Rather, the larger proportion of calcium channels in osteoclast plasma membrane may be calcium operated, and this may form a primary means of osteoclast regulation (Zaidi et al, 1989). One may accordingly draw parallels with the modulation by extracellular calcium of muscle function, with the proviso that osteoclast regulation may be primarily mediated through such ligand binding by extracellular [Ca 2+ ], rather than primarily through changes in membrane potential. The latter notion would not be consistent with an action of perchlorate through its lyotropic effects as these would only influence voltage-dependent membrane processes. This exclusion is reinforced by the contrasting absence of any effect on osteoclast activity of thiocyanate even when applied at comparable concentrations that would be expected to give similar adsorptive properties. Nevertheless, even in skeletal muscle, perchlorate and the sterically similar dichromate ion influenced contractile activation yet spared other voltage dependent processes such as Na and K channel activation (Gomella at al, 1983). Perchlorate was effective at concentrations (1 - 10 mM) very similar to those employed in striated muscle. Thus, perchlorate may have a more specific action than simple electrostatic adsorption onto membranes in either system. The effect of perchlorate in vivo resembled that of calcitonin in that the maximal fall in plasma [Ca 2+ ] , to around 2.2 mM, took place within 20-30 min of injection, and this effect fully decayed between 45-90 min after administration. Perchlorate also mimicked the effects of a number of agents known to elevate intracellular [Ca 2+ ] , such as calcitonin, elevated extracellular [Ca 2+ ] (Datta et al, 1989), ionomycin and high micromolar concentrations of verapamil (Zaidi et al, 1990) on bone resorption in vitro. Thus it markedly reduced the total bone area resorbed and the number of excavations attributable to osteoclastic activity. These comparisons reinforce the notion that elevation of intracellular [Ca 2+ ] may inhibit osteoclastic function. However, perchlorate produced a considerably more transient increase in intracellular [Ca ] than that elicited by the agents cited above (Zaidi et al, 1989). Yet, this more transient intracellular [Ca 2+ ] response sufficed for the sustained cell retraction, and the relatively long period that followed in which bone resorption was inhibited. A further contrast is offered by recent reports of the inhibition of acid phosphatase release that accompanied the marked cell retraction and inhibition of osteoclastic bone resorption produced by the sustained elevations of intracellular [Ca 2+ ] (Zaidi et al, 1989). Acid phosphatase may have an essential role in the resorptive process, possibly through a role in removing pyrophosphate (Moonga et al, 1990). In contrast, even prolonged incubations with perchlorate di not effect acid phosphatase release. These comparisons suggest that perchlorate differs from the former agents in that it does not prevent the secretion of enzymes involved in the resorptive process such as acid phosphatase (cf. Zaidi et al, 1989). Rather, one might consider a simpler explanation in terms of effects on cell retraction by a calcium transient. In any event, the experiments here demonstrate that perchlorate is a potent antihypercalcaemic agent which seems to act by arresting osteoclastic bone resorption. This influence appeared at least partially specific to osteoclasts insofar as some other highly differentiated cells, including macrophages and lymphocytes, did not show such a response. Thus a formulation of this anion (optionally with other active substances that influence intracellular ) may provide the basis of developing novel therapeutic approaches for osteoclastic bone disease, including Paget's disease, malignancy-associate hypercalcaemia, or even osteoporosis. In view of the few antihypercalcaemic regimes available apart from those using mithramycin, aminohydroxyprolidine diphosphonate. and combinations of calcitonin and corticosteroids, this would be of considerable clinical interest.

Claims: CLAIMS : 1. A method of inhibiting bone resorption in vivo, which comprises administering to a subject a therapeutically effective amount of a composition containing the perchlorate (C10 ~) ion. 2. A composition possessing inhibitory properties towards bone resorption, characterised in that the composition contains the perchlorate (C10 4 ~) ion. 3. A method for use in the treatment of diseases or conditions characterised by bone resorption, e.g. osteoporosis, a pharmaceutical composition containing, as active ingredient, the perchlorate (C10 ** ) ion. 4. A method of preventing and/or treating inflammatory osteolysis. which comprises administering a therapeutically effective amount of the perchlorate (C10 4 -) ion.

Inventors: ZAIDI MONE, ZAIDI, MONE
Applicants: GAINCORP LTD, GAINCORP LIMITED
Application year: 1991
Main classification: A61K33
IPC codes: A61K33