[Basic Studies on the Mechanism, Prevention, and Treatment of Osteonecrosis of the Jaw Induced by Bisphosphonates].

Since the first report in 2003, bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been increasing, without effective clinical strategies. Osteoporosis is common in elderly women, and bisphosphonates (BPs) are typical and widely used anti-osteoporotic or anti-bone-resorptive drugs. BRONJ is now a serious concern in dentistry. As BPs are pyrophosphate analogues and bind strongly to bone hydroxyapatite, and the P-C-P structure of BPs is non-hydrolysable, they accumulate in bones upon repeated administration. During bone-resorption, BPs are taken into osteoclasts and exhibit cytotoxicity, producing a long-lasting anti-bone-resorptive effect. BPs are divided into nitrogen-containing BPs (N-BPs) and non-nitrogen-containing BPs (non-N-BPs). N-BPs have far stronger anti-bone-resorptive effects than non-N-BPs, and BRONJ is caused by N-BPs. Our murine experiments have revealed the following. N-BPs, but not non-N-BPs, exhibit direct and potent inflammatory/necrotic effects on soft-tissues. These effects are augmented by lipopolysaccharide (the inflammatory component of bacterial cell-walls) and the accumulation of N-BPs in jawbones is augmented by inflammation. N-BPs are taken into soft-tissue cells via phosphate-transporters, while the non-N-BPs etidronate and clodronate inhibit this transportation. Etidronate, but not clodronate, has the effect of expelling N-BPs that have accumulated in bones. Moreover, etidronate and clodronate each have an analgesic effect, while clodronate has an anti-inflammatory effect via inhibition of phosphate-transporters. These findings suggest that BRONJ may be induced by phosphate-transporter-mediated and infection-promoted mechanisms, and that etidronate and clodronate may be useful for preventing and treating BRONJ. Our clinical trials support etidronate being useful for treating BRONJ, although additional clinical trials of etidronate and clodronate are needed.

Crystallization and preliminary neutron diffraction experiment of human farnesyl pyrophosphate synthase complexed with risedronate.

Nitrogen-containing bisphosphonates (N-BPs), such as risedronate and zoledronate, are currently used as a clinical drug for bone-resorption diseases and are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). X-ray crystallographic analyses of FPPS with N-BPs have revealed that N-BPs bind to FPPS with three magnesium ions and several water molecules. To understand the structural characteristics of N-BPs bound to FPPS, including H atoms and hydration by water, neutron diffraction studies were initiated using BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ). FPPS-risedronate complex crystals of approximate dimensions 2.8 × 2.5 × 1.5 mm (∼3.5 mm(3)) were obtained by repeated macro-seeding. Monochromatic neutron diffraction data were collected to 2.4 Šresolution with 98.4% overall completeness. Here, the first successful neutron data collection from FPPS in complex with N-BPs is reported.

Autoradiographic studies of rhenium-188-hydroxyethylidine diphosphonate in normal skeleton and osteoblastic bone metastases in a rat model of metastatic prostate cancer.

AIM: The quantitative distribution of bone-seeking radiopharmaceuticals in trabecular bone, cortical bone and in skeletal metastases is required for calculation of radiation-absorbed dose in radionuclide therapy. An animal model of intraosseous tumor cell administration was developed to simulate osteoblastic metastases for autoradiographic study of radionuclide localization. METHODS: In 45 Copenhagen rats R3327-MATLyLu syngeneic prostate cancer cells were given intraosseously in both the femori. Rhenium-188-hydroxyethylidine diphosphonate (HEDP) was administered intravenously 17+/-1 days after cells instillation and these animals were euthanized at 4, 24 and 48 h after injection of the radiopharmaceutical. The uptake of radiopharmaceutical was estimated in normal skeleton and the bone metastases by means of region of interest analysis using autoradiography. The tumor to nontumor ratio and the fractional uptake in cortical bone and trabecular bone were quantified. RESULTS: The uptake of rhenium-188-HEDP in cortical bone was 33.5% and in trabecular bones was 66.5% after 4 h, 34.6 and 65.4% after 24 h, and 35.9 and 64.1% after 48 h, respectively. Assuming a theoretic cortical-trabecular distribution of 50-50%, (MIRDOSE) calculation, radiation-absorbed dose to bone marrow was underestimated by 26%. In bone metastases, an inhomogeneous distribution with a minimal and maximal tumor to nontumor ratio of 3 : 1 and 14 : 1 after 4 h, 5 : 1 and 14 : 1 after 24 h, and 5 : 1 and 16 : 1 after 48 h was observed. CONCLUSION: The MIRDOSE model underestimates the radiation-absorbed dose to the bone marrow because of demonstrable differences in the uptake of rhenium-188-HEDP in cortical and trabecular bone and inhomogeneous uptake in skeletal metastases.

Fluorescently labeled risedronate and related analogues: "magic linker" synthesis.

We report synthesis of the first fluorescently labeled conjugates of risedronate (1), using an epoxide linker strategy enabling conjugation of 1 via its pyridyl nitrogen with the label (carboxyfluorescein). Unlike prior approaches to create fluorescent bisphosphonate probes, the new linking chemistry did not abolish the ability to inhibit protein prenylation in vitro, while significantly retaining hydroxyapatite affinity. The utility of a fluorescent 1 conjugate in visualizing osteoclast resorption in vitro was demonstrated.

Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells.

Bisphosphonates (BPs) target bone due to their high affinity for calcium ions. During osteoclastic resorption, these drugs are released from the acidified bone surface and taken up by osteoclasts, where they act by inhibiting the prenylation of small GTPases essential for osteoclast function. However, it remains unclear exactly how osteoclasts internalise BPs from bone and whether other cells in the bone microenvironment can also take up BPs from the bone surface. We have investigated this using a novel fluorescently-labelled alendronate analogue (FL-ALN), and by examining changes in protein prenylation following treatment of cells with risedronate (RIS). Confocal microscopic analysis showed that FL-ALN was efficiently internalised from solution or from the surface of dentine by resorbing osteoclasts into intracellular vesicles. Accordingly, unprenylated Rap1A accumulated to the same extent whether osteoclasts were cultured on RIS-coated dentine or with RIS in solution. By contrast, J774 macrophages internalised FL-ALN and RIS from solution, but took up comparatively little from dentine, due to their inability to resorb the mineral. Calvarial osteoblasts and MCF-7 tumour cells internalised even less FL-ALN and RIS, both from solution and from the surface of dentine. Accordingly, the viability of J774 and MCF-7 cells was drastically reduced when cultured with RIS in solution, but not when cultured on dentine pre-coated with RIS. However, when J774 macrophages were co-cultured with rabbit osteoclasts, J774 cells that were adjacent to resorbing osteoclasts frequently internalised more FL-ALN than J774 cells more distant from osteoclasts. This was possibly a result of increased availability of BP to these J774 cells due to transcytosis through osteoclasts, since FL-ALN partially co-localised with trancytosed, resorbed matrix protein within osteoclasts. In addition, J774 cells occupying resorption pits internalised more FL-ALN than those on unresorbed surfaces. These data demonstrate that osteoclasts are able to take up large amounts of BP, due to their ability to release the BP from the dentine surface during resorption. By contrast, non-resorbing cells take up only small amounts of BP that becomes available due to natural desorption from the dentine surface. However, BP uptake by non-resorbing cells can be increased when cultured in the presence of resorbing osteoclasts.

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase.

T4 RNA ligase catalyzes the synthesis of ATP beta,gamma-bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH(2)p) (100), clodronate (pCCl(2)p) (52), and etidronate (pC(OH)(CH(3))p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH(2)-CH(2)-NH(2))p), and alendronate (pC(OH)(CH(2)-CH(2)-CH(2)-NH(2))p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E-AMP in a concentration dependent manner. The K(m) values and the rate of synthesis (k(cat)) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26+/-0.05 mM (0.28+/-0.05 s(-1)); clodronate, 0.54+/-0.14 mM (0.29+/-0.05 s(-1)); and etidronate, 4.3+/-0.5 mM (0.028+/-0.013 s(-1)). In the presence of GTP, and ATP or AppCCl(2)p the relative rate of synthesis of adenosine 5',5'''-P(1),P(4)-tetraphosphoguanosine (Ap(4)G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH(2)p) was a very poor substrate for the synthesis of Ap(4)G. To our knowledge this report describes, for the first time, the synthesis of ATP beta,gamma-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases.

Enthalpy versus entropy-driven binding of bisphosphonates to farnesyl diphosphate synthase.

We report the results of an ITC (isothermal titration calorimetry) investigation of the binding of six bisphosphonates to the enzyme farnesyl diphosphate synthase (FPPS; EC 2.5.1.10) from Trypanosoma brucei. The bisphosphonates investigated were zoledronate, risedronate, ibandronate, pamidronate, 2-phenyl-1-hydroxyethane-1,1-bisphosphonate, and 1-(2,2-bisphosphonoethyl)-3-iodo pyridinium. At pH = 7.4, both risedronate and the phenylethane bisphosphonate bind in an enthalpy-driven manner (DeltaH approximately -9 to 10 kcal mol-1), but the other four bisphosphonates bind in an entropy-driven manner (DeltaS varying from 31.2 to 55.1 cal K-1 mol-1). However, at pH = 8.5, zoledronate binding switches from entropy to enthalpy-driven. The DeltaG results are highly correlated with FPPS inhibition results obtained using a radiochemical assay (R2 = 0.85, N = 11, P < 0.001). The DeltaH and DeltaS results are interpreted in terms of a model in which bisphosphonates with charged side chains have positive DeltaH values, due to the enthalpic cost of desolvation (due to strong ion-dipole interactions) and, likewise, a positive DeltaS, due to an increase in water entropy (both ligand and protein associated) on ligand binding to FPPS: the hydrophobic effect. For the neutral side chains (risedronate at pH 7.4, 8.5 and zoledronate at pH 8.5, as well as the phenylethane bisphosphonate), binding is overwhelmingly enthalpy-driven, with the enhanced activity of the basic side chain containing species being attributable to their becoming protonated in the active site. Given the large size of the bisphosphonate market and the potential importance of the development of these compounds for cancer immunotherapy and anti-parasitic chemotherapy, these results are of broad general interest in the context of the development of new, potent, and selective FPPS inhibitors.

Clinical trial of risedronate in Japanese volunteers: a study on the effects of timing of dosing on absorption.

Because of its chemical structure, risedronate was thought to form a complex with divalent cations, e.g., Ca(2+), and to be likely to show changes in the efficiency of absorbance from the gastrointestinal tract according to the presence of food. Therefore, we conducted a crossover study using healthy Japanese adults to examine the effects of food intake on absorption after the oral administration of risedronate and to choose the best timing of regimen for risedronate. Using single doses of 5 mg risedronate, the following four dose times were investigated: (a) in the morning under a fasting condition without breakfast; (b) 30 min before breakfast; (c) 30 min after breakfast; and (d) 3 h after breakfast. The results showed that the C(max) and AUC(0-24) of the plasma risedronate concentrations and its cumulative urinary excretions decreased in the following order: fasting without breakfast >>30 min before breakfast >>3 h after breakfast >>30 min after breakfast. In other words, it was demonstrated that the absorption of risedronate decreases due to the effects of food. Several adverse events, whose causality with risedronate was unknown or possibly related, were observed, including headaches, diarrhea, increased CK-BB, and an increased urinary Beta(2)-microglobulin excretion rate, but none of these events was clinically significant, and none differed in frequency or severity from the events after a single oral administration. In consideration of the optimal practical timings required to administer risedronate for Japanese patients, therefore, it was found that ingesting the drug immediately after waking up in the morning, when the stomach is empty, was optimal, and that it was necessary to refrain from eating and drinking for at least 30 min after drug ingestion. Therefore, we determined that the optimal time for risedronate to be administered in Japanese patients is 30 min before breakfast.

[Prophylaxis of the unfavorable shifts in mineral turnover in bones during extended exposure to the conditions reproducing the physiological effects of microgravity]. / Profilaktika neblagopriiatnykh izmenenii mineral'nogo metabolizma v kostnoi tkani pri dlitel'nom prebyvanii v usloviiakh, modeliruiushchikh fizioligicheskie éffekty mikrogravitatsii.

Effectiveness of the countermeasures and methods for mitigation of the unfavorable effects of microgravity on mineral turnover and properties of bones was evaluated in simulation experiments with animals (penal immobilization, suspension) and with participation of human subjects (37 to 120-d head-down bed rest). The experiments with rats were aimed at testing pharmaceutical preparations (bisphosphonates), determining doses, plan and way of administration. Preparations of this group differ considerably in their action on osteogenesis, mostly because of specifics of the chemical structure. Nonetheless, optimization of the course of treatment masked significantly side effects without detriment to the preventive action. Head-down bed rest was used to test effectiveness of physical exercises (PE) as a main countermeasure against the microgravity impacts. Both standard and experimental PE regimens were tested. Several groups of test-subjects combined PE with consumption of xidifon (a bisphosphonate). In addition, mineral supplements (potassium and calcium) were used. The PE + xidifon combination and regulation of mineral intake with foods was found to reduce calcium losses during 370-d bed rest and prevent a material degradation of strength of bone samples, and essentially rehabilitate shifts in the hormonal regulation of calcium turnover and calciuretic function of the kidney.

Involvement of vacuolar H+ -ATPase in incorporation of risedronate into osteoclasts.

Although osteoclasts incorporate bisphosphonates during bone resorption, the mechanism of this incorporation by osteoclasts is not known. We previously reported that bisphosphonates disrupt the actin rings (clear zones) formed in normal osteoclasts, but did not disrupt actin rings in osteoclasts derived from osteosclerotic oc/oc mice, which have a defect in the gene encoding vacuolar H(+)-ATPase (V-ATPase). The present study showed that V-ATPase is directly involved in the incorporation of risedronate, a nitrogen containing bisphosphonate, into osteoclasts. Treatment of osteoclasts with risedronate disrupted actin rings and inhibited pit formation by osteoclasts on dentine slices. Bafilomycin A(1), a V-ATPase inhibitor, inhibited the pit-forming activity of osteoclasts but did not disrupt actin rings. Risedronate failed to disrupt actin rings in the presence of bafilomycin A(1). E-64, a lysosomal cysteine proteinase inhibitor, showed no inhibitory effect on the demineralization of dentine by osteoclasts but inhibited the digestion of dentine matrix proteins without disrupting actin rings. Risedronate disrupted actin rings even in the presence of E-64. Treatment of osteoclasts placed on plastic plates with risedronate also disrupted actin rings. Bafilomycin A(1) but not E64 prevented the disruption of actin rings in osteoclasts treated with risedronate on plastic plates. Inhibition of V-ATPase with bafilomycin A(1) also prevented disruption of actin rings by etidronate, a non-nitrogen-containing bisphosphonate. These results suggest that V-ATPase induced acidification beneath the ruffled borders of osteoclasts and subsequent bone demineralization triggers the incorporation of both nitrogen-containing and non-nitrogen-containing bisphosphonates into osteoclasts.

Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces some biomechanical properties in dog rib.

It has been hypothesized that suppression of bone remodeling allows microdamage to accumulate, leading to increased bone fragility. This study evaluated the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties of cortical bone in the dog rib. Thirty-six female beagles, 1-2 years old, were divided into three groups. The control group (CNT) was treated daily for 12 months with saline vehicle. The remaining two groups were treated daily with risedronate (RIS) at a dose of 0.5 mg/kg per day or alendronate (ALN) at 1.0 mg/kg per day orally. After sacrifice, the right ninth rib was assigned to cortical histomorphometry or microdamage analysis. The left ninth rib was tested to failure in three-point bending. Total cross-sectional bone area was significantly increased in both RIS and ALN compared with CNT, whereas cortical area did not differ significantly among groups. One-year treatment with RIS or ALN significantly suppressed intracortical remodeling (RIS, 53%; ALN, 68%) without impairment of mineralization and significantly increased microdamage accumulation in both RIS (155%) and ALN (322%) compared with CNT. Although bone strength and stiffness were not significantly affected by the treatments, bone toughness declined significantly in ALN (20%). Regression analysis showed a significant nonlinear relationship between suppressed intracortical bone remodeling and microdamage accumulation as well as a significant linear relationship between microdamage accumulation and reduced toughness. This study showed that suppression of bone turnover by high doses of bisphosphonates is associated with microdamage accumulation and reduced some mechanical properties of bone.

Binding and antiresorptive properties of heterocycle-containing bisphosphonate analogs: structure-activity relationships.

To define structure-activity relationships for bisphosphonate activity, we examined the bone binding and antiresorptive properties of heterocycle-containing analogs of risedronate, a pyridylbisphosphonate, in cultures of mouse fetal bone explants. Our studies indicated that hydroxybisphosphonates with the nitrogen molecule in the pyridyl ring were very potent inhibitors of osteoclastic resorption. Changing the place of the nitrogen in the ring structure of risedronate or its methylation did not significantly alter antiresorptive potency in relation to risedronate. Extension of the R2 chain, however, reduced efficacy. In binding experiments, we found that all heterocyclic bisphosphonates with a hydroxyl group in R1 had comparable affinity for bone mineral and inhibited calcium incorporation into bone explants to a similar extent. The affinity of a risedronate analog without R1 was markedly reduced. We also examined the properties of a risedronate analog (NE-10790) belonging to the group of phosphonocarboxylates in which one of the phosphonate groups is substituted by a carboxyl group. NE-10790 had strongly reduced binding affinity, but still retained some antiresorptive activity. Interestingly, the continuous presence of NE-10790 in cultures of fetal mouse metacarpal bones increased its antiresorptive efficacy by about 40-fold compared with 24 h preincubation, whereas, under the same conditions, the potency of high-affinity hydroxybisphosphonates did not change or only slightly increased. This may be explained by the differences in pharmacokinetic behavior between compounds of high and of low affinity for bone mineral. These data show that, as with alkylbisphosphonates, heterocycle-containing bisphosphonates with a nitrogen functionality in the R2 chain are potent antiresorptive agents and a hydroxyl substitution in the R1 chain confers high affinity for bone mineral, probably due to tridentate configuration. The group of phosphonocarboxylates, with strongly reduced bone affinity, provides an interesting therapeutic option.

Entamoeba histolytica: computer-assisted modeling of phosphofructokinase for the prediction of broad-spectrum antiparasitic agents.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) is the rate-limiting glycolytic enzyme found in the pathogenic protists Entamoeba histolytica, Giardia lamblia, Toxoplasma gondii, Trichomonas vaginalis, and Naegleria fowleri. The enzyme differs significantly from ATP-dependent phosphofructokinases found in humans and as such represents an important drug target. Current therapy for infections caused by these pathogens is inadequate, especially for children, pregnant women, and the immune compromised. The development of more selective, safer agents in imperative, as parasitic infections are currently a significant health threat worldwide and will likely become increasingly common agents of disease in the future. For the purpose of designing drugs to treat parasitic infections, we have constructed a model of PPi-PFK from E. histolytica based on the three-dimensional structure of the ATP-dependent PFK from Bacillus stearothermophilus. The model was used with the computer program Dock 3.5 (University of California, San Francisco) to predict the binding of pyrophosphate and selected bisphosphonates to the enzyme. The predicted drug-enzyme interactions suggested that two of these compounds would be competitive inhibitors of pyrophosphate. These drugs were tested against E. histolytica and inhibited the growth of amebae in vitro. This class of compounds may have broad-spectrum antiparasitic activity and, in the future, may facilitate the treatment of serious parasitic infections.

Bisphosphonates: preclinical aspects and use in osteoporosis.

The bisphosphonates are synthetic compounds characterized by a P-C-P bond. They have a strong affinity to calcium phosphates and hence to bone mineral. In vitro they inhibit both formation and dissolution of the latter. Many of the bisphosphonates inhibit bone resorption, the newest compounds being 10,000 times more active than etidronate, the first bisphosphonate described. The antiresorbing effect is cell mediated, partly by a direct action on the osteoclasts, partly through the osteoblasts, which produce an inhibitor of osteoclastic recruitment. When given in large amounts, some bisphosphonates can also inhibit normal and ectopic mineralization through a physical-chemical inhibition of crystal growth. In the growing rat the inhibition of resorption is accompanied by an increase in intestinal absorption and an increased balance of calcium. Bisphosphonates also prevent various types of experimental osteoporosis, such as after immobilization, ovariectomy, orchidectomy, administration of corticosteroids, or low calcium diet. The P-C-P bond of the bisphosphonates is completely resistant to enzymatic hydrolysis. The bisphosphonates studied up to now, such as etidronate, clodronate, pamidronate, and alendronate, are absorbed, stored, and excreted unaltered. The intestinal absorption of the bisphosphonates is low, between 1% or less and 10% of the amount ingested. The newer bisphosphonates are at the lower end of the scale. The absorption diminishes when the compounds are given with food, especially in the presence of calcium. Bisphosphonates are rapidly cleared from plasma, 20%-80% being deposited in bone and the remainder excreted in the urine. In bone, they deposit at sites of mineralization as well as under the osteoclasts. In contrast to plasma, the half-life in bone is very long, partially as long as the half-life of the bone in which they are deposited. In humans, bisphosphonates are used successfully in diseases with increased bone turnover, such as Paget's disease, tumoural bone disease, as well as in osteoporosis. Various bisphosphonates, such as alendronate, clodronate, etidronate, ibandronate, pamidronate, and tiludronate, have been investigated in osteoporosis. All inhibit bone loss in postmenopausal women and increase bone mass. Furthermore, bisphosphonates are also effective in preventing bone loss both in corticosteroid-treated and in immobilized patients. The effect on the rate of fractures has recently been proven for alendronate. In humans, the adverse effects depend upon the compound and the amount given. For etidronate, practically the only adverse effect is an inhibition of mineralization. The aminoderivatives induce for a period of 2-3 days a syndrome with pyrexia, which shows a similitude with an acute phase reaction. The more potent compounds can induce gastrointestinal disturbances, sometimes oesophagitis, when given orally. Bisphosphonates are an important addition to the therapeutic possibilities in the prevention and treatment of osteoporosis.

Identification of adenine nucleotide-containing metabolites of bisphosphonate drugs using ion-pair liquid chromatography-electrospray mass spectrometry.

Bisphosphonates are synthetic pyrophosphate analogues, which are used as therapeutic drugs for the treatment of metabolic bone disorders. Some of these bisphosphonates can be metabolised in cells into non-hydrolysable nucleotide analogues. In this paper, we describe an ion-pairing high-performance liquid chromatography method that is compatible with negative ion electrospray mass spectrometry for the separation of these metabolites. Tandem mass spectrometry and collision-induced dissociation (CID) were used for identification of the metabolites. The CID mass spectra of bisphosphonate-adenine nucleotide adducts are very informative, because major fragment ions are formed by cleavage of the bisphosphonate moiety from the conjugate. The method was used for detection of the nucleotide metabolites of clodronate, tiludronate and etidronate in extracts from mammalian cells after treatment with bisphosphonates.

Dissociation of binding and antiresorptive properties of hydroxybisphosphonates by substitution of the hydroxyl with an amino group.

The purpose of this study was to examine the role of the R1 moiety of bisphosphonates in binding to bone mineral and for antiresorptive action. For this, the R1 chain of three clinically useful hydroxybisphosphonates (etidronate, pamidronate, and olpadronate) was substituted with an amino group. The effects of the aminosubstituted bisphosphonates were compared with those of their hydroxy counterparts in a crystal growth assay and in fetal mouse long bone cultures which are representative of bisphosphonate actions in vivo. It was found that all three aminosubstituted compounds and their hydroxy analogs bound with similar affinity to bone mineral and inhibited the growth of calcium oxalate crystals to the same extent. Surprisingly, the antiresorptive effect of olpadronate was totally abolished by the amino substitution of the hydroxyl group while that of pamidronate was reduced by about six-fold and that of etidronate did not change. These studies demonstrate the involvement of the entire bisphosphonate molecule in the cellular mechanism of antiresorptive action. In addition, the aminosubstituted analog of olpadronate, which lacks any antiresorptive action but retains all other properties of olpadronate, provides an excellent tool for the study of specific cellular effects involved in bisphosphonate action.

Growth inhibition of macrophage-like and other cell types by liposome-encapsulated, calcium-bound, and free bisphosphonates in vitro.

Bisphosphonates effectively inhibit osteoclastic bone resorption in diseases characterized by excessive bone loss. Liposome-encapsulated clodronate (dichloromethylene bisphosphonate) also is known to inactivate phagocytic cells in vivo, and inhibit the growth of macrophage-like RAW 264 cells in vitro. The macrophage suppressive effect of liposomal clodronate is of interest in autoimmune diseases, like rheumatoid arthritis, in which phagocytic cells are involved in inflammatory processes. Earlier in vivo studies suggested that liposomal clodronate is a far more potent inactivator of macrophages than liposomal forms of two other bisphosphonate compounds, pamidronate (3-amino-1-hydroxypropylidene bisphosphonate), and etidronate (1-hydroxyethylidene-1,1-bisphosphonate). We examined the growth inhibitory properties of these three bisphosphonates with macrophage-like RAW 264 cells and with other types of cells in vitro. All three bisphosphonates encapsulated in liposomes effectively inhibited the growth of RAW 264 and CV1-P cells, while free drugs were 20-1000 times less potent growth inhibitors. Also, high extracellular calcium concentrations enhanced the potency of bisphosphonates for RAW 264 cells, indicating that, in addition to liposomes, the uptake of bisphosphonates by macrophages is mediated also by calcium. In all formulations, pamidronate was the most potent compound for the cells, with the exception of CV1-P cells, for which liposomal clodronate was the most potent. The effects of liposomal drugs were selective for highly endocytotic cells. The results suggest that liposome-encapsulated bisphosphonates could provide a specific tool to affect the function of macrophages and all three of these bisphosphonates are potentially effective as macrophage suppressors in autoimmune diseases.

The effect of low-dose cyclical etidronate and calcium on bone mass in early postmenopausal women.

This 2-year study was carried out in 36 healthy women of mean age 53.9 +/- 3.8 (SD) years and 3.4 +/- 2.3 years postmenopausal. Bone mineral density (BMD) in the spine, measured by single-energy quantitative computed tomographic scanning, gave a mean initial value of 110 +/- 26 mg/ml. The women were divided randomly into group 1 (n = 11), calcium 600 mg/day; group 2 (n = 15), calcium plus etidronate sodium 400 mg/day for 14 days every 3 months; and group 3 (n = 10), calcium plus etidronate plus phosphate, the 14-day etidronate course being preceded by phosphate 1 g twice daily for 3 days. During the first year of the study BMD decreased by 6.0 +/- 5.8% (p < 0.005) in group 1 subjects and increased by 4.5 +/- 7.8% (p < 0.005) in the combined etidronate-treated groups (difference between control and treated p < 0.001). Inclusion of phosphate in the regimen did not affect the response to etidronate. In the second year there was no significant mean change in BMD in any of the three groups. However, whilst there was little change in BMD values for most of the group 1 subjects, there was considerable variation in individual response within the etidronate-treated groups, with some subjects gaining and some losing bone. The change in BMD during the second year in the subjects as a whole was highly correlated with the change in plasma calcium after 3 months of treatment (r = 0.60, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)