Dichloromethane Diphosphonate Research Articles

Pharmacokinetics of clodronate in renal failure.

The pharmacokinetic parameters describing the fate of one intravenous clodronate (disodium dichloromethane diphosphonate) dose was studied in 24 normal subjects and in 24 patients with different degrees of renal insufficiency. The aim of the study was to derive data for adjustment of dosage in relation to renal function. Disodium clodronate in serum and urine samples was analyzed by capillary gas chromatography with mass-selective detection. The renal clearance (CLR) of clodronate was highly dependent on renal function and declined successively with declining glomerular filtration rate (GFR). Plasma clearance (CLP) declined, too, but to a lesser degree than CLR. The impairment of renal function resulted in decreased cumulative urinary elimination of clodronate and increased total areas under the serum concentration-time curve (AUC0-infinity). Hence, as the renal elimination of clodronate diminishes with decreasing GFR, there is a related retention of the substance. As a result of the present study, the following dosages are recommended: creatinine clearance (CLCr) from 50 to 80 ml/minute, 75-100% of normal dose; CLCr 12-50 ml/minute, 50-75% of normal dose; and ClCr < 12 ml/minute, 50% of normal dose. The results must be interpreted with caution in patients with malignancy and severe skeletal disease, in whom the nonrenal clearance may vary markedly.

Cytotoxicity of dichloromethane diphosphonate and of 1-hydroxyethane-1,1-diphosphonate in the amoebae of the slime mould Dictyostelium discoideum: A 31P NMR study

Two pyrophosphate analogues, dichloromethane diphosphonate (Cl 2MDP), and 1-hydroxy-ethane-1,1-diphosphonate (EHDP), at concentrations of 0.5–1 mM, efficiently inhibited the growth of amoebae of the slime mould Dictyostelium discoideum. Cell viability decreased markedly upon incubation with the diphosphonates. The mechanism of tenacity was investigated by in vivo 31P NMR spectroscopy and the formation of analogues of ATP [adenosine 5'-(β,γ-dichloromethane triphosphate) and adenosine 5'-(β,γ-1-hydroxyethane triphosphate)] was demonstrated. These two compounds were identified from their 31P NMR spectra in perchloric add extracts prepared from amoebae poisoned with C1 2MDP or EHDP and may have been synthesized by reversible pyrophosphate exchange catalysed by cytosolic aminoacyl-tRNA synthetases.

Characterization of the Early Ultrastructural and Biochemical Events Occurring in Dichloromethane Diphosphonate Nephrotoxicity

A chelator, dichloromethane diphosphonate (Cl2MDP), used to treat for malignancy-induced hypercalcemia, has nephrotoxic potential. An acute animal model developed to examine the mechanism was used to further characterize the renal effects. NAG enzymuria appears to be an early premonitor of injury. Ultrastructurally, an increase in size and number of protein-containing phagolysosomal reabsorption droplets in proximal convoluted tubules associated with proteinuria precedes advent of tubular cell necrosis indicating these organelles to be a potential target site for Cl2MDP in the kidney. In vitro studies using rabbit cortical tubules and rat brush border membrane vesicle preparations suggest that the renal toxicity is not due to perturbation of phosphate transport or oxidative metabolism. An operational hypothesis emerges indicating that Cl2MDP may be protein bound affecting carrier protein charge facilitating glomerular leakage with tubular accumulation via protein transport. Cl2MDP may induce critical cation perturbation at the subcellular level as the mechanism of cell death.

Characterization of the Early Ultrastructural and Biochemical Events Occurring in Dichloromethane Diphosphonate Nephrotoxicity

Characterization of the Early Ultrastructural and Biochemical Events Occurring in Dichloromethane Diphosphonate Nephrotoxicity

The Beneficial Effects of Diphosphonate and Piroxicam on the Osteolytic and Metastatic Spread of Rat Prostate Carcinoma Cells

Transplantable rat prostate adenocarcinoma III cells produce local tumors and osteolytic and osteoplastic lesions and they metastasize through defined lymphatic channels to the lungs in which they produce expanding focal tumors. The bone lesions were prevented by treatments with dichloromethane diphosphonate (C12MDP). Treatment of rats with piroxicam, a nonsteroidal antiinflammatory drug, suppressed to some extent tumor growth, bone destruction, and metastasis. However, simultaneous treatments of rats with both drugs (C12MDP and piroxicam) prevented bone damage and suppressed tumor growth and metastatic spread very significantly without evidence of toxicity.

The beneficial effects of diphosphonate and piroxicam on the osteolytic and metastatic spread of rat prostate carcinoma cells.

Transplantable rat prostate adenocarcinoma III cells produce local tumors and osteolytic and osteoplastic lesions and they metastasize through defined lymphatic channels to the lungs in which they produce expanding focal tumors. The bone lesions were prevented by treatments with dichloromethane diphosphonate (Cl2MDP). Treatment of rats with piroxicam, a nonsteroidal antiinflammatory drug, suppressed to some extent tumor growth, bone destruction, and metastasis. However, simultaneous treatments of rats with both drugs (Cl2MDP and piroxicam) prevented bone damage and suppressed tumor growth and metastatic spread very significantly without evidence of toxicity.

Influence of bivalent cations, phosphate and complexing substances on inorganic pyrophosphate in the microsomal fraction of isolated rat odontoblasts

The influence of three diphosphonates, ethane-1-hydroxy 1.1. diphosphonate, methane-diphosphonate and dichloromethane diphosphonate, on inorganic pyrophosphatase (PP iase, E.G. 3.6.1.1.) was investigated. The inhibition by the diphosphonates appeared to be due to their complexation with a bivalent cation, probably Zn 2+, which acts as a cofactor for the enzyme. The influence of calcium and inorganic phosphate (P i) on the PP iase activity was studied in the presence and absence of several complexing substances, including the diphosphonates. Ca 2+ alone inhibited the enzyme whereas, in the presence of EDTA, a Ca 2+ stimulation of the PP iase was observed which was substrate-dependent and had an optimal activity at a Ca 2+:PP i 4− ratio of 1:1. There was a small inhibition of the PP iase activity by P i which was not influenced by the substrate used.

Secondary hyperparathyroidism associated with dichloromethane diphosphonate treatment of Paget's disease.

Six patients with extensive polyostotic Paget's disease were treated for 3 months with dichloromethane diphosphonate (CL2MDP; 1600 mg/day, orally). Serum alkaline phosphatase and urinary hydroxyproline excretion decreased 60-80% in each patient. Blood ionized calcium (ca++) and immunoreactive PTH (iPTH) were measured weekly during the first month of therapy and monthly thereafter. As an index of parathyroid function, PTH secretory reserve was assessed by EDTA infusions before treatment, at the end of treatment, and 3 months after Cl2 MDP therapy was stopped. Before therapy, iPTH and Ca++ were normal in all patients. During treatment, Ca++ decreased and iPTH increased in all patients; mean iPTH approximately doubled (95% confidence limits, 1.5- to 2.7-fold increase). At the end of 3 months of treatment, EDTA infusion raised iPTH in each patient to a level higher than that in the control infusion, indicating augmented PTH secretory reserve. Ca++, iPTH, and the iPTH response to EDTA-induced hypocalcemia returned toward baseline by 3 months after the end of CL2MDP treatment. The results indicate that secondary hyperparathyroidism developed as a result of Cl2MDP therapy. The cause of the parathyroid-gland adaptation is not known; the hyperparathyroidism is, however, at least partly reversible. Cl2MDP inhibits bone resorption while allowing bone mineralization to continue; this differential effect could lead to the hypocalcemia and parathyroid hyperfunction.

Inhibition of post-extraction alveolar ridge resorption in rats by dichloromethane diphosphonate.

The therapeutic effect of dichloromethane diphosphonate (C12MDP) on postdental extraction alveolar bone resorption was studied in rats. Rais were injected with 10 mg Cl2MDP/kg/day s.c. for varying intervals from seven days prior to, through 27 days after extraction of maxillary molar teeth. Results demonstrated that C12MDP effectively inhibited rapid alveolar bone loss even after a brief interval of therapy (seven days of therapy prior to extraction), suggesting that the efficacy is not immediately reversible.

Relationship of inorganic pyrophosphatase and para-nitrophenylphosphatase activities of alkaline phosphatase in the microsomal fraction of isolated odontoblasts.

Some properties of inorganic pyrophosphatase (PPiase EC 3.6.1.1.) and para-nitrophenylphosphatase (p-NPPase EC 3.1.3.1) in the microsomal fraction of odontoblasts were investigated. The ratio of Mg2+:p-NPP and Mg2+:PPi for optimal enzyme activities was 1:1. A mutual substrate competition for PPiase and p-NPPase was described. In the presence of 0.1 mM EDTA, Mg2+ alone was not able to reactivate p-NPPase or PPiase. Instead, Zn2+ and Co2+ reactivated the PPiase, indicating they might act as cofactors for the enzyme. Mg2+ increased the PPiase activity, probably because Mg PP2-i was the true substrate for the enzyme. The diphosphonates ethane-1-hydroxy 1,1 diphosphonate (EHDP), methane diphosphonate (MDP) and dichloromethane diphosphonate (Cl2MDP) inhibited the PPiase activity.

Gut-mediated hypercalcemia in rabbits bearing VX2 carcinoma: new mechanism for tumor-induced hypercalcemia.

The VX2 carcinoma-bearing rabbit is an animal model for tumor-induced hypercalcemia, thought to be due to increased bone destruction effected by prostaglandin E2. The present experiments suggest that the pathophysiology of the hypercalcemia differs from that previously proposed. Tumor was transplanted intramuscularly into 2.5- to 3-kg male New Zealand White rabbits, which were conditioned to a 1.5% calcium diet and treated with daily subcutaneous injections of dichloromethane diphosphonate (10 mg . kg-1 . day-1), a potent inhibitor of bone resorption, or 0.9% NaCl (2 ml . kg-1 . day -1). The diphosphonate had no significant effect on plasma Ca2+ in either group. After day 31, half the animals of each group were fed a calcium-free diet. This normalized the plasma Ca2+ in each VX2-bearing rabbit within 3 to 4 days but had little effect in control rabbits. In a second series of experiments, VX2-bearing rabbits maintained on standard rabbit chow were treated for 11 days with parenteral indomethacin (30--60 mg/day) or 0.9% NaCl. Although indomethacin normalized the markedly elevated urinary excretion of prostaglandin E2, both treatment groups became severely hypercalcemic. Dietary calcium restriction promptly restored to normal the plasma Ca2+ concentration. In a third series of experiments, rabbits were fed standard rabbit chow and treated with oral indomethacin (40 mg/day) while control-rabbits were pair fed in identical chow. Transplantation of VX2 tumor into both groups caused hypercalcemia. We conclude that the hypercalcemia produced by this tumor strain is indomethacin resistant and dependent on an increase in gastrointestinal calcium absorption, not on skeletal calcium mobilization.

Human parathyroid hormone 1-34-mediated hypercalcemia in a rat model, and its inhibition by dichloromethane diphosphonate.

The purpose of these experiments was twofold: (a) to see if a continuous hPTH-(1-34) infusion could sustain hypercalcemia for 1–2 weeks; and (b) to test the efficacy of dichloromethane diphosphonate (Cl2MDP) in controlling the hypercalcemia of bone origin. First, 150-g Sprague-Dawley rats underwent parathyroid transplant-thyroidectomy and were equilibrated on a calcium-free diet prior to treatment. To evaluate the model, animals received either the hPTH-(1-34) infusion (12 units/rat/h), or 0.1 cc 0.9% NaCl per 100 g body weight (b.w.) per day. Thereafter, similar animals received 1 of 4 treatments: (a) hPTH-(1-34); (b) Cl2MDP (10 mg drug per kg b.w. per day); (c) hPTH-(1-34) plus Cl2MDP; or (d) saline (controls). The peptide was administered by subcutaneously implanted osmotic minipumps; the Cl2MDP and saline by once-daily subcutaneous injections. Plasma calcium and inorganic phosphate were measured for up to 15.5 days.

Effect of Dichloromethane Diphosphonate on Cortisol-Induced Bone Loss in Young Adult Rabbits

Cortisol, 2.5 mg/kg, dichloromethane diphosphonate (Cl2MDP), 0.1, 1.0 and 4.0 mg/kg, and combinations of the two were administered daily for 42 days to young adult rabbits (3.9 kg) to determine if Cl2MDP could inhibit development of cortisol-induced osteopenia. A beginning and a terminal control group were included in the experiment. In the terminal controls compared with the beginning controls, the proximal tibia lost cortical and trabecular bone, the distal femur gained cortical bone and the lumbar vertebral body lost trabecular bone. The administration of Cl2MDP alone inhibited bone resorption at those sites where bone loss was occurring. Two cortical bone sites showed gains in bone. The 1.0 mg/kg dose was most effective. In rabbits administered cortisol, bone formation was inhibited; the number of osteoclasts was markedly increased, cortical bone was lost in all the bones examined, and there was no marked effect on trabecular bone. Cl2MDP administered with cortisol partially inhibited cortisol-induced bone loss. The concomitant administration of Cl2MDP did not alter the number of osteoclasts and osteoblasts. The 1.0 mg/kg dose was most effective. The results of this study clearly demonstrated the need to evaluate several bones and different sites within individual bones if erroneous conclusions are to be avoided when studying agents that affect bone metabolism.

Effects of pyrophosphate and diphosphonates on the dissolution of hydroxyapatites using a flow system.

Pyrophosphate and diphosphonate ions have been said to diminish the dissolution of hydroxyapatite crystals, because they lower the equilibrium concentrations of calcium and phosphate ions in the bulk solution around hydroxyapatite crystals in a closed system. However, in a closed system these effects are not necessarily due to an effect on dissolution alone. In this paper we have used a continuous flow system to study the effects of pyrophosphate and two diphosphonates, ethane-1-hydroxy-1,1,-diphosphonate and dichloromethane diphosphonate, on the dissolution of hydroxyapatite. All three compounds decreased markedly the rate of dissolution of hydroxyapatite as well as the exchangeable pools of calcium and phosphate ions around the crystals.

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasing substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg 2+ (0.01–10.0 mM) and Mn 2+ (0.1–0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, P i, CN −, Zn 2+ and l-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. P i is a competitive inhibitor with a K i of 10.0 mM. Levamisole and Zn 2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be 2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, l-cysteine, phenylmethylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCl, KCl and Na 2SO 4 at 0.5–1.0 M inhibit the enzyme. At ph 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg 2+ and depressed by concentrations higher than 1 mM. Ca 2+ show only inhibition. Similar effects of Mg 2+ and Ca 2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.

Comparative antiinflammatory and bone protective effects of two diphosphonates in adjuvant arthritis

The effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) and disodium dichloromethane diphosphonate (Cl2MDP) were evaluated in the rat adjuvant model of arthritis to directly compare their ability to inhibit arthritic processes. The results of the experiment indicated that both diphosphonates inhibited osseous changes, pedal inflammation, and the change in body weight gain patterns which are characteristic of this model. The latter effects suggest that diphosphonates may have antinflammatory activity that is not related to their previously known actions on bone. Cl2MDP appeared to be more effective than EHDP in this particular model when all aspects were considered. The relationship of these results to the potential safety of these compounds in clinical situations is discussed.

The effect of pyrophosphate and diphosphonates on calcium transport in red cells

The effect of 0.5 mM pyrophosphate (PPi), disodium ethane-4-hydroxy-1,1-diphosphonate (EHDP) and disodium dichloromethane diphosphonate (Cl2MDP) on the ATP-dependent Ca2+ extrusion from the human red cell ghosts was studied. PPi and Cl2MDP had no effect, when introduced into the cells or added outside to the medium. EHDP slightly increased the calcium concentration in the released cells and slightly decreased the rate constant of the calcium transport, having opposite effects when it was inside or outside the cells. PPi and the 2 diphosphonates were not found to move easily across the red cell membrane.

The inhibition of calcium hydroxyapatite crystal growth by polyphosphonates and polyphosphates

The formation of crystalline calcium hydroxyapatite from solutions of calcium and phosphate ions and the inhibition of calcium hydroxyapatite crystal growth by polyphosphonates and polyphosphates have been studied. The polyphosphonates, disodium ethane-1-hydroxy-1,1-diphosphonate and disodium dichloromethane diphosphonate, are effective inhibitors of calcium hydroxyapatite crystal growth. The polyphosphates are also effective inhibitors of calcium hydroxyapatite crystal growth as long as the required level of intact polyphosphate is present in the system. However, because of their hydrolytic instability, which is enhanced by high temperature, low pH, and certain enzymes, the concentration of the polyphosphate decreases with timein vitro, and its activity as an inhibitor is lost. In contrast to the polyphosphates, the polyphosphonates are hydrolytically stable. The polyphosphonates are chemisorbed on the surface of the microcrystallites of calcium hydroxyapatite and, in the manner of other known crystal growth poisons, thus prevent further crystal growth. The stability of the polyphosphonates and their chemisorption on apatite suggest their use in medical and dental applications involving pathological calcium and phosphate metabolism.