Ethane-1-hydroxy-1,1-diphosphonate Research Articles

Biological Effects of Phosphate on Fibroblast-Like Synoviocytes

This study sought to examine the expression of genes implicated in phosphate transport and pathological calcification in osteoarthritis (OA) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and investigate the biological effects of phosphate. Results revealed that several genes, which were implicated in phosphate transport and pathological calcification, were differentially expressed in OA FLS and RA FLS. Phosphate stimulated the expression of matrix metalloproteinse-1, matrix metalloproteinse-3, cyclooxygenase-2, and interleukin-1βin a dose-dependent manner. Phosphate also induced OA FLS cell death but not RA FLS cell death at higher concentration. Calcification inhibitors, phosphocitrate (PC), and ethane-1-hydroxy-1,1-diphosphonate (EHDP), effectively inhibited these detrimental biological effects of phosphate. These findings suggest that abnormal expression of genes implicated in phosphate transport and pathological calcification may contribute to the progression of OA through the induction of extracellular matrix-degrading enzymes, proinflammatory cytokines, cell death, and calcium deposits. Calcification inhibitors such as PC and EHDP are potent inhibitors of these detrimental biological effects of phosphate.

Effects of high Doses of Ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on Alkaline and Acid Phosphatase Activities

Effects of high Doses of Ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on Alkaline and Acid Phosphatase Activities

Etidronate (EHDP) inhibits osteoclastic-bone resorption, promotes apoptosis and disrupts actin rings in isolate-mature osteoclasts.

Bisphosphonates, therapeutic reagents against tumoral bone diseases (Paget's disease or osteoporosis), are potent inhibitors of bone resorption. The mechanisms by which they directly act on mature osteoclasts remain unclear. Using a recently developed technique for isolation of highly purified mammalian mature osteoclasts, we demonstrated that etidronate [ethane-1-hydroxy-1,1-diphosphonate (EHDP), 1-hydroxy-1,1-ethylidenebisphosphonate], inhibited directly osteoclastic bone-resorbing activity by pit assay. In addition, EHDP also directly induced apoptosis and disrupted actin rings in osteoclasts. The data support previous data on non-purified osteoclasts and results in vivo.

Does elimination of Kupffer cells prolong survival time in liver transplantation?

Inorganic pyrophosphate (PPi) and two diphosphonates, ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2MDP), were found to inhibit in vitro mineralization induced by matrix vesicle-enriched fractions from chicken epiphyseal cartilage. Inhibitor concentrations from 0.20 to 20 μM caused a dosage-dependent decrease in 45Ca and 32Pi uptake by the vesicle fraction. These inhibitors were also tested in a hydroxyapatite (HA)-seeded system to help distinguish between effects on the mineral vs nonmineral portions of the vesicle fraction. The order of inhibition of the HA-seeded system was EHDP > PPi > C2MDP, except for inhibitor concentrations of 0.20 μm where EHDP was the least inhibitory. This variation may be due to differences in the binding of the inhibitors to HA crystals.In general, inhibition of HA mineralization was greatest during later time periods, whereas vesicle ion uptake was affected more during early stages of incubation. The differential effects of the three inhibitors were most obvious at the 2.0 μM concentration. With PPi, substantial inhibition of HA-seeded mineralization was observed even in late stages of the study; in contrast, with time the vesicle fraction overcame this inhibition. This suggests that alkaline phosphatase, an enzyme notably enriched in matrix vesicles, catalyzed the hydrolysis of PPi, reducing its concentration to a level where mineralization could proceed.Our findings show that matrix vesicle-induced mineralization differs significantly from apatite-induced mineralization. The data support the concept that vesicle alkaline phosphatase acts, at least in part, to remove physiological crystal growth inhibitors.

Vitamin D and enterocyte brush border membrane calcium transport and fluidity in the rat

Prior studies of vitamin D repletion showed a threefold increase in the maximum rate (Vmax) for calcium uptake by brush border membrane vesicles, but did not differentiate saturable and nonsaturable uptake components. We studied the calcium uptake and fluidity response of intestinal brush border vesicles to vitamin D by treatment with 1α,25-dihydroxy-24,24-difluorocholecalciferol (24,24-F-1,25-(OH) 2 D 3). Treatment responses were measured by effects on (1) saturable and nonsaturable initial uptake rates of calcium by rat proximal small intestinal brush border membrane vesicles; (2) transmucosal calcium transport by everted duodenal sac; and (3) fluorescence anisotropy. Treatment of vitamin D-depleted weanlings increased the Vmax by 50% ( P < .05) in vesicles from the proximal 12 cm of small intestine from rats injected with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), but there was no response in rats not injected with EHDP or in vesicles from the proximal 30 cm of small intestine. Vitamin D-depleted weanlings were D-deficient based on serum 25-hydroxycalciferol (25-OH-D) concentration, but to produce 1α,25-dihydroxycalciferol [1,25-(OH) 2D] depletion, EHDP injection was required. Treatment of vitamin D-replete adult rats caused a 20% ( P < .05) increase in Vmax. Treatment did not affect the calcium concentration at half-Vmax ( K T ), the rate constant for nonsaturable uptake ( K D ), or vesicle fluidity measured as fluorescence anisotropy. Contrasting with these minimal effects of treatment on brush border Vmax, treatment increased transmucosal calcium transport by everted duodenal sac almost threefold in vitamin D-depleted weanlings administered EHDP. Thus, vitamin D actions on enterocyte calcium transport (1) at the brush border increase saturable but not nonsaturable uptake, and (2) produce the major transport response distal to the brush border. Despite previously described changes in membrane lipid, brush border fluidity is unaffected by vitamin D treatment.

Investigation of the acute and chronic anti‐inflammatory properties of diphosphonates using a broad spectrum of immune and non‐immune inflammatory reactions

AbstractDiphosphonates are known to affect skeletal and soft tissue mineralization; however, several studies have shown that these compounds effectively suppress adjuvant‐induced polyarthritis in the rat, reducing the edematous inflammatory component as well as skeletal changes. Therefore, we investigated the specific anti‐inflammatory potential of two diphosphonates, ethane‐1‐hydroxy‐1,1 diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2MDP), using several different models of acute, non‐immune‐ and immune‐driven inflammation in the pleural cavity of rats and guinea pigs which afforded both a qualitative and quantitative assessment of leukocyte infiltration and exudate fluid (edema) formation. EHDP and Cl2MDP, administered at doses which suppressed the chronic inflammatory lesions induced in adjuvant arthritis, failed to inhibit acute inflammation regardless of the stimulus used (carrageenan; calcium pyrophosphate dihydrate crystals; hydroxyapatite crystals; immune complexes (Arthus reaction); delayed‐type hypersensitivity to purified protein derivate). At the highest dose tested (15 mg/kg subcutaneously [sc]) both EHDP and Cl2MDP exacerbated hydroxyapatite‐ and delayed‐type hypersensitivity‐induced leukocyte infiltration; hydroxyapatite crystals precoated with EHDP or Cl2MDP caused a small but significant inhibition of the chronic inflammatory response to sc cotton pellet implantation. We conclude that EHDP and Cl2MDP may be effective in chronic rather than acute inflammation. Our observations suggest that further studies are warranted on the potential of diphosphonates as novel therapeutics for chronic inflammatory disease. © 1993 Wiley‐Liss, Inc.

Inhibition of the formation of oral calcium phosphate precipitates: beneficial effects of Chinese traditional (Kampo) medicines

Screening tests on the formation of calcium phosphate precipitates using 23 different kinds of Kampo medicines (Chinese traditional medicines) were carried out, at concentrations where the effects of chelation are not significant. Four of them, Hochu-ekki-to (TJ-41), Kyuki-kyogai-to (TJ-77), Oren-to (TJ-120) and Inchin-ko-to (TJ-135) showed an inhibitory effect on the formation of amorphous calcium phosphate (ACP). The inhibitory effect on the induction time and the rate of transformation to hydroxyapatite (HAP) varied greatly among the 23 Kampo medicines. We classified them according to their effects on increasing the induction time and/or decreasing the rate of HAP transformation. Ethane-1-hydroxy-1, 1-diphosphonate (EHDP) was used as the standard. This compound is a common toothpaste additive which decreases dental calculus formation. Two of the 23 Kampo medicines showed little or no inhibition either on the induction time or on the rate of HAP transformation. Twelve of them reduced the rate of HAP transformation by 20-40% and with 1.9- to 4.0-fold increases in the induction time. The remaining nine showed even greater activity. Keishi-ninjin-to (TJ-82), Dai-kenchu-to (TJ-100), Toki-to (TJ-102), Rikko-san (TJ-110) and San'o-shashin-to (TJ-113) showed the same inhibitory effect as EHDP. Shigyaku-san (TJ-35;5.2-fold), Dai-kanzo-to (TJ-84;4.9-fold), Oren-gedoku-to (TJ-15;12.7-fold) and Inchin-ko-to (TJ-135;9.5-fold) had a greater effect on the increase of induction time than EHDP and reduced the rate of HAP transformation by 50-60%. These results suggest that these nine kinds of Kampo medicines may have potential as anticalculus agents in toothpastes and mouthwashes.

Effects of ethane-1-hydroxy-1, 1-diphosphonate on cell differentiation, and proteoglycan and calcium metabolism, in the proximal tibia of young rats

The effects of ethane-1-hydroxy-1, 1-diphosphonate(EHDP) on cell differentiation, and on the metabolism of proteoglycan and calcium in the epiphyseal plate and metaphysis of rats were investigated through histology and autoradiograms of [ 35S]-sulfate, 45Ca, and [ 3H]-thymidine. Suppression of bone resorption in the metaphysis due to low dose EHDP administration was associated with a proliferation of osteoclasts with an increased number of nuclei. High dose EHDP induced enlargement of the hypertrophie zone of the epiphyseal plate and suppression of calcification of the cartilage matrix. This change had a significant association not only with the suppression of chondroitin sulfate synthesis and the degradation in the cartilage matrix, but also with the suppression of growth and differentiation of chondrocytes. Calcification was also inhibited in the metaphysis, and growth and differentiation from undifferentiated mesenchymal cells to osteoblasts, osteocytes, and osteoclasts were also suppressed by high dose EHDP.

Ethane 1-Hydroxy-1, 1-Diphosphonate (EHDP) Counteracts the Inhibitory Effect of Uranyl Nitrate on Bone Formation

The beneficial effect of ethane 1-hydroxy-1, 1-diphosphonate (EHDP) in restoring the inhibition of bone formation in cases of acute uranium intoxication is presented. Bone formation was studied histomorphometrically in a model of alveolar bone healing. After tooth extraction, 40 rats were divided into 4 groups that received (1) no further treatment, (2) 10 daily intraperitoneal injections of 7.5 mg/kg of body weight of EHDP, (3) an intraperitoneal injection of 2.0 mg/kg of body weight of uranyl nitrate, and (4) the same treatment as was provided rats in groups 2 and 3. The results showed that the healing of bone did not occur in exposed animals, whereas healing in EHDP-treated exposed animals did not differ from that of nonexposed controls. This effect might result from a blocking and/or competitive action of EHDP and/or the stimulation that EHDP elicits at the doses and in the administration period studied.

Fibrodysplasia Ossificans Progressiva An 11‐Year‐Old Boy Treated with a Diphosphonate

Fibrodysplasia ossificans progressiva (FOP) is a severe, rare, autosomal dominant, ectopic ossifying condition, with primary involvement of the skeletal muscles associated with skeletal abnormalities. This report concerns an 11-year-old boy suffering from FOP, who presented significant modification of the musculoskeletal structure of the thorax and problems with articular movements. The patient showed progress after treatment with ethane-1-hydroxy-1,1-diphosphonate (EHDP). In fact, using a scintiscanner we were able to observe a significant improvement in symptoms and a recovery of some of the active sites of ossification.

Effects of Propane-2, 2-Diphosphonate (PDP) on Matrix-Induced Ectopic Bone Formation in Comparison to Ethane- 1 -Hydroxy- 1, 1 -Diphosphonate (EHDP)

The most prominent effect of propane-2, 2-diphosphonate (PDP) and ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on matrix-induced ectopic bone in the rat was a dose-dependent inhibition of osteogenesis in the early phases of development. The delay was seen as a consequence of osteoprogenitor cell inhibition. Additionally, later phases of bone maturation were disturbed by interference with the mineralization and remodeling processes. However, direct effects on the calcium phosphates of bone are only of minor additional value, which remains of lesser importance in comparison to the cellular impairment. After withdrawal of diphosphonates, the effects were nearly completely remitted. Neither PDP nor EHDP, even given in high doses, resulted in a lasting reduction in ectopic mass. The remission may be referred to the recovery of cell activities, whereas the mineral impregnation of osteoidosis was, if at all, of little importance. For treatment of ectopic osteogenesis PDP proved inefficient.

Comparative Study of the Effects of Radiation, Indomethacin, Prednisolone, and Ethane-1-Hydroxy-1,1-Diphosphonate (EHDP) in the Prevention of Ectopic Bone Formation

Forty male New Zealand white rabbits were treated with immobilization and forcible manipulation of the right knee known to cause ectopic bone formation in the quadriceps muscle. The animals were also treated with either: A) A total radiation dose of 1840 rad (18.4 Gy); B) A 0.5 mg/kg/day oral dose of prednisolone; C) 10 mg/kg/day of indomethacin, given in two oral doses; D) Ethane-1-hydroxy-1,1-diphosphonate (EHDP) given in a 20-mg/kg/day oral dose; or E) with the vehicle given alone as a control. The experimental period was four weeks, and the amount of ectopic bone was determined by planimetry of serial transverse sections of the femur. The degree of knee stiffness was recorded during the experiment. The amount (mean +/- SEM) of ectopic bone was 1.412 +/- 0.264 cm3 in controls; it was insignificantly higher in the EHDP-treated group, but significantly lower (p less than 0.01) in rabbits treated with radiation (0.390 +/- 0.094 cm3), prednisolone (0.181 +/- 0.076 cm3), and indomethacin (0.314 +/- 0.112 cm3). In control animals and those given EHDP, the treatment invariably led to almost complete stiffness of the right knee. The rabbits treated with prednisolone and indomethacin offered the least resistance to the manipulations, and the group treated with radiation showed intermediate stiffness. Semiquantitative histologic evaluation demonstrated less inflammation in the group treated with prednisolone than in the control group.

The effect of disodium ethane-1-hydroxy-1,1-diphosphonate on the metabolism of calcitriol in chicks.

Decreased intestinal absorption of Ca2+ occurs in response to treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP). The effect is due to decreased 1-hydroxylation of calcidiol (25-hydroxycholecalciferol) in the kidney. In an attempt to establish whether impairment of vitamin D metabolism at steps beyond kidney hydroxylation occurs due to treatment with EHDP, chicks were depleted of vitamin D and were treated with calcitriol (1,25-dihydroxycholecalciferol) as their sole source of the vitamin. The chicks were then divided into two groups, one being treated with EHDP while the second group served as control. Intestinal absorption of Ca2+ in the EHDP-treated group was found to be impaired, along with decreases in concentrations of calbindin D28K (the 28,000-Mr vitamin D-dependent Ca2+-binding protein). When the chicks were dosed with [3H]calcitriol, significantly lower concentrations of the sterol were detected in the duodena of EHDP-treated birds. Measurement of levels of receptors for calcitriol in duodena showed no difference between groups, but levels of calcitriol in sera were considerably lower in the EHDP-treated group along with the elevated biliary and urinary excretion of glucuronidated conjugates. It is therefore concluded that treatment with EHDP results in increased catabolism of calcitriol in addition to the known suppression of the renal production of the hormone.

Dynamic Radiologic Patterns of Paget??s Disease of Bone

Within the diaphyseal cortex, the primary resorption phase of Paget's disease is often limited either to the endosteum or to the central layers of the cortex. This results in primary resorption fronts that are usually discrete, both radiologically and scintigraphically. The subsequent activation of the subperiosteal cortex may be delayed, leading to secondary expanding fronts associated with subperiosteal new bone formation. Sequential radiographs of 19 untreated patients followed 6.4 +/- 1.2 years showed that the mean extension rate of the lesions within cortical bone was 8 +/- 0.5 mm/year per advancing front. This extension rate showed no significant change in 15 patients treated with calcitonin (CT) and/or ethane-1-hydroxy-1,1-diphosphonate (EHDP), but was significantly decreased in 14 patients treated with 3-amino-1-hydroxypropilidene-1,1-diphosphonate (APD). The increased remodeling rate of pagetic bone magnifies the radiologic changes due to mechanical, dystrophic, and metabolic interferences. Thus, a sclerotic pattern of the disease may rapidly change into a mixed or even a lytic pattern under the influence of any rarefying factor. Conversely, lytic pagetic bone may transform into dense bone through the administration of antiosteoclastic medications such as CT and APD. The reconstructive action of any new therapeutic regimen should be monitored radiologically.

Procollagen-III peptide serum levels in Paget's disease of the bone.

A commercially available radioimmunoassay kit was used to determine aminoterminal procollagen-III peptide (pNcoll III) serum levels in patients with Paget's disease of the bone and control subjects. In patients with Paget's disease pNcoll III concentrations were significantly elevated. They decreased to varying degrees under chronic therapy with human and salmon calcitonin, disodium ethane 1-hydroxy 1,1-diphosphonate (EHDP), or a combination therapy of EHDP and human calcitonin. The results were compared with the effect on traditional biochemical markers of disease activity: serum alkaline phosphatase and urinary hydroxyproline excretion, both of which reacted more acutely to the various therapies than pNcoll III, although pretreatment correlations were close. The most probable source of pNcoll III is not the Pagetic bone per se, but the vascular, fibrous connective tissue replacing normal bone marrow.

外傷性骨化性筋炎に対するＥＨＤＰ治療の小経験

We experienced traumatic myositis ossificans affecting triceps on the left elbow in a 17-year-old boy. His elbow joint was dislocated by a fall in Judo. After one week, he was treated by over-vigorous massage and passive stretching. Ectopic calcification and ossification were induced on his elbow three months after dislocation, and he visited our hospital. To this patient, we administered 1000mg EHDP (Disodium ethane-1-hydroxy-1, 1-diphosphonate) for two months and 600mg EHDP for next one month. During the treatment his elbow joint motion was gradually improved, and serial X-rays showed a resolution of ectopic calcification. He showed no side effects during and after EHDP administration.

Effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on interleukin 1 production by macrophages.

The effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on the in vitro functions of guinea pig macrophages were studied. A high dose (1 mg/ml) of EHDP inhibited interleukin 1 (IL 1) production by oil-induced peritoneal macrophages stimulated with muramyl dipeptide (MDP), lipopolysaccharide (LPS), phorbol myristic acetate (PMA), heat-aggregated IgG2 or calcium ionophore A23187. On the other hand, low doses (less than 0.125 mg/ml) of EHDP augmented the MDP induced IL 1 production by macrophages. This biphasic effect was also observed when macrophages were exposed to EHDP at 37 C for 24 hr and then stimulated with IL 1 inducers. Superoxide anion generation induced by formyl peptide or PMA was not affected by preincubation of the macrophages with doses of EHDP up to 1 mg/ml. Adherence and spreading of macrophages was inhibited by EHDP in a dose dependent manner without affecting cell viability. These results demonstrated that EHDP acted on macrophages directly and modulated IL 1 production in vitro.

Effect of diphosphonate on the prevention of X-radiation-induced inhibition of bone formation in rats.

A histologic and histometric study of the effect of EHDP (ethane-1-hydroxy-1,1-diphosphonate) on the alveolar wound healing of irradiated animals is presented. This drug was selected based on its osteosclerotic effect when applied at low doses. The three left mandibular molars of 80 male Wistar rats were extracted. The rats were grouped into 4 sets of 20 rats each. Group I received no further treatment; Group II received daily intraperitoneal injections of EHDP (7.5 mg/Kg of body weight during 10 days); Group III: 15 Gy of X-radiation were applied to the head; and Group IV were irradiated and injected with EHDP as were animals of Groups III and II. Half of the animals were killed after 14 days and the other half 30 days post-extraction. Sections were made at the level of the mesial socket of the first molar in a bucco-lingual orientation, on which histometric determinations of bone activity were made. The results showed the attenuation of the inhibitory effect of radiation on bone formation when the animals were treated with EHDP.

The calcium antagonist diltiazem inhibits calcification enhanced by calcitonin in growth cartilage of rats in ethane-1-hydroxy-1,1-diphosphonate (EHDP)-induced rickets

In an animal model of human rickets developed by giving a short-term administration of large doses of EHDP to young rats, concomitant administration of [Asu1,7]eel calcitonin (CT) with EHDP resulted in the promotion of calcification in growth cartilage. In an attempt to clarify the mechanisms related to the accelerated calcification due to CT, the effects of diltiazem, a calcium antagonist, were studied. Diltiazem suppressed, in a dose-dependent manner, the accelerated calcification due to CT in the growth cartilage, as determined by findings on the soft X-ray photos, contact microradiograph and light microscopic histology of the proximal region of the tibia. This suppression was only evident when diltiazem and CT were given concomitantly. If it is assumed that diltiazem inhibits the entry of calcium ion into the cells of growth cartilage, in the same manner as seen in case of smooth muscle and myocardial cells, then our results indicate that intracellular concentrations of calcium might play an important role in the occurrence of accelerated calcification due to CT.

Effects of ethane-1-hydroxy-1, 1-diphosphonate on ectopic bone formation induced by murine osteosarcoma-derived bone-inducing substance

The effects of ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on ectopic bone formation were studied qualitatively and quantitatively in an experimental system for ectopic bone formation induced by murine osteosarcoma-derived bone-inducing substance. At a low dose of EHDP (3 mg/kg per day i.p.), histologic sequelae of ectopic bone formation were normal, and the size of the induced bone mass was unaffected. At a high dose of EHDP (30 mg/kg per day i.p.), an unmineralized bone matrix with hematopoietic bone marrow was formed without evidence of retardation. This osteoid tissue showed no radiologic and histologic evidence of mineralization during the period of EHDP administration. When EHDP was withdrawn, its inhibitory effect on mineralization was reversed. The induced bone mass was almost the same size as that in controls. These results suggest that EHDP might not prevent ectopic bone matrix formation, but its mineralization and withdrawal of EHDP might lead to the formation of a normal bone similar in size to that formed without EHDP treatment.