Quantitative morphometric evaluation of the inhibitory activity of new aminobisphosphonates on bone resorption in the rat

Summary Three new bisphosphonates of various chain length, namely, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP), 5-amino-1-hydroxypentylidene-1,1-bisphosphonate (AHPeBP), and 6-amino-1-hydroxyhexylidene-1,1-bisphosphonate (AHHexBP) were compared with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP), and dichloromethylene bisphosphonate as to their effect on metaphyseal bone remodeling in the rat. The parameters assessed were growth in length, mineralization, metaphyseal density, mean trabecular diameter, and number of trabeculae. Both metaphyseal density, an index of metaphyseal Ca balance, and number of trabeculae, an index of bone resorption, showed the following sequence of potency: AHBuBP>AHPrBP=AHPeBP=AHHexBP>Cl₂MBP. All compounds decreased trabecular diameter somewhat, an index of bone formation, with AHBuBP decreasing the least. AHBuBP and possibly AHHexBP appear to be interesting new bisphosphonates for future clinical use.     

Structure-activity relationships of various bisphosphonates

Summary A variety of bisphosphonates with aliphatic side chains of increasing length, as well as 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP, formerly APD), dichloromethylenebisphosphonate (Cl₂MBP, formerly Cl₂MDP), and dibromomethylene bisphosphonate (Br₂MBP, formerly Br₂MDP), were compared in vitro and in vivo to find (a) a possible relationship between structure and activity in order to give some indication about their mechanism(s) of action on bone and (b) the most efficient and safe compound having an effect on bone resorption. Some relationship was found between inhibition of calcium phosphate precipitation in vitro and of mineralization in vivo. No correlation existed, however, between any parameter measured and bone resorption. The number of calvaria cells in culture was decreased by compounds with a chain length greater than 5-C, by AHPrBP, Cl₂MBP, and Br₂MBP. Lactate production by these cells in vitro was increased by the long chain bisphosphonates and AHPrBP, and was decreased by Cl₂MBP. No good correlation existed between the inhibition of bone resorption measured in vitro on calvaria and that seen in vivo on rat tibiae metaphyses. The latter was inhibited the most efficiently by the bisphosphonates longer than 5-C and by AHPrBP; these were 10 times more effective than Cl₂MBP. Taking into account all factors, 1-hydroxypentylidene-1,1-bisphosphonate and AHPrBP seem to be the most active compounds to inhibit bone resorption.     

Cytotoxic and migration inhibitory effects of bisphosphonates on macrophages

Summary Twoin vitro model systems were developed to facilitate investigation of the mechanisms by which bisphosphonates block bone resorption. These systems assess the cytotoxic and the migration inhibitory activities of bisphosphonates using mouse peritoneal macrophages as osteoclast surrogates. Several bisphosphonates, 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP), dichloromethylene bisphosphonate (Cl₂MBP), 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), 1-hydroxybutylidene-1,1-bisphosphonate (HBBP), 1-hydroxyhexylidene-1,1-bisphosphonate (HHBP), and 1-hydroxyoctylidene-1,1-bisphosphonate (HOBP), possess the same relative activities in these systems as they do in bone resorption systems. Calcium ion replacement studies using these systems demonstrated that bisphosphonates do not derive all their activity from sequestration of calcium ions from cells by chelation. Whereas calcium ion replacement abrogated the activity of EDTA, a nonbisphosphonate calcium chelator active in both systems, it failed to abrogate either the cytotoxic or the migration inhibitory effects of the bisphosphonates tested. Calcium ion replacement increased the migration inhibitory activity of all the bisphosphonates tested. Further, calcium ion replacement increased the cytotoxicity of HHBP and HOBP; however, it decreased the cytotoxicity of HEBP, HBBP, AHPrBP, and Cl₂MBP.     

Role of bone and kidney in parathyroid hormone-related peptide-induced hypercalcemia in rats

A protein responsible for the biochemical syndrome similar to primary hyperparathyroidism associated with certain tumors has been recently characterized and its effects at the level of bone and kidney reported. However, the relative role of tubular reabsorption of calcium (Ca) and bone resorption in the pathogenesis of hypercalcemia induced by this factor is still debated. We investigated the effects of a synthetic amino-terminal fragment of parathyroid hormone-related protein [PTHrP-(1-34)] administered chronically by intraperitoneal osmotic minipumps in thyroparathyroidectomized (TPTX) rats. Clearance studies performed on day 6 of treatment after a 24 h fast revealed an increase in renal tubular reabsorption of Ca and a decrease in renal tubular reabsorption of phosphate (Pi), accompanied by an increase in cAMP excretion. PTHrP-(1-34) (90 pmol/h) stimulated bone resorption as evaluated by an increment in fasting urinary Ca excretion. Although the bone resorption inhibitor aminopropylidene diphosphonate fully corrected urinary Ca excretion and reduced plasma Ca from 3.04 +/- 0.07 to 2.44 +/- 0.21 mM (p less than 0.05), this latter value remained considerably higher than in TPTX control rats (1.54 +/- 0.12 mM, p less than 0.01). In contrast, when the agent WR-2721, which is known to decrease the renal tubular reabsorption of Ca by a PTH-independent mechanism, was given, a further drop in plasma Ca and an increase in urinary Ca excretion were observed. These findings are similar to those found in animals implanted with the hypercalcemic Leydig cell tumor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of bone resorption by the bisphosphonate BM 21.0955 is not associated with an alteration of the renal handling of calcium in rats infused with parathyroid hormone-related protein.

Hypercalcaemia of malignancy is determined by an increase of bone resorption and/or renal tubular reabsorption of calcium (Ca). However, this latter component has been found to vary in certain patients during therapy with bone resorption inhibitors such as bisphosphonates. We investigated the possible effects of the highly potent bisphosphonate BM 21.0955 on the renal handling of Ca in thyroparathyroidectomized rats made hypercalcaemic by the stimulation of both bone resorption and renal tubular reabsorption of Ca induced by the chronic infusion of parathyroid hormone-related protein (PTHrP). Dose-dependent inhibition of bone resorption by BM 21.0955, as indicated by the decrease in fasting urinary Ca excretion from 64.0 +/- 7.3 to 6.7 +/- 3.1 nmol/ml GFR, was associated with a change in plasma Ca from 2.97 +/- 0.10 to 2.63 +/- 0.16 mmol/l. However, the relationship between urinary Ca excretion and plasma Ca was not altered, either at endogenous plasma Ca concentration or during the acute infusion of Ca. Similarly, an index of renal tubular reabsorption of Ca calculated from the slope of the linear portion of the relationship between urinary Ca and plasma Ca, which was increased by PTHrP administration, was not influenced by BM 21.0955 therapy (2.59 +/- 0.15 vs. 2.55 +/- 0.11 mmol/l GFR). These results indicate that BM 21.0955, which is one of the most potent bisphosphonates inhibiting bone resorption, did not affect the renal tubular reabsorption of Ca enhanced by PTHrP.     

The acute-phase response after bisphosphonate administration

Summary In patients who have never previously received bisphosphonate therapy, the intravenous administration of 4-amino-1-hydroxybuthilidene-1,1-bisphosphonate (AHButBP), 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP), or 6-amino-1-hydroxyhexylidene-1,1-bisphosphonate (AHHexBP) induced an acute-phase response (APR) irrespective of the underlying disease, manifested by a fall in circulating lymphocyte number and serum zinc concentration and in a rise in C-reactive protein (CRP); a febrile reaction occurred in 30% of the patients. The APR was maximally expressed within 28–36 hours of i.v. administration of the bisphosphonates and disappeared 2–3 days later despite continuous treatment. These effects were dose dependent and the lowest doses necessary for an APR were 10 mg of AHButBP and AHPrBP and 75 mg of AHHexBP. Doses up to 1,000 mg/day i.v. of dichloromethanebisphosphonate (Cl₂MBP) were devoid of these side effects. In patients treated with either a single i.v. dose of amino-bisphosphonates which resulted in an APR or with a suboptimal dose, a subsequent challenge 12–160 days later of the high dose failed to cause a rise in CRP or a fall in the lymphocyte count. The desensitization to AHButBP or AHPrBP was also seen following pretreatment with Cl₂MBP. These findings suggest that bisphosphonates interact with macrophage-like cells resident in the skeleton and stimulate interleukin-1 release which is responsible for the appearance of the APR. At the same time, however, the bisphosphonates render these cells insensitive to further stimulation for several months. This latter observation might be relevant to the long-lasting suppression of bone resorption observed after bisphosphonate therapy.     

Bisphosphonates inhibit 1,25-dihydroxyvitamin D3-induced increase of osteocalcin in plasma of ratsin vivo and in culture medium of rat calvariain vitro

Summary In order to test whether bisphosphonates, which are potent inhibitors of osteoclastic bone resorption, may also act upon osteoblasts, we studied the effect of dichloromethylenebisphosphonate (Cl₂MBP) and 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP) onin vivo levels andin vitro release of osteocalcin, a bone-specific protein produced by osteoblasts. In rats, 161 μmol/kg of Cl₂MBP or 1.61 μmol/kg AHBuBP strongly inhibited the increase of plasma osteocalcin induced by 1,25(OH)₂D₃. The inhibition was measurable within 24 hours after the administration of bisphosphonate and was independent of any change in bone resorption. The effect upon osteocalcin release was also present in calvaria cultures. 250 μM Cl₂MBP strongly inhibited the osteocalcin release induced by 10⁻⁸ M 1,25(OH)₂D₃. In the presence of 1,25(OH)₂D₃, protein synthesis and DNA synthesis were also decreased, whereas in the absence of 1,25(OH)₂D₃, protein synthesis was increased. Thus, bisphosphonates affect the production of a bone-specific protein by osteoblasts in addition to their inhibitory action on osteoclasts.     

Effects of four bisphosphonates on bone resorption, lysosomal enzyme release, protein synthesis and mitotic activities in mouse calvarial bones in vitro

The effects of 3-amino-1-hydroxy-propylidene-1,1-bisphosphonate (AHPrBP), 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), dichloromethylenebisphosphonate (Cl2MBP) and azacycloheptylidene-2,2-bisphosphonate (AHBP) on bone were examined in organ culture using newborn mice calvaria. AHPrBP, HEBP and Cl2MBP caused a dose-dependent inhibition of PTH-stimulated (10 nmol/l) release of 45Ca from the calvaria, at and above a concentration of 3 mumol/l, whereas AHBP only caused a slight inhibition, at and above 100 mumol/l. AHPrBP inhibited PTH-stimulated release of 3H from bones prelabelled with [3H]-proline. AHPrBP (30 mumol/l) diminished the stimulatory effect of 1 alpha(OH)vitamin D3 (10 nmol/l), prostaglandin E2 (0.1 mumol/l) and renal tumor conditioned media on 45Ca release. AHPrBP and Cl2MBP, at and above 3 mumol/l, decreased PTH-stimulated mobilization of Ca2+ and Pi and in parallel the release of beta-glucuronidase without affecting the release of lactate dehydrogenase. The inhibitory effect of AHPrBP (30 mumol/l) on PTH-induced 45Ca release was irreversible. The inhibition by AHPrBP (30 mumol/l) on spontaneous and PTH-stimulated release of 45Ca can be seen first after 24 h of culture. Similarly the inhibitory effect by HEBP (30 mumol/l) and Cl2MBP (30 mumol/l) was delayed and could be observed after 36 and 24 h of culture, respectively. PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase was reduced by AHPrBP first after 24 h of culture. AHPrBP, HEBP and Cl2MBP, at concentrations which are inhibitory on bone resorption, do not affect protein synthesis and mitotic activities in mouse calvaria. These data show that AHPrBP, HEBP and Cl2MBP inhibit bone resorption in vitro and in parallel decrease lysosomal enzyme release by a mechanism, which is not related to cytotoxicity. In addition, the delayed inhibitory effect on bone resorption and lysosomal enzyme release by all the compounds suggest that bisphosphonates inhibit bone resorption indirectly and not by a direct effect on existing osteoclasts. The delayed inhibition by bisphosphonates on bone resorption may be due to decreased recruitment of new osteoclasts as a consequence of an inhibitory action on mononuclear osteoclast precursor cells.     

Short- and long-term effects of a single dose of bisphosphonates on retinoid-induced bone resorption in thyroparathyroidectomized rats

Summary The inhibitory effect of a single subcutaneous (s.c.) dose of three different bisphosphonates (Bps)—4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP), 2-(2-pyridinyl)-ethylidene-1,1-bisphosphonate (2-PEBP), and dichloromethylene-bisphosphonate (Cl₂MBP)—was studied in a model of retinoid-induced bone resorption, which consists of assessing the hypercalcemic effect of the arotinoid Ro 13-6298 given s.c. for three days in thyroparathyroidectomized (TPTX) rats. The retinoid was given on day 0, 1, and 2. Bps were administered together with or at different times prior to the first dose of retinoid. A dose-dependent inhibition was obtained with all three compounds. AHBuBP produced complete inhibition which remained for 3 weeks at 0.1 mg P/kg. The dose-response curves were identical when the compound was given on the first day of retinoid administration (day 0) or 6 days earlier. With 2-PEBP, the dose-response curve was the same as that for AHBuBP when given on day 0. When given 6 days earlier, the curve was shifted to 30 times less potency. Cl₂MBP was about 100 times less potent than AHBuBP when given on day 0, with 3 mg P/kg producing complete inhibition. When given 6 days earlier, the curve was also shifted to 10 times less potency, and even 30 mg P/kg failed to produce complete inhibition. With 10 mg P/kg, the inhibitory effect was maintained partially for up to 3 weeks. This study shows that in this model of bone resorption the inhibitory effect of a single dose of certain Bps is effective for at least 3 weeks and that the compounds vary in their activity over time.     

Inhibition of bone resorption by bisphosphonates: Interactions between bisphosphonates,osteoclasts, and bone

Summary Bisphosphonates are nonbiodegradable pyrophosphate analogues that are being used increasingly to inhibit bone resorption in disorders characterized by excessive bone loss. We have previously found that dichloromethylene bisphosphonate (Cl₂MBP) inhibits bone resorption through injury to the cells that resorb Cl₂MBP-contaminated surfaces. 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) is a more potent inhibitor of bone resorptionin vivo, and we have attempted to identify a step in the resorptive pathway that accounts for this increased potency. We found that when osteoclasts, isolated from neonatal rat long bones, were incubated on bone slices in the presence of bisphosphonates, AHPrBP was less, rather than more potent as a resorption-inhibitor than Cl₂MBP. The greater sensitivity of resorption to AHPrBPin vivo could neither be attributed to an effect of AHPrBP on the ability of osteoblastic cells to stimulate resorption in response to calcium-regulating hormonesin vitro nor to an effect on osteoclast generation: osteoclast formation was unaffected by concentrations of AHPrBP 10-fold higher than those of Cl₂MBP which inhibit bone resorption in the bone slice assay. We also found no evidence for impaired osteoclast generationin vivo in AHPrBP-treated rats. These results suggest that the comparisons of potencyin vitro do not include all the factors responsible for determining bisphosphonate potencyin vivo. Because bisphosphonates owe the specificity of their actions to their ability to bind to bone surfaces, we performed experiments using bone slices that had been immersed in bisphosphonates before use. Bone resorption was virtually abolished on bone slices preincubated in 10⁻³ M AHPrBP. Inhibition was associated with degenerative changes in osteoclasts and a more rapid decrease in the number remaining on the bone surface than occurred with Cl₂MBP. The effect was specific for osteoclasts, could be prevented if bone resorption was suppressed by calcitonin, and was not seen in osteoclasts incubated in AHPrBP on plastic coverslips. These observations suggest that AHPrBP inhibits bone resorption through injury to osteoclasts when they solubilize bisphosphonate-contaminated bone. We found that the concentration of AHPrBP used in the preincubation phase could be reduced by an order of magnitude if the volume of the AHPrBP solution was correspondingly increased. This implies that the concentration of bisphosphonate is less relevant to potency comparisons than the density of bisphosphonate on the bone surface. The latter will be strongly influencedin vivo not only by affinity for bone but by the pharmacokinetic and other properties of the compound.     

The effects of dichloromethylene diphosphonate on hypercalcemia and other parameters of the humoral hypercalcemia of malignancy in the rat leydig cell tumor

Summary There is a high frequency of Leydig cell tumors associated with hypercalcemia in the aged Fischer 344 rat. We studied a transplantable tumor cell line (Rice D-6) which is associated with hypercalcemia, hypercalciuria, hypophosphatemia, renal phosphate wasting, increased urinary cyclic adenosine monophosphate (AMP) excretion, absence of bone metastases, increased osteoclastic bone resorption, and suppressed immunoreactive parathyroid hormone (iPTH) concentrations. We examined the ability of dichloromethylene diphosphonate (Cl₂MDP) to lower serum calcium and decrease the parameters of increased bone resorption. We used this drug also as a pharmacologic tool to determine the relationship of hypercalcemia and increased bone resorption to the abnormalities in renal tubular function associated with the humoral hypercalcemia of malignancy. Daily administration of Cl₂MDP before development of hypercalcemia, in doses from 2.5–40 mg/kg body weight subcutaneously, delayed and suppressed both the hypercalcemia and hypercalciuria. There was an increase in bone mass and decrease in both osteoclast number and activity compared with bones from untreated tumor-bearing animals. The urinary hydroxyproline excretion in treated animals declined towards the normal range. There were no significant effects on serum phosphorus, urine phosphorus, or urine cyclic AMP excretion. These data suggest that Cl₂MDP reverses the increased bone resorption that occurs in the humoral hypercalcemia of malignancy, and confirms that diphosphonates are effective agents in the prevention and treatment of increased bone resorption associated with malignant disease. They also suggest that renal phosphate wasting and increased urinary cyclic AMP excretion are not directly related to the hypercalcemia.     

Apposition and resorption of bone during oral treatment with (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD)

The effects of 1.5-2 years oral administration of disodium (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) on bone metabolism were studied in male and female rats. APD was mixed in the food at levels of 500, 2,000 and 10,000 ppm. A dose-dependent increase in metaphyseal bone was found, indicative of continued inhibition of bone and cartilage resorption. APD did not affect mineralization of bone and cartilage, primary bone formation, or periosteal apposition. A short-term metabolic balance study was performed to compare the effects of oral with subcutaneous APD. Absorption of APD was in the order of 0.2%. Oral APD increased absorption of phosphate, probably by complexation of calcium with APD. The excess absorbed phosphate increased phosphaturia and decreased urinary calcium.     

Bisphosphonates and extrarenal acid buffering capacity

Summary Both the intracellular compartment and bone mineral are supposed to play a role in acid-base balance by contributing to the extrarenal acid buffering capacity. Bisphosphonates could affect extrarenal acid buffering capacity by interfering with the formation and/or dissolution of bone mineral. In the present study, rats were pretreated with either 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP, 10 mg/kg.day sc), with prevailing inhibitory action on bone mineral formation, or dichloromethylene bisphosphonate (Cl₂MBP, 10 mg p/kg.day sc) with prevailing action on bone resorption, or NaCl injections (controls) for 7 days. In intact rats, blood acid-base variables were influenced by neither HEBP, nor Cl₂MBP. Two hours after nephrectomy and before acute acid loading, HEBP-but not Cl₂MBP-pretreated rats displayed a significant increase in both blood HCO₃ ⁻ and PCO₂. After HCl infusion (2,5 mEq/kg), the relative decrement in blood HCO₃ ⁻ (difference in blood HCO₃ ⁻ before and after acid loading) was transiently more important in the two bisphosphonate pretreated groups than in controls. After a 24 hour fasting period, nephrectomized animals pretreated with Cl₂MBP displayed significantly lower blood HCO₃ ⁻ and pH values than controls or HEBP-pretreated rats. These results suggest that bisphosphonates influence extrarenal buffering capacity according to their prevailing inhibitory action on either bone mineral formation and/or dissolution. These compounds could interfere with the release rate of bone proton buffers. However, in the presence of normal renal function, this effect does not disturb the blood acid-base equilibrium.     

Apposition and resorption of bone during oral treatment with (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD)

Summary The effects of 1.5–2 years oral administration of disodium (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) on bone metabolism were studied in male and female rats. APD was mixed in the food at levels of 500, 2,000 and 10,000 ppm. A dose-dependent increase in metaphyseal bone was found, indicative of continued inhibition of bone and cartilage resorption. APD did not affect mineralization of bone and cartilage, primary bone formation, or periosteal apposition. A short-term metabolic balance study was performed to compare the effects of oral with subcutaneous APD. Absorption of APD was in the order of 0.2%. Oral APD increased absorption of phosphate, probably by complexation of calcium with APD. The excess absorbed phosphate increased phosphaturia and decreased urinary calcium. The long-term toxicology study was performed at the Henkel toxicology department, Düsseldorf, West Germany. The metabolic balance study was performed in Leiden.     

Effect of diphosphonates and calcitonin on the chemistry and quantitative histology of rat bone

Young male rats were treated with one of three diphosphonates, disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), disodium dichloromethylene-diphosphonate (Cl₂MDP), or disodium methane-1-cyclohexyl-1-hydroxy-1,1-diphosphonate (MCHDP), or with porcine calcitonin. Changes in the length weight, calcium, phosphorus, and ash of the femora and in plasma calcium, phosphate and alkaline phosphatase were measured. Bone formation rate was measured histologically in the tibial diaphysis using tetracycline markers. The lower dose (0.01–1mg P/kg/day s.c.) of the phosphonates, which block immobilization osteoporosis and reduce bone turnover measured by⁴⁵Ca kinetics, did not cause marked changes in the composition of the femora or in plasma values. This suggests that, at these doses in intact animals, the diphosphonates similarly reduce both resorption and mineralization rates. Higher doses of EHDP and MCHDP (10 or 30 mg P/kg/day s.c.) impaired bone growth both in length and width and inhibited mineralization of new cartilage and osteoid. The total calcium content of the bones decreased but the plasma values remained unchanged. Cl₂MDP did not impair mineralization at equivalent doses. EHDP, unlike Cl₂MDP, therefore seems to have two effects on bone. At lower doses it reduces bone turnover rate; but at higher doses it also directly prevents the full mineralization of new matrix. This difference between the effects of EHDP and Cl₂MDP may be important.     

Parathyroid hormone increases the expression level of matrix metalloproteinase-13 in vivo

Parathyroid hormone (PTH) increases serum calcium (Ca) by enhancing bone resorption and renal Ca reabsorption. However, detailed mechanisms of enhanced bone resorption by PTH remain to be elucidated. Although PTH has been shown to increase the expression level of osteoblastic matrix metalloproteinase (MMP)-13 in vitro, only limited results are available regarding the in vivo regulation of MMP expression. In the present study, we have examined expression levels of MMPs in PTH-infused rats. Infusion of 1.5 or 2.0 nmol/kg/day rat PTH(1–34) for 3 days resulted in a dose-dependent increase in serum Ca. PTH infusion also decreased serum phosphate levels and increased urinary excretion of Ca and phosphate. Infusion of PTH for 7 days resulted in less severe hypercalcemia and hypophosphatemia. Urinary Ca and phosphate excretion in rats infused for 7 days was less than that in rats infused for 3 days. Northern blot analysis showed that PTH infusion increased the expression level of MMP-13 in calvaria, although it did not affect MMP-2 expression. Furthermore, the time-course and severity of hypercalcemia and hypercalciuria correlated with the expression level of MMP-13. In situ hybridization also showed that PTH infusion increased the expression level of MMP-13 in femora. These results indicate that PTH enhances MMP-13 expression in vivo and suggest that PTH stimulates bone resorption at least partly by enhancing MMP-13 expression. Received: June 5, 2000 / Accepted: January 12, 2001     

Effects of bisphosphonates on the incorporation of calcium-45 and 3H-proline in orthotopic and in demineralized matrix-induced bone in rats

The influence of the two bisphosphonates, 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) and dichloromethylene bisphosphonate (Cl2MBP), on bone formation was studied in demineralized matrix-induced bone in young rats. Cl2MBP was given in doses of 0.3 and 3 mg P/kg per day and HEBP 2,4, and 8 mg P/kg per day starting immediately after implantation of the bone matrix and continued until sacrifice at 3 weeks. Rates of collagen synthesis and mineralization of implants, femurs, and incisor teeth were quantified by assay of 3H-proline and 45Ca injected 24 h before death. New bone formation in implants was also evaluated by histologic examinations. Implants from Cl2MBP-treated animals had wider bone trabeculae than controls but were colonized by normal appearing hematopoietic tissue. Implants and femurs from both Cl2MBP groups had a higher ash content than controls, but uptake of the two isotopes was not affected. These results indicate that Cl2MBP, at these dose levels, inhibits bone resorption without affecting bone formation in rats. The uptake of 45Ca in implants decreased with increasing doses of HEBP; almost no ash or 45Ca activity was encountered with the highest dose. The bone in implants from this group appeared less mature, and only scanty areas of bone marrow were seen. The uptake of 3H-proline was not affected by HEBP. It appears that, in rats, HEBP primarily inhibits mineralization and resorption, whereas matrix formation remains largely unaffected.     

Evaluation of bone resorption and renal tubular reabsorption of calcium and phosphate in malignant and nonmalignant hypercalcemia

Tubular reabsorption of calcium (Ca) is becoming recognized as a determinant of malignant hypercalcemia. However, its importance as compared to increased bone resorption has not yet been widely investigated. We determined Ca fluxes of bone resorption and tubular reabsorption in 141 rehydrated patients with hypercalcemia of malignant or benign origin, before any specific treatment. Bone resorption (BRI) was evaluated by fasting urinary Ca excretion and Ca tubular reabsorption using an index (TRCaI) calculated from a nomogram relating fasting urinary Ca excretion and calcemia. The relationship between alterations in TRCaI and in the tubular capacity to reabsorb inorganic phosphate (Pi), as judged by TmPi/GFR, was also examined for each cause of hypercalcemia. Among 101 cases with malignancy, 67% had overt bone metastases, but all displayed increased BRI. Calcemia was highest in breast cancer and lowest in prostate carcinoma. BRI was markedly increased in breast cancer, lymphoma, and multiple myeloma, whereas it was slightly elevated in lung squamous cell, renal, and liver carcinomas. TRCaI was increased in 49% of malignant hypercalcemia, particularly in epidermoid (above the upper normal limit in 71% of the cases), renal, and liver carcinomas. It was elevated in 54% of breast cancer and normal in multiple myeloma and prostate cancer. In nonmalignant hypercalcemia, BRI was markedly increased in vitamin D intoxication, sarcoidosis, and immobilization. In primary hyperparathyroidism (PHP), BRI was moderately increased. TRCaI was abnormally elevated in PHP, but normal in vitamin D intoxication, sarcoidosis, and immobilization. In malignant hypercalcemia, TmPi/GFR was low in 77% of patients and in all types of tumors, except in prostate carcinoma. The index ratio [TRCaI/(TmPi/GFR)] gave a better discrimination of PHP from other causes of nonmalignant hypercalcemia than the use of either TRCaI or TmPi/GFR taken alone. Thus, in malignant hypercalcemia, increased bone resorption is associated with an elevation in tubular Ca reabsorption in half the patients surveyed, whereas low tubular Pi reabsorption is observed in more than 75%. Increased TRCaI is restricted to some types of tumor, whereas decreased TmPi/GFR is observed in all types except prostate carcinoma. In nonmalignant hypercalcemia, a significant increase in mean TRCaI was only observed in PHP, of which individual cases can be fully discriminated from other conditions by using a new index taking into account alteration in the renal transport capacity of both Ca and Pi.     

No short-term effects of 24,25-dihydroxycholecalciferol in healthy subjects

Seven healthy volunteers were given 25 μg of 24,25-DHCC for one week to study the effects on calcium and bone metabolism. Mean plasma 24,25-DHCC concentration increased from 2.2 ± 1.7 μg/l to 10.8 ± 6.1 μg/l (p < 0.001). No significant change was seen in the fasting plasma concentrations of Ca, Ca^{+ +}, PTH and alkaline phosphatase activity and in urinary excretion of calcium and hydroxyproline and in tubular reabsorption of phosphate. The area under the curve for plasma ionized calcium concentration and urinary excretion of calcium during a standard calcium infusion of 10 mg/kg of Ca in 2 h did not change by 24,25-DHCC. We conclude that in healthy subjects no effect of 24,25-DHCC on the steady state parameters of calcium and bone metabolism, on renal calcium handling and on the handling of an intravenous calcium challenge by the homeostatic system could be demonstrated.     

Differential response of bone cells isolated by sequential digestion to dichloromethylenebisphonate in culture

Summary Dichloromethylenebisphosphonate (Cl₂MBP), a compound structurally related to inorganic pyrophosphate but resistant to hydrolysis of endogenous phosphatase to yield inorganic phosphate, inhibits bone resorption and soft tissue mineralizationin vivo. Previously, we have shown that bone cells isolated from rat calvaria respond profoundly to the exposure of Cl₂MBP. To determine whether the cellular effects evoked by Cl₂MBP are confined to a particular bone cell type, calvaria from 1 day postnatal rats were subjected to a sequential time-dependent enzyme digestion, yielding five bone cell populations marked by differences in PTH response, alkaline phosphatase activity and collagen, as well as hyaluronic acid synthesis. Culturing these bone cell populations with Cl₂MBP revealed that previously observed results found with mixed bone cells (inhibition of cell proliferation, diminution of hyaluronic acid synthesis, and increase in alkaline phosphatase) were limited to cell populations which, according to the isolation scheme, stem from the outer tissue layer(s) of the calvaria. Collagen synthesis, however, was found to be equally increased regardless of cell type. These present results indicate that the action of Cl₂MBP on bone may be cell specific.