This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints, Permissions and Rights Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fleisch, H. Search for Related Content PubMed PubMed Citation Articles by Fleisch, H.

Bisphosphonates: Mechanisms of Action

Department of Pathophysiology, University of Berne, CH-3010 Berne, Switzerland

	Abstract

Top Abstract I. Introduction II. Chemistry III. Effects in Vivo IV. Mechanisms of Action V. Pharmacokinetics VI. Animal Toxicology and... VII. Conclusion References

 

I. Introduction

II. Chemistry

III. Effects in Vivo

A. Inhibition of calcification

B. Inhibition of bone resorption

C. Effects on bone formation

D. Effects on noncalcified tissues

IV. Mechanisms of Action

A. Calcification

B. Bone resorption

C. Other effects

V. Pharmacokinetics

VI. Animal Toxicology and Human Adverse Events

A. Animal toxicology

B. Human adverse events

VII. Conclusion

	I. Introduction

Top Abstract I. Introduction II. Chemistry III. Effects in Vivo IV. Mechanisms of Action V. Pharmacokinetics VI. Animal Toxicology and... VII. Conclusion References

 
The bisphosphonates have been known to chemists since the middle of the 19th century, when the first synthesis occurred in 1865 in Germany (1). Etidronate, the first bisphosphonate to be used to treat a human disease (2), was synthesized exactly 100 yr ago (3). Bisphosphonates were used in industry, mainly as corrosion inhibitors or as complexing agents in the textile, fertilizer, and oil industries. Their ability to inhibit calcium carbonate precipitation, similar to polyphosphates, was put to good use in the prevention of scaling (4). Only in the past three decades have bisphosphonates been developed as drugs for use in various diseases of bone, tooth, and calcium metabolism.

Our knowledge of the biological characteristics of bisphosphonates dates back 30 yr. The first report was done by the author’s group and published in 1968 (5). The concept was derived from our earlier studies on inorganic pyrophosphate. We had found that plasma and urine contained compounds that inhibit calcium phosphate precipitation and that part of this inhibitory activity was due to inorganic pyrophosphate, a compound that had not been described previously in the scientific literature (6). Pyrophosphate was then shown to impair in vitro the formation and dissolution of calcium phosphate crystals. This effect was therefore similar to that on calcium carbonate and, for this reason, had been used in washing powders. Since pyrophosphate was able to inhibit ectopic calcification in vivo, it was suggested that it might act as a physiological regulator of calcification and perhaps also of decalcification in vivo, its local concentration being determined by the activity of local pyrophosphatases (7).

Because of its failure to act when given orally and its rapid hydrolysis when given parenterally, pyrophosphate was used therapeutically only in scintigraphy and against dental calculus. This prompted us to search for analogs that showed similar physicochemical activity but resisted enzymatic hydrolysis and, therefore, would not be degraded metabolically. The bisphosphonates fulfilled these conditions.

This review will deal with the mechanisms of action of these compounds. In vitro results, as well as results both in animals and humans, will be integrated in an attempt to deduce the current state of the art. Various reviews have been published recently on bisphosphonates and may be consulted also for information on other aspects (8, 9, 10, 11, 12, 13, 14). Since the literature in this field is plentiful, selective citation was necessary. Priority is given to papers dealing with the mechanisms of action. Since many papers often deal with the same finding, in most cases only the first ones are quoted. Subsequent papers are quoted only if they convey new knowledge.

	II. Chemistry

Top Abstract I. Introduction II. Chemistry III. Effects in Vivo IV. Mechanisms of Action V. Pharmacokinetics VI. Animal Toxicology and... VII. Conclusion References

 
Bisphosphonates, erroneously called diphosphonates in the past, are compounds characterized by two C-P bonds. If the two bonds are located on the same carbon atom, the compounds are called geminal bisphosphonates and are analogs of pyrophosphate, containing an oxygen instead of a carbon atom (Fig. 1). In the literature these compounds are usually called bisphosphonates. Although this is not entirely correct since nongeminal bisphosphonates are also bisphosphonates, we shall nevertheless adopt this nomenclature for simplicity’s sake.

View larger version (12K): [in this window] [in a new window]   Figure 1. Chemical structure of pyrophosphate and bisphosphonates.

View larger version (50K): [in this window] [in a new window]   Figure 2. Chemical structure of the bisphosphonates investigated for their effects in humans. *, Commercially available. [From H. Fleisch (14).]

	III. Effects in Vivo

Top Abstract I. Introduction II. Chemistry III. Effects in Vivo IV. Mechanisms of Action V. Pharmacokinetics VI. Animal Toxicology and... VII. Conclusion References

 
The bisphosphonates have two fundamental biological effects: inhibition of calcification, when given at high doses, and inhibition of bone resorption.

A. Inhibition of calcification
The first rationale for the search for analogs of polyphosphates was to find compounds that would inhibit the formation of calcium phosphate salts without being destroyed by enzymes, therefore making them useful in treating diseases with ectopic mineralization. One possible application was to administer the compounds systemically in diseases such as atherosclerosis; another application was as an addition to toothpastes to fight against dental calculus.

1. Ectopic mineralization and ossification.
a. In animals.
Bisphosphonates can efficiently inhibit ectopic calcification in vivo. Thus, among others, they prevent experimentally induced calcification of many soft tissues when given both parenterally and orally (15, 16). In contrast to pyrophosphate, which acts only when given parenterally, they are also active when administered orally. They decrease not only mineral deposits but also the accumulation of cholesterol, elastin, and collagen in the arteries (17, 18).

Bisphosphonates can also inhibit the calcification of bioprosthetic heart valves. Thus, etidronate administered subcutaneously inhibits the calcification of aortic valves implanted subcutaneously in rats (19). The bisphosphonate is also active when it is released locally from various matrices (20, 21). Certain results suggest that the bisphosphonates can be bound covalently to the valves (22). These results open an interesting field of application in heart surgery.

Bisphosphonates also decrease the formation of experimental urinary stones (23). Unfortunately, the dose has to be such that normal mineralization is impaired, as well.

As originally hypothesized, topical administration can lead to a decreased formation of dental calculus (24). This effect is currently used to prevent tartar formation in humans by the addition of bisphosphonates to toothpastes.

Finally, certain bisphosphonates also inhibit ectopic ossification when given systemically (25) or locally (26). It appears that the process is mainly an impairment of the calcification process because the deposition of matrix is not impaired, at least in the beginning.

b. In humans.
One of the bisphosphonates, etidronate, has been used in humans to prevent ectopic calcification and ossification. Unfortunately, with respect to calcification, the results so far have been disappointing. In conditions such as scleroderma, dermatomyositis, and calcinosis universalis, results are inconclusive (27). In urolithiasis, the dose that might be effective is such that normal mineralization is inhibited (28). Better effects are seen with topical applications to prevent dental calculus (29, 30), and toothpastes containing bisphosphonates are marketed in some countries. More published reports are available in ectopic ossification, especially fibrodysplasia ossificans progressiva (31), and ossification after spinal cord injury, cranial trauma, and especially after total hip replacement (32, 33). However, the efficacy of etidronate has still not been proven beyond a doubt, although the results are promising (34).

2. Normal mineralization. The results cited above raised the hope that bisphosphonates might indeed be used clinically to inhibit various types of calcifications. Unfortunately, however, when administered in doses approximating those that inhibit soft tissue calcification, bisphosphonates can impair the mineralization of normal calcified tissues such as bone and cartilage (35, 36, 37) and, when given in higher amounts, also dentine (38), enamel (39, 40), and cementum (41). In the latter case, their administration can lead to a reduction of the extraction force.

While the different compounds vary greatly in their activity in bone resorption, they do not vary greatly in the inhibition of mineralization. For most species the effective daily dose is on the order of 5–20 mg of compound phosphorus per kg, administered parenterally. Interestingly, clodronate inhibits normal mineralization to a lesser degree than etidronate. The inhibition is eventually reversed after discontinuation of the drug (37). The inhibition of mineralization can lead to impaired fracture healing (42).

Since the inhibition is not corrected by 1,25-(OH)₂D₃ or 24,25-(OH)₂D₃ (43), it shows that the defect is not due to a decrease in this hormone. The decrease in calcitriol, which is sometimes observed when large amounts of etidronate are given (44, 45), and which is accompanied by a decrease in intestinal calcium absorption (46), is most probably secondary to the inhibition of mineralization. The decrease represents a homeostatic mechanism that adapts intestinal calcium absorption to the needs of the organism to maintain calcium homeostasis (47). When bisphosphonates are given in amounts small enough to decrease bone resorption without inhibiting mineralization, an increase in both plasma calcitriol and intestinal calcium absorption is observed (48).

Bisphosphonates also inhibit calcification of bone in humans when given in larger amounts (49, 50, 51, 52) (see Section VI).

The propensity to inhibit the calcification of normal bone has hampered the therapeutic use of bisphosphonates in ectopic calcification.

B. Inhibition of bone resorption
Bisphosphonates can be very powerful inhibitors of bone resorption, their potency varying according to their structure. This was shown in vitro in cell and organ culture, as well as in vivo in both animals and humans. The effect is present in normal animals as well as in experimental conditions in which resorption is increased. Similarly, bone resorption is decreased in normal individuals as well as in patients afflicted with a series of conditions accompanied by increased bone resorption, such as Paget’s disease, tumoral osteolysis, hyperparathyroidism, and osteoporosis.

1. Effects in vivo. Bisphosphonates inhibit bone resorption both in intact animals and in those with experimentally increased resorption.

a. Intact animals.
In growing intact rats, the bisphosphonates block the degradation of both bone and cartilage, thus arresting the remodeling of the metaphysis, which becomes club-shaped and radiologically denser than normal (36). This is similar to observations in animals with congenital osteopetrosis (53). These various changes are all secondary to the inhibition of bone resorption. This effect is used as a model with which to study the potency of new compounds (54).

The inhibition of endogenous bone resorption has also been documented by ⁴⁵Ca kinetic studies (55, 56) and by markers of bone resorption (55). The effect occurs within 24–48 h (57) and is therefore slower than that of calcitonin.

In view of the accumulation of the bisphosphonates in bone, it is of great clinical interest that the inhibition of bone resorption reaches a certain steady level even when the compounds are given continuously (58). This level depends on the administered dose. This has also been described in humans (59). These results show that there is no accumulation of effect with time and suggest that the bisphosphonate buried in the bone is inactive, at least as long as it remains buried there. They also show that, at the therapeutic dosage, there is no danger of a continuous decrease in bone turnover in the long run, coupled with an increase in bone fragility, as seen in osteopetrosis.

The decrease in resorption is accompanied by an increase in calcium balance (55, 56) and in mineral content of bone. This is possible because of an increase in intestinal absorption of calcium (55, 56) consequent to an elevation of 1,25-(OH)₂ vitamin D. This increased balance is the reason for administering these compounds to humans suffering from osteoporosis. However, the increase is smaller than predicted, considering the dramatic decreases in bone resorption and bone formation (55, 56), possibly due to the so-called "coupling" between formation and resorption. This will be discussed in a later section.

Similar results are found in humans. Bisphosphonates decrease both resorption and formation, as described in numerous studies (for reviews, see Refs. 12 and 14).

b. Animals with experimentally increased resorption including osteoporosis.
Bisphosphonates can also prevent experimentally induced increases in bone resorption. They impair resorption induced by agents such as PTH (60, 61), 1,25-(OH)₂ vitamin D, and retinoids. The effect on retinoid-induced hypercalcaemia has been used to develop a powerful and rapid screening assay for new compounds (62).

The bisphosphonates are also effective in preventing bone destruction in a number of disease models.

i. Osteoporosis. Many osteoporosis models have been investigated, including sciatic nerve section [which was the first model investigated (63)], spinal cord section, hypokinesis, ovariectomy (64, 65), orchidectomy (66), heparin, lactation (67), low calcium diet, and corticosteroids (68). All bisphosphonates investigated, i.e., alendronate, clodronate, etidronate, ibandronate, incadronate, olpadronate, pami-dronate, risedronate, tiludronate, and YH 529, have been effective.

Bisphosphonates also decrease bone loss and actually increase bone mineral density in humans with postmenopausal osteoporosis (69, 70, 71, 72, 73, 74) and corticosteroid-induced bone loss (75). Alendronate and tiludronate also prevent bone loss in healthy postmenopausal women (76, 77).

The effect of bisphosphonates upon the mechanical properties of the skeleton has been addressed only recently. This issue is important since longlasting, strong inhibition of bone resorption can lead to increased bone fragility and, therefore, to fractures caused by an inability to replace old bone by young bone and to repair microcracks. Such an effect of bisphosphonates is present when very large amounts of bisphosphonates are administered to animals. Thus, mice given such a treatment from birth develop a radiological and morphological bone appearance similar to that seen in congenital osteopetrosis (53). Dogs develop an increase in fractures if given very large amounts of etidronate or clodronate over a year (37). In contrast, doses of risedronate 5 and 20 times the anticipated clinical dose did not induce any increase in microdamage of the bones of dogs, despite the fact that the activation frequency, an index of bone turnover, was decreased between 53% and 94% (78).

It is now clear that, if not given in excess, bisphosphonates improve biomechanical properties both in normal animals and in experimental models of osteoporosis. This is the case with alendronate, clodronate, etidronate, incadronate, neridronate, olpadronate, pamidronate, tiludronate, and YH 529. This effect is seen in various animals such as the rat, the chick, and the baboon (65, 79, 80, 81, 82). Note, however, that the effect is more ambiguous with etidronate, since at higher doses it is obscured by an inhibition of mineralization.

Recent human data show that alendronate actually decreases the incidence of both vertebral and nonvertebral fractures (72, 83). However, it will always be prudent to administer a dose that does not induce too profound an inhibition of turnover. In treating osteoporosis, the general aim is to attain levels that correspond to those observed before the menopause. This is obtained, for example, with 10 mg daily of alendronate (59).

ii. Tumor bone disease. Bisphosphonates partially or entirely correct the increase in bone resorption in experimental tumor bone disease. Etidronate and clodronate inhibit the bone resorption induced by supernatants of tumor cultures in vitro (84, 85). In vivo, various bisphosphonates partially correct the hypercalcemia induced in rats by subcutaneously implanted Walker 256 carcinomas (86, 87) or Leydig tumors (88). For calciuria, the effect is generally more pronounced than for calcemia. This is explained by the fact that hypercalcaemia is often due to the systemic production of PTH-related peptide, which increases both bone resorption and tubular reabsorption of calcium (89), with bisphosphonates acting only on the former. Bone resorption secondary to actual tumor invasion is also retarded, as shown by numerous models using different tumor cells. The bisphosphonates shown to be active were, among others, clodronate, etidronate, incadronate, pamidronate, and risedronate (for review see Ref.90). Of great clinical interest is the fact that not only osseous metastases but also tumor burden is decreased, at least with risedronate (91). On the other hand, an increase in the burden has been described with a different bisphosphonate and another type of cell (92). The mechanism of the decrease in tumor burden is still debated. The decrease may be due to the diminished release of growth factors that are present in bone matrix and may stimulate tumor cell growth during bone resorption (93). Another possibility would be less space in bone, which might prevent the tumor cells from developing.

In humans, bisphosphonates inhibit tumor-induced bone resorption, correct hypercalcemia, reduce pain, prevent development of new osteolytic lesions, prevent the occurrence of fractures and, consequently, improve the quality of life (94, 95, 96, 97, 98, 99). They are now the treatment of choice in hypercalcemia of malignancy.

iii. Periodontal disease. Another interesting future use is in alveolar bone resorption. Bisphosphonates have been shown to decrease the bone destruction in various animal models (100, 101, 102).

2. Effects in organ and cell culture. Bisphosphonates block bone resorption induced by various means in organ culture (60, 61, 103, 104). For many years it was not possible to obtain a good correlation between the results obtained in vitro and those found in vivo. Recently, however, such a correlation was obtained using the mouse calvaria system (105).

An inhibition can also be found when the effect of isolated osteoclasts on various mineralized matrices is investigated in vitro (106, 107, 108). Under bisphosphonate treatment, the osteoclasts form fewer erosion cavities, which are of smaller size. However, only certain models show the same sequence of potency as that found in vivo (109).

3. Potency of various bisphosphonates on bone resorption. One of the aims of bisphosphonate research has been to develop compounds with a more powerful antiresorptive activity but without a higher inhibition of mineralization. This is possible since the activity of bisphosphonates on bone resorption varies greatly from compound to compound. Compounds have now been developed that are 5,000–10,000 times more powerful than etidronate in inhibiting bone resorption. The gradation of potency evaluated in the rat corresponds quite well with that found in humans (Table 1).

Until recently it was thought that only geminal compounds (i.e., compounds with only one carbon between the two P atoms) were effective. In 1995 it was reported that longer chain compounds could be made effective both on the inhibition of calcification in vitro and in vivo, as well as on bone resorption, if a keto group in the -positions near the phosphoric functions was added (115). Again, as for the bisphosphonates, the chain length is important. These bisacylphosphonates might be of interest in the future.

C. Effects on bone formation
Until recently, bisphosphonates were considered not to affect bone formation directly but to increase bone balance merely by inhibiting bone resorption. However, new results suggest that this may not be entirely true. Morphological data on the basic structural unit suggest a possible increase in formation in the bone multicellular unit (BMU), implying that some stimulating effect on bone formation might be present (see Section IV.B.1.) (65, 116, 117).

It is noteworthy that incadronate administered at toxic doses orally for 13 weeks was found to produce intramembranous intramedullary bone formation (118). No explanation has yet been found for this unique phenomenon.

At the cellular level bisphosphonates have been shown to increase in vitro the proliferation of osteoblasts (119, 120) and cartilage cells (121), as well as the biosynthesis of collagen and osteocalcin by bone cells (119, 122, 123) and proteoglycans by cartilage cells (124). The effect on collagen may be partially due to impaired intracellular collagenolysis (125). Alendronate can increase colony formation of osteoblasts (119) and the formation of mineralized nodules in human cell cultures in vitro, a phenomenon that is accompanied by an increased formation of basic fibroblast growth factor (126). It has been suggested that some of these effects may be mediated through protein-tyrosine phosphatases (120).

Thus it is possible that bisphosphonates could, under certain circumstances, also act by increasing bone formation. This possibility, although far from being established, is of enough potential interest to deserve a thorough investigation.

D. Effects on noncalcified tissues
Bisphosphonates also have some effects in vivo that are not necessarily related to the effects on bone. Often, however, these effects occur after very large doses, so that any relevance to pharmacological doses is doubtful. The effects on the immune system are discussed in Section IV.B.5.b. Of possible clinical interest is an increase in plasma high-density lipoproteins. This, and the fact that bisphosphonates and phosphonosulfonates linked to an isoprene chain are potent inhibitors of squalene synthase and hence cholesterol-lowering agents in animals (127) may open some interesting new therapeutic applications for these drugs.

A clinically important effect, the mechanism of which is not yet understood, is their influence on mucosa. It has been known for a long time that bisphosphonates can induce gastrointestinal disturbances (128). These appeared to be more pronounced for the aminobisphosphonates. It is now known that pamidronate (129), as well as alendronate (130), can, when given orally, induce serious adverse esophageal effects such as esophagitis, erosions, and ulcerations.

	IV. Mechanisms of Action

Top Abstract I. Introduction II. Chemistry III. Effects in Vivo IV. Mechanisms of Action V. Pharmacokinetics VI. Animal Toxicology and... VII. Conclusion References

 
A. Calcification
The mechanism of the inhibition of both normal and ectopic mineralization is most likely due, in part if not entirely, to a physicochemical mechanism. There is a close relationship between the ability of an individual bisphosphonate to inhibit calcium phosphate in vitro and its effectiveness on calcification in vivo (15, 47, 131); therefore, the mechanism is likely to be a physicochemical one. It is of interest that, in contrast to what occurs in bone resorption, the bisphosphonate must be continuously present to exert this effect both in vitro (131) and in vivo (36, 132).

The physicochemical effects of most of the bisphosphonates are very similar to those of pyrophosphate. Thus, they inhibit the formation and aggregation of calcium phosphate crystals from clear solutions, even at very low concentrations (15), block the transformation of amorphous calcium phosphate into hydroxyapatite (133, 134), and delay the aggregation of apatite crystals (135).

Bisphosphonates also delay the dissolution of calcium phosphate crystals (60, 61, 136). This effect was one of the reasons for investigating the action of these compounds on bone resorption in vivo. While they indeed proved to be good inhibitors of bone resorption, the mechanism is now thought not to be physicochemical but rather biological.

All of these effects appear to be related to the marked affinity of these compounds for the surface of solid-phase calcium phosphate where they bind onto the calcium by chemisorption (137), presumably chiefly at screw dislocations and kink sites of growth, and then act as a crystal poison on both growth and dissolution. The binding can be of two types (138, 139): bidentate or tridentate. In bidentate binding, an oxygen atom from each phosphonate group binds onto a calcium of the hydroxyapatite. Clodronate is an example of this type of binding. Most of the bisphosphonates that are now used clinically are tridentate. They bind at a third location, such as the oxygen of a hydroxyl group on the central carbon. This tridentate binding displays a better binding strength, which explains why clodronate is relatively less bound. A nitrogen atom can take the place of the hydroxyl group, as in incadronate. There is a positive relation between the binding of various bisphosphonates and their inhibitory effect on crystallization (131), giving strong support to the theory that the inhibition of mineralization in vivo is due to a physicochemical mechanism.

To date, there is no indication that the bisphosphonates are incorporated into the crystal lattice of hydroxyapatite. They are, however, incorporated into the bone because the crystals, along with bisphosphonate, on their surface become trapped by new crystals formed on top of them.

Bisphosphonates also inhibit the formation (23, 140) and the aggregation (141) of calcium oxalate crystals. These effects on calcium phosphate and oxalate crystal formation raised the hope that bisphosphonates might be used to prevent urinary lithiasis. This proved not to be possible since the dose necessary to inhibit crystallization in urine also induces an inhibition of normal mineralization, leading to the development of osteomalacia (28).

While these results point to a physicochemical mechanism in the inhibition of calcification, an effect on matrix formation cannot be totally excluded. When etidronate is given in doses that produce mineralization defects, changes in glycosaminoglycan synthesis are seen in teeth (142) and growth plate cartilage (143). Furthermore, collagen synthesis seems to be effected in dentine (38, 144, 145) and heterotopic bone (25, 146). These changes, as well as those observed in arteries (17, 18), could be a consequence of the inhibition of mineralization. However, it is interesting that changes are seen also in nonmineralized tissues such as articular cartilage (147).

B. Bone resorption
First of all, it must be stressed that, while the effects on calcification are probably explained by a physicochemical mechanism on the crystals, this is not the case for bone resorption. The inhibition of bone resorption can actually be explained largely, if not entirely, by cellular mechanisms. The latter can be considered at three levels: tissue, cellular, and molecular. The effect may be directly on the osteoclasts and may be mediated, at least partially, by other cells such as osteoblastic lineage cells and macrophages.

1. Physical chemistry. The earliest hypothesis for the action of bisphosphonates on bone proposed physicochemical effects on mineral dissolution. Bisphosphonates, like pyrophosphate, do indeed inhibit mineral dissolution (7, 60, 61, 136). However, the concentrations of bisphosphonates required to inhibit bone resorption with the newer, more potent compounds are so low that they are unlikely to have a significant impact on mineral dissolution. Moreover, structure/activity studies on a large array of compounds showed no correlation between the inhibition of mineral dissolution in vitro and the pharmacological activity on bone resorption in vitro (131) or in vivo (110). It is therefore accepted by most investigators that the effect on bone resorption is essentially cellular.

2. Tissue level. At this level, the action of the active bisphosphonates appears to be the same for all, i.e., a reduction in bone turnover. This is shown by a decrease in both bone resorption and bone formation, as assessed in animals as and humans by calcium-⁴⁵ kinetics (55, 56), biochemical markers (59), and morphology (36, 65, 116, 117).

Under normal conditions, destroyed bone is replaced by bone formation. In adults this occurs mostly at the sites of remodeling in both the trabeculae and the cortex. The morphological dynamic unit of the turnover is the BMU. The remodeling process in this unit starts with the erosion of a certain amount of bone through osteoclasts on the surface of the trabeculae, as well as on the surface or the interior of the cortex. The resorption follows a linear path, forming a canal within the cortex and a trench on the surface. The destruction is followed by a refilling of the excavation by the osteoblasts within a tight temporal sequence. This explains why every decrease in resorption is accompanied by a secondary decrease in formation, since there is less need for a bone defect to be replenished. The final morphological entity is called the bone structural unit (BSU). It corresponds to an osteon within the cortex and has of late been termed a hemiosteon when it is at the surface of the bone (148). The total bone resorption and formation will therefore depend upon the number of BMUs present at any time which, in turn, will depend upon both the number of BMUs formed and the length of time they are active (for reviews, see Refs. 148–150).

Under normal conditions, the amount of bone formed in each BMU equals the amount destroyed, so that the balance is zero. In osteoporosis, however, a greater amount of bone is resorbed than formed, leading to a negative balance. Thus, while a change in turnover has no influence on the total calcium balance in normal people, there is a local negative bone balance in osteoporosis because more bone is destroyed than formed. Therefore, in this disease a decrease in turnover per se will slow down the total bone loss. This is why a high turnover after menopause, when such imbalance is present, is a good indicator for bone loss and the occurrence of osteoporosis in the future. This is also why all inhibitors of turnover, including bisphosphonates, will diminish bone loss in osteoporosis. In the case of bisphosphonates, it is probably the main mode of action in all types of osteoporosis. However, it must be stressed that there are conditions in which an increase in bone turnover is not necessarily accompanied by a negative balance. The growing animal is an obvious example, as well as certain cases of Paget’s disease in humans.

In addition, the bisphosphonates also act at the individual BMU level by decreasing the depth of the resorption site (65, 116, 117). Since the amount of new bone formed in the BMU is not decreased, but possibly even increased (65, 116, 117), the local and consequently the whole body bone balance will be less negative or might even be positive.

The effect both on the general turnover and the local balance will lead to less trabecular thinning, a decreased number of trabecular perforations, a decreased reduction in connectivity (151), and a smaller erosion of the cortex, thus slowing down the decrease in bone strength and the occurrence of fractures.

Of crucial importance in the final effect is the behavior of the formation. As mentioned above, the total amount of bone formed is decreased because of the decrease in turnover, as shown by calcium⁴⁵ kinetics, biochemical markers such as serum alkaline phosphatase and osteocalcin, and by a reduction in the bone formation surface assessed morphologically (55, 65, 116, 117). This reduction reflects reduced remodeling only. There is no evidence for reduced osteoblastic activity at individual bone formation sites, as judged by the amount of bone produced per unit time. On the contrary, the amount of bone formed at each individual basic structural unit (BSU), as measured by the thickness of the newly formed bone, is, if anything, increased (65, 116, 117). This effect is modest and needs to be confirmed. If present, however, such an effect could not be detected by any current technique measuring total bone formation in the body, such as biomechanical markers, since it would be obscured by the decrease in remodeling.

It is now generally accepted that bisphosphonates can lead to a positive calcium and bone balance, both in animals (55, 56) and in humans (69, 70, 71, 72, 73, 74, 75, 76, 77, 152). There are several explanations for this gain. One is inherent to bone turnover. Therefore, a decrease in bone resorption is not immediately followed by the diminution of formation, so that a temporary increase in balance through a reduction in the so-called remodeling space occurs. The second explanation is that, after the decrease in turnover, the new BSU formed will be remodeled later than it would be normally. It therefore has more time to finish the lengthy process of mineralization. This will lead to a higher calcium content and, therefore, a higher bone mineral density and content. However, it will not lead to an increase in actual bone mass, a fact that is often forgotten. Third, if the decrease in resorption depth at individual remodeling sites is not matched by a decrease in formation in the individual BMU, which seems to be the case, the local bone balance in the BMU will be positive. The last possibility is an increase in the amount formed at the level of the BMU (Fig. 3).

View larger version (32K): [in this window] [in a new window]   Figure 3. Possible effect of bisphosphonates at the level of the individual BMU. [From H. Fleisch (14).]

Another question has been whether bisphosphonates could display an additive effect together with another inhibitor of bone resorption. One report suggests this to be the case with estrogen in humans (159).

3. Cellular level. There is now general agreement that the final target of bisphosphonate action is the osteoclast. Four mechanisms appear to be involved: 1) inhibition of osteoclast recruitment; 2) inhibition of osteoclastic adhesion; 3) shortening of the life span of osteoclasts; and 4) inhibition of osteoclast activity. The first three mechanisms will lead to a decrease in the number of osteoclasts, which is observed in humans and often, although not always, in animals. All four effects could be due either to a direct action on the osteoclast or its precursors or indirectly through action on cells that modulate the osteoclast.

1. Several bisphosphonates inhibit osteoclast differentiation in various culture systems of both cells (160) and bones (104, 112). Bisphosphonates are also powerful inhibitors of macrophage proliferation, cells that are of the same lineage as osteoclasts (161). In the hemopoietic series, the effect appears to be specific, or at least specially pronounced, for the mononuclear phagocyte lineage (162). Furthermore, the potency rank of bisphosphonates, when assessed in vitro, correlates with effects in vivo only when systems are used that detect osteoclast recruitment and not activity alone (104, 112). Some experiments suggest that the effect occurs at the terminal step of the differentiation process (163). Other recent results (109, 164) also support the effect on differentiation. Thus, a correlation between the number of osteoclasts and osteoclastic cavity formation, on one hand, and the effect in vivo, on the other hand, occurs only if other cells, probably osteoblasts but not osteoclasts, are exposed to the bisphosphonates (see SectionIV.B.5.a). Finally, when a system involving osteoclast differentiation is used (104, 112), the dose necessary to inhibit resorption is low only for aminobisphosphonates, but not for etidronate and clodronate, which are less powerful inhibitors of resorption. This suggests that two mechanisms may be operating, one on osteoclast recruitment and one with a direct effect on osteoclast activity.

2. The second possibility would be a decreased osteoclastic adhesion to the mineralized matrix. Whether this takes place is still uncertain since the results are ambiguous. One recent study reports such an effect (165). However, there is now excellent evidence that bisphosphonates can inhibit the adhesion of some cells, mainly tumor cells, in vitro (166).

3. The third possibility is a shortening of the lifespan of the osteoclast. It has been proposed that this might be due to a toxic effect, but the results were obtained at very high concentrations. Recently it was reported that bisphosphonates induce osteoclast programmed cell death (apoptosis), both in vitro and in vivo, and both in normal mice and in mice with increased bone resorption (167). The ranking of effectiveness of clodronate, pamidronate, and risedronate was the same as seen in vivo. The effect was not due to toxic cell death. Whether this is a direct effect on osteoclasts, or an indirect one through the effect on other cells, is not known. A similar effect occurs in macrophage-like cells in vitro and is nitric oxide independent (168).

4. The last possibility is an inhibition of osteoclast activity after the bisphosphonate has been taken up by the osteoclasts. Indeed, several facts suggest that the inhibition of recruitment is not the only mode of action of bisphosphonates in vivo. Thus, after bisphosphonate administration, the number of multinucleated osteoclasts on the bone surface often increases initially, despite a reduced bone resorption (36, 169, 170); however, the cells appear inactive (36). It is only later, after chronic administration, that the osteoclast number decreases. The cause for the initial increase is unknown. One possibility is that it could reflect a stimulation of osteoclast formation to compensate for the decrease in osteoclast activity.

A direct effect on the osteoclasts is supported by the finding that, under bisphosphonates, osteoclasts can show changes in morphology both in vitro (107, 170) and in vivo (36, 132, 169). These include changes in the cytoskeleton, especially actin (107, 171, 172) and vinculin (172), and the ruffled border (132, 169, 173). One study (171) showed that the morphological changes occurred only when the cells were actively resorbing the calcified matrix, or if the bisphosphonate was injected into the cells. No changes occurred when the osteoclasts were not active, showing that they have to be taken up with the resorbed mineral. As mentioned earlier, bisphosphonates inhibit the formation of resorption cavities by isolated osteoclasts deposited on calcified matrices in vitro (106, 107, 108). A direct action on osteoclasts is also supported by the fact that, under certain conditions, bisphosphonates can enter cells (174), particularly those of the macrophage lineage. The concentration of the bisphosphonate can also attain very high values under the osteoclasts, probably 100 µM or more, partly because they deposit preferentially under these cells (173, 175) and are then released from the mineral at the acid pH prevailing at this location.

4. Molecular level. The events leading to either osteoclast inactivation or diminished osteoclast formation by bisphosphonates have not yet been fully elucidated. It may be worth introducing this section by reiterating some general facts.

The circulating levels of pharmacologically active bis-phosphonates are usually extremely low. This implies that uniform circulating levels are not necessary for continu-ous activity. This is supported by the fact that a single administration of these compounds can lead to a sustained inhibition of bone resorption which, e.g., in patients with Paget’s disease, can last over years. This suggests either that some cells are affected over a long time or, more likely, that the bisphosphonate taken up by the bone is released in very low amounts over time at areas of high turnover, thus affecting resorption locally. The latter would explain the high efficacy of these compounds in diseases with focal resorption, such as Paget’s disease or metastases.

The other interesting fact is the low concentrations necessary for activity, which suggests either some sort of "receptor" or some cellular binding site, which induces a cellular transduction mechanism. Until now no such active receptor or binding site has been identified. However, the fact that osteoblasts exposed for only 5 min to very low concentrations of bisphosphonates are being stimulated into augmenting the release of an osteoblast recruitment inhibitor (100, 154) speaks in favor of their presence as a linking site. Since bisphosphonates enter the cell via fluid pinocytosis or adsorptive pinocytosis, the latter could be within the cell and might be an enzyme, a pump, or some other intracellular protein involved in the signaling cascade.

It has long been known that bisphosphonates decrease acid production of various cells (121) and of calvaria (176). In 1990, it was reported that bisphosphonates decrease the proton accumulation and the protein synthesis by osteoclasts in vitro (177). More recently, bisphosphonates were shown to decrease the extrusion of acid through a sodium-independent mechanism by true osteoclasts (178). Possibly part of this effect is due to the decrease of the proton transport by the vacuolar-type proton ATPase, which is inhibited by tiludronate, but surprisingly not by other bisphosphonates (179). However, until now no correlation between the effect in vitro on acid production and in vivo on bone resorption was evident. Some bisphosphonates, such as pamidronate or long-chain bisphosphonates, actually increase lactic acid production, possibly due to a toxic action (110, 180).

Various bisphosphonates, especially clodronate, inhibit lysosomal enzymes in vitro (181), in cultured calvaria (176, 182), or in vivo (180). Certain bisphosphonates, such as clodronate and etidronate, also inhibit prostaglandin synthesis by bone cells or calvaria, both in vitro and in vivo (183, 184). Since prostaglandins are involved in bone resorption, this inhibition may play a role in the resorption process.

Some data indicate that still other mechanisms may come into play. Thus, both in osteoporosis and in Paget’s disease, bisphosphonates induce a decrease in urinary cross-links. This reflects the decrease in bone resorption. Surprisingly, in opposition to what occurs with estrogens, the effect is almost solely on peptide-bound collagen cross-links and not on free cross-links (185). This suggests that the bisphosphonates might influence the degradation process of collagen.

In view of the homology between pyrophosphate and bisphosphonates, various enzymes involving pyrophosphate or ATP have been examined. Phosphatases and pyrophosphatases were influenced only at relatively high concentrations (181, 186) or not influenced at all (187). However, PTP, a protein-tyrosine phosphatase present in osteoclasts, is inhibited in vitro by alendronate with an IC₅₀ of only 3 µM, while etidronate is active at 2 µM (187). Another protein-tyrosine phosphatase, PTP, which is present both in osteoclasts and osteoblasts, is also inhibited by alendronate and etidronate with an IC₅₀ of 0.5 µM and 0.2 µM, respectively (120). Other protein-tyrosine phosphatases such as CD45 are also inhibited. These effects might be relevant since protein-tyrosine phosphorylation is important in the signal transduction pathways that control cell growth, differentiation, and activity. Furthermore, not only the bisphosphonates but also orthovanadate and phenylarsine oxide inhibit PTPs at very low concentrations and inhibit the formation of osteoclasts in vitro (187). Unfortunately, the potency to inhibit the PTPs of various bisphosphonates tested so far has no relationship to their pharmacological potency, since alendronate is about 1000 times more effective than etidronate on bone resorption in vivo, while their potency in vitro was of similar magnitude.

It was shown recently that various bisphosphonates, excluding clodronate, inhibit posttranslational modification of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups in J774 macrophages. Furthermore, alendronate-induced apoptosis could be prevented in these cells by farnesylpyrophosphate or geranylgeranylpyrophosphate (M. J. Rogers, S. P. Luckman, F. P. Coxon, and R. G. G. Russell, submitted). This suggests that at least some bisphosphonates cause apoptosis through a mechanism involving prenylation of proteins. Whether this is true for osteoclasts must still be proven.

Another interesting observation is that both macrophage-like cells and human MG63 osteosarcoma cells metabolize primary clodronate to a nonhydrolysable ATP analog, adenosine 5'-(ß,-dichloromethylene)triphosphate (189). This is not the case for other bisphosphonates. It has been suggested, therefore, that clodronate might act through this mechanism to induce apoptosis and necrotic cell death and therefore to inhibit bone resorption.

One of the conclusions based on the various biochemical results is that no single individual mechanism shows a good correlation with the potency in vivo when different bisphosphonates of various potencies are investigated. This suggests that, if any of the above mechanisms is relevant for bone resorption, it is not relevant for all bisphosphonates.

The various cellular modes of action are summarized in Table 2.

a. Osteoblast-lineage cells.
It is now generally accepted that cells of osteoblastic lineage control the recruitment and activity of osteoclasts under physiological and pathological conditions. This control was proposed to be due to the production of an as yet unknown activity, generated by osteoblast-lineage cells, and modulating bone resorption (190), and this modulation was thought to be an activation of resorption (191, 192, 193, 194).

It has been shown that bisphosphonates may also act through the modulation of the osteoclast-osteoblast interrelation. It has been known for quite some time that, when assessed in vitro, various bisphosphonates can inhibit the destruction of the mineralized matrix, but that all those tested have a similar activity despite the fact that in vivo their antiresorbing effect varies from 1 to 1000 (107). This result suggests that the conditions created did not represent those operating in vivo. It was then discovered that this lack of correlation is only present when the bisphosphonates are added to the mineral before the osteoclasts, but not when the cell population containing the osteoclasts added to the matrix are treated for a time as short as 5 min, at concentrations as low as 10^-11 M, before allowing them to adhere to the ivory (109). When doing this, five different bisphosphonates with potencies ranging from 1 to 10,000 showed a stringent correlation between the results in vitro and in vivo (109). Therefore, the best conditions are not when the bisphosphonates are on the mineral, as a direct effect on osteoclasts would imply, but when they are in contact with the cells.

This effect appears to be due to the osteoblasts present in the unpurified osteoclastic cell population. Thus, pretreating pure osteoblastic cell populations for 5 min with the bisphosphonates alendronate and ibandronate, and then coculturing the osteoblasts with the osteoclasts, prevented the usual increase in resorption (109). In contrast, adding osteoblastic cells to osteoclasts pretreated with the bisphosphonate had no effect. This result is supported by previous findings that, when assayed in a coculture of bone and osteoclast precursors, the bisphosphonates do not act directly on the precursors, but need the presence of a cell in the bone (104).

The inhibitory effect is not due to a decrease in the osteoclast-stimulating activity, but to the synthesis by the osteoblasts into the culture medium of an inhibitor of osteoclastic resorption. The latter is labile to heat and proteinase and has a molecular mass of approximately 3–4 kDa (164). The inhibitor has not been characterized, so that it is not possible to speculate as to what family it belongs.

The resorption cavities are reduced parallel to the reduction of the number of tartrate-resistant acid phosphatase-positive multi- and mononuclear cells, which are thought to be osteoclasts and their precursors. In contrast, the mean area resorbed by cavity remains unchanged, suggesting that the inhibitor affects osteoclast formation but not osteoclast activity (164). Other cells such as fibroblasts and preosteoblasts do not produce such an inhibitor. The question of which cells of the osteoblastic lineage are able to mediate this effect has not yet been answered. Recently, it has been postulated that lining cells play a role in the osteoblast-osteoclast relation (195) (Fig. 4).

View larger version (43K): [in this window] [in a new window]   Figure 4. Indirect effect of the bisphosphonates on the osteoclasts mediated by the osteoblasts. [From H. Fleisch (14).]

b. Cells of the mononuclear phagocyte and immune systems.
The other possible candidates are the cells of the mononuclear phagocyte system and of the immune system. Since they produce a variety of bone-resorbing cytokines, it is possible that they may play a role in the cascade involved in the inhibition of bone resorption induced by a bisphosphonate.

There are numerous reports on the effect of bisphosphonates on these cells, both in vitro and in vivo. Unfortunately, these studies are often performed with high concentrations, so that the described effects might be secondary only to a toxic action. Low concentrations often give an effect contrary to that of higher concentrations, which might also reflect toxicity. Thus it is not possible at this time to state whether or not they are implied in the inhibition of bone resorption by bisphosphonates.

i. In vitro. It seems clear that the cells of the mononuclear phagocytic lineage are specially sensitive to bisphosphonates since other marrow populations are either much less or not at all influenced, at least in vitro (161, 162). The multiplication (161, 162) as well as the activity (199, 200) are both decreased. In addition, bisphosphonates have been reported to depress accessory function of monocytes (111), inhibit the action of mitogens on mononuclear function and on the lymphoblastic response (201), influence the effect of antilymphocyte serum on T lymphocytes (202), and inhibit migratory activities of macrophages (200). They also inhibit the proliferation of human peripheral blood mononuclear cells induced by various means. It has been suggested that this effect is mediated by the antigen-presenting cells (203).

With respect to cytokine production, clodronate inhibits lipopolysaccharide-induced interleukin (IL)-1ß, IL-6, and tumor necrosis factor- (TNF) production by a macrophage-like cell line (RAW 264) (204, 205). Alendronate inhibits, in a dose-dependent fashion, the production of these three cytokines by activated human monocytes (203). Pamidronate, however, increases the production of IL-6 (205). Clodronate and pamidronate, but not alendronate, also decrease the production of nitric oxide and the expression of inducible nitric oxide synthase in the RAW 264 cells (206). When clodronate is encapsulated into liposomes, its effect is increased while that of pamidronate is decreased.

ii. In vivo. The following effects on the immune system have been described: decrease in the formation of antibody-secreting cells and impaired delayed and immediate hypersensitivity (207); inhibition of passive cutaneous anaphylaxis (208); atrophy of the thymus (209); disappearance of certain thymus-dependent macrophages (210); disappearance of natural killer cells (211); and diminished response of the T lymphocytes to mitogens (209) in newborn mice. All these effects were obtained at very high dosages, some of which led to an osteopetrotic condition, so that the relevance to what occurs with clinical regimens is far from being proven. Indeed, none of these effects have been seen in humans.

The sensitivity of macrophages to bisphosphonates, especially to clodronate, has been made use of to selectively destroy macrophages in vivo. Thus, if bisphosphonates are administered encapsulated in liposomes, they are taken up by the macrophages mostly in the spleen and the liver, and the macrophages are then destroyed within 2 days (212). This technique has been used to study repopulating kinetics of macrophages and the role of macrophages in the organism.

An effect on macrophages, or possibly on other cells, might be the explanation for the acute phase response in humans. Thus, some patients who receive an amino-bisphosphonate intravenously for the first time show a transient pyrexia of 1–2 degrees C, sometimes more, accompanied by flu-like symptoms (111, 213). This episode is accompanied by a decrease in peripheral lymphocytes, especially the CD3+ T cells (214), an increase in C-reactive protein, and a decrease in serum zinc. Interestingly, this reaction occurs only once in a lifetime, even if the treatment is discontinued and restarted later. This raises the possibility, among others, that a specific cell population involved in the development of the acute phase reaction is influenced over longer periods. Recently, the pyrexia was shown to be accompanied by an increase in circulating IL-6 bioactivity (215). Furthermore, olpadronate but not clodronate stimulated the release in vitro of IL-6 from fetal mouse explants. In addition to IL-6, TNF is also increased in the blood after treatment with pamidronate but not clodronate (216). The effect is not seen with etidronate, clodronate, or tiludronate. It is not known why only compounds that are potent inhibitors of bone resorption and contain a nitrogen atom in their structure show this effect.

Of clinical interest is that some bisphosphonates, including etidronate, clodronate, tiludronate, risedronate, and zole-dronate, inhibit local bone and cartilage resorption, preserve the joint architecture, and decrease the inflammatory reaction in experimental arthritis induced by Freund’s adjuvant, carrageenin and, to a smaller extent, collagen (217, 218, 219, 220, 221). The effect on the joints is especially pronounced when the bisphosphonates are encapsulated in liposomes (222, 223). The fact that not only bone resorption, but also the inflammatory reaction in the joint and in the paw itself, is diminished (223, 224) suggests that mechanisms other than those in bone, possibly involving the mononuclear phagocyte system, are operating. These results open the exciting possibility of using bisphosphonates in inflammatory arthritis, given either systemically or locally, possibly encapsulated in liposomes.

c. Tumor cells.
As described in Section III.B.1.b.ii, bisphosphonates inhibit the bone resorption induced by various tumors both in animals (84, 85, 86, 87, 88, 91, 94) and in humans (96, 97, 98, 99, 225, 226, 227). This is generally explained by the inhibition of bone resorption. The inhibited development of metastases can have various causes. One is that, since less bone has been destroyed, the place for tumoral expansion is limited. Another explanation is that, as a consequence to a decrease in bone resorption, the release of matrix or osteoclastic cytokines that would stimulate the multiplication of tumor cells may be decreased (91). In contrast, the bisphosphonates do not seem to inhibit directly the multiplication of tumor cells. Furthermore, there is now excellent evidence that bisphosphonates can inhibit the adhesion of tumor cells in vitro (166). The effect is specific for mineralized matrices, and the potency of various bisphosphonates is well correlated with the potency to inhibit bone resorption in vivo. It might explain in part the bisphosphonate-induced decrease in the development of tumor burden in animals (91).

C. Other effects
A great number of other cellular or biochemical effects have been described. They are confusing and can sometimes go in opposite directions with different compounds, or even with the same compound at different concentrations. With one or two exceptions, there is no indication that they are involved in bone resorption, and those most likely to play a role in the inhibition of bone resorption have been described earlier in this article. These other effects include the following: increase of fatty acid oxidation (228) and amino acid oxidation (180); stimulation of the citric acid cycle (180); increase in cellular content of glycogen (229); increase in production of alkaline phosphatase (230); inhibition of the 1,25-(OH)₂D₃-induced production of osteocalcin in vivo (231); contradictory effects on cAMP production (232, 233); decrease or increase in cellular multiplication (121, 234); inhibition of DNA polymerase (235); and inhibition of amoebal phosphofructokinase (236). A few results point to an effect on cellular calcium handling, e.g., reduced release of calcium from kidney mitochondria in vitro (237) and increase in calcium of mitochondria in vivo (238); inhibition in vitro of calcium-induced contraction of smooth muscle, possibly through inhibition of intracellular Ca mobilization and influx of extracellular Ca (239); protection of the kidney from ischemic damage, possibly by preventing intracellular Ca accumulation (240). Considering this, it is interesting that nongeminal bisphosphonates act in a manner similar to Ca channel blockers (241). Finally, squalene synthase is inhibited (127).

It is interesting that bisphosphonates inhibit the growth of the slime mold amoeba Dictyostelium discoideum, and that some of them can form nonhydrolyzable methylene analogs of ATP (242, 243). The effect on growth of these organisms is of interest because of the presence of a remarkable correlation with a great number of different bisphosphonates between the effects found on this system using the growth of a slime mold and the bone resorption in vivo (244, 245). It suggests that this system might give us further insight in what occurs in bone resorption, which is supported by the fact that human cells can also perform such a transformation (189).

	V. Pharmacokinetics

Top Abstract I. Introduction II. Chemistry III. Effects in Vivo IV. Mechanisms of Action V. Pharmacokinetics VI. Animal Toxicology and... VII. Conclusion References

 
Bisphosphonates can enter mammalian cells. This has been confirmed by studies in vitro both for etidronate and clodronate (121, 174). The cellular uptake is mostly in the cytosol, and the concentration expressed in terms of cellular water can be several fold higher than in the medium (174). Cells with phagocytic properties display special avidity if the compounds are bound to apatite crystals (199).

Nevertheless, the bisphosphonates have a very low bioavailability, from a few percent for clodronate, etidronate, and tiludronate, which are given in larger amounts, to below 1% for the newer ones, which are given in low quantities. This is partly explained by their low lipophilicity, which hampers transcellular transport, and their high negative charge, which hampers paracellular transport. Furthermore, they are probably partly in an insoluble form in the gut, due to chelation to calcium. It is thought that the absorption in the intestine follows mainly a paracellular route (246). The latter is under the influence of calcium, which tightens the junctional complex. This explains why the administration of EDTA, a strong calcium chelator, increases the absorption of bisphosphonate (247) and why high doses of bisphosphonates, which also chelate calcium, will lead to an increase in their own absorption (248). Why a higher intestinal pH increases absorption while orange juice and coffee decrease it (249) is not known.

Some uncertainty still exists as to the state of bisphosphonates in the circulation. They are indeed only partially ultrafilterable in aqueous solutions as well as in plasma (250), possibly because of the formation of polynuclear aggregate complexes (251, 252, 253). In plasma they are bound to proteins, whereby this binding varies between compounds and between animals (254). The binding is pH and calcium dependent, whereby calcium and increasing pH augment it (255). There are also displacers of the binding in the plasma of, for instance, the dog (254). The role this binding could have on the action and the pharmacokinetics of bisphosphonates has never been investigated despite the fact that it may be conspicuous. For example, the assumption that bisphosphonates are not actively secreted in the kidney is probably wrong. Indeed, most renal studies were not corrected for the binding so that the filtered load was overestimated. If a correction is done, the results point to a secretory mechanism (256, 257).

Once in the blood, bisphosphonates disappear very rapidly, mostly to bone (258). This might be explained by the fact that they are characterized by a rapid and strong binding to the hydroxyapatite crystals (137). The rate of entry into bone is very fast, similar to that of calcium and phosphate. It has been calculated that the bone clearance is compatible with a complete extraction from the skeleton after the first passage (258), so that skeletal uptake might be determined above all by the vascularization of the bone. Consequently, soft tissues are exposed to these compounds for only short periods, explaining their bone-specific effects and their low toxicity.

The various bisphosphonates display some differences in their affinity for the hydroxyapatite surface. This reflects itself in the binding of bisphosphonates to bone in vivo. Thus, at least 50% of most of the hydroxylbisphosphonates distribute themselves to bone (259), whereas in the case of clodronate (260, 261) it is only about 20–40%. Their preferred location in the skeleton is bone with a high turnover, namely trabecular bone.

The binding of polyphosphates and bisphosphonates to calcified tissues is the basis for the use of these compounds as skeletal markers in nuclear medicine when linked to ^99mtechnetium. However, it is important to note that the handling of the technetium-labeled compounds is not identical with that of the bisphosphonates (262), so that caution must be given in extrapolating data from one to the other.

It was generally thought that the bisphosphonates deposit in those locations within the bone where new bone is formed. Recently, however, they were found to deposit under the osteoclasts as well (173). The distribution of the amount deposited at bone formation and bone resorption sites depends upon the amount of bisphosphonate administered (263). When small amounts are given, they deposit mostly under the osteoclasts while larger amounts go to both bone-forming and bone-resorbing sites. This would explain the results with ^99mtechnetium-labeled compounds, thought to go to formation sites, since larger amounts are usually injected. However, the fact that the erosion locations seen in multiple myeloma do not take up any visible radioactive ^99mtechnetium-labeled bisphosphonates has not yet been explained.

The fact that bisphosphonates are targeted to bone may be used in the future to administer drugs to the skeleton. Initial results with methotrexate in rats are encouraging (264).

Usually bisphosphonates do not deposit in soft tissues. However, some of them, especially pamidronate, can at times deposit in other organs such as the stomach (265), liver, and spleen (266, 267, 268), the deposition being proportionally greater when large amounts of compounds are given. Part of this extraosseous deposition appears to be due to the formation of complexes with iron (hemolysis) and calcium because of too high and too rapid an intravenous injection. The insoluble aggregate is then phagocytized by the macrophages of the reticuloendothelial system. Thus, results obtained with large amounts of labeled compounds given rapidly intravenously must be interpreted with caution. The danger of too rapid an infusion of large amounts of bisphosphonate exists also in humans where this procedure has led to renal failure (269) because of the formation of insoluble calcium aggregates in the blood.

Once the bisphosphonates are buried in the skeleton, they will be released only when the bone is destroyed in the course of the turnover. The skeletal half-life of various bisphosphonates is between 3 months and 1 yr for mice and rats (266, 267, 268) and is much longer, sometimes more than 10 yr, for humans (270).

The bisphosphonates are not metabolized in vivo. This is due to the stability of their P-C-P bond to heat and most chemical reagents, as well as to their resistance to hydrolysis by the enzymes found in the body. To date, all the bisphosphonates investigated were excreted unaltered. However, it is quite possible in the future that some compounds will be metabolized in their side chain, especially in the gut, so that it cannot be generally stated that bisphosphonates are not metabolized in vivo.

	VI. Animal Toxicology and Human Adverse Events

Top Abstract I. Introduction II. Chemistry III. Effects in Vivo IV. Mechanisms of Action V. Pharmacokinetics VI. Animal Toxicology and... VII. Conclusion References

 
A. Animal toxicology
Published animal toxicological data are scant. Acute, subacute, and chronic administration of bisphosphonates has in general revealed little toxicity. This is explained by their rapid incorporation into calcified tissue and hence their short presence in the circulation.

Acute toxicity is mostly due to hypocalcemia, which is induced by the formation of complexes or aggregates with calcium, leading to a decrease in ionized calcium.

The nonacute, nonskeletal toxicity is usually manifested, as is the case with many phosphates and polyphosphate, first in the kidney (271, 272). This occurs, however, only at doses substantially larger than those administered in humans. At still higher doses, other organs can show cellular alterations. The mechanisms leading to these changes are not known. In the skeleton and in teeth an inhibition of normal mineralization occurs, as mentioned earlier, usually at parenteral doses of approximately 10 mg/kg daily (35, 36, 37, 38, 39, 40, 41). As discussed earlier, this inhibition is explained by a physicochemical impairment of crystal growth. Large doses of bisphosphonates can inhibit mineralization to such a level, which by itself can lead to an increased fragility and fractures (37). Finally, very large doses of bisphosphonates can lead to fetal abnormality of the skeleton and the kidney (273).

B. Human adverse events
As in animals, studies in humans have revealed only a few significant adverse events. Caution must be taken with all intravenous administrations of large amounts of bisphosphonates since rapid injection has led to renal failure (269), probably because the bisphosphonate is forming a solid phase in the blood, which is then retained in the kidney. No such events have occurred since care is taken to administer all bisphosphonates in large amounts by slow infusion in plenty of fluids.

The oral administration of bisphosphonates, especially those with a primary amine, can be accompanied by esophageal and gastrointestinal side effects such as nausea, dyspepsia, vomiting, gastric pain, and diarrhea, and sometimes even ulceration (129, 130, 274). These adverse events have decreased since patients began ingesting the drug with adequate water and without reclining after its intake to minimize esophageal reflux.

As seen in animals, etidronate, when given at daily oral doses of 400–800 mg, can produce an inhibition of normal skeletal mineralization, leading to a clinical and histological picture of osteomalacia. This condition regresses after discontinuation of therapy (31, 49, 50). Similar results have been seen with pamidronate in Paget’s disease when given intravenously at doses equal to or higher than 180 mg per year (51, 52).

The last commonly seen effect, which has been mentioned earlier in this paper, is observed after intravenous administration of more potent bisphosphonates containing a nitrogen atom. This is not observed with etidronate, clodronate, or tiludronate. After intravenous administration, a transient pyrexia of usually 1–2°C, sometimes more, accompanied by flu-like symptoms, may occur. It is maximal within 24–48 h and disappears after approximately 3 days, in spite of continued treatment. It is usually observed only once, even if treatment is continued and restarted later (213). The mechanism of these changes, which resemble an acute phase response, seems to involve the stimulation of macrophages to release IL-6 and TNF (215, 216), both of which increase in plasma.

Most of the other adverse events are seen only occasionally, and it is not certain to what extent they are actually related to the drugs.

	VII. Conclusion

Top Abstract I. Introduction II. Chemistry III. Effects in Vivo IV. Mechanisms of Action V. Pharmacokinetics VI. Animal Toxicology and... VII. Conclusion References

 
Since the discovery of their effects on biological tissues in 1968, much progress has been made in our understanding of the mechanisms of action of the bisphosphonates. While the effects on mineralization appear to be physicochemical by inhibiting crystal growth, those on resorption are cellular. However, we still do not know the molecular mechanisms leading to the inhibition of resorption. The consensus is that the final effect is through the osteoclasts, but we do not know how much is via the inhibition of their activity and how much is due to a decrease in their number. It is also unknown how much of the effect is direct or indirect through other cells, such as the osteoblasts. It is agreed that the bisphosphonates need the P-C-P bond to target themselves to the mineral; however, the effect on cells occurs in part even when no mineral is present while they are exposed to the drug. Thus, the cells may be modulated by the bisphosphonate liberated from the mineral, their potency being determined by the structure of the lateral chain. Finally, we have practically no knowledge as to which part of the molecule is responsible for the effect, nor what the optimal structure of a compound for this effect is. The latter is regrettable since such knowledge would not only allow us to synthesize new and better inhibitors, but also give us an insight into the mechanisms of bone resorption in general. Further research in this direction is therefore desirable.

Current clinical applications for the inhibition of bone resorption are Paget’s disease, tumor bone disease, and osteoporosis. Future applications could be, among others, Sudeck’s atrophy, fibrous dysplasia, loosening of bone implants, and alveolar resorption. As to their property of inhibiting calcification, only etidronate is currently used with variable success for ectopic calcification and ossification.

	Footnotes

 
Address reprint requests to: Herbert Fleisch, M.D., Effingerstr 40, CH-3008 Berne, Switzerland. e-mail: fleisch@sams.ch

	References

Top Abstract I. Introduction II. Chemistry III. Effects in Vivo IV. Mechanisms of Action V. Pharmacokinetics VI. Animal Toxicology and... VII. Conclusion References

 

1. Menschutkin N 1865 Ueber die Einwirkung des Chloracetyls auf phosphorige Säure. Ann Chem Pharm 133:317–320
2. Bassett CAL, Donath A, Macagno F, Preisig R, Fleisch H, Francis MD 1969 Diphosphonates in the treatment of myositis ossificans. Lancet 2:845[Medline]
3. Von Baeyer H, Hofmann KA 1897 Acetodiphosphorige Säure. Beitr Dtsch Chem Ges 30:1973–1978
4. Blomen LJMJ 1995 History of the bisphosphonates: discovery and history of the non-medical uses of bisphosphonates. In: Bijvoet OLM, Fleisch HA, Canfield RE, Russell RGG (eds) Bisphosponate on Bones. Elsevier, Amsterdam, pp 111–124
5. Fleisch H, Russell RGG, Bisaz S, Casey PA, Mühlbauer RC 1968 The influence of pyrophosphate analogues (diphosphonates) on the precipitation and dissolution of calcium phosphate in vitro and in vivo. Calcif Tissue Res 2:10–10A
6. Fleisch H, Bisaz S 1962 Isolation from urine of pyrophosphate, a calcification inhibitor. Am J Physiol 203:671–675[Abstract/Free Full Text]
7. Fleisch H, Russell RGG, Straumann F 1966 Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis. Nature 212:901–903
8. Geddes AD, D’Souza SM, Ebetino FH, Ibbotson KJ 1994 Bisphosphonates: structure-activity relationships and therapeutic implications. In: Heersche JNM, Kanis JA (eds) Bone and Mineral Research. Elsevier, Amsterdam, vol 8: 265–306
9. Sietsema WK, Ebetino FH 1994 Bisphosphonates in development for metabolic bone disease. Exp Opin Invest Drugs 3:1255–1276[CrossRef]
10. Felix R 1995 Studies with isolated cells and cell systems. In: Bijvoet OLM, Fleisch HA, Canfield RE, Russell RGG (eds) Bisphosphonate on Bones. Elsevier, Amsterdam, pp 189–204
11. Mühlbauer RC 1995 Mechanisms of action of bisphosphonates: information from animal models. In: Bijvoet OLM, Fleisch HA, Canfield RE, Russell RGG (eds) Bisphosphonate on Bones. Elsevier, Amsterdam, pp 171–187
12. Fleisch H 1996 Bisphosphonates: mechanisms of action and clinical use. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, pp 1037–1052
13. Rodan GA, Fleisch H 1996 Bisphosphonates: mechanisms of action. J Clin Invest 97:2692–2696[Medline]
14. Fleisch H 1997 Bisphosphonates in bone disease. From the Laboratory to the Patient, ed 3. The Parthenon Publishing Group, New York
15. Fleisch H, Russell RGG, Bisaz S, Mühlbauer RC, Williams DA 1970 The inhibitory effect of phosphonates on the formation of calcium phosphate crystals in vitro and on aortic and kidney calcification in vivo. Eur J Clin Invest 1:12–18[Medline]
16. Rosenblum IY, Black HE, Ferrell JF 1977 The effects of various diphosphonates on a rat model of cardiac calciphylaxis. Calcif Tissue Res 23:151–159[CrossRef][Medline]
17. Hollander W, Prusty S, Nagraj S, Kirkpatrick B, Paddock J, Colombo M 1978 Comparative effects of cetaben (PHB) and dichlor-methylenediphosphonate (Cl₂ MBP) on the development of atherosclerosis in the cynomolgus monkey. Atherosclerosis 31:307–325[CrossRef][Medline]
18. Kramsch DM, Chan CT 1978 The effect of agents interfering with soft tissue calcification and cell proliferation on calcific fibrous-fatty plaques in rabbits. Circ Res 42:562–571[Free Full Text]
19. Levy RJ, Schoen FJ, Lund SA, Smith MS 1987 Prevention of leaflet calcification of bioprosthetic heart valves with diphosphonate injection therapy. J Thorac Cardiovasc Surg 94:551–557[Abstract]
20. Levy RJ, Wolfrum J, Schoen FJ, Hawley MA, Lund SA, Langer R 1985 Inhibition of calcification of bioprosthetic heart valves by local controlled-release diphosphonate. Science 228:190–192[Abstract/Free Full Text]
21. Golomb G, Langer R, Schoen FJ, Smith MS, Choi YM, Levy RJ 1986 Controlled release of diphosphonate to inhibit bioprosthetic heart valve calcification: dose-response and mechanistic studies. J Contr Rel 4:181–194[CrossRef]
22. Webb CL, Benedict JJ, Schoen FJ, Linden JA, Levy RJ 1987 Inhibition of bioprosthetic heart valve calcification with covalently bound aminopropanehydroxydiphosphonate. ASAIO Trans 33:592–595[Medline]
23. Fraser D, Russell RGG, Pohler O, Robertson WG, Fleisch H 1972 The influence of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on the development of experimentally induced urinary stones in rats. Clin Sci 42:197–207[Medline]
24. Briner WW, Francis MD, Widder JS 1971 The control of dental calculus in experimental animals. Int Dent J 21:61–73[Medline]
25. Plasmans CMT, Kuypers W, Slooff TJJH 1978 The effect of ethane-1-hydroxy-1,1-diphosphonic acid (EHDP) on matrix induced ectopic bone formation. Clin Orthop 132:233–243
26. Ahrengart L, Lindgren U 1986 Prevention of ectopic bone formation by local application of ethane-1-hydroxy-1,1-diphosphonate (EHDP): an experimental study in rabbits. J Orthop Res 4:18–26[CrossRef][Medline]
27. Fleisch H 1988 Bisphosphonates: a new class of drugs in diseases of bone and calcium metabolism. In: Baker PF (ed) Handbook of Experimental Pharmacology. Springer-Verlag, New York, vol 83:440–466
28. Baumann JM, Bisaz S, Fleisch H, Wacker M 1978 Biochemical and clinical effects of ethane-1-hydroxy-1,1-diphosphonate in calcium nephrolithiasis. Clin Sci Mol Med 54:509–516[Medline]
29. Mühlemann HR, Bowles D, Schatt A, Bernimoulin JP 1970 Effect of diphosphonate on human supragingival calculus. Helv Odont Acta 14:31–33[Medline]
30. Sturzenberger OP, Swancar JR, Reiter G 1971 Reduction of dental calculus in humans through the use of a dentifrice containing a crystal-growth inhibitor. J Periodontol 42:416–419[Medline]
31. Reiner M, Sautter V, Olah A, Bossi E, Largiadèr U, Fleisch H 1980 Diphosphonate treatment in myositis ossificans progressiva. In: Caniggia A (ed) Etidronate. Istituto Gentili, Pisa, pp 237–241
32. Slooff TJJH, Feith R, Bijvoet OLM, Nollen AJG 1974 The use of a diphosphonate in para-articular ossifications after total hip replacement. A clinical study. Acta Orthop Belg 40:820–828[Medline]
33. Finerman GAM, Stover SL 1981 Heterotopic ossification following hip replacement or spinal cord injury. Two clinical studies with EHDP. Metab Bone Dis Relat Res 4:337–342
34. Thomas BJ, Amstutz HC 1985 Results of the administration of diphosphonate for the prevention of heterotopic ossification after total hip arthoplasty. J Bone Joint Surg [Am] 67:400–403[Abstract/Free Full Text]
35. King WR, Francis MD, Michael WR 1971 Effect of disodium ethane-1-hydroxy-1,1-diphosphonate on bone formation. Clin Orthop 78:251–270[CrossRef][Medline]
36. Schenk R, Merz WA, Mühlbauer R, Russell RGG, Fleisch H 1973 Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl₂ [scap]mDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif Tissue Res 11:196–214[Medline]
37. Flora L, Hassing GS, Parfitt AM, Villanueva AR 1980 Comparative skeletal effects of two diphosphonates in dogs. Metab Bone Dis Rel Res 2:389–407
38. Larsson A 1974 The short-term effects of high doses of ethylene-1-hydroxy-1,1-diphosphonates upon early dentin formation. Calcif Tissue Res 16:109–127[CrossRef][Medline]
39. Ogawa Y 1980 Disturbances in enamel and dentin formation of rat incisor following injection with sodium fluoride, strontium (II) chloride and EHDP. Jpn J Oral Biol 22:199–226
40. Weile V, Josephsen K, Fejerskov O 1990 Effects of single doses of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on the mineralizing front of rat incisor enamel: a microradiographic and scanning electron microscopic study. Arch Oral Biol 35:857–867[CrossRef][Medline]
41. Alatli I, Hammarström L 1996 Root surface defects in rat molar induced by 1-hydroxyethylidene-1,1-bisphosphonate. Acta Odontol Scand 54:59–65[Medline]
42. Lenehan TM, Balligand M, Nunamaker DM, Wood Jr FE 1985 Effect of EHDP on fracture healing in dogs. J Orthop Res 3:499–507[CrossRef][Medline]
43. Atkin I, Ornoy A, Pita JC, Muniz OE, Agundez A, Castiglione G, Howell DS 1988 EHDP-induced rachitic syndrome in rats is not reversed by vitamin D metabolites. Anat Rec 220:22–30[CrossRef][Medline]
44. Hill LF, Lumb GA, Mawer EB, Stanbury SW 1973 Indirect inhibition of the biosynthesis of 1,25-dihydroxycholecalciferol in rats treated with a diphosphonate. Clin Sci 44:335–347[Medline]
45. Baxter LA, DeLuca HF, Bonjour JP, Fleisch H 1974 Inhibition of vitamin D metabolism by ethane-1-hydroxy-1,1-diphosphonate. Arch Biochem Biophys 164:655–662[CrossRef][Medline]
46. Bonjour JP, Russell RGG, Morgan DB, Fleisch H 1973 Intestinal calcium absorption, Ca-binding protein, and CA-ATPase in diphosphonate-treated rats. Am J Physiol 224:1011–1017
47. Trechsel U, Schenk R, Bonjour JP, Russell RGG, Fleisch H 1977 Relation between bone mineralization, Ca absorption, and plasma Ca in phosphonate-treated rats. Am J Physiol 232:E298–E305
48. Guilland D, Trechsel U, Bonjour JP, Fleisch H 1975 Stimulation of calcium absorption and apparent increased intestinal, 1,25-dihydroxycholecalciferol in rats treated with low doses of ethane-1-hydroxy-1,1-diphosphonate. Clin Sci Mol Med 48:157–160[Medline]
49. Jowsey J, Riggs BL, Kelly PJ, Hoffman DL, Bordier P 1971 The treatment of osteoporosis with disodium ethane-1-hydroxy-1,1-diphosphonate. J Lab Clin Med 78:574–584[Medline]
50. Boyce BF, Smith L, Fogelman I, Johnston E, Ralston S, Boyle IT 1984 Focal osteomalacia due to low-dose diphosphonate therapy in Paget’s disease. Lancet 1:821–824[Medline]
51. Adamson BB, Gallacher SJ, Byars J, Ralston SH, Boyle IT, Boyce BF 1993 Mineralisation defects with pamidronate therapy for Paget’s disease. Lancet 342:1459–1460[CrossRef][Medline]
52. Liens D, Delmas PD, Meunier PJ 1994 Long-term effects of intravenous pamidronate in fibrous dysplasia of bone. Lancet 343:953–954[CrossRef][Medline]
53. Reynolds JJ, Murphy H, Mühlbauer RC, Morgan DB, Fleisch H 1973 Inhibition by diphosphonates of bone resorption in mice and comparison with grey lethal osteopetrosis. Calcif Tissue Res 12:59–71[CrossRef][Medline]
54. Schenk R, Eggli P, Fleisch H, Rosini S 1986 Quantitative morphometric evaluation of the inhibitory activity of new aminobisphosphonates on bone resorption in the rat. Calcif Tissue Int 38:342–349[Medline]
55. Gasser AB, Morgan DB, Fleisch HA, Richelle LJ 1972 The influence of two diphosphonates on calcium metabolism in the rat. Clin Sci 43:31–45[Medline]
56. Fleisch H 1996 The bisphosphonate ibandronate, given daily as well as discontinuously, decreases bone resorption and increases calcium retention as assessed by ⁴⁵Ca kinetics in the intact rat. Osteoporos Int 6:166–170[Medline]
57. Mühlbauer RC, Fleisch H 1990 A method for continual monitoring of bone resorption in rats: evidence for a diurnal rhythm. Am J Physiol 259:R679–R689
58. Reitsma PH, Bijvoet OLM, Verlinden-Ooms H, van der Wee-Pals LJA 1980 Kinetic studies of bone and mineral metabolism during treatment with (3-amino-1-hydroxy-propylidene)-1,1-bisphosphonate (APD) in rats. Calcif Tissue Int 32:145–157[CrossRef][Medline]
59. Garnero P, Shih WJ, Gineyts E, Karpf DB, Delmas PD 1994 Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to alendronate treatment. J Clin Endocrinol Metab 79:1693–1700[Abstract]
60. Fleisch H, Russell RGG, Francis MD 1969 Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture and in vivo. Science 165:1262–1264[Abstract/Free Full Text]
61. Russell RGG, Mühlbauer RC, Bisaz S, Williams DA, Fleisch H 1970 The influence of pyrophosphate, condensed phosphates, phosphonates and other phosphate compounds on the dissolution of hydroxyapatite in vitro and on bone resorption induced by parathyroid hormone in tissue culture and in thyroparathyroidectomised rats. Calcif Tissue Res 6:183–196[CrossRef][Medline]
62. Trechsel U, Stutzer A, Fleisch H 1987 Hypercalcemia induced with an arotinoid in thyroparathyroidectomized rats. A new model to study bone resorption in vivo. J Clin Invest 80:1679–1686
63. Mühlbauer RC, Russell RGG, Williams DA, Fleisch H 1971 The effects of diphosphonates, polyphosphates and calcitonin on "immobilisation osteoporosis" in rats. Eur J Clin Invest 1:336–344[Medline]
64. Wronski TJ, Dann LM, Scott KS, Crooke LR 1989 Endocrine and pharmacological suppressors of bone turnover protect against osteopenia in ovariectomized rats. Endocrinology 125:810–816[Abstract/Free Full Text]
65. Balena R, Toolan BC, Shea M, Markatos A, Myers ER, Lee SC, Opas EE, Seedor JG, Klein H, Frankenfield D, Quartuccio H, Fioravanti C, Clair J, Brown E, Hayes WC, Rodan GA 1993 The effects of 2-year treatment with the aminobisphosphonate alen-dronate on bone metabolism, bone histomorphometry, and bone strength in ovariectomized nonhuman primates. J Clin Invest 92:2577–2586
66. Wink CS, Onge MS, Parker B 1985 The effects of dichloromethylene bisphosphonate on osteoporotic femora of adult castrate male rats. Acta Anat (Basel) 124:117–121[Medline]
67. Brommage R, Baxter DC 1990 Inhibition of bone mineral loss during lactation by Cl₂ MBP. Calcif Tissue Int 47:169–172[Medline]
68. Jee WSS, Black HE, Gotcher JE 1981 Effect of dichloromethane diphosphonate on cortisol-induced bone loss in young adult rabbits. Clin Orthop 156:39–51
69. Watts NB, Harris ST, Genant HK, Wasnich RD, Miller PD, Jackson RD, Ligata AA, Ross P, Woodson III GC, Yanover MJ, Mysiw WJ, Kohse L, Rao MB, Steiger P, Richmond B, Chesnut III CH 1990 Intermittent cyclical etidronate treatment of postmenopausal osteoporosis. N Engl J Med 323:73–79[Abstract]
70. Harris ST, Watts NB, Jackson RD, Genant HK, Wasnich RD, Ross P, Miller PD, Licata AA, Chesnut III CH 1993 Four-year study of intermittent cyclic etidronate treatment of postmenopausal osteoporosis: three years of blinded therapy followed by one year of open therapy. Am J Med 95:557–567[CrossRef][Medline]
71. Reid IR, Wattie DJ, Evans MC, Gamble GD, Stapleton JP, Cornish J 1994 Continuous therapy with pamidronate, a potent bisphosphonate, in postmenopausal osteoporosis. J Clin Endocrinol Metab 79:1595–1599[Abstract]
72. Liberman UA, Weiss SR, Bröll J, Minne HW, Quan H, Bell NH, Rodriguez-Portales J, Downs Jr RW, Dequeker J, Favus M, Seeman E, Recker RR, Capizzi T, Santora AC II, Lombardi A, Shah RV, Hirsch LJ, Karpf DB 1995 Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. New Engl J Med 333:1437–1443[Abstract/Free Full Text]
73. Filipponi P, Cristallini S, Rizzello E, Policani G, Fedeli L, Gregorio F, Boldrini S, Troiani S, Massoni C 1996 Cyclical intravenous clodronate in postmenopausal osteoporosis: results of a long-term clinical trial. Bone 18:179–184[Medline]
74. Ravn P, Clemmesen B, Riis BJ, Christiansen C 1996 The effect on bone mass and bone markers of different doses of ibandronate: a new bisphosphonate for prevention and treatment of postmenopausal osteoporosis: a 1-year, randomized, double-blind, placebo-controlled dose-finding study. Bone 19:527–533[Medline]
75. Reid IR, King AR, Alexander CJ, Ibbertson HK 1988 Prevention of steroid-induced osteoporosis with (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD). Lancet 1:143–146[Medline]
76. Reginster JY, Lecart MP, Deroisy R, Sarlet N, Denis D, Ethgen D, Collette J, Franchimont P 1989 Prevention of postmenopausal bone loss by tiludronate. Lancet 2:1469–1471[CrossRef][Medline]
77. Hosking DJ, McClung MR, Ravn P, Wasnich RD, Thompson DE, Daley MS, Yates AJ 1996 Alendronate in the prevention of osteoporosis: EPIC study two-year results. J Bone Miner Res 11:S133
78. Forwood MR, Burr DB, Takano Y, Eastman DF, Smith PN, Schwardt JD 1995 Risedronate treatment does not increase microdamage in the canine femoral neck. Bone 16:643–650[Medline]
79. Toolan BC, Shea M, Myers ER, Borchers RE, Seedor JG, Quartuccio H, Rodan G, Hayes WC 1992 Effects of 4-amino-1-hydroxybutylidene bisphosphonate on bone biomechanics in rats. J Bone Miner Res 7:1399–1406[Medline]
80. Ammann P, Rizzoli R, Caverzasio J, Shigematsu T, Slosman D, Bonjour JP 1993 Effects of the bisphosphonate tiludronate on bone resorption, calcium balance, and bone mineral density. J Bone Miner Res 8:1491–1498[Medline]
81. Ferretti JL, Delgado CJ, Capozza RF, Cointry G, Montuori E, Roldán E, Pérez Lloret A, Zanchetta JR 1993 Protective effects of disodium etidronate and pamidronate against the biochemical repercussion of betamethasone-induced osteopenia in growing rat femurs. Bone Miner 20:265–276[Medline]
82. Motoie H, Nakamura T, O’Uchi N, Nishikawa H, Kanoh H, Abe T, Kawashima H 1995 Effects of the bisphosphonate YM175 on bone mineral density, strength, structure, and turnover in ovariectomized beagles on concomitant dietary calcium restriction. J Bone Miner Res 10:910–920[Medline]
83. Black DM, Cummings SR, Karpf DB, Cauley JA, Thompson DE, Nevitt MC, Bauer DC, Genant HK, Haskell WL, Marcus R, Ott SM, Torner JC, Quandt SA, Reiss TF, Ensrud KE 1996 Randomized trial of the effect of alendronate on the risk of fracture in women with existing vertebral fractures. Lancet 438:1535–1541
84. Galasko CSB, Samuel AW, Rushton S, Lacey E 1980 The effect of prostaglandin synthesis inhibitors and diphosphonates on tumour-mediated osteolysis. Br J Surg 67:493–496[Medline]
85. Jung A, Mermillod B, Barras C, Baud M, Courvoisier B 1981 Inhibition by two diphosphonates of bone lysis in tumor conditioned media. Cancer Res 41:3233–3237[Abstract/Free Full Text]
86. Johnson KY, Wesseler MA, Olson HM, Martodam RR, Poser JW 1982 The effects of diphosphonates on tumor-induced hypercalcemia and osteolysis in Walker carcinosarcoma 256 (W-256) of rats. In: Donath A, Courvoisier B (eds) Diphosphonates and Bone. Editions Médecine et Hygiène, Genèva, pp 386–389
87. Jung A, Bornand J, Mermillod B, Edouard C, Meunier PJ 1984 Inhibition by diphosphonates of bone resorption induced by the Walker tumor of the rat. Cancer Res 44:3007–3011[Abstract/Free Full Text]
88. Martodam RR, Thornton KS, Sica DA, D’Souza SM, Flora L, Mundy GR 1983 The effects of dichloromethylene diphosphonate on hypercalcemia and other parameters of the humoral hypercalcemia of malignancy in the rat Leydig cell tumor. Calcif Tissue Int 35:512–519[CrossRef][Medline]
89. Rizzoli R, Caverzasio J, Fleisch H, Bonjour JP 1986 Parathyroid hormone-like changes in renal calcium and phosphate reabsorption induced by Leydig cell tumor in thyroparathyroidectomized rats. Endocrinology 119:1004–1009[Abstract/Free Full Text]
90. Fleisch H 1991 Bisphosphonates. Pharmacology and use in the treatment of tumour-induced hypercalcaemic and metastatic bone disease. Drugs 42:919–944[Medline]
91. Sasaki A, Boyce BF, Story B, Wright KR, Chapman M, Boyce R, Mundy GR, Yoneda T 1995 Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice. Cancer Res 55:3551–3557[Abstract/Free Full Text]
92. Kostenuik PJ, Orr FW, Suyama K, Singh G 1993 Increased growth rate and tumor burden of spontaneously metastatic Walker 256 cancer cells in the skeleton of bisphosphonate-treated rats. Cancer Res 53:5452–5457[Abstract/Free Full Text]
93. Mundy GR, Yoneda T 1995 Facilitation and suppression of bone metastasis. Clin Orthop 312:34–44
94. Jung A 1982 Comparison of two parenteral diphosphonates in hypercalcemia of malignancy. Am J Med 72:221–226[CrossRef][Medline]
95. Dunn CJ, Fitton A, Sorkin EM 1994 Etidronic acid. A review of its pharmacological properties and therapeutic efficacy in resorptive bone disease. Drugs Aging 5:446–474[Medline]
96. Plosker GL, Goa KL 1994 Clodronate. A review of its pharmacological properties and therapeutic efficacy in resorptive bone disease. Drugs 47:945–982[Medline]
97. Berenson JR, Lichtenstein A, Porter L, Dimopoulos MA, Bordoni R, George S, Lipton A, Keller A, Ballester O, Kovacs MJ, Blacklock HA, Bell R, Simeone J, Reitsma DJ, Heffernan M, Seaman J, Knight RD 1996 Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. N Engl J Med 334:488–493[Abstract/Free Full Text]
98. Hortobagyi GN, Theriault RL, Porter L, Blayney D, Lipton A, Sinoff C, Wheeler H, Simeone JF, Seaman J, Knight RD, Heffernan M, Reitsma DJ 1996 Efficacy of pamidronate in reducing skeletal complications in patients with breast cancer and lytic bone metastases. N Engl J Med 335:1785–1791[Abstract/Free Full Text]
99. Pecherstorfer M, Herrmann Z, Body JJ, Manegold C, Degardin M, Clemens MR, Thürlimann B, Tubiana-Hulin M, Steinhauer EU, van Eijkeren M, Huss HJ, Thiébaud D 1996 Randomized phase II trial comparing different doses of the bisphosphonate ibandronate in the treatment of hypercalcemia of malignancy. J Clin Oncol 14:268–276[Abstract]
100. Gotcher JE, Jee WSS 1981 The progress of the periodontal syndrome in the rice rat II. The effects of a diphosphonate on the periodontium. J Periodont Res 16:441–455[CrossRef][Medline]
101. Brunsvold MA, Chaves ES, Kornman KS, Aufdemorte TB, Wood R 1992 Effects of a bisphosphonate on experimental periodontitis in monkeys. J Periodontol 63:825–830[Medline]
102. Shoji K, Horiuchi H, Shinoda H 1995 Inhibitory effects of a bisphosphonate (risedronate) on experimental periodontitis in rats. J Periodont Res 30:277–284[CrossRef][Medline]
103. Reynolds JJ, Minkin C, Morgan DB, Spycher D, Fleisch H 1972 The effect of two diphosphonates on the resorption of mouse calvaria in vitro. Calcif Tissue Res 10:302–313[CrossRef][Medline]
104. Boonekamp PM, van der Wee-Pals LJA, van Wijk-van Lennep MML, Thesing CW, Bijvoet OLM 1986 Two modes of action of bisphosphonates on osteoclastic resorption of mineralized matrix. Bone Miner 1:27–39[Medline]
105. Green JR, Müller K, Jaeggi KA 1994 Preclinical pharmacology of CGP 42'446, a new, potent, heterocyclic bisphosphonate compound. J Bone Miner Res 9:745–751[Medline]
106. Flanagan AM, Chambers TJ 1989 Dichloromethylenebisphosphonate (Cl₂ MBP) inhibits bone resorption through injury to osteoclasts that resorb Cl₂ MBP-coated bone. Bone Miner 6:33–43[CrossRef][Medline]
107. Sato M, Grasser W 1990 Effects of bisphosphonates on isolated rat osteoclasts as examined by reflected light microscopy. J Bone Miner Res 5:31–40[Medline]
108. Piper K, Boyde A, Jones SJ 1994 The effect of 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) on the resorptive function of osteoclasts of known nuclear number. Calcif Tissue Int 54:56–61[CrossRef][Medline]
109. Sahni M, Guenther HL, Fleisch H, Collin P, Martin TJ 1993 Bisphosphonates act on rat bone resorption through the mediation of osteoblasts. J Clin Invest 91:2004–2011
110. Shinoda H, Adamek G, Felix R, Fleisch H, Schenk R, Hagan P 1983 Structure-activity relationships of various bisphosphonates. Calcif Tissue Int 35:87–99[CrossRef][Medline]
111. Bijvoet OLM, Frijlink WB, Jie K, van der Linden H, Meijer CJLM, Mulder H, van Paassen HC, Reitsma PH, te Velde J, de Vries E, van der Wey JP 1980 APD in Paget’s disease of bone. Role of the mononuclear phagocyte system? Arthritis Rheum 23:1193–1204[Medline]
112. Boonekamp PM, Löwik CWGM, van der Wee-Pals LJA, van Wijk-van Lennep MLL, Bijvoet OLM 1987 Enhancement of the inhibitory action of APD on the transformation of osteoclast precursors into resorbing cells after dimethylation of the amino group. Bone Miner 2:29–42[Medline]
113. Mühlbauer RC, Bauss F, Schenk R, Janner M, Bosies E, Strein K, Fleisch H 1991 BM 21.0955, a potent new bisphosphonate to inhibit bone resorption. J Bone Miner Res 6:1003–1011[Medline]
114. Takeuchi M, Sakamoto S, Yoshida M, Abe T, Isomura Y 1993 Studies on novel bone resorption inhibitors. I. Synthesis and pharmacological activities of aminomethylenebisphosphonate derivatives. Chem Pharm Bull (Tokyo) 41:688–693[Medline]
115. Van Gelder JM, Breuer E, Ornoy A, Schlossman A, Patlas N, Golomb G 1995 Anticalcification and antiresorption effects of bisacylphosphonates. Bone 16:511–520[Medline]
116. Storm T, Steiniche T, Thamsborg G, Melsen F 1993 Changes in bone histomorphometry after long-term treatment with intermittent, cyclic etidronate for postmenopausal osteoporosis. J Bone Miner Res 8:199–208[Medline]
117. Boyce RW, Paddock CL, Gleason JR, Sletsema WK, Eriksen EF 1995 The effects of risedronate on canine cancellous bone remodeling: three-dimensional kinetic reconstruction of the remodeling site. J Bone Miner Res 10:211–221[Medline]
118. Nii A, Fujimoto R, Okazaki A, Narita K, Miki H 1994 Intramembranous and endochondral bone changes induced by a new bisphosphonate (YM175) in the beagle dog. Toxicol Pathol 22:536–544[Abstract/Free Full Text]
119. Tsuchimoto M, Azuma Y, Higuchi O, Sugimoto I, Hirata N, Kiyoki M, Yamamoto I 1994 Alendronate modulates osteogenesis of human osteoblastic cells in vitro. Jpn J Pharmacol 66:25–33[Medline]
120. Endo N, Rutledge SJ, Opas EE, Vogel R, Rodan GA, Schmidt A 1996 Human protein tyrosine phosphatase-: alternative splicing and inhibition by bisphosphonates. J Bone Miner Res 11:535–543[Medline]
121. Fast DK, Felix R, Dowse C, Neuman WF, Fleisch H 1978 The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture. Biochem J 172:97–107[Medline]
122. Guenther HL, Guenther HE, Fleisch H 1981 The effects of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on collagen synthesis by rabbit articular chondrocytes and rat bone cells. Biochem J 196:293–301[Medline]
123. Guenther HL, Guenther HE, Fleisch H 1981 The influence of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on lysine hydroxylation and cross-link formation in rat bone, cartilage and skin collagen. Biochem J 196:303–310[Medline]
124. Guenther HL, Guenther HE, Fleisch H 1979 Effects of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on rabbit articular chondrocytes in culture. Biochem J 184:203–214[Medline]
125. Gallagher JA, Guenther HL, Fleisch H 1982 Rapid intracellular degradation of newly synthesized collagen by bone cells. Effect of dichloromethylenebisphosphonate. Biochim Biophys Acta 719:349–355[Medline]
126. Giuliani N, Girasole G, Pedrazzoni M, Passeri G, Gatti C, Passeri M 1995 Alendronate stimulates b-FGF production and mineralized nodule formation in human osteoblastic cells and osteoblastogenesis in human bone marrow cultures. J Bone Miner Res 10:S171 (Abstract 129)
127. Ciosek CP, Magnin DR, Harrity TW, Logan JVH, Dickson JK, Gordon EM, Hamilton KA, Jolibois KG Kunselman LK, Lawrence RM, Mookhtiar KA, Rich LC, Slusarchyk DA, Sulsky RB, Biller SA 1993 Lipophilic 1,1-bisphosphonates are potent squalene synthase inhibitors and orally active cholesterol lowering agents in vivo. J Biol Chem 268:24832–24837[Abstract/Free Full Text]
128. Harinck HIJ, Papapoulos SE, Blanksma HJ, Moolenaar AJ, Vermeij P, Bijvoet OLM 1987 Paget’s disease of bone: early and late responses to three different modes of treatment with aminohydroxypropylidene bisphosphonate (APD). Br Med J 295:1301–1305
129. Lufkin EG, Argueta R, Whitaker MD, Cameron AL, Wong VH, Egan KS, O’Fallon WM, Riggs BL 1994 Pamidronate: an unrecognized problem in gastrointestinal tolerability. Osteoporosis Int 4:320–322[CrossRef][Medline]
130. De Groen PC, Lubbe DF, Hirsch LJ, Daifotis A, Stephenson W, Freedholm D, Pryor-Tillotson S, Seleznick MJ, Pinkas H, Wang KK 1996 Esophagitis associated with the use of alendronate. N Engl J Med 355:1016–1021
131. Van Beek E, Hoekstra M, van de Ruit M, Löwik C, Papapoulos S 1994 Structural requirements for bisphosphonate actions in vitro. J Bone Miner Res 9:1875–1882[Medline]
132. Plasmans CMT, Jap PHK, Kuijpers W, Slooff TJJH 1980 Influence of a diphosphonate on the cellular aspect of young bone tissue. Calcif Tissue Int 32:247–256[CrossRef][Medline]
133. Francis MD 1969 The inhibition of calcium hydroxyapatite crystal growth by polyphosphonates and polyphosphates. Calcif Tissue Res 3:151–162[CrossRef][Medline]
134. Francis MD, Russell RGG, Fleisch H 1969 Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vivo. Science 165:1264–1266[Abstract/Free Full Text]
135. Hansen NM, Felix R, Bisaz S, Fleisch H 1976 Aggregation of hydroxyapatite crystals. Biochim Biophys Acta 451:549–559[Medline]
136. Evans JR, Robertson WG, Morgan DB, Fleisch H 1980 Effects of pyrophosphate and diphosphates on the dissolution of hydroxyapatites using a flow system. Calcif Tissue Int 31:153–159[CrossRef][Medline]
137. Jung A, Bisaz S, Fleisch H 1973 The binding of pyrophosphate and two diphosphonates by hydroxyapatite crystals. Calcif Tissue Res 11:269–280[Medline]
138. Uchtman VA 1972 Structural investigations of calcium binding molecules. II. The crystal and molecular structures of calcium dihydrogen ethane-1-hydroxy-1,1-diphosphonate dihydrate, CaC(CH₃)(OH)(PO₃H)₂.2H₂O; implications for polynuclear complex formation. J Phys Chem 76:1304–1310[CrossRef]
139. Barnett BL, Strickland LC 1979 Structure of disodium dihydrogen 1-hydroxyethylidene-diphosphonate tetrahydrate: a bone growth regulator. Acta Crystallogr B 35:1212–1214[CrossRef]
140. Meyer JL, Lee KE, Bergert JH 1977 The inhibition of calcium oxalate crystal growth by multidentate organic phosphonates. Calcif Tissue Res 28:83–86[CrossRef]
141. Robertson WG, Peacock M, Nordin BEC 1973 Inhibitors of the growth and aggregation of calcium oxalate crystals in vitro. Clin Chim Acta 43:31–37[CrossRef][Medline]
142. Larsson SE 1976 The metabolic heterogeneity of glycosaminoglycans of the different zones of the epiphyseal growth plate and the effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) upon glycosaminoglycan synthesis in vivo. Calcif Tissue Res 21:67–82[Medline]
143. Howell DS, Muniz OE, Blanco LN, Pita JC 1980 A micropuncture study of growth cartilage in phosphonate (EHDP) induced rickets. Calcif Tissue Int 30:35–42[CrossRef][Medline]
144. Van den Bos T, Beertsen W 1987 Effects of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on the synthesis of dentine matrix proteins in the mouse. Collagen Rel Res 7:135–147
145. Ogawa Y, Adachi Y, Hong S, Yagi T 1989 1-hydroxyethylidene-1,1-bisphosphonate (HEPB) simultaneously induces two distinct types of hypomineralization in the rat incisor dentine. Calcif Tissue Int 44:46–60[Medline]
146. Ostrowski K, Wojtowicz A, Dziedzic-Goclawska A, Rozycka M 1988 Effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl₂MBP) on the structure of the organic matrix of heterotopically induced bone tissue. Histochemistry 88:207–212[CrossRef][Medline]
147. Palmoski M, Brandt K 1978 Effects of diphosphonates on glycosaminoglycan synthesis and proteoglycan aggregation in normal adult articular cartilage. Arthritis Rheum 21:942–949[Medline]
148. Parfitt AM 1992 The physiologic and pathogenetic significance of bone histomorphometric data. In: Coe FL, Favus MJ (eds) Disorders of Bone and Mineral Metabolism. Raven Press, New York, pp 475–489
149. Eriksen EF, Vesterby A, Kassem M, Melsen F, Mosekilde L 1993 Bone remodeling and bone structure. In: Mundy GR, Martin TJ (eds) Physiology and Pharmacology of Bone. Handbook of Experimental Pharmacology. Springer-Verlag, Berlin, vol 107:67–109
150. Parfitt AM, Mundy GR, Roodman GD, Hughes DE, Boyce BF 1996 A new model for the regulation of bone resorption, with particular reference to the effects of bisphosphonates. J Bone Miner Res 11:150–159[Medline]
151. Boyce RW, Wronski TJ, Ebert DC, Stevens ML, Paddock CL, Youngs TA, Gundersen HJG 1995 Direct stereological estimation of three-dimensional connectivity in rat vertebrae: effect of estrogen, etidronate and risedronate following ovariectomy. Bone 16:209–213[Medline]
152. Giannini S, D’Angelo A, Malvasi L, Castrignano R, Pati T, Tronca R, Liberto L, Nobile M, Crepaldi G 1993 Effects of one-year cyclical treatment with clodronate on postmenopausal bone loss. Bone 14:137–141[Medline]
153. Delmas PD, Vergnaud P, Arlot ME, Pastoureau P, Meunier PJ, Nilssen MHL 1995 The anabolic effect of human PTH (1–34) on bone formation is blunted when bone resorption is inhibited by the bisphosphonate tiludronate-Is activated resorption a prerequisite for the in vivo effect of PTH on formation in a remodeling system? Bone 16:603–610[Medline]
154. Li M, Mosekilde Li Sogaard CH, Thomsen JS, Wronski TJ 1995 Parathyroid hormone monotherapy and cotherapy with antiresorptive agents restore vertebral bone mass and strength in aged ovariectomized rats. Bone 16:629–635[Medline]
155. Jee WSS, Lin BY, Ma YF, Ke HZ 1995 Extra cancellous bone induced by combined prostaglandin E₂ and risedronate administration is maintained after their withdrawal in older female rats. J Bone Miner Res 10:963–970[Medline]
156. Cheng PT, Chan C, Müller K 1995 Cyclical treatment of osteopenic ovariectomized adult rats with PTH(1–34) and pamidronate. J Bone Miner Res 10:119–126[Medline]
157. Ma Y, Jee WSS, Chen Y, Gasser J, Ke HZ, Li XJ, Kimmel DB 1995 Partial maintenance of extra cancellous bone mass by antiresorptive agents after discontinuation of human parathyroid hormone (1–38) in right hindlimb immobilized rats. J Bone Miner Res 10:1726–1734[Medline]
158. Takano Y, Tanizawa T, Mashiba T, Endo N, Nishida S, Takahashi HE 1996 Maintaining bone mass by bisphosphonate incadronate disodium (YM175) sequential treatment after discontinuation of intermittent human parathyroid hormone (1–34) administration in ovariectomized rats. J Bone Miner Res 11:169–177[Medline]
159. Wimalawansa SJ 1995 Combined Therapy with estrogen and eti-dronate has an additive effect on bone mineral density in the hip and vertebrae: four-year randomized study. Am J Med 99:36–42[CrossRef][Medline]
160. Hughes DE, MacDonald BR, Russell RGG, Gowen M 1989 Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow. J Clin Invest 83:1930–1935
161. Cecchini MG, Felix R, Fleisch H, Cooper PH 1987 Effect of bisphosphonates on proliferation and viability of mouse bone marrow-derived macrophages. J Bone Miner Res 2:135–142[Medline]
162. Cecchini MG, Fleisch H 1990 Bisphosphonates in vitro specifically inhibit, among the hematopoietic series, the development of the mouse mononuclear phagocyte lineage. J Bone Miner Res 5:1019–1027[Medline]
163. Löwik CWGM, van der Pluijm G, van der Wee-Pals LJA, Bloys van Treslong-de Groot H, Bijvoet OLM 1988 Migration and phenotypic transformation of osteoclast precursors into mature osteoclasts: the effect of a bisphosphonate. J Bone Miner Res 3:185–192[Medline]
164. Vitté C, Fleisch H, Guenther HL 1996 Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption. Endocrinology 137:2324–2333[Abstract]
165. Colucci S, Minielli V, Zambonin G, Grano M 1995 Etidronate inhibits osteoclast adhesion to bone surfaces but does not interfere with their specific recognition of single bone proteins. Ital J Miner Electrolyte Metab 9:159–164
166. Van der Pluijm G, Vloedgraven H, van Beek E, van der Wee-Pals L, Löwik C, Papapoulos S 1996 Bisphosphonates inhibit the adhesion of breast cancer cells to bone matrices in vitro. J Clin Invest 98:698–705[Medline]
167. Hughes DE, Wright KR, Uy HL, Sasaki A, Yoneda T, Roodman GD, Mundy GR, Boyce BF 1995 Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res 10:1478–1487[Medline]
168. Rogers MJ, Chilton KM, Coxon FP, Lawry J, Smith MO, Suri S, Russell RGG 1996 Bisphosphonates induce apoptosis in mouse macrophage-like cells in vitro by a nitric oxide-independent mechanism. J Bone Miner Res 11:1482–1491[Medline]
169. Miller SC, Jee WSS 1979 The effect of dichloromethylenediphosphonate, a pyrophosphate analog, on bone and bone cell structure in the growing rat. Anat Rec 193:439–462[CrossRef][Medline]
170. Endo Y, Nakamura M, Kikuchi T, Shinoda H, Takeda Y, Nitta Y, Kumagai K 1993 Aminoalkylbisphosphonates, potent inhibitors of bone resorption, induce a prolonged stimulation of histamine synthesis and increase macrophages, granulocytes, and osteoclasts in vivo. Calcif Tissue Int 52:248–254[CrossRef][Medline]
171. Murakami H, Takahashi N, Sasaki T, Udagawa N, Tanaka S, Nakamura I, Zhang D, Barbier A, Suda T 1995 A possible mechanism of the specific action of bisphosphonates on osteoclasts: tiludronate preferentially affects polarized osteoclasts having ruffled borders. Bone 17:137–144[Medline]
172. Selander K, Lehenkari P, Väänänen HK 1994 The effects of bisphosphonates on the resorption cycle of isolated osteoclasts. Calcif Tissue Int 55:368–375[CrossRef][Medline]
173. Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, Golub E, Rodan GA 1991 Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 88:2095–2105
174. Felix R, Guenther HL, Fleisch H 1984 The subcellular distribution of [¹⁴C] dichloromethylenebisphosphonate and [14C] 1-hydroxyethylidene-1,1-bisphosphonate in cultured calvaria cells. Calcif Tissue Int 36:108–113[CrossRef][Medline]
175. Azuma Y, Sato H, Oue Y, Okabe K, Ohta T, Tsuchimoto M, Kiyoki M 1995 Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia models. Bone 16:235–245[Medline]
176. Morgan DB, Monod A, Russell RGG, Fleisch H 1973 Influence of dichloromethylene diphosphonate (Cl₂ [scap]mDP) and calcitonin on bone resorption, lactate production and phosphatase and pyrophosphatase content of mouse calvaria treated with parathyroid hormone in vitro. Calcif Tissue Res 13:287–294[CrossRef][Medline]
177. Carano A, Teitelbaum SL, Konsek JD, Schlesinger PH, Blair HC 1990 Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro. J Clin Invest 85:456–461
178. Zimolo Z, Wesolowski G, Rodan GA 1995 Acid extrusion is induced by osteoclast attachment to bone: inhibition by alendronate and calcitonin. J Clin Invest 96:2277–2283
179. David P, Nguyen H, Barbier A, Baron R 1996 The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H⁺-ATPase. J Bone Miner Res 11:1498–1507[Medline]
180. Ende JJ 1979 Effects of Some Diphosphonates on the Metabolism of Bone in Vivo and in Vitro. PhD. Thesis, University of Leiden, Leiden, The Netherlands
181. Felix R, Russell RGG, Fleisch H 1976 The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes. Biochim Biophys Acta 429:429–438[Medline]
182. Delaissé JM, Eeckhout Y, Vaes G 1985 Bisphosphonates and bone resorption: effects on collagenase and lysosomal enzyme excretion. Life Sci 37:2291–2296[CrossRef][Medline]
183. Felix R, Bettex JD, Fleisch H 1981 Effect of diphosphonates on the synthesis of prostaglandins in cultured calvaria cells. Calcif Tissue Int 33:549–552[CrossRef][Medline]
184. Ohya K, Yamada S, Felix R, Fleisch H 1985 Effect of bisphosphonates on prostaglandin synthesis by rat bone cells and mouse calvaria in culture. Clin Sci 69:403–411[Medline]
185. Garnero P, Gineyts E, Arbault P, Christiansen C, Delmas PD 1995 Different effects of bisphosphonate and estrogen therapy on free and peptide-bound bone cross-links excretion. J Bone Miner Res 10:641–649[Medline]
186. Felix R, Fleisch H 1975 Properties of inorganic pyrophosphatase of pig scapula cartilage. Biochem J 147:111–118[Medline]
187. Schmidt A, Rutledge SJ, Endo N, Opas EE, Tanaka H, Wesolowski G, Leu CT, Huang Z, Ramachandaran C, Rodan SB, Rodan GA 1996 Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate. Proc Natl Acad Sci USA 93:3068–3073[Abstract/Free Full Text]
188. Deleted in proof
189. Frith JC, Mönkkönen J, Blackburn GM, Russell RGG, Rogers MJ 1977 Clodronate and liposome-encapsulated clodronate are metabolised to a toxic ATP analogue, adenosine 5'(ß,-dichloromethylene)triphosphate, by mammalian cells in vitro. J Bone Miner Res 12:1358–1367
190. Rodan GA, Martin TJ 1981 Role of osteoblasts in hormonal control of bone resorption: a hypothesis. Calcif Tissue Int 33:349–351[CrossRef][Medline]
191. McSheehy PMJ, Chambers TJ 1986 Osteoblast-like cells in the presence of parathyroid hormone release soluble factor that stimulates osteoclastic bone resorption. Endocrinology 119:1654–1659[Abstract/Free Full Text]
192. Perry HM, Skogen W, Chappel J, Kahn AJ, Wilner G, Teitelbaum SL 1989 Partial characterization of a parathyroid hormone-stimulated resorption factor(s) from osteoblast-like cells. Endocrinology 125:2075–2082[Abstract/Free Full Text]
193. Morris CA, Mitnick ME, Weir EC, Horowitz H, Kreider BL, Isogna KL 1990 The parathyroid hormone-related protein stimulates human osteoblast-like cells to secrete a 9,000 Dalton bone resorbing protein. Endocrinology 126:1783–1785[Abstract/Free Full Text]
194. Collin P, Guenther HL, Fleisch H 1992 Constitutive expression of osteoclast-stimulating activity by normal clonal osteoblast-like cells: effects of parathyroid hormone and 1,25-dihydroxyvitamin D₃. Endocrinology 131:1181–1187[Abstract/Free Full Text]
195. Parfitt AM, Mundy GR, Roodman GD, Hughes DE, Boyce BF 1996 A new model for the regulation of bone resorption, with particular reference to the effects of bisphosphonates. J Bone Miner Res 11:150–159
196. Ishii T, Saito T, Morimoto K, Takeuchi Y, Asano S, Kumegawa M, Ogata E, Matsumoto T 1993 Estrogen stimulates the elaboration of cell/matrix surface-associated inhibitory factor of osteoclastic bone resorption from osteoblastic cells. Biochem Biophys Res Commun 191:495–502[CrossRef][Medline]
197. Yu X, Schøller J, Foged NT 1996 Interaction between effects of parathyroid hormone and bisphosphonate on regulation of osteoclast activity by the osteoblast-like cell line UMR-106. Bone 19:339–345[Medline]
198. Nishikawa M, Akatsu T, Katayama Y, Yasutomo Y, Kado S, Kugai N, Yamamoto M, Nagata N 1996 Bisphosphonates act on osteoblastic cells and inhibit osteoclast formation in mouse marrow cultures. Bone 18:9–14[Medline]
199. Chambers TJ 1980 Diphosphonates inhibit bone resorption by macrophages in vitro. J Pathol 132:255–262[CrossRef][Medline]
200. Stevenson PH, Stevenson JR 1986 Cytotoxic and migration inhibitory effects of bisphosphonates on macrophages. Calcif Tissue Int 38:227–233[Medline]
201. De Vries E, van der Weij JP, van der Veen CJP, van Paassen HC, Jager MJ, Sleeboom HP, Bijvoet OLM, Cats A 1982 In vitro effect of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonic acid (APD) on the function of mononuclear phagocytes in lymphocyte proliferation. Immunology 47:157–163[Medline]
202. Zernov IN, Stefani DV, Vel’tishchev YE 1979 Assessment of the protective action of diphosphonate compounds against damage to T-lymphocytes by antilymphocytic serum. Bull Exp Biol Med 87:253–254
203. Sansoni P, Passeri G, Fagnoni F, Mohagheghpour N, Snelli G, Brianti V, Engleman EG 1995 Inhibition of antigen-presenting cell function by alendronate in vitro. J Bone Miner Res 10:1719–1725[Medline]
204. Mönkkönen J, Pennanen N, Lapinjoki S, Urtti A 1994 Clodronate (dichloromethylene bisphosphonate) inhibits LPS- stimulated IL-6 and TNF production by RAW 264 cells. Life Sci 54:229–234
205. Pennanen N, Lapinjoki S, Urtti A, Mönkkönen J 1995 Effect of liposomal and free bisphosphonates on the IL-1ß, IL-6 and TNF secretion from RAW 264 cells in vitro. Pharmacol Res 12:916–922
206. Makkonen N, Hirvonen MR, Teräväinen T, Savolainen K, Mönkkönen J 1996 Different effects of three bisphosphonates on nitric oxide production by RAW 264 macrophage-like cells in vitro. J Pharmacol Exp Ther 277:1097–1102[Abstract/Free Full Text]
207. Komissarenko SV, Zhuravskii NI, Karlova NP, Gulyi MF 1977 Inhibition of hypersensitivity of delayed and immediate types in guinea pigs by methylenediphosphonic acid. Bull Exp Biol Med 84:1322–1323[CrossRef]
208. Barbier A, Brelière JC, Paul R, Roncucci R 1985 Comparative study of etidronate and SR 41319, a new diphosphonate, on passive cutaneous anaphylaxis and phospholipase A₂ activity. Agents Actions 16:41–42[CrossRef][Medline]
209. Milhaud G, Labat ML, Moricard Y 1983 (Dichloromethylene) diphosphonate-induced impairment of T-lymphocyte function. Proc Natl Acad Sci USA 80:4469–4473[Abstract/Free Full Text]
210. Labat ML, Tzehoval E, Moricard Y, Feldman M, Milhaud G 1983 Lack of a T-cell dependent subpopulation of macrophages in (dichloromethylene)diphosphonate-treated mice. Biomed Pharmacother 37:270–276[Medline]
211. Labat ML, Florentin I, Davigny M, Moricard Y, Milhaud G 1984 Dichloromethylene diphosphonate (Cl₂ MDP) reduces natural killer (NK) cell activity in mice. Metab Bone Dis Rel Res 5:281–287[CrossRef]
212. Van Rooijen N, van Nieuwmegen R 1984 Elimination of phagocytic cells in the spleen after intravenous injection of liposome-encapsulated dichloromethylene diphosphonate: an enzyme-histochemical study. Cell Tissue Res 238:355–358[Medline]
213. Adami S, Bhalla AK, Dorizzi R, Montesanti F, Rosini S, Salvagno G, Lo Cascio V 1987 The acute-phase response after bisphosphonate administration. Calcif Tissue Int 41:326–331[Medline]
214. Lioté F, Boval-Boizard B, Fritz P, Kuntz D 1995 Lymphocyte subsets in pamidronate-induced lymphopenia. Br J Rheumatol 34:993–995[Free Full Text]
215. Schweitzer DH, Oostendorp-van de Ruit M, van der Pluijm G, Löwik CWGM, Papapoulos SE 1995 Interleukin-6 and the acute phase response during treatment of patients with Paget’s disease with the nitrogen-containing bisphosphonate dimethylaminohydroxypropylidene bisphosphonate. J Bone Miner Res 10:956–962[Medline]
216. Sauty A, Pecherstorfer M, Zimmer-Roth I, Fioroni P, Juillerat L, Markert M, Ludwig H, Leuenberger P, Burckhardt P, Thiébaud D 1996 Interleukin-6 and tumor necrosis factor levels after bisphosphonate treatment in vitro and in patients with malignancy. Bone 18:133–139[Medline]
217. Francis MD, Flora L, King WR 1972 The effects of disodium ethane-1-hydroxy-1,1-diphosphonate on adjuvant induced arthritis in rats. Calcif Tissue Res 9:109–121[CrossRef][Medline]
218. Flora L 1979 Comparative antiinflammatory and bone protective effects of two diphosphonates in adjuvant arthritis. Arthritis Rheum 22:340–346[Medline]
219. Francis MD, Hovancik K, Boyce RW 1989 NE-58095: a diphosphonate which prevents bone erosion and preserves joint architecture in experimental arthritis. Int J Tissue React 11:239–252[Medline]
220. Dunn CJ, Galinet LA, Wu H, Nugent RA, Schlachter ST, Staite ND, Aspar DG, Elliott GA, Essani NA, Rohloff NA Smith RJ 1993 Demonstration of novel anti-arthritic and anti-inflammatory effects of diphosphonates. J Pharmacol Exp Ther 266:1691–1698[Abstract/Free Full Text]
221. Österman T, Kippo K, Lauren L, Hannuniemi R, Sellman R 1995 Effect of clodronate on established collagen-induced arthritis in rats. Inflamm Res 44:258–263[CrossRef][Medline]
222. Kinne RW, Schmidt-Weber CB, Hoppe R, Buchner E, Palombo-Kinne E, Nürnberg E, Emmrich F 1995 Long-term amelioration of rat adjuvant arthritis following systemic elimination of macrophages by clodronate-containing liposomes. Arthritis Rheum 38:1777–1790[Medline]
223. Van Lent PLEM, van den Bersselaar LAM, Holthuyzen AEM, van Rooijen N, van de Putte LBA, van den Berg WB 1994 Phagocytic synovial lining cells in experimentally induced chronic arthritis: down-regulation of synovitis by CL₂ MDP-liposomes. Rheumatol Int 13:221–228
224. Österman T, Kippo K, Laurén L, Hannuniemi R, Sellman R 1994 Effect of clodronate on established adjuvant arthritis. Rheumatol Int 14:139–147[CrossRef][Medline]
225. Elomaa I, Blomqvist C, Gröhn P, Porkka L, Kairento AL, Selander K, Lamberg-Allardt C, Holmström T 1983 Long-term controlled trial with diphosphonate in patients with osteolytic bone metastases. Lancet 1:146–149[Medline]
226. Van Holten-Verzantvoort AT, Bijvoet OLM, Cleton FJ, Hermans J, Kroon HM, Harinck HIJ, Vermey P, Elte JWF, Neijt JP, Beex LVAM, Blijham G 1987 Reduced morbidity from skeletal metastases in breast cancer patients during long-term bisphosphonate (APD) treatment. Lancet 2:983–985[Medline]
227. Paterson AHG, Powles TJ, Kanis JA, McCloskey E, Hanson J, Ashley S 1993 Double-blind controlled trial of oral clodronate in patients with bone metastases from breast cancer. J Clin Oncol 11:59–65[Abstract]
228. Felix R, Fleisch H 1981 Increase in fatty acid oxidation in calvaria cells cultured with diphosphonates. Biochem J 196:237–245[Medline]
229. Felix R, Fast DK, Sallis JD, Fleisch H 1980 Effect of diphosphonates on glycogen content of rabbit ear cartilage cells in culture. Calcif Tissue Int 30:163–166[CrossRef][Medline]
230. Felix R, Fleisch H 1979 Increase in alkaline phosphatase activity in calvaria cells cultured with diphosphonates. Biochem J 183:73–81[Medline]
231. Stronski SA, Bettschen-Camin L, Wetterwald A, Felix R, Trechsel U, Fleisch H 1988 Bisphosphonates inhibit 1,25-dihydroxyvitamin D₃-induced increase of osteocalcin in plasma of rats in vivo and in culture medium of rat calvaria in vitro. Calcif Tissue Int 42:248–254[Medline]
232. Pilczyk R, Sutcliffe H, Martin TJ 1972 Effects of pyrophosphate and diphosphonates on parathyroid hormone- and fluoride-stimulated adenylate cyclase activity. FEBS Lett 24:225–228[CrossRef][Medline]
233. Gebauer U, Russell RGG, Touabi M, Fleisch H 1976 Effect of diphosphonates on adenosine 3':5' cyclic monophosphate in mouse calvaria after stimulation by parathyroid hormone in vitro. Clin Sci Mol Med 50:473–478[Medline]
234. Evéquoz V, Trechsel U, Fleisch H 1985 Effect of bisphosphonates on production of interleukin 1-like activity by macrophages and its effect on rabbit chondrocytes. Bone 6:439–444[Medline]
235. Talanian RV, Brown NC, McKenna CE, Ye TG, Levy JN, Wright GE 1989 Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase. Biochemistry 28:8270–8274[CrossRef][Medline]
236. Eubank WB, Reeves RE 1982 Analog inhibitors for the pyrophosphate-dependent phosphofructokinase of entamoeba histolytica and their effect on culture growth. J Parasitol 68:599–602[CrossRef][Medline]
237. Guilland DF, Sallis JD, Fleisch H 1974 The effect of two diphosphonates on the handling of calcium by rat kidney mitochondria in vitro. Calcif Tissue Res 15:303–314[CrossRef][Medline]
238. Guilland DF, Fleisch H 1974 The effect of in vivo treatment with EHDP and/or 1,25-DHCC on calcium uptake and release in isolated kidney mitochondria. Biochem Biophys Res Commun 61:906–911[CrossRef][Medline]
239. Paspaliaris V, Leaver DD 1990 The bisphosphonate, clodronate, inhibits calcium-induced contraction in vascular smooth muscle. Eur J Pharmacol 183:1257[CrossRef]
240. Greif F, Anais D, Frei L, Arbeit L, Sorroff HS 1990 Blocking the calcium cascade in experimental acute renal failure. Isr J Med Sci 26:301–305[Medline]
241. Rossier JR, Cox JA, Niesor EJ, Bentzen CL 1989 A new class of calcium entry blockers defined by 1,3-diphosphonates. J Biol Chem 264:16598–16607[Abstract/Free Full Text]
242. Klein G, Martin JB, Satre M 1988 Methylenediphosphonate, a metabolic poison in dictyostelium discoideum. 31P NMR evidence for accumulation of adenosine 5'-(ß,-methylenetriphosphate) and diadenosine 5',5'''-P¹,P⁴-(P²,P³-methylenetetraphosphate). Biochemistry 27:1897–1901[CrossRef]
243. Rogers MJ, Russell RGG, Blackburn GM, Williamson MP, Watts DJ 1992 Metabolism of halogenated bisphosphonates by the cellular slime mold Dictyostelium discoideum. Biochem Biophys Res Commun 189:414–423[CrossRef][Medline]
244. Rogers MJ, Watts DJ, Russell RGG, Ji X, Xiong X, Blackburn GM, Bayless AV, Ebetino FH 1994 Inhibitory effects of bisphosphonates on growth of amoebae of the cellular slime mold Dictyostelium discoideum. J Bone Miner Res 9:1029–1039[Medline]
245. Rogers MJ, Xiong X, Brown RJ, Watts DJ, Russell RGG, Bayless AV, Ebetino FH 1995 Structure-activity relationships of new heterocycle-containing bisphosphonates as inhibitors of bone resorption and as inhibitors of growth of Dictyostelium discoideum amoebae. Mol Pharmacol 47:398–402[Abstract]
246. Boulenc X, Marti E, Joyeux H, Roques C, Berger Y, Fabre G 1993 Importance of the paracellular pathway for the transport of a new bisphosphonate using the human CACO-2 monolayers model. Biochem Pharmacol 46:1591–1600[CrossRef][Medline]
247. Janner M, Mühlbauer RC, Fleisch H 1991 Sodium EDTA enhances intestinal absorption of two bisphosphonates. Calcif Tissue Int 49:280–283[Medline]
248. Boulenc X, Roques C, Joyeux H, Berger Y, Fabre G 1995 Bisphosphonates increase tight junction permeability in the human intestinal epithelial (Caco-2) model. Int J Pharmacol 123:13–24[CrossRef]
249. Gertz BJ, Holland SD, Kline WF, Matuszewski BK, Freeman A, Quan H, Lasseter KC, Mucklow JC, Porras AG 1995 Studies of the oral bioavailability of alendronate. Clin Pharmacol Ther 58:288–298[CrossRef][Medline]
250. Wiedmer WH, Zbinden AM, Trechsel U, Fleisch H 1983 Ultrafiltrability and chromatographic properties of pyrophosphate, 1-hydroxyethylidene-1,1-bisphosphonate, and dichloromethylenebisphosphonate in aqueous buffers and in human plasma. Calcif Tissue Int 35:397–400[CrossRef][Medline]
251. Grabenstetter RJ, Cilley WA 1971 Polynuclear complex formation in solutions of calcium ion and ethane-1-hydroxy-1,1-diphosphonic acid. I. Complexometric and pH titrations. J Phys Chem 75:676–682[CrossRef]
252. Lamson ML, Fox JL, Huguchi WI 1984 Calcium and 1-hydroxyethylidene-1,1-bisphosphonic acid: polynuclear complex formation in the physiological range of pH. Int J Pharm 21:143–154[CrossRef]
253. Lamson ML, Fox JL, Higuchi WI 1985 Model selection for complex systems involving polynuclear species. Polyhedron 4:133–141[CrossRef]
254. Lin JH, Chen I-W, DeLuna FA 1994 Nonlinear kinetics of alen-dronate. Plasma protein binding and bone uptake. Drug Metab Dispos 22:400–405[Abstract]
255. Lin JH, Chen IW, DeLuna FA, Hichens M 1993 Role of calcium in plasma protein binding and renal handling of alendronate in hypo- and hypercalcemic rats. J Pharmacol Exp Ther 267:670–675[Abstract/Free Full Text]
256. Troehler U, Bonjour JP, Fleisch H 1975 Renal secretion of diphosphonates in rats. Kidney Int 8:6–13[Medline]
257. Lin JH 1996 Bisphosphonates: a review of their pharmacokinetic properties. Bone 18:75–85[Medline]
258. Bisaz S, Jung A, Fleisch H 1978 Uptake by bone of pyrophosphate, diphosphonates and their technetium derivatives. Clin Sci Mol Med 54:265–272[Medline]
259. Fitton A, McTavish D 1991 Pamidronate. A review of its pharmacological properties and therapeutic efficacy in resorptive bone disease. Drugs 41:289–318[Medline]
260. Yakatan GJ, Poynor WJ, Talbert RL, Floyd BF, Slough CL, Ampulski RS, Benedict JJ 1982 Clodronate kinetics and bioavailability. Clin Pharmacol Ther 31:402–410[Medline]
261. Conrad KA, Lee SM 1981 Clodronate kinetics and dynamics. Clin Pharmacol Ther 30:114–120[Medline]
262. Daley-Yates PT, Bennett R 1988 A comparison of the pharmacokinetics of ¹⁴C-labelled APD and ^99mTc-labelled APD in the mouse. Calcif Tissue Int 43:125–127[Medline]
263. Masarachia P, Weinreb M, Balena R, Rodan GA 1996 Comparison of the distribution of ³H-alendronate and ³H-etidronate in rat and mouse bones. Bone 19:281–290[Medline]
264. Sturtz G, Couthon H, Fabulet O, Mian M, Rosini S 1993 Synthesis of gem-bisphosphonic methotrexate conjugates and their biological response towards Walker’s osteosarcoma. Eur J Med Chem 28:899–903[CrossRef]
265. Larsson A, Rohlin M 1980 In vivo distribution of ¹⁴C-labeled ethylene-1-hydroxy-1,1-diphosphonate in normal and treated young rats. An autoradiographic and ultrastructural study. Toxicol Appl Pharmacol 52:391–399[CrossRef][Medline]
266. Wingen F, Schmähl D 1985 Distribution of 3-amino-1-hydroxypropane-1,1-diphosphonic acid in rats and effects on rat osteosarcoma. Arzneimittelforschung 35:1565–1571[Medline]
267. Mönkkönen J 1988 A one year follow-up study of the distribution of 14C-clodronate in mice and rats. Pharmacol Toxicol 62:51–53[Medline]
268. Mönkkönen J, Koponen HM, Ylitalo P 1990 Comparison of the distribution of three bisphosphonates in mice. Pharmacol Toxicol 66:294–298[Medline]
269. Bounameaux HM, Schifferli J, Montani JP, Jung A, Chatelanat F 1983 Renal failure associated with intravenous diphosphonates. Lancet 1:471
270. Kasting GB, Francis MD 1992 Retention of etidronate in human, dog, and rat. J Bone Miner Res 7:513–522[Medline]
271. Alden CL, Parker RD, Eastman DF 1989 Development of an acute model for the study of chloromethanediphosphonate nephrotoxicity. Toxicol Pathol 17:27–32[Medline]
272. Cal JC, Daley-Yates PT 1990 Disposition and nephrotoxicity of 3-animo-1-hydroxypropylidene-1,1-bisphosphonate (APD) in rats and mice. Toxicology 65:179–197[CrossRef][Medline]
273. Eguchi M, Yamaguchi T, Shiota E, Handa S 1982 Fault of ossification and calcification and angular deformities of long bone in th mouse fetuses caused by high doses of ethane-1-hydroxy-1,1-diphosphonate (EHDP) during pregnancy. Cong Anom 22:47–52
274. Van Breukelen FJM, Bijvoet OLM, Frijlink WB, Sleeboom HP, Mulder H, van Oosterom AT 1982 Efficacy of amino-hydroxypropylidene bisphosphonate in hypercalcemia: observations on regulation of serum calcium. Calcif Tissue Int 34:321–327[CrossRef][Medline]

This article has been cited by other articles:

				G. SPIZZO, A. SEEBER, and M. MITTERER Routine Use of Pamidronate in NSCLC Patients with Bone Metastasis: Results from a Retrospective Analysis Anticancer Res, December 1, 2009; 29(12): 5245 - 5249. [Abstract] [Full Text] [PDF]

				S. N. Gebara and H. Moubayed Risk of osteonecrosis of the jaw in cancer patients taking bisphosphonates Am. J. Health Syst. Pharm., September 1, 2009; 66(17): 1541 - 1547. [Abstract] [Full Text] [PDF]

				C. Panaroni, R. Gioia, A. Lupi, R. Besio, S. A. Goldstein, J. Kreider, S. Leikin, J. C. Vera, E. L. Mertz, E. Perilli, et al. In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta Blood, July 9, 2009; 114(2): 459 - 468. [Abstract] [Full Text] [PDF]

				R. Villa-Bellosta and V. Sorribas Phosphonoformic Acid Prevents Vascular Smooth Muscle Cell Calcification by Inhibiting Calcium-Phosphate Deposition Arterioscler Thromb Vasc Biol, May 1, 2009; 29(5): 761 - 766. [Abstract] [Full Text] [PDF]

				F. LUMACHI, A. BRUNELLO, A. ROMA, and U. BASSO Cancer-induced Hypercalcemia Anticancer Res, May 1, 2009; 29(5): 1551 - 1555. [Abstract] [Full Text] [PDF]

				T. TANAKA, H. KAWASHIMA, K. OHNISHI, K. MATSUMURA, R. YOSHIMURA, M. MATSUYAMA, K. KURATSUKURI, and T. NAKATANI Indirect Antitumor Effects of Bisphosphonates on Prostatic LNCaP Cells Co-cultured with Bone Cells Anticancer Res, April 1, 2009; 29(4): 1089 - 1094. [Abstract] [Full Text] [PDF]

				N. D. Toussaint, G. J. Elder, and P. G. Kerr Bisphosphonates in Chronic Kidney Disease; Balancing Potential Benefits and Adverse Effects on Bone and Soft Tissue Clin. J. Am. Soc. Nephrol., January 1, 2009; 4(1): 221 - 233. [Abstract] [Full Text] [PDF]

				P. D. Ottewell, H. Monkkonen, M. Jones, D. V. Lefley, R. E. Coleman, and I. Holen Antitumor Effects of Doxorubicin Followed by Zoledronic Acid in a Mouse Model of Breast Cancer J Natl Cancer Inst, August 20, 2008; 100(16): 1167 - 1178. [Abstract] [Full Text] [PDF]

				N Walji, A K Chan, and D R Peake Common acute oncological emergencies: diagnosis, investigation and management Postgrad. Med. J., August 1, 2008; 84(994): 418 - 427. [Abstract] [Full Text] [PDF]

				T. B. Dodson, N. S. Raje, P. A. Caruso, and A. E. Rosenberg Case 9-2008 -- A 65-Year-Old Woman with a Nonhealing Ulcer of the Jaw N. Engl. J. Med., March 20, 2008; 358(12): 1283 - 1291. [Full Text] [PDF]

				L. Tauchmanova, A. Colao, G. Lombardi, B. Rotoli, and C. Selleri REVIEW: Bone Loss and Its Management in Long-Term Survivors from Allogeneic Stem Cell Transplantation J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4536 - 4545. [Abstract] [Full Text] [PDF]

				Y. Hacchou, T. Uematsu, O. Ueda, Y. Usui, S. Uematsu, M. Takahashi, T. Uchihashi, Y. Kawazoe, T. Shiba, S. Kurihara, et al. Inorganic Polyphosphate: a Possible Stimulant of Bone Formation Journal of Dental Research, September 1, 2007; 86(9): 893 - 897. [Abstract] [Full Text] [PDF]

				L. V. Virkki, J. Biber, H. Murer, and I. C. Forster Phosphate transporters: a tale of two solute carrier families Am J Physiol Renal Physiol, September 1, 2007; 293(3): F643 - F654. [Abstract] [Full Text] [PDF]

				R. A. Cahill, D. Wenkert, S. A. Perlman, A. Steele, S. P. Coburn, W. H. McAlister, S. Mumm, and M. P. Whyte Infantile Hypophosphatasia: Transplantation Therapy Trial Using Bone Fragments and Cultured Osteoblasts J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 2923 - 2930. [Abstract] [Full Text] [PDF]

				S. Ravera, L. V. Virkki, H. Murer, and I. C. Forster Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements Am J Physiol Cell Physiol, August 1, 2007; 293(2): C606 - C620. [Abstract] [Full Text] [PDF]

				E. L. Kaijzel, G. van der Pluijm, and C. W.G.M. Lowik Whole-Body Optical Imaging in Animal Models to Assess Cancer Development and Progression Clin. Cancer Res., June 15, 2007; 13(12): 3490 - 3497. [Abstract] [Full Text] [PDF]

				M. J. Favus Diabetes and the Risk of Osteonecrosis of the Jaw J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 817 - 818. [Full Text] [PDF]

				S Chiandussi, M Biasotto, F Dore, F Cavalli, M. Cova, and R Di Lenarda Clinical and diagnostic imaging of bisphosphonate-associated osteonecrosis of the jaws. Dentomaxillofac. Radiol., July 1, 2006; 35(4): 236 - 243. [Abstract] [Full Text] [PDF]

				L. F. Massa, V. Bradaschia-Correa, and V. E. Arana-Chavez Immunocytochemical Study of Amelogenin Deposition during the Early Odontogenesis of Molars in Alendronate-treated Newborn Rats J. Histochem. Cytochem., June 1, 2006; 54(6): 713 - 725. [Abstract] [Full Text] [PDF]

				T. R. Halfdanarson, W. J. Hogan, and T. J. Moynihan Oncologic Emergencies: Diagnosis and Treatment Mayo Clin. Proc., June 1, 2006; 81(6): 835 - 848. [Abstract] [Full Text] [PDF]

				S.-B. Woo, J. W. Hellstein, and J. R. Kalmar Systematic Review: Bisphosphonates and Osteonecrosis of the Jaws Ann Intern Med, May 16, 2006; 144(10): 753 - 761. [Abstract] [Full Text] [PDF]

				M. Caraglia, D. Santini, M. Marra, B. Vincenzi, G. Tonini, and A. Budillon Emerging anti-cancer molecular mechanisms of aminobisphosphonates. Endocr. Relat. Cancer, March 1, 2006; 13(1): 7 - 26. [Abstract] [Full Text] [PDF]

				M Bevilacqua, L J Dominguez, S Rosini, and M Barbagallo Bisphosphonates and atherosclerosis: why? Lupus, September 1, 2005; 14(9): 773 - 779. [Abstract] [PDF]

				L. M. Ward, A. E. Denker, A. Porras, S. Shugarts, W. Kline, R. Travers, C. Mao, F. Rauch, A. Maes, P. Larson, et al. Single-Dose Pharmacokinetics and Tolerability of Alendronate 35- and 70-Milligram Tablets in Children and Adolescents with Osteogenesis Imperfecta Type I J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4051 - 4056. [Abstract] [Full Text] [PDF]

				A. A Licata Discovery, Clinical Development, and Therapeutic Uses of Bisphosphonates Ann. Pharmacother., April 1, 2005; 39(4): 668 - 677. [Abstract] [Full Text] [PDF]

				C. V. Odvina, J. E. Zerwekh, D. S. Rao, N. Maalouf, F. A. Gottschalk, and C. Y. C. Pak Severely Suppressed Bone Turnover: A Potential Complication of Alendronate Therapy J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1294 - 1301. [Abstract] [Full Text] [PDF]

				A. F. Stewart Hypercalcemia Associated with Cancer N. Engl. J. Med., January 27, 2005; 352(4): 373 - 379. [Full Text] [PDF]

				Y. Mori, N. Shimizu, M. Dallas, M. Niewolna, B. Story, P. J. Williams, G. R. Mundy, and T. Yoneda Anti-{alpha}4 integrin antibody suppresses the development of multiple myeloma and associated osteoclastic osteolysis Blood, October 1, 2004; 104(7): 2149 - 2154. [Abstract] [Full Text] [PDF]

				A. Lipton, A. Colombo-Berra, R. M. Bukowski, L. Rosen, M. Zheng, and G. Urbanowitz Skeletal Complications in Patients with Bone Metastases from Renal Cell Carcinoma and Therapeutic Benefits of Zoledronic Acid Clin. Cancer Res., September 15, 2004; 10(18): 6397S - 6403S. [Abstract] [Full Text] [PDF]

				J. Barrett, E. Worth, F. Bauss, and S. Epstein Ibandronate: A Clinical Pharmacological and Pharmacokinetic Update J. Clin. Pharmacol., September 1, 2004; 44(9): 951 - 965. [Abstract] [Full Text] [PDF]

				T. Hiraga, P. J. Williams, A. Ueda, D. Tamura, and T. Yoneda Zoledronic Acid Inhibits Visceral Metastases in the 4T1/luc Mouse Breast Cancer Model Clin. Cancer Res., July 1, 2004; 10(13): 4559 - 4567. [Abstract] [Full Text] [PDF]

				R. Rizzoli Bisphosphonates and reduction of skeletal events in patients with bone metastatic breast cancer Ann. Onc., May 1, 2004; 15(5): 700 - 701. [Full Text] [PDF]

				H. D. Danenberg, G. Golomb, A. Groothuis, J. Gao, H. Epstein, R. V. Swaminathan, P. Seifert, and E. R. Edelman Liposomal Alendronate Inhibits Systemic Innate Immunity and Reduces In-Stent Neointimal Hyperplasia in Rabbits Circulation, December 2, 2003; 108(22): 2798 - 2804. [Abstract] [Full Text] [PDF]

				M. Nakamura, N. Udagawa, S. Matsuura, M. Mogi, H. Nakamura, H. Horiuchi, N. Saito, B. Y. Hiraoka, Y. Kobayashi, K. Takaoka, et al. Osteoprotegerin Regulates Bone Formation through a Coupling Mechanism with Bone Resorption Endocrinology, December 1, 2003; 144(12): 5441 - 5449. [Abstract] [Full Text] [PDF]

				E. Alvarez, M. Westmore, R. J. S. Galvin, C. L. Clapp, E. L. Considine, S. J. Smith, K. Keyes, P. W. Iversen, D. M. Delafuente, S. Sulaimon, et al. Properties of Bisphosphonates in the 13762 Rat Mammary Carcinoma Model of Tumor-Induced Bone Resorption Clin. Cancer Res., November 15, 2003; 9(15): 5705 - 5713. [Abstract] [Full Text] [PDF]

				M. Bezzi, M. Hasmim, G. Bieler, O. Dormond, and C. Ruegg Zoledronate Sensitizes Endothelial Cells to Tumor Necrosis Factor-induced Programmed Cell Death: EVIDENCE FOR THE SUPPRESSION OF SUSTAINED ACTIVATION OF FOCAL ADHESION KINASE AND PROTEIN KINASE B/Akt J. Biol. Chem., October 31, 2003; 278(44): 43603 - 43614. [Abstract] [Full Text] [PDF]

				M. Coco, D. Glicklich, M. C. Faugere, L. Burris, I. Bognar, P. Durkin, V. Tellis, S. Greenstein, R. Schechner, K. Figueroa, et al. Prevention of Bone Loss in Renal Transplant Recipients: A Prospective, Randomized Trial of Intravenous Pamidronate J. Am. Soc. Nephrol., October 1, 2003; 14(10): 2669 - 2676. [Abstract] [Full Text] [PDF]

				L. Zeitlin, F. Rauch, H. Plotkin, and F. H. Glorieux Height and Weight Development During Four Years of Therapy With Cyclical Intravenous Pamidronate in Children and Adolescents With Osteogenesis Imperfecta Types I, III, and IV Pediatrics, May 1, 2003; 111(5): 1030 - 1036. [Abstract] [Full Text] [PDF]

				F. Rauch, H. Plotkin, R. Travers, L. Zeitlin, and F. H. Glorieux Osteogenesis Imperfecta Types I, III, and IV: Effect of Pamidronate Therapy on Bone and Mineral Metabolism J. Clin. Endocrinol. Metab., March 1, 2003; 88(3): 986 - 992. [Abstract] [Full Text] [PDF]

				J. R. Berenson, B. E. Hillner, R. A. Kyle, K. Anderson, A. Lipton, G. C. Yee, and J. S. Biermann American Society of Clinical Oncology Clinical Practice Guidelines: The Role of Bisphosphonates in Multiple Myeloma J. Clin. Oncol., September 1, 2002; 20(17): 3719 - 3736. [Abstract] [Full Text] [PDF]

				O. Johnell, W. H. Scheele, Y. Lu, J.-Y. Reginster, A. G. Need, and E. Seeman Additive Effects of Raloxifene and Alendronate on Bone Density and Biochemical Markers of Bone Remodeling in Postmenopausal Women with Osteoporosis J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 985 - 992. [Abstract] [Full Text] [PDF]

				R. Marcus, M. Wong, H. Heath III, and J. L. Stock Antiresorptive Treatment of Postmenopausal Osteoporosis: Comparison of Study Designs and Outcomes in Large Clinical Trials with Fracture as an Endpoint Endocr. Rev., February 1, 2002; 23(1): 16 - 37. [Abstract] [Full Text] [PDF]

				P. A. Price, J. R. Buckley, and M. K. Williamson The Amino Bisphosphonate Ibandronate Prevents Vitamin D Toxicity and Inhibits Vitamin D-Induced Calcification of Arteries, Cartilage, Lungs and Kidneys in Rats J. Nutr., November 1, 2001; 131(11): 2910 - 2915. [Abstract] [Full Text] [PDF]

				P. A. Price, H. H. June, J. R. Buckley, and M. K. Williamson Osteoprotegerin Inhibits Artery Calcification Induced by Warfarin and by Vitamin D Arterioscler Thromb Vasc Biol, October 1, 2001; 21(10): 1610 - 1616. [Abstract] [Full Text] [PDF]

				Bisphosphonates for osteoporosis DTB, September 1, 2001; 39(9): 68 - 72. [Abstract] [Full Text] [PDF]

				B. M. Lange, R. E.B. Ketchum, and R. B. Croteau Isoprenoid Biosynthesis. Metabolite Profiling of Peppermint Oil Gland Secretory Cells and Application to Herbicide Target Analysis Plant Physiology, September 1, 2001; 127(1): 305 - 314. [Abstract] [Full Text] [PDF]

				S. M. Ali, F. J. Esteva, G. Hortobagyi, H. Harvey, J. Seaman, R. Knight, L. Costa, and A. Lipton Safety and Efficacy of Bisphosphonates Beyond 24 Months in Cancer Patients J. Clin. Oncol., July 15, 2001; 19(14): 3434 - 3437. [Abstract] [Full Text] [PDF]

				N. R. Biermasz, N. A. T. Hamdy, Y. J. H. Janssen, and F. Roelfsema Additional Beneficial Effects of Alendronate in Growth Hormone (GH)-Deficient Adults with Osteoporosis Receiving Long-Term Recombinant Human GH Replacement Therapy: A Randomized Controlled Trial J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3079 - 3085. [Abstract] [Full Text] [PDF]

				P. A. Price, S. A. Faus, and M. K. Williamson Bisphosphonates Alendronate and Ibandronate Inhibit Artery Calcification at Doses Comparable to Those That Inhibit Bone Resorption Arterioscler Thromb Vasc Biol, May 1, 2001; 21(5): 817 - 824. [Abstract] [Full Text] [PDF]

				G. G. Reinholz, B. Getz, L. Pederson, E. S. Sanders, M. Subramaniam, J. N. Ingle, and T. C. Spelsberg Bisphosphonates Directly Regulate Cell Proliferation, Differentiation, and Gene Expression in Human Osteoblasts Cancer Res., November 1, 2000; 60(21): 6001 - 6007. [Abstract] [Full Text]

				D. M. Nierman and J. I. Mechanick Biochemical Response to Treatment of Bone Hyperresorption in Chronically Critically Ill Patients Chest, September 1, 2000; 118(3): 761 - 766. [Abstract] [Full Text] [PDF]

				J. Hunziker, T. Wronski, and S.C. Miller Mandibular Bone Formation Rates in Aged Ovariectomized Rats Treated with Anti-resorptive Agents Alone and in Combination with Intermittent Parathyroid Hormone Journal of Dental Research, June 1, 2000; 79(6): 1431 - 1438. [Abstract] [PDF]

				H. Plotkin, F. Rauch, N. J. Bishop, K. Montpetit, J. Ruck-Gibis, R. Travers, and F. H. Glorieux Pamidronate Treatment of Severe Osteogenesis Imperfecta in Children under 3 Years of Age J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 1846 - 1850. [Abstract] [Full Text]

				S. C. Manolagas Birth and Death of Bone Cells: Basic Regulatory Mechanisms and Implications for the Pathogenesis and Treatment of Osteoporosis Endocr. Rev., April 1, 2000; 21(2): 115 - 137. [Abstract] [Full Text]

				P. Ravn, S. R. Weiss, J. A. Rodriguez-Portales, M. R. McClung, R. D. Wasnich, N. L. Gilchrist, P. Sambrook, I. Fogelman, D. Krupa, A. J. Yates, et al. Alendronate in Early Postmenopausal Women: Effects on Bone Mass during Long-Term Treatment and after Withdrawal J. Clin. Endocrinol. Metab., April 1, 2000; 85(4): 1492 - 1497. [Abstract] [Full Text]

				B. E. Hillner, J. N. Ingle, J. R. Berenson, N. A. Janjan, K. S. Albain, A. Lipton, G. Yee, J. S. Biermann, R. T. Chlebowski, and D. G. Pfister American Society of Clinical Oncology Guideline on the Role of Bisphosphonates in Breast Cancer J. Clin. Oncol., March 13, 2000; 18(6): 1378 - 1391. [Abstract] [Full Text] [PDF]

				P. Ravn, M. Bidstrup, R. D. Wasnich, J. W. Davis, M. R. McClung, A. Balske, C. Coupland, O. Sahota, A. Kaur, M. Daley, et al. Alendronate and Estrogen-Progestin in the Long-Term Prevention of Bone Loss: Four-Year Results from the Early Postmenopausal Intervention Cohort Study: A Randomized, Controlled Trial Ann Intern Med, December 21, 1999; 131(12): 935 - 942. [Abstract] [Full Text] [PDF]

				A. A. Reszka, J. M. Halasy-Nagy, P. J. Masarachia, and G. A. Rodan Bisphosphonates Act Directly on the Osteoclast to Induce Caspase Cleavage of Mst1 Kinase during Apoptosis. A LINK BETWEEN INHIBITION OF THE MEVALONATE PATHWAY AND REGULATION OF AN APOPTOSIS-PROMOTING KINASE J. Biol. Chem., December 3, 1999; 274(49): 34967 - 34973. [Abstract] [Full Text] [PDF]

				H. L. Benford, J. C. Frith, S. Auriola, J. Mönkkönen, and M. J. Rogers Farnesol and Geranylgeraniol Prevent Activation of Caspases by Aminobisphosphonates: Biochemical Evidence for Two Distinct Pharmacological Classes of Bisphosphonate Drugs Mol. Pharmacol., July 1, 1999; 56(1): 131 - 140. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints, Permissions and Rights Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fleisch, H. Search for Related Content PubMed PubMed Citation Articles by Fleisch, H.