Diphosphonates: History and mechanisms of action

Metab.

Bone

Dis. 8 Rel. Res. 4 8 5, 279-288

@ 1981 Pergamon Press Ltd and S.N.P.M.D.

(1981)

H; FLEISCH. Department

of Pathophysiology,

University

of Berne,

Murtensfrasse

Abstract The history of dlphosphonajes began wf#i studies of inorgmlc ~yEophosPh*. This compound was iound to occur in many bio#ogicaf fiufds and Inhibi4d the precipitation of caicium p-hates. it &so siowed the tram of amot$hous caiciuni phosphate to its c-tine form, and +hibited crystql aggregation and dissoiutfon. These observations. suggested that it might be a compoMd of ~hysiologicai or. patho~hysiofogicaf significance, perhaps in, hypopho+ phatasia and in renai iithiiasfs. Diphosphonates are compounds where the P-O&P bond of PyrePhosPhate is @aced by a P-C-P bond. Many difioqhonates have been ryntheeind and teated and:aome reia tionship of their structure to the spectrum of bidogicai effects has been obeerved. These analagues have similar ProPHues to -hate, buMniike pyrophosphate fhey are reststant to emzymlc degradation. Their eetai propeHies have led tq.their ciinicai~asbonescanningagent@andinthe treatment of disorders of ectopic mineralization and increased bone resorption, Key Words : Pyrophosphate - Diphosphonates - Mineralization - Bone resorption - Crystal formation Growth and aggregation.

Intro&&Ion The development of the diphosphonates is a good example of the fruitfulness of an interdisciplinary approach, and the interdependance of the so-called u basic w and u applied m research. It shows how an approach, which might at a first glance appear rather far from biological, can lead to the development of a new physiological and pathophysiological concept and to the synthesis of a new class of drugs.

35, 3010 Berne,

Switzerlarrd.

ned why bone collagen mineralizes, it did not explain why soft tissue collagen does not. Indeed, collagen with nucleating activity had been extracted both from mineralizing and non-mineralizing tissues. The old question of u why do certain tissues mineralize ? * had therefore become Q why do ail collagenous tissues not mineralize ? * In 1960, while visiting the laboratory of Dr. W.F. Neuman in Rochester for a postdoctoral year, we investigated the possibility that inhibitors of calcification might exist which would be destroyed locally at the site of mineralization. We found that plasma did indeed contain inhibitory activity against calcium phosphate precipitation and that part of this activity could be destroyed by alkaline phosphatase (Fleisch and Neuman, 1961). Among the possible phosphate compounds which could ,be responsible for this inhibition, polyphosphates, which are characterized by a P-O-P- bond, were good candidates. They had been known for a long time to inhibit the precipitation of calcium carbonate (Biihrer and Reitemeyer, 1940 ; Reitemeyer and Biihrer, 1940) and had been used therefore, to ,prevent scaling in water installations. We found that they indeed possessed a very powerful inhibitory activity on calcium phosphate precipitation at concentrations as low as 1O-6 M (Fleisch and Neuman, 1961). Later, in collaboration with Dr. Bisaz, we were able to verify the original hypothesis and elucidate in part the nature and the role of this inhibitory activity. We found that urine also had inhibitory activity, and in much higher amounts than plasma (Fleisch and Bisaz, 1962). From urine we isolated one of the inhibitors present which proved to be inorganic pyrophosphate (Fleisch and Bisaz, X162), OH OIH

Pyrophoephate and poiyphosphate The possibility that diphosphonates influenced calcium metabolism originated from ear,lier work on inorganic pyrophosphate and polyphosphates. In the early sixties the concept was forwarded that the organic matrix, particularly collagen, played an important role in mineralization by inducing the precipitation of calcium phosphate from the supersaturated extracellular fluid (Neuman and Neuman, 1959 ; Glimcher, 1959). This induction was thought to take place by heterogenous nucleation at specific sites of the collagen molecule. Although this theory explai-

O=P-0 OH

-P=O

inorganic pyrophosphate

I OH

a compound which had not been previously identified in biological fluids. We subsequently found that pyrophosphate (PPi) was present in plasma (Fleisch and Bisaz, 1962 ; Russell et al., 1971), saliva (Hausmann et al., 1970) and synovial fluid (Russell et al., 1970).

280

H. Fleisch : Diphosphonates : History and Mechanisms of Action

A more thorough investigation of the physico-chemical action of pyrophosphate showed that not only did it inhibit crystallization of calcium phosphate from solution, but that it also slowed the transformation of amorphous calcium phosphate to its crystallized form (Fleisch et al., 1966) and inhibited the process of aggregation of calcium phosphate crystals into larger clusters (Hansen et al., 1976). Finally, pyrophosphate a,lso inhibited the dissolution of hydroxyapatite crystals (Fleisch et al., 1966). All these effects are probably related to the high affinity of this compound for hydroxyapatite (Jung et al., 1973). In a further series of studies it was found that PPi also inhibited calcification in living tissue. Thus, it prevented calcification of chick embryo femurs incubated in tissue culture (Fleisch et al., 1966) and, when given subcutaneously to rats, inhibited aortic and kidney calcification induced by large doses of vitamin D (F,leisch et al., 1965 ; Schibler et al., 1968) and skin calcification induced by dihydrotachysterol and other means (Gabbiani, 1966 ; Schibler and Fleisch, 1966). These effects were not obtained when the compound was administered orally. Interestingly, no effect was found either on calcification or on resorption of bone. The development of quantitative techniques for measuring ,pyro:phosphate showed that its concentration in biological fluids was high ,enough to be effective on cal,cium phosphate crystals in viva, since the levels in urine are between 10 and 80 pM (Fleisch and Bisaz, 1963), in plasma between 1 and 6 r*M (Russell et al., 1971), in saliva between 0.1 and 1 pM (Hausmann et al., 1970), and between 2 and 7 pM in articular fluid (Russell et al., 1970). These results suggested that pyrophosphate could have both physiological and pathophysiological significance (Fleisch, 1964 ; Fleisch et al., 1966). It might protect soft tissues from mineralization ; and in bone might influence the rate of calcification as well as the rate of dissolution of preformed mineral. The regulation of ‘local concentrations could be performed by enzymes such as alkaline phosphatase and lysosomal acid phosphatase, which both possess pyrophosphatase activity. Circumstantial evidence for this theory was obtained later by Dr. Russell in his studies of the disease hypophosphatasia. In this condition, which is caracterized by a deficiency of alkaline phosphatase and a failure of bone to mineralize normally, pyrophosphate is increased both in plasma (Russell et al., 1971) and urine (Russell, 1965). Pyrophosphate might also have an important role in urine. Thus, this compound also prevents precipitation (Fleisch and Bisaz, 1964) and aggregation (Robertson et al., 1973) of calcium oxalate, and this at concentrations found in urine, so that it could be an inhibitor of stone formation. Since the urinary excretion of pyrophosphate is increased by the oral administration of orthophosphate (Fleisch et al., 1964 ; Russell et al., 1964), it is possible that the beneficial effects of phosphate administration reported in recurrent calcium stone formers is due to the increased excretion of pyrophosphate. The question arose whether these various {properties of ,pyrophosphate could be exploited in diseases characterized by ectopic calcification or increased

bone resor’ption. The failure of ‘pyrophosphate to act and its failure to inhibit #bone resorption ‘made such an application unlikely. Since this lack of effect is probably due to its rapid hydrolysis in vivo o;ne possibility was to find analogues of pyrophosphate, which would have similar actions, but be resistant to enzymatic breakdown. in vivo when given orally,

Diphosphonates In 1966 I was invited by Dr. Francis to give a lecture at the Procter and Gamble Co. in Cincinnati. This company had had a long standing interest in the dental field and Dr. Francis had been working with analogues of pyrophosphate, the diphosphonates, in order to develop a topical agent for use against dental calculus. The diphosphonates resemble polyphosphates but have a P-C-P bond instead of the P-O-P. He had found that one such diphosphonate, ethane1-hydroxy-l,l-diphosphonate (EHDP) OH I 0 = PI OH

CH3 I cI OH

OH P=O I OH

EHDP

was a powerful inhibitor of calcium phosphate crystallization. This visit led to a collaboration with the Procter and Gamble now more than 15 years, to investigate the biological effects and possible use of diphosphonates in diseases of calcium metabolism. The first phase, done in collaboration with Dr. Russe,ll who was working in our laboratory at this time, was to confirm that the diphosphonates had physico-chemica1 effects similar to pyrophosphate. This proved to be the case and we could confirm Dr. Francis’ results using different techniques. In addition, we found that the diphosphonates also inhibited calcium phosphate dissolution. At the same time we investigated their possible biological effects and in 1968 (Fleisch et al., 1968) we reported our first results : these compounds were able to prevent ectopic calcification. In addition and in contrast to pyrophosphate, they also inhibited bone resorption. Furthermore, unlike pyrophosphate, diphosphonates were active when given either parenterally or orally. The way to their clinical use was set.

Physico-chemical

effects

The diphosphonates inhibit the ,preci,pitation of cal8ciu.m‘phosph’ate from clear solutions CFleisch et al., 1970) and when ,precipitation is induced by various means (Meyer and Nancollas, 1973 ; Boskey et al., 1979), they block the transformation of amorphous calcium phosphate into hydroxyapatite (Francis, 1969 ; Francis et al., 1969) and delay the aggregation of apatite crystals into larger clusters (Hansen et al., 1976). They also disaggregate apatite crystal clusters l(Bisaz et al., 1976) and transform the crystals into a colloidal state, a phenomenon called peptiz&ion (Robertson et al., 1972). Furthermore, like pyrophosphate, diphosphonates slow down the dissolution of crystals (Fleisch et al., 1969 ; Russell et al., 1970 ; Evans et al., 1980). All these effects seem to be related to the marked affinity of the diphosphonates for hydroxyapatite (Jung et al., 1973).

281

H. Ffeisch : Diphosphonates : History and Mechanisms of A&ion

In addition to the effects on calcium phosphate, the diphosphonates also inhibit the formation (Fraser et al., 1972 ; Meyer et al., 1977) and aggregation (Robertson et al., 1973 ; Felix et al., 1977) of calcium oxalate crystals.

those on cartilage. In children, therefore, lack of X-ray changes at the epiphyseal plate during treatment with EHDP does not necessarily mean that the drug has not affected bone mineralization. Apart from bone and cartilage, the calcification of dentine is also inhibited (Larsson, 1974).

Effect on soft tissue calcification

No marked correlation is seen between the effects of di,phosphonates on crystallization in vitro and the inhibition of hard tissue calcification. Although all compounds tested which ehowed an action in vivo also inhibited crystal growth in vitro, certain other compounds were observed to be good inhi,bitors in vitro, but not active in Oivo (Trechsel et al., dichloromethyl,ene Ndiphosphonate 1977). Thus, (ClzMDP)

Like pyrophosphate, diphosphonates very efficiently inhibit experimental soft tissue calcification. Thus they prevent aortic and renal calcification induced by large amounts of vitamin Ds (Fleisch et al., 1970) and other types of ectopic calcification (Casey et al., 1972 ; Francis et al., 1972 ; Rayssiguier et al., 1973 ; Hollander et al., 1974 ; Rosenblum et al., 1975 ; Russell et al., 1975 ; Rosenblum et al., 1977 ; Matthews et al., 1978 ; Potokar and Schmidt-Dunker, 1978) including experimentat urinary stones (Fraser et al., 1972). In the arteries, the accumulation of cholesterol, elastin and collagen is also inhibited under certain conditions (Hollander et al., 1978 ; Kramsch and Chan, 1978 ; Hollander et al., 1979) by mechanisms which are not known. Finally, topical administration a,lso diminishes the formation of dental calculus (Muhlemann et al., 1970 ; Briner et al., 1971). There is a close relationship between the ability of individual diphosphonates to isnhilbit crystal growth in vitro #and their effect in vivo (Fleisch et al., 1970), suggesting that the latter is explicable by a physicochemical mechanism. The most effective compounds are characterized by the P-C-P bond, structures containing C-P or P-C-C-P bonds being less effective or ineffective. Conversely, under certain conditions and with very high doses, diphosphonates can themselves induce mineralization in specific locations such as the injection sites, and in the stomach or the kidney (Larsson and Rohlin, 1980). The nature of the mineral deposited is however not yet clear.

Effect on calcification of hard tissues Diphosphonates not only inhibit ectopic calcification, but also, under certain conditions, prevent the normal mineralization of hard tissues. Thus, in growing animals certain diphosphonates, including EHDP, can inhisbit the mineralization of cartilage and bone (Jowsey et al., 1970 ; King et al., 1971 ; Russell et al., 1973 ; Schenk et al., 1973 ; Rosenblum, 1974 ; Larsson and Larsson, 1976). The doses required vary according to the species, the length of treatment, and the route of administration. Interestingly, the inhibition seems to be reduced by the administration of calcitonin (Boris et al., 1979). In growing anima,ls, the X-rays of the skeletal lesions resemble rickets caused by vitamin D deficiency, with a widening of the epiphyseal plate (Schenk et al., 1973). Histologically, however, the two conditions differ fundamentally, at least in the chick, since in vitamin D deficiency the epiphyseal plate is comprised mainly of proliferating cells, whereas after EHDP it consists mainly of hypertrophic cells (Bisaz et al., 1975). Inhibitory effects on bone occur at lower doses than

OH

Cl

I

I

I

cI Cl

P=O I OH

0 = PI OH

OH

. ClzMDP

despite its strong activity on crystal growth and on soft tissue calcification (Fleisch et al., 1970), has only limited interchange activity towards bone mineralization, the minimal effective dose being more than 10 times above that of EHDP (Schenk et al., 1973). One possible explanation for this discrepancy is that EHDP and ClzMDP have a different distribution in the body. Another possibility is that diphosphonates act by mechanisms other than crystal growth, for example by an effect on the state of aggregation of proteoglycans, as suggested by Howell and Pita (1977).

Effect on bone resorption In contrast to pyrophosphate, diphosphonates are extremely active in inhibiting bone resorption. In CL& ture they decrease both endogenous resorption of bone as well as resorption induced by various agents (Fleisch et al., 1969 ; Russell et al., 1970 ; Hausmann et al., 1972 ; Reynolds et al., 1972 ; Minkin et al., 1974 ; Rowe and Hausmann, 1976 ; Howell and Pita, 1977 ; Galasko et al., 1980 ; Gebauer and Fleisch, 1980). Of al.1 the compounds tested ClzMDP is the most potent. Powerful effects are also seen in vivo. Thus when given to growing rats many of the diphosphonates arrest remodelling in the metaphysis so that it becomes club-shaped and radiologically more dense than normal (Schenk et al., 1973). It appears that not only is the degradation of bone inhibited but so too is that of cartilage. The effect appears to be accompanied by a decrease in vascular invasion (Larsson and Larsson, 1979). The effect of diphosphonates is especially apparent in newborn mice, where ChMDP, given in large doses, impairs normal bone remodetling to such an extent that the bones resemble those of the greylethal strain of congenital osteopetrotic mice, and the animals develop symptoms akin to those of this disease (Reynolds et al., 1973).

H. Fleisch

282

An inhibition of bone resorption is also seen when resorption is measured by ‘%a kinetics (Gasser et al., 1972) urinary excretion of hydroxyproline (Gasser et al., 1972 ; Goulding and McChesney, 1977 ; Reitsma et al., 1980) and by other techniques (Lemkes et al., 1978 ; Reitsma et al., 1980). The decrease in resorption is sometimes accompanied by an increase in calcium ,balance (Gasser et al., 1972 ; Reitsma et al., 1980) and mineral content of bone (Michael et al., 1971 ; Miihl,bauer et al., 1971 ; Gasser et al., 1972). However, this increase is relatively small, since bone formation decreases almost in parallel with the change in resorption. The main ,effect of the diphosphonates is thus to induce a decrease in bone turnover, a conclusion which is supported by morphological observations (Evans et al., 1979). Diphosphonates also prevent an increase in bone resorption induced by various agents. Thus, they prevent changes in blood calcium induced by PTH (Fleisch et al., 1989 ; Russell et al., 1970) and other hypercalcemic as well as hypocalcemic challenges (Bonjour et al., 1973 ; Yarrington et al., 1978 ; Yarrington et al., 1977 ; Yarrington et al., 1977). ClzMDP and to a lesser degree EHDP also prevents various types of experimental osteoporosis (Cabanela and Jowsey, 1971 ; Michael et al., 1971 ; Miihlbauer et al., 1971 ; Lane and Steinberg, 1973 ; Hahnel et al., 1973 ; Hahnel et al., 1978 ; Black and Jee, 1977). They also inhibit resorption found in experimental renal osteodystrophy (Russell et al., 1975) the peridontal destruction seen in rice rats (Leonard et al., 1979), and the destruction of implants of bones from treated animals (Rosenquist and Baylink, 1978). An interesting exception is that bone loss induced by a low calcium diet is not prevented (Jowsey and Halley, 1973 ; Morgan et al., 1975). A great number of diphosphonates have been investigated for their inhibitory effect of bone resorption. It appears that increasing the chain length of the C-backbone

o=p-

yH , OH

i 7 -

qH Fi -

(CH&

0

long chain drphosphonates X = H, OH

OH

I CHa increases activity until a length of about 9 carbon atoms is reached (Shinoda et al., 1979). .Adding a hydroxyl group at position 1 also increases the effect (Shinoda et al., 1979). The amino-derivatives such as the &amino-1-hydroxypropane-l,l-diphosphonate (AHPDP) OH I O’P

--I” OH

OH OH I I -P=O I fH” F” NH,

OH

AHPDP

: Diphosphonates

: History

and

Mechanisms

of Action

are also very active (Lemkes et al., 1978 ; Shinoda et al., 1979 ; Reitsma et al., 1980). The relative activity of some of the diphosphonates tested is as follows : AHPDP > long chain 1-hydroxy diphosphonates > ClzMDP > EHDP (Shinoda et al., 1979). No correlation has ,been found jbetween activity in viva and the inhi,bition of crystal dissolution in vitro (Shinoda et al., 1979). This lack of relationship suggests that the ‘action on bone resorption is mediated through mechanisms other than the jphysico,chemrcal effects on apatite. Thus an extensive effort has been devoted to investigate possible cellular effects. Such cellular ,effects are suggested by the observations that diphosphonates alter the morphology of the osteoclasts, both in culture (Rowe and Hausm,ann, 1978) and when administered in vivo (Schenk et al., 1973 ; Miller and.Jee, 1975 ; Miller et al., 1977 ; Miller and Jee, 1979 ; Plastnans et al., 1980). Biochemical effects In recent years numerous biochemical effects have ibeen, described. ,However, th,eh relevance to the activity of the diphosphonates in vivo ,is still unclear. Effects of diphosphonates on the formation of cAMlP have been, described (Pilczyk et al., 1972 ; Eisman et al., 1974 ; Goulding et al., 1976 ; Plasmans et al., 1980) ,but does not correlate with their activity on bone resorption (Gebauer et al., 1978). Diphosphon+tes, particularly C1zM.D.Pinhibit various lysosomal enzymes in vitro, including acid ,phosphatase and pyrophosphatase (Felix et al., 1978). They also prevent :PTH-induced increases of these enzymes in calvaria culture (Morgan et al., 1973) anmd diminish the acid phosphatase activity of bone cells when given in vivo (Doty et al., 1972 ; SEnde, 1979). EH,DP:and ClrMDP slow down the release of calcium in vitio from kid,ney mitochondria (Guilland et al., 1974 ; Guilland and Fleisch, 1974), an observation which is ,supported by the increase in calcium content of mitochondria in Ibone cells in vivo (Plasmans et al., 1980). ClaMDP, and to a lesser extent EHDP, decrease the rate of glycolysis as dudged by a decrease in glucose consumption and lactic acid production both in cultured calvaria and cartilage cells (Fast et al., 1978 ; Ende, 1979) .and itn who1.e calvaria in culture (Morgan ,ei al., 1973 ; ,Ende, 1979). ,Since lactate (production is generalmly thought to be related to bone resorption, it ‘is tempting to suggest that one way in which the diphosphonates decrease resorption Is by their effect on lactic acid production. However, this explanation can ‘not a,pply to all compounds of this class, since AIHPDP and long chain diphosphon,ates, which are very effective inhibitors of bone resorptlon, have either ,no eff,ect, or even mcrease lactic acid #production l(End,e, 1979 ; Shinoda et al., 1979). Di,phos,phonates mcrease fatty acid oxidation (Felix and Fleisch, 1979) and amino acid oxidation (Ende, 19$9), and stimulate the citric acid cycle (Ende, 1979). ‘EHDP arrd ClzMDP also increase the cellular content of glycogen in vitro (Felix et al., 1980) and ClaMDP increases the production oaf al,kal,ine phosphatase in bone cells ,more than 30 fold (Felix and ‘Fleisch, 1979). The latt,er effect has also ,been observed in vivo (Weisbrode et al., 1978). Imnterestingly, EHDP is almost inactive in this respect. ClzMDP but

283

H. Fleisch : Diphosphonates : History and Mechanisms of Action

not EHDP also increases the biosynthesis of bone and cartilage collagen, both when injected into animals or when added to isolated cells in culture (Guenther et al., 1981 ; Guenther et al., 1981). ClzMDP also stimulates the biosynthesis of proteoglycans by isolated chondrocytes (Guenther et al., 1979) but a decrease in glycosaminoglycan synthesis has been observed under certain conditions (Larsson, 1976). Recently ChMDP, and to a lesser extent EHDP, have been found to inhibit prostaglandin synthesis when added to bone cells in vitro (Felix et al., submitted), whereas AHPDP and long chain diphosphonates stimulate its production. Diphosphonates have been found to have effects on the immune system. Thus methylene diphosphonate decreases the formation of antibody secreting cells in response to immunization and decreases hypersensitivity of delayed and immediate types (Komissarenko et al., 1977). EHDP inhibits the activity of antilymphocyte serum on T lymphocytes (Zemskov et al., 1979). Furthermore certain diphosphonates inhibit the effect in vitro of a mitogen on mononuclear phagocyte function and lymphoblastic response (Bijvoet et al., 1980) and AHPDP induces a transient lymphopenla and changes in acute phase proteins in man (Bijvoet et al., 1980). Whether these results are related to the action on bone resorption or to the possible antiinflammatory effect (Flora, 1979) is uncertain. Finally an antimutagenic activity (Veltischev and Seleznev, 1978), an inhibition of viral DNA polymerase and an inhibition of plaque formation in cell culture have been described (Eriksson et al., 1980). EHDP has been reported to lower plasma cholesterol in man (Caniggia and Gennari, 1977 ; Caniggia et al., 1979). This effect on plasma lipids has, however, not been confirmed by other investigators (Mellies et al., 1979). All these effects suggest that the diphosphonates are taken up (by ceils. This has been confirmed in vitro, in the case of EHDP and ClzMDP (Fast et al., 1978). Indeed the cellular concentration, expressed in terms.of cellular water, can be severalfold higher than in the medium, suggesting that it is accumulated within specific cellular compartments. Cells with phagocytic properties take up the diphosphonates with special avidity if they are‘bound to apatite crystals (Chambers and Path, 1980). This might explain why osteoclasts are target cells for these compounds. In the light of all these results, it appears astonishing that diphosphonates act only on bone and not on other tissues. The answer probably lies in the affinity of these compounds for calcium phosphate crystals (Jung et al., 1973) which causes them to be cleared very rapidly from blood and incorporated into bone. Indeed, the half-life in the circulation is in the order of minutes, the rate constant of entry into bone being similar to that of calcium or phosphate (Bisaz et al., 1978). Soft tissues will therefore be exposed to the compounds for only short periods. In bone, however, the concentration of the diphosphonates is much higher, especially at the sites of resorption, when the apatite crystals are dissolved and the diphosphonates released. The compounds may at these locations be taken up by bone resorbing cells either as free compounds or bound onto the apatite crystals (Felix et al., submitted), thereafter exerting their cel-

lular effects. It appears likely that the P-C-P part of the molecule gives the diphosphonate the affinity for mineralized tissues, whereas the specific activity will depend on the molecule as a whole.

Other effects of diphosphonates A number of other effects of diphosphonates have been described. Some of these are probably not direct effects but secondary to their action on bone. Thus, large doses of EHDP decrease the intestinal absorption of calcium (Gasser et al., 1972 ; Bonjour et al., 1973) because of a decrease in the formation of 1,2!%dihydroxycholecalciferol (1,25(OH)zD3). Lower doses of EHDP on the contrary lead to an increase in 1,25(OH)zD3 (Guilland et al., 1975). It is likely that the decrease in 1,25(OH)zD3 production with high doses is due to an indirect homeostatic mechanism caused by the block of mineralization. Thus in renal cell culture, EHDP is ineffective in decreasing the 1-alpha-hydroxylase (Trechsel et al., 1979). The inhibition of mineralization by EHDP is not, however, due to a decrease of the vitamin D metabolite, since it precedes this decrease, has a different histological appearance than D-deficient rickets (Bisaz et al., 1975), and is not (Bonjour et al., 1975) or only partially (Boris et al., 1978 ; Baxter et al., 1979) reversed by 1,25(OH)zD3. Another effect in the rat, which is probably also indirect, is the ability of EHDP to increase the capacity of the kidney to excrete phosphate (Bonjour et al., 1978). Since the kidney has been shown to adapt very efficiently to the needs of the organism, this effect of EHDP could be another homeostatic regulatory response. The effect in the rat is in contrast to that seen in humans, where EHDP induces a rise in plasma phosphate accompanied by an increase in renal reabsorption of phosphate (Reeker et al., 1973).

Metabolism While certain monophosphonates are found in animals and man, diphosphonates have not yet been shown to occur naturally nor have any enzymes capable of cleaving the P-C-P bond yet been described. As far as we know diphosphonates are absorbed, stored, and excreted unchanged in mammals. Most of the pharmacokinetic data on diphosphonates have been collected with EHDP. Its absorption is very variable both between and within species and lies between i and 10 O/O of an oral dose. It seems generally somewhat higher in the young (Michael et al., 1972 ; Reeker and Saville, 1973). Absorption occurs partly in the small intestine (Wasserman et al., 1973) and depends on the size of the diphosphonate. Absorption is lower for compounds of large molecular weight and some of the larger diphosphonates are virtually non-absorbable (Anbar et al., 1973). Approximately half of the EHDP absorbed enters bone, and half is excreted in the urine (Michael et al., 1972). The renal clearance of EHDP and ClzMDP can exceed that of inulin, indicating the existence of a secretory pathway (Troehler et al., 1975). The retained EHDP is hearly all found in bone, only very small amounts are found in soft tissues (Michael et al., 1972). The half-time for bone retention will depend on the turnover rate of the skeleton.

284

H. Fleisch

: Diphosphonates

: History

and

Mechanisms

of Action

Conclusion

destruction. Furthermore, their affinity for apatite has made it possible to bring other compounds, such as 99mT~ to calcified tissues by linking them to the diphosphonates. It would not be surprising if the first compounds tested are by no means optimal and that a further exploration of other types of diphosphonates could lead to a fruitful future development of these novel compounds.

The diphosphonates are very potent inhibitors of mineralization and bone resorption. These characteristics have opened the way to the clinical use of these compounds in disorders of mineral metabolism such as ectopic calcification and increased bone

Acknowledgments : The work reported in this review has been carried out with the support of the Swiss National Science Foundation (grant 3.324.79), the Procter and Gamble Company, Cincinnati, U.S.A., and the Ausbildungsund Forderungsfonds der Arbeits gemeinschaft fur Osteosynthese (AO), Chur, Switzerland.

Little is known of the distrisbution of other diphosphonates. More attention will have to be directed to this problem, since it is possible that their pharmacokinetics vary, a fact which could well affect their biological activity.

References Anbar M.. Newell G.W. and St. John G.A. : Fate and toxicity of orally administered polyethylene polyphosphonates. Fed. Cosmet Toxicd. 11 : 1001-1010, 1973. Baxter L.A., DeLuca H.F., Bonjour J.-P. and Fleisch H. : Inhibition of vitamin D metabolism by ethane-l-hydroxy-l,l-diphosphonate. Arch. Biochem. Biophys. 164 : 655-662, 1974. Baxter L.A., Canty D.J., Bednar G.J., Stern L., DeLuca H.F., Ginn D.L., Flora L. and Hassing G.S. : Effect of ethane-lhydroxy-l,l-diphosphonate and vitamin D on bone mineralization. Calcif. Tissue Int. 26 : 73-76, 1979. Bijvoet O.L.M., Frijlink W.B., Jie K., van der Linden H., Meijer C.J.L.M., Mulder H., van Paassen H.C., Reitsma P.H., teVelde J., deVries E. and van der Wey J.P. : APD in Paget’s disease of bone : role of the mononuclear phagocyte system ? Arthritis Rheum. 23 : 1193-1204, 1960. Bisaz S., Jung A. and Fleisch H. : Uptake by bone of pyrophosphate, diphosphonates and their technetium derivatives. Clin.

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H. Fleisch

: Diphosphonates

: History and Mechanisms

of Action

Felix R., Fast D.K., Sallis J.D. and Fleisch H. : Effect of diphosphonate on glycogen content of rabbit ear cartilage cells in culture. Cakif. Tissue Int. 30 : 183-166, 1980. Felix R. and Fleisch H. : Increase in fatty acid oxidation in calvaria cells cultured with diphosphonates. Biochem. J. 196 : 237-245, 1981. Felix R., Bettex J.D. and Fleisch H. : Effect of diphosphonates on the synthesis of prostaglandins in cultured caivaria Cells. Calc. Tbs. Int., in press. Fleisch H. and Neuman W.F. : Mechanisms Of calcification : role of collagen, polyphosphates, and phosphatase. Am. J. Physiol. 200 : 1296-1300, 1961. Fleisch H. and Bisaz S. : Isolation from urine of pyrophosphate, a calcification inhibitor. Am. J. Physiol. 203 : 671875, 1962. Fleisch H. and Bisaz S. : Mechanism of calcification : inhibitory role of pyrophosphate. Nature 195 : 911, 1962. Fleisch H. and Bisaz S : Die Pyrophosphatausscheidung im Harn beim gesunden Menschen. Helv. Physiof. PharmaCd. Acta 21 : 88-94, 1963. Fleisch H. : Role of nucleation and inhibition in calcification. C/in. O&O/J. 32 : 170-180, 1964. Fleisch H. and Bisaz S. : The inhibitory effect of pyrophosphate on calcium oxalate precipitation and its relation to urolithiasis. fxperientia 29 : 276, 1964. Fleisch H., Bisaz S. and Care A.D. : Effect of orthophosphate on urinary pyrophosphate excretion and the prevention of urolithiasis. Lancet i : 1065-1067, 1964. Fleisch H., Schibler D., Maerki J. and Frossard 1. : Inhibition of aortic calcification by means of pyrophosphate and polyphosphates. Nature 207 : 1300-1301, 1965. Fleisch H., Maerki J. and Russell R.G.G. : Effect of pyrophosphate on dissolution of hydroxyapatite and its possible importance in calcium homeostasis. Proc. SOC. Exp. Bio/. Med. 122 : 317-320, 1966. Fleisch H.. Straumann F., Schenk R., Bisaz S. and Allgijwer M. : Effect of condensed phosphates on calcification of chick embryo femurs in tissue culture. Am. J. Physiol. 211 : 821825, 1966. Fleisch H., Russell R.G.G. and Straumann F. : Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis. Nature 212 : 901-903, 1966. Fleisch H., Russell R.G.G., Bisaz S., Termine J.D. and Posner A.S. : Influence of pyrophosphate on the transformation of amorphous to crystalline calcium phosphate. Calcif. Tissue Res. 2 : 49-59, 1968. Fleisch H., Russell R.G.G., Bisaz S., Casey P.A. and Miihlbauer R. : The influence of pyrophosphate analogues (diphosphonates) on the precipitation and dissolution of calcium phosphate in vitro and in vivo. Calcif. Tissue Res. 2 : Suppl. IO-lOA, 1968. Fleisch H., Russell R.G.G. and Francis M.D. : Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorptiOn in tissue culture and in viva. Science 165 : 1282-1264, 1969. Fleisch H., Russell R.G.G., Bisaz S., Miihlbauer R.C. and Williams D.A. : The inhibitory effect of phosphonates on the formation of calcium phosphate crystals in vitro and on aortic and kidney calcification in vivo. Eur. J. C/in. Invest. 1 : 12-18, 1970. Flora L. : Comparative antiinflammatory and bone protective effects of two diphosphonates in adjuvant arthritis. Arthritis Rheum. 22 : 340-346, 1979. Francis M.D. : The inhibition of calcium hydroxyapatite crystal growth by polyphosphates. Calcif. Tissue Res. 3 : X1-162, 1969. Francis M.D., Russell R.G.G. and Fleisch H. : Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vfvo. Science 165 : 1284-1266, 1969. Francis M.D., Flora L.F. and King W.F. : The effects of disodium ethane-1-hydroxy-l,l-diphosphonate on adjuvant induced arthritis in rats. Calcif. Tissue Res. 9 : 109-121, 1972. Fraser D.. Russelsl R.G.G., Pohler 0.. Robertson W.G. and Flelsch H. : The influence of disodium ethane-l-hydroxy-l,ldiphosphonate (EHDP) on the development of experimentally induced urinary stones In rats. C/in. SC/. 42 : 197-207. 1972. Gabbiani G. : Effect of phosphates upon experimental skin calCinOSis. Can. J. Physiol. Pharmacol. 44 : 203-207, 1966.

285

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Hollander W., Prusty S., Nagraj S., Kirkpatrick B., Paddock J. and Colombo M. : Comparative effects of cetaben (PHB) and dichloromethylene diphosphonate (Cl2MDP) on the development of atherosclerosis in the cynomolgus monkey. Atherosclerosis 31 : 307-325, 1978. Hollander W., Paddock J., Nagraj S., Colombo M. and Kirkpatrick B. : Effects of anticalcifying and antifibrobrotic drugs on pre-established atherosclerosis in the rabbit. Atherosclerosis 33 : 111-123, 1979. Howell D.S. and Pita J.C. : Role of proteoglycans in calcification of cartilage. Upsala J. Med. Sci. 82 : 97-98, 1977. Jung A., Bioaz S. and Fleisch H. : The binding of pyrophosphate and two diphosphonates on hydroxyapatite crystals. Calcif. Tissue Res. 11 : 264280, 1973. Jowsey J., Halley K.E. and Linman J.W. : Effect of sodium etidronate in adult cats. J. Lab. C/in. Med. 76 : 126-133, 1970. Jowsey J. and Halley K.E. : Influence of diphosphonates on progress of experimentally induced osteoporosis. J. Lab. Clin. Med. 62 : 567-575, 1973. King W.R., Francis M.D. and Michael W.R. : Effect of disodium ethane-l-hydroxy-l,l-diphosphonate on bone formation. C/in. Orthop. 76 : 251-270, 1971. Komissarenko S.V., Zhuravskii N.I., Karlova N.P. and Guly~ M.F. : Inhibition of hypersensitivity of delayed and immediate types in guinea pigs by methylenediphosphonic acid. Byull. Eksp. Biol. Med. 64 : 1322-1323, 1977. Kramsch D.M. and Ghan CT. : The effect of agents interfering with soft tissue calcification and cell proliferation on calcific fibrous fatty plaques in rabbits. Circ. Res. 42 : 562. 57l( 1970. in Lane J.M. and Steinberg M.E. : The role diphosphonates : osteoporosis of disuse. J. Trauma 13 : 863-869. 1973. Larsson A. : The short-term effects of high doses of ethylene-lhydroxy-l,l-diphosphonates upon early dentin formation. Calcif. Tissue Res. 16 : 109-127, 1974. Larsson A. and Larsson ‘S.E. : Light microscopic and ultrastructural observations on the short-term effects of ethglene-1-hydroxy-l,l-diphosphonate (EHDP) on iat tibia epiphysis. Acts Path. hffcrobfof. Scan& 64 : 17-27, 1976. Larsson S.E. : The metabolic heterogeneity of glycosaminoglycans of the different zones of the epiphyseal growth plate and the effect of ethanedl-hydroxy-l,l+phosphonat6 (EHDP) upon glycosaminoglycefi synihe6is in vi@% CalcM. Tissue Res. 21 : W-02, 1976. Larsson A. and Larsson SE. : The effects of ethane-1-hydroxy-1, I-diphosphonate on cellular traqsformation and organic m;l trix’ of the epiphy&%l growth ,plate of the rat : a light microsc#c lnd ultrastructural study. Acta Patho/. Microbid. Stand. 86 : 211-223, 1978. Larsson A. and Rohlin M. : In v&o distributio? of 14C-labelled ethylene-I-hydroxy-l,l-diphosphonate in normel and treated y&ii9 rats. Ah autoradiographic and ultrastructural study. Toxicd. Appl. Pharmacol. 62 : 391-3QQ. 1980. Lemkes H.H.P.J., Reitsma P.H., Frijlink W., Verlinden-Ooms H. and Bijvoet O.L.M. : A new diphosphonate : dissociation between effects on cells and mineral in rats and a preliminary trial in Paget’s disease. Adv. exp. Med. B/o/. 163 : 459-469, 1976. Leonard E.P., Reese W.V. and Mandel E.J. : Comparison of the effects of ethane-1-hydroxy-l,l-diphosphonate and dichloromethane diphosphonate upon periodontal bone resorption in rice rats. Arch. Oral Biol. 24 : 707-708, 1979. Matthews J.L.. Martin J.H. and Cal’Son F.L. : Ultrastructure of calciphylaxis in skin. Metab. Bone Dis. and Rel. Res. 1 : 219226, 1978. Mellies M.J., Boggs D.. VanDussey B., Michael R.S., Clarke Ficken G., Radcliff R. and Glueck C.J. : Effects of etidronate disodium (EHDP) on plasma lipids and lipoproteins in familial endogenous hypertriglyceridemia. Artery 6 : 38-49. 1979. Meyer J.L. and Nanoollas G.H. : The influence of multidentale organic phosphonates on the crystal growth of hydroxyapatite. Caicif. Tissue Res. 10 : 295-303, 1973. Meyer J.L., Lee K.E. and Eergert J.H. : The inhibition of calcium oxalate crystal growth by multidentate organic phosphonates. Calcif. Tissue Res. 23 : 63-66. 1977.

: Diphosphonates

: History

and

Mechanisms

of Action

Michael W.R., King W.R. and Francis M.D. : Effectiveness of diphosphonates in preventing * osteoporosis * of diguse 1971. in the rat. C/in. Orthop. Rel. Res. 73 : 271476, Michael W.R., King W.R. and Wakim J.M. : Metabolism of disodium ethane-1-hydroxy-l,l-diphosphonate (disodium etidrQnate) in the rat, rabbit, dog and monkey. Toxic Appl. Phermacol. 21 : 503-515, 1972. Miller S.C. and Jee W.S.S. : Ethane-l-hydroxy-i,l-diphosphonate (EHDP) : Effects on growth and modeling of the rat tibia. Calcif. Tissue Res. la: 215-231, 1975. Miller SC., Jee W.S.S., Kimmel D.B. and Woodbury L. : EthaneI-hydroxy-I,+diphosphonata (EHDP) effects on incorporation a?d accumulation of osteoclast nuclei. Calcif. Tissue Res. 22 : 243-252, 1977. Miller SC. aid Jee W.S.S. : The effect of dichloromethylene diphosphonate, a pyrophosphate analog, on bone and bone cell structure in the growing rat. Anat. Rec. lQ3 : 439-482, 1979. Minkin C., Rabadjija L. and Goldhaber P. : Bone remodelling in vitro : the effect of tJIo diphosphonates in osteoid syqthesis and bone resorption in mouse chlvaria. Calcif, Tissue Res. 14 : 161-168, 1974. Morgan D.B., Monod A., Russell R.G.G. and Fleisch H. : lni( fluence of dichloromethylene diphosphonate (CI,MDP) and calcitonin on bone resorption, lactate production and phoephatase and pyrophosphatase content of mouse caivarla treated With parathyroid hormone in vitro. Calclf. Tissue Res. 13 : 287-294, 1973. Morgan D.B., Gasser A., Largiadbr U., Jung A. and Fleisch H. : Effects Of a diphosphonate on calcium metabolism in calcium-deprived rats. Am. J. Physibl. 226 : 1750-1756, 1976. MShlbauer R.C., Russell R.G.G., Williams D.A. and Fleisch H. : The effects of diphosphonates, pQlyphosphates, and calcitonin on (L immobilisation osteoporosis * in rats. Eur. J. Clin. Invest. 1 : 336344,1971. MShlemann H.R., Bowles D., Schatt A. and Bernimoulin J.P. : Effect of diphosphonate: ori human supragingival calculus. /-&Iv. Odont. Acta 14 : 31-33, 1970. Neuman W.F. and Neuman M.W. : The chemical dynamics qf bone mineral. Univ. Chic;lgo Press, Chicago, 1958. Pilczyk R., Sutcliffe H. and Martin’T.J. : effects of pyrophosphate and diphosphonates on parathyroid hormone- and fluoride-stimulated ada?ylate cyclase activity. FEBS Letters P4 : Z&5-228, 1972. Plasmans C.M.T.! Jap P.H.K., Kujipers W. and Slooff T.J.J.H. : Influence of diphosphonate on the cellular aspect of young bone tissue. &t&if. Tissue Inf. 32 : 247-256, 1660. Potokar M. and Schmidt-Dunker M. : The. inhibitory effect of new diphosphonic acids on Bortic kidney calcification in vivo. Atherosolerosis 30 : 313-320, 1978. Rayssiguier Y., Larvor P. and Barlet J.P. : Etude de I’lnfluence d’un phosphonate de sodium sur le taux r&ml de calcium chez le rat carenc6 en magn&ium. C.I?. AC&Y.’SC/. Par/s 276 : 2035-2038, 1973. Reeker RR., Hassing G.S., Lau J.R. and Saville P.D. : The hyperphosphatemic effect of disodium ethane-l-hydroxy-l,l-dlphosphonates (EHDPTM) : renal handling of phosphorus and the renal response to parathyroid hormone. J. Lab. CM. Med. 67 : 258-266, 1973. Reeker R.R. and Saville P.D. : Intestinal absorption of disodium etidronate) ethane-1-hydroxy-1,1-diphosphonate (disodium using a deconvolution technique. 70x/c Appl. Pharmacol. 24 : 580-589, 1973. Reitemeyer R. and Biihrer T. : The inhibiting action of minute amounts of sodium hexametaphosphate on the precipitation of calcium carbonate from ammoniacal solutions. J. Phys. Chem. 44 : 535-551, 1940. Reitsma P.H.. Bijvoet O.L.M., Verlinden-Ooms, H. and van der Wee-Pals L.J.A. : Kinetic studies of bone and mineral metabolism during treatment with @-amino-l-hydroxypropylidene)-l,i-bisphosphonate (APD) in rats. Calcif. Tissue Int. 32 : 145.147. 1980. Reynolds J.J., Minkin C., Morgan D.B., Spycher D. and Fleisch H. : The effect of two diphosphonates on the resorption of mouse calvaria in vitro. Calcif. Tissue Res. 10 : 302-313, 1972.

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RESUME L’histoire daa diphoaphonatas a dBbut6 avec lea 6tudea aur lo pyrophoaphata inorganique. Ca compor6 a 6t6 trouvb dana de nombreux liquidea bioiogiquea at inhibe la pr&&pWion dea phoaph&ea de calcium. ii raientit auaai la tranaformstian du phoaphata de caicium emorphe en aa forme criataiiine, et lnhibe i’agrbgatfen et la diaaoktion wiataiknea. Cea obaewationa ont aug&I% qua ie pyrophoaphate inorganisfue pour&t avoir un r8le physiokgiquo ou phyaiopathoiogique aignifkatif, peut4tre dana I’hypophoaphateale et k Bthiaae r&ale. k diphoapkmtea aont dea compoak oit ia Raiaon P-O-P du pyrophoe~ eat rempie&e par une Uaison P-C-P. Da nombreux disatea ant W aynthWa6a et teatie et certainea rektiow entre ieur at~ctura at IYventaii de ieurs effets bioiogiquea aont apparuaa. Cea anaiogues unt dea proprMt&a abniiairea B ceilea du pyrophoaphata. maia contrakement B ce demiar, lie tiaiatent il la d6gredation enxymatique. Cea propriMe exp6rinwWaa ont conduit a ieur dbvaloppement dinique en tant que tracawa oaaeux pour la acintigraphia oaaeuae et en tent qu’agenta Mrapautiques dana la6 caicifh~Hon6 ectopiquaa et lea aituationa pathologiquea corn-t una tiaorption oaaeuaa accrue.