Metab. Bone Dis. 8 Rel. Res.

4 8 5, 279-288 (1981) @ 1981

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

H; FLEISCH.

Department of Pathophysiology, University of Berne, Murtensfrasse 35, 3010 Berne, Switzerlarrd.

Abstract ned why bone collagen mineralizes, it did not explain

why soft tissue collagen does not. Indeed, collagen
The history of dlphosphonajes began wf#i studies of with nucleating activity had been extracted both from
inorgmlc ~yEophosPh*. This compound was iound mineralizing and non-mineralizing tissues. The old
to occur in many bio#ogicaf fiufds and Inhibi4d the question of u why do certain tissues mineralize ? *
precipitation of caicium p-hates. it &so siowed had therefore become Q why do ail collagenous
the tram of amot$hous caiciuni phosphate tissues not mineralize ? *
to its c-tine form, and +hibited crystql aggrega-
tion and dissoiutfon. These observations. suggested In 1960, while visiting the laboratory of Dr. W.F. Neu-
that it might be a compoMd of ~hysiologicai or. pa- man in Rochester for a postdoctoral year, we inves-
tho~hysiofogicaf significance, perhaps in, hypopho+ tigated the possibility that inhibitors of calcification
phatasia and in renai iithiiasfs. Diphosphonates are might exist which would be destroyed locally at the
compounds where the P-O&P bond of PyrePhosPhate site of mineralization. We found that plasma did
is @aced by a P-C-P bond. Many difioqhonates indeed contain inhibitory activity against calcium
have been ryntheeind and teated and:aome reia phosphate precipitation and that part of this activity
tionship of their structure to the spectrum of bidogi- could be destroyed by alkaline phosphatase (Fleisch
cai effects has been obeerved. These analagues have and Neuman, 1961). Among the possible phosphate
similar ProPHues to -hate, buMniike pyro- compounds which could ,be responsible for this inhi-
phosphate fhey are reststant to emzymlc degradation. bition, polyphosphates, which are characterized by
Their eetai propeHies have led tq.their ciini- a P-O-P- bond, were good candidates. They had
cai~asbonescanningagent@andinthe been known for a long time to inhibit the precipi-
treatment of disorders of ectopic mineralization and tation of calcium carbonate (Biihrer and Reitemeyer,
increased bone resorption, 1940 ; Reitemeyer and Biihrer, 1940) and had been
used therefore, to ,prevent scaling in water installa-
Key Words : Pyrophosphate - Diphosphonates - Mi- tions. We found that they indeed possessed a very
neralization - Bone resorption - Crystal formation - powerful inhibitory activity on calcium phosphate
Growth and aggregation. precipitation at concentrations as low as 1O-6 M
(Fleisch and Neuman, 1961).

Later, in collaboration with Dr. Bisaz, we were able

Intro&&Ion to verify the original hypothesis and elucidate in part
The development of the diphosphonates is a good the nature and the role of this inhibitory activity. We
example of the fruitfulness of an interdisciplinary found that urine also had inhibitory activity, and in
approach, and the interdependance of the so-called much higher amounts than plasma (Fleisch and
u basic w and u applied m research. It shows how an Bisaz, 1962). From urine we isolated one of the inhi-
approach, which might at a first glance appear rather bitors present which proved to be inorganic pyro-
far from biological, can lead to the development of a phosphate (Fleisch and Bisaz, X162),
new physiological and pathophysiological concept
and to the synthesis of a new class of drugs.
OH
Pyrophoephate and poiyphosphate OIH
inorganic
The possibility that diphosphonates influenced cal- O=P-0 -P=O pyrophosphate
cium metabolism originated from ear,lier work on
inorganic pyrophosphate and polyphosphates. In the I
early sixties the concept was forwarded that the OH OH
organic matrix, particularly collagen, played an im-
portant role in mineralization by inducing the preci- a compound which had not been previously identi-
pitation of calcium phosphate from the supersatu- fied in biological fluids. We subsequently found that
rated extracellular fluid (Neuman and Neuman, 1959 ; pyrophosphate (PPi) was present in plasma (Fleisch
Glimcher, 1959). This induction was thought to take and Bisaz, 1962 ; Russell et al., 1971), saliva (Haus-
place by heterogenous nucleation at specific sites mann et al., 1970) and synovial fluid (Russell et al.,
of the collagen molecule. Although this theory explai- 1970).
280 H. Fleisch : Diphosphonates : History and Mechanisms of Action

A more thorough investigation of the physico-chemi- bone resor’ption. The failure of ‘pyrophosphate to act
cal action of pyrophosphate showed that not only in vivo when given orally, and its failure to inhibit
did it inhibit crystallization of calcium phosphate #bone resorption ‘made such an application unlikely.
from solution, but that it also slowed the transforma- Since this lack of effect is probably due to its rapid
tion of amorphous calcium phosphate to its crystalli- hydrolysis in vivo o;ne possibility was to find analo-
zed form (Fleisch et al., 1966) and inhibited the pro- gues of pyrophosphate, which would have similar
cess of aggregation of calcium phosphate crystals actions, but be resistant to enzymatic breakdown.
into larger clusters (Hansen et al., 1976). Finally,
pyrophosphate a,lso inhibited the dissolution of hy-
droxyapatite crystals (Fleisch et al., 1966). All these Diphosphonates
effects are probably related to the high affinity of
this compound for hydroxyapatite (Jung et al., 1973). In 1966 I was invited by Dr. Francis to give a lecture
In a further series of studies it was found that PPi at the Procter and Gamble Co. in Cincinnati. This
also inhibited calcification in living tissue. Thus, it company had had a long standing interest in the
prevented calcification of chick embryo femurs incu- dental field and Dr. Francis had been working with
bated in tissue culture (Fleisch et al., 1966) and, when analogues of pyrophosphate, the diphosphonates, in
given subcutaneously to rats, inhibited aortic and order to develop a topical agent for use against den-
kidney calcification induced by large doses of vita- tal calculus. The diphosphonates resemble polyphos-
min D (F,leisch et al., 1965 ; Schibler et al., 1968) and phates but have a P-C-P bond instead of the P-O-P.
skin calcification induced by dihydrotachysterol and He had found that one such diphosphonate, ethane-
other means (Gabbiani, 1966 ; Schibler and Fleisch, 1-hydroxy-l,l-diphosphonate (EHDP)
1966). These effects were not obtained when the
compound was administered orally. Interestingly, no OH CH3 OH
effect was found either on calcification or on resorp- I I
tion of bone. 0 = P- c- P=O EHDP
I I I
The development of quantitative techniques for OH OH OH
measuring ,pyro:phosphate showed that its concen-
tration in biological fluids was high ,enough to be was a powerful inhibitor of calcium phosphate crys-
effective on cal,cium phosphate crystals in viva, since tallization. This visit led to a collaboration with the
the levels in urine are between 10 and 80 pM (Fleisch Procter and Gamble now more than 15 years, to
and Bisaz, 1963), in plasma between 1 and 6 r*M investigate the biological effects and possible use of
(Russell et al., 1971), in saliva between 0.1 and 1 pM diphosphonates in diseases of calcium metabolism.
(Hausmann et al., 1970), and between 2 and 7 pM in The first phase, done in collaboration with Dr. Russe,ll
articular fluid (Russell et al., 1970). who was working in our laboratory at this time, was
to confirm that the diphosphonates had physico-che-
These results suggested that pyrophosphate could mica1 effects similar to pyrophosphate. This proved
have both physiological and pathophysiological si- to be the case and we could confirm Dr. Francis’
gnificance (Fleisch, 1964 ; Fleisch et al., 1966). It results using different techniques. In addition, we
might protect soft tissues from mineralization ; and in found that the diphosphonates also inhibited calcium
bone might influence the rate of calcification as well phosphate dissolution. At the same time we investi-
as the rate of dissolution of preformed mineral. The gated their possible biological effects and in 1968
regulation of ‘local concentrations could be perfor- (Fleisch et al., 1968) we reported our first results :
med by enzymes such as alkaline phosphatase and these compounds were able to prevent ectopic cal-
lysosomal acid phosphatase, which both possess cification. In addition and in contrast to pyrophos-
pyrophosphatase activity. Circumstantial evidence phate, they also inhibited bone resorption. Further-
for this theory was obtained later by Dr. Russell in more, unlike pyrophosphate, diphosphonates were
his studies of the disease hypophosphatasia. In this active when given either parenterally or orally. The
condition, which is caracterized by a deficiency of way to their clinical use was set.
alkaline phosphatase and a failure of bone to mine-
ralize normally, pyrophosphate is increased both in
plasma (Russell et al., 1971) and urine (Russell, Physico-chemical effects
1965).
The diphosphonates inhibit the ,preci,pitation of cal-
Pyrophosphate might also have an important role in 8ciu.m‘phosph’ate from clear solutions CFleisch et al.,
urine. Thus, this compound also prevents precipita- 1970) and when ,precipitation is induced by various
tion (Fleisch and Bisaz, 1964) and aggregation (Ro- means (Meyer and Nancollas, 1973 ; Boskey et al.,
bertson et al., 1973) of calcium oxalate, and this at 1979), they block the transformation of amorphous
concentrations found in urine, so that it could be an calcium phosphate into hydroxyapatite (Francis,
inhibitor of stone formation. Since the urinary excre- 1969 ; Francis et al., 1969) and delay the aggregation
tion of pyrophosphate is increased by the oral admi- of apatite crystals into larger clusters (Hansen et
nistration of orthophosphate (Fleisch et al., 1964 ; al., 1976). They also disaggregate apatite crystal
Russell et al., 1964), it is possible that the beneficial clusters l(Bisaz et al., 1976) and transform the crystals
effects of phosphate administration reported in recur- into a colloidal state, a phenomenon called pepti-
rent calcium stone formers is due to the increased z&ion (Robertson et al., 1972). Furthermore, like
excretion of pyrophosphate. pyrophosphate, diphosphonates slow down the dis-
solution of crystals (Fleisch et al., 1969 ; Russell et
The question arose whether these various {properties al., 1970 ; Evans et al., 1980). All these effects seem
of ,pyrophosphate could be exploited in diseases to be related to the marked affinity of the diphospho-
characterized by ectopic calcification or increased nates for hydroxyapatite (Jung et al., 1973).
H. Ffeisch : Diphosphonates : History and Mechanisms of A&ion 281

In addition to the effects on calcium phosphate, the those on cartilage. In children, therefore, lack of
diphosphonates also inhibit the formation (Fraser et X-ray changes at the epiphyseal plate during treat-
al., 1972 ; Meyer et al., 1977) and aggregation (Ro- ment with EHDP does not necessarily mean that the
bertson et al., 1973 ; Felix et al., 1977) of calcium drug has not affected bone mineralization. Apart
oxalate crystals. 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
Like pyrophosphate, diphosphonates very efficiently compounds tested which ehowed an action in vivo
inhibit experimental soft tissue calcification. Thus also inhibited crystal growth in vitro, certain other
they prevent aortic and renal calcification induced by compounds were observed to be good inhi,bitors
large amounts of vitamin Ds (Fleisch et al., 1970) and in vitro, but not active in Oivo (Trechsel et al.,
other types of ectopic calcification (Casey et al., 1977). Thus, dichloromethyl,ene Ndiphosphonate
1972 ; Francis et al., 1972 ; Rayssiguier et al., 1973 ; (ClzMDP)
Hollander et al., 1974 ; Rosenblum et al., 1975 ;
Russell et al., 1975 ; Rosenblum et al., 1977 ; Mat- OH Cl OH
thews et al., 1978 ; Potokar and Schmidt-Dunker,
1978) including experimentat urinary stones (Fraser
I I I .
0 = P- c- P=O ClzMDP
et al., 1972). In the arteries, the accumulation of
I I I
cholesterol, elastin and collagen is also inhibited
OH Cl OH
under certain conditions (Hollander et al., 1978 ;
Kramsch and Chan, 1978 ; Hollander et al., 1979) by
mechanisms which are not known. Finally, topical despite its strong activity on crystal growth and on
administration a,lso diminishes the formation of den- soft tissue calcification (Fleisch et al., 1970), has
tal calculus (Muhlemann et al., 1970 ; Briner et al., only limited interchange activity towards bone mine-
1971). ralization, the minimal effective dose being more
than 10 times above that of EHDP (Schenk et al.,
There is a close relationship between the ability of 1973). One possible explanation for this discrepancy
individual diphosphonates to isnhilbit crystal growth is that EHDP and ClzMDP have a different distribu-
in vitro #and their effect in vivo (Fleisch et al., 1970), tion in the body. Another possibility is that diphos-
suggesting that the latter is explicable by a physico- phonates act by mechanisms other than crystal
chemical mechanism. The most effective compounds growth, for example by an effect on the state of
are characterized by the P-C-P bond, structures aggregation of proteoglycans, as suggested by
containing C-P or P-C-C-P bonds being less effective Howell and Pita (1977).
or ineffective.

Conversely, under certain conditions and with very

high doses, diphosphonates can themselves induce Effect on bone resorption
mineralization in specific locations such as the injec-
tion sites, and in the stomach or the kidney (Larsson In contrast to pyrophosphate, diphosphonates are
and Rohlin, 1980). The nature of the mineral depo- extremely active in inhibiting bone resorption. In CL&
sited is however not yet clear.
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
Effect on calcification of hard tissues 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,
Diphosphonates not only inhibit ectopic calcification, 1980). Of al.1 the compounds tested ClzMDP is the
but also, under certain conditions, prevent the nor- most potent.
mal mineralization of hard tissues. Thus, in growing
animals certain diphosphonates, including EHDP,
can inhisbit the mineralization of cartilage and bone Powerful effects are also seen in vivo. Thus when
(Jowsey et al., 1970 ; King et al., 1971 ; Russell et al., given to growing rats many of the diphosphonates
1973 ; Schenk et al., 1973 ; Rosenblum, 1974 ; arrest remodelling in the metaphysis so that it beco-
Larsson and Larsson, 1976). The doses required vary mes club-shaped and radiologically more dense than
according to the species, the length of treatment, normal (Schenk et al., 1973). It appears that not only
and the route of administration. Interestingly, the is the degradation of bone inhibited but so too is
inhibition seems to be reduced by the administration that of cartilage. The effect appears to be accompa-
of calcitonin (Boris et al., 1979). In growing anima,ls, nied by a decrease in vascular invasion (Larsson and
the X-rays of the skeletal lesions resemble rickets Larsson, 1979).
caused by vitamin D deficiency, with a widening of
the epiphyseal plate (Schenk et al., 1973). Histolo- The effect of diphosphonates is especially apparent
gically, however, the two conditions differ fundamen- in newborn mice, where ChMDP, given in large
tally, at least in the chick, since in vitamin D defi- doses, impairs normal bone remodetling to such an
ciency the epiphyseal plate is comprised mainly of extent that the bones resemble those of the grey-
proliferating cells, whereas after EHDP it consists lethal strain of congenital osteopetrotic mice, and
mainly of hypertrophic cells (Bisaz et al., 1975). Inhi- the animals develop symptoms akin to those of this
bitory effects on bone occur at lower doses than disease (Reynolds et al., 1973).
 282 H. Fleisch : Diphosphonates : History and Mechanisms of Action

An inhibition of bone resorption is also seen when are also very active (Lemkes et al., 1978 ; Shinoda et
resorption is measured by ‘%a kinetics (Gasser et al., 1979 ; Reitsma et al., 1980). The relative activity
al., 1972) urinary excretion of hydroxyproline (Gas- of some of the diphosphonates tested is as follows :
ser et al., 1972 ; Goulding and McChesney, 1977 ; AHPDP > long chain 1-hydroxy diphosphonates
Reitsma et al., 1980) and by other techniques (Lem- > ClzMDP > EHDP (Shinoda et al., 1979).
kes et al., 1978 ; Reitsma et al., 1980). The decrease
in resorption is sometimes accompanied by an in- No correlation has ,been found jbetween activity
crease in calcium ,balance (Gasser et al., 1972 ; in viva and the inhi,bition of crystal dissolution in vitro
Reitsma et al., 1980) and mineral content of bone (Shinoda et al., 1979). This lack of relationship
(Michael et al., 1971 ; Miihl,bauer et al., 1971 ; Gasser suggests that the ‘action on bone resorption is media-
et al., 1972). However, this increase is relatively ted through mechanisms other than the jphysico-
small, since bone formation decreases almost in ,chemrcal effects on apatite. Thus an extensive effort
parallel with the change in resorption. The main has been devoted to investigate possible cellular
,effect of the diphosphonates is thus to induce a effects. Such cellular ,effects are suggested by the
decrease in bone turnover, a conclusion which is observations that diphosphonates alter the mor-
supported by morphological observations (Evans et phology of the osteoclasts, both in culture (Rowe and
al., 1979). Hausm,ann, 1978) and when administered in vivo
(Schenk et al., 1973 ; Miller and.Jee, 1975 ; Miller et
Diphosphonates also prevent an increase in bone al., 1977 ; Miller and Jee, 1979 ; Plastnans et al.,
resorption induced by various agents. Thus, they 1980).
prevent changes in blood calcium induced by PTH
(Fleisch et al., 1989 ; Russell et al., 1970) and other
hypercalcemic as well as hypocalcemic challenges Biochemical effects
(Bonjour et al., 1973 ; Yarrington et al., 1978 ; Yarring- In recent years numerous biochemical effects have
ton et al., 1977 ; Yarrington et al., 1977). ClzMDP and ibeen, described. ,However, th,eh relevance to the
to a lesser degree EHDP also prevents various types activity of the diphosphonates in vivo ,is still unclear.
of experimental osteoporosis (Cabanela and Jowsey, Effects of diphosphonates on the formation of
1971 ; Michael et al., 1971 ; Miihlbauer et al., 1971 ; cAMlP have been, described (Pilczyk et al., 1972 ;
Lane and Steinberg, 1973 ; Hahnel et al., 1973 ; Eisman et al., 1974 ; Goulding et al., 1976 ; Plasmans
Hahnel et al., 1978 ; Black and Jee, 1977). They also et al., 1980) ,but does not correlate with their activity
inhibit resorption found in experimental renal osteo- on bone resorption (Gebauer et al., 1978). Diphos-
dystrophy (Russell et al., 1975) the peridontal des- phon+tes, particularly C1zM.D.Pinhibit various lyso-
truction seen in rice rats (Leonard et al., 1979), and somal enzymes in vitro, including acid ,phospha-
the destruction of implants of bones from treated tase and pyrophosphatase (Felix et al., 1978). They
animals (Rosenquist and Baylink, 1978). An interes- also prevent :PTH-induced increases of these enzy-
ting exception is that bone loss induced by a low mes in calvaria culture (Morgan et al., 1973) anmd
calcium diet is not prevented (Jowsey and Halley, diminish the acid phosphatase activity of bone cells
1973 ; Morgan et al., 1975). when given in vivo (Doty et al., 1972 ; SEnde, 1979).
EH,DP:and ClrMDP slow down the release of calcium
A great number of diphosphonates have been inves- in vitio from kid,ney mitochondria (Guilland et al.,
tigated for their inhibitory effect of bone resorption. 1974 ; Guilland and Fleisch, 1974), an observation
It appears that increasing the chain length of the which is ,supported by the increase in calcium con-
C-backbone tent of mitochondria in Ibone cells in vivo (Plasmans
et al., 1980).

ClaMDP, and to a lesser extent EHDP, decrease the

yH i qH long chain
rate of glycolysis as dudged by a decrease in glucose
o=p- , 7 - Fi - 0 drphosphonates consumption and lactic acid production both in cul-
tured calvaria and cartilage cells (Fast et al., 1978 ;
OH (CH& OH X = H, OH Ende, 1979) .and itn who1.e calvaria in culture (Morgan
,ei al., 1973 ; ,Ende, 1979). ,Since lactate (production is
I generalmly thought to be related to bone resorption,
CHa it ‘is tempting to suggest that one way in which the
diphosphonates decrease resorption Is by their effect
increases activity until a length of about 9 carbon on lactic acid production. However, this explanation
atoms is reached (Shinoda et al., 1979). .Adding a can ‘not a,pply to all compounds of this class, since
hydroxyl group at position 1 also increases the effect AIHPDP and long chain diphosphon,ates, which are
(Shinoda et al., 1979). The amino-derivatives such very effective inhibitors of bone resorptlon, have
as the &amino-1-hydroxypropane-l,l-diphosphonate either ,no eff,ect, or even mcrease lactic acid #pro-
(AHPDP) duction l(End,e, 1979 ; Shinoda et al., 1979). Di,phos-
,phonates mcrease fatty acid oxidation (Felix and
OH OH OH Fleisch, 1979) and amino acid oxidation (Ende,
I I I 19$9), and stimulate the citric acid cycle (Ende,
-P=O AHPDP 1979). ‘EHDP arrd ClzMDP also increase the cellular
O’P --I” I content of glycogen in vitro (Felix et al., 1980) and
ClaMDP increases the production oaf al,kal,ine phos-
OH fH” OH phatase in bone cells ,more than 30 fold (Felix and
‘Fleisch, 1979). The latt,er effect has also ,been obser-
F” ved in vivo (Weisbrode et al., 1978). Imnterestingly,
NH, EHDP is almost inactive in this respect. ClzMDP but
H. Fleisch : Diphosphonates : History and Mechanisms of Action 283

not EHDP also increases the biosynthesis of bone lular effects. It appears likely that the P-C-P part of
and cartilage collagen, both when injected into the molecule gives the diphosphonate the affinity for
animals or when added to isolated cells in culture mineralized tissues, whereas the specific activity
(Guenther et al., 1981 ; Guenther et al., 1981). ClzMDP will depend on the molecule as a whole.
also stimulates the biosynthesis of proteoglycans by
isolated chondrocytes (Guenther et al., 1979) but a
decrease in glycosaminoglycan synthesis has been Other effects of diphosphonates
observed under certain conditions (Larsson, 1976).
Recently ChMDP, and to a lesser extent EHDP, have A number of other effects of diphosphonates have
been found to inhibit prostaglandin synthesis when been described. Some of these are probably not
added to bone cells in vitro (Felix et al., submitted), direct effects but secondary to their action on bone.
whereas AHPDP and long chain diphosphonates Thus, large doses of EHDP decrease the intestinal
stimulate its production. 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
Diphosphonates have been found to have effects on doses of EHDP on the contrary lead to an increase
the immune system. Thus methylene diphosphonate in 1,25(OH)zD3 (Guilland et al., 1975). It is likely that
decreases the formation of antibody secreting cells
the decrease in 1,25(OH)zD3 production with high
in response to immunization and decreases hyper- doses is due to an indirect homeostatic mechanism
sensitivity of delayed and immediate types (Komis- caused by the block of mineralization. Thus in renal
sarenko et al., 1977). EHDP inhibits the activity of cell culture, EHDP is ineffective in decreasing the
antilymphocyte serum on T lymphocytes (Zemskov 1-alpha-hydroxylase (Trechsel et al., 1979). The inhi-
et al., 1979). Furthermore certain diphosphonates bition of mineralization by EHDP is not, however, due
inhibit the effect in vitro of a mitogen on mononu- to a decrease of the vitamin D metabolite, since it
clear phagocyte function and lymphoblastic response precedes this decrease, has a different histological
(Bijvoet et al., 1980) and AHPDP induces a transient appearance than D-deficient rickets (Bisaz et al.,
lymphopenla and changes in acute phase proteins 1975), and is not (Bonjour et al., 1975) or only par-
in man (Bijvoet et al., 1980). Whether these results tially (Boris et al., 1978 ; Baxter et al., 1979) reversed
are related to the action on bone resorption or to by 1,25(OH)zD3.
the possible antiinflammatory effect (Flora, 1979) is
uncertain. Finally an antimutagenic activity (Veltis- Another effect in the rat, which is probably also
chev and Seleznev, 1978), an inhibition of viral DNA indirect, is the ability of EHDP to increase the capa-
polymerase and an inhibition of plaque formation in city of the kidney to excrete phosphate (Bonjour et
cell culture have been described (Eriksson et al., al., 1978). Since the kidney has been shown to adapt
1980). very efficiently to the needs of the organism, this
effect of EHDP could be another homeostatic regu-
EHDP has been reported to lower plasma choleste- latory response. The effect in the rat is in contrast
rol in man (Caniggia and Gennari, 1977 ; Caniggia et to that seen in humans, where EHDP induces a rise
al., 1979). This effect on plasma lipids has, however, in plasma phosphate accompanied by an increase in
not been confirmed by other investigators (Mellies renal reabsorption of phosphate (Reeker et al., 1973).
et al., 1979).

All these effects suggest that the diphosphonates Metabolism

are taken up (by ceils. This has been confirmed
in vitro, in the case of EHDP and ClzMDP (Fast et al., While certain monophosphonates are found in ani-
1978). Indeed the cellular concentration, expressed mals and man, diphosphonates have not yet been
in terms.of cellular water, can be severalfold higher shown to occur naturally nor have any enzymes ca-
than in the medium, suggesting that it is accumulated pable of cleaving the P-C-P bond yet been described.
within specific cellular compartments. Cells with As far as we know diphosphonates are absorbed,
phagocytic properties take up the diphosphonates stored, and excreted unchanged in mammals.
with special avidity if they are‘bound to apatite crys-
tals (Chambers and Path, 1980). This might explain Most of the pharmacokinetic data on diphosphonates
why osteoclasts are target cells for these compounds. have been collected with EHDP. Its absorption is very
In the light of all these results, it appears astonishing variable both between and within species and lies
that diphosphonates act only on bone and not on between i and 10 O/O of an oral dose. It seems gene-
other tissues. The answer probably lies in the affinity rally somewhat higher in the young (Michael et al.,
of these compounds for calcium phosphate crystals 1972 ; Reeker and Saville, 1973). Absorption occurs
(Jung et al., 1973) which causes them to be cleared partly in the small intestine (Wasserman et al., 1973)
very rapidly from blood and incorporated into bone. and depends on the size of the diphosphonate. Ab-
Indeed, the half-life in the circulation is in the order sorption is lower for compounds of large molecular
of minutes, the rate constant of entry into bone being weight and some of the larger diphosphonates are
similar to that of calcium or phosphate (Bisaz et al., virtually non-absorbable (Anbar et al., 1973). Approxi-
1978). Soft tissues will therefore be exposed to the mately half of the EHDP absorbed enters bone, and
compounds for only short periods. In bone, however, half is excreted in the urine (Michael et al., 1972).
the concentration of the diphosphonates is much The renal clearance of EHDP and ClzMDP can
higher, especially at the sites of resorption, when the exceed that of inulin, indicating the existence of a
apatite crystals are dissolved and the diphospho- secretory pathway (Troehler et al., 1975). The retai-
nates released. The compounds may at these loca- ned EHDP is hearly all found in bone, only very small
tions be taken up by bone resorbing cells either as amounts are found in soft tissues (Michael et al.,
free compounds or bound onto the apatite crystals 1972). The half-time for bone retention will depend on
(Felix et al., submitted), thereafter exerting their cel- the turnover rate of the skeleton.
284 H. Fleisch : Diphosphonates : History and Mechanisms of Action

Little is known of the distrisbution of other diphos- destruction. Furthermore, their affinity for apatite
phonates. More attention will have to be directed to has made it possible to bring other compounds, such
this problem, since it is possible that their pharma- as 99mT~ to calcified tissues by linking them to the
cokinetics vary, a fact which could well affect their diphosphonates. It would not be surprising if the first
biological activity. compounds tested are by no means optimal and that
a further exploration of other types of diphospho-
nates could lead to a fruitful future development of
Conclusion these novel compounds.

Acknowledgments : The work reported in this review has

The diphosphonates are very potent inhibitors of
been carried out with the support of the Swiss National
mineralization and bone resorption. These characte- Science Foundation (grant 3.324.79), the Procter and
ristics have opened the way to the clinical use of Gamble Company, Cincinnati, U.S.A., and the Ausbildungs-
these compounds in disorders of mineral metabolism und Forderungsfonds der Arbeits gemeinschaft fur Osteo-
such as ectopic calcification and increased bone synthese (AO), Chur, Switzerland.

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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 phoa-
phata 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 rem-
pie&e par une Uaison P-C-P. Da nombreux disatea ant W aynthWa6a et teatie et certainea rektiow entre ieur at~c-
tura at IYventaii de ieurs effets bioiogiquea aont apparuaa. Cea anaiogues unt dea proprMt&a abniiairea B ceilea du pyrophoa-
phata. 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.