Bone 38 (2006) 617 627

www.elsevier.com/locate/bone

Novel insights into actions of bisphosphonates on bone: Differences in

interactions with hydroxyapatite
G.H. Nancollas a , R. Tang a , R.J. Phipps b , Z. Henneman a , S. Gulde a , W. Wu a ,
A. Mangood a , R.G.G. Russell c , F.H. Ebetino b,
a

Department of Chemistry, Natural Sciences Complex, University at Buffalo, The State University of New York, Buffalo, NY 14260, USA
Procter and Gamble Pharmaceuticals, Inc., Health Care Research Center, 8700 Mason Montgomery Road, Mason, OH 45040-9462, USA
c
The Oxford University Institute of Musculoskeletal Sciences (The Botnar Research Centre), Nuffield Department of Orthopaedic Surgery,
Nuffield Orthopaedic Centre, Headington, Oxford OX3 7LD, UK

Received 19 October 2004; revised 18 April 2005; accepted 13 May 2005

Available online 20 July 2005

Abstract
Bisphosphonates are now the most widely used drugs for diseases associated with increased bone resorption, such as osteoporosis. Although
bisphosphonates act directly on osteoclasts, and interfere with specific biochemical processes such as protein prenylation, their ability to adsorb to
bone mineral also contributes to their potency and duration of action.
The aim of the present study was to compare the binding affinities for hydroxyapatite (HAP) of 6 bisphosphonates currently used clinically and
to determine the effects of these bisphosphonates on other mineral surface properties including zeta potential and interfacial tension.
Affinity constants (KL) for the adsorption of bisphosphonates were calculated from kinetic studies on HAP crystal growth using a constant
composition method at 37C and at physiological ionic strength (0.15 M). Under conditions likely to simulate bisphosphonate binding onto bone,
there were significant differences in K L among the bisphosphonates for HAP growth (pH 7.4) with a rank order of
zoledronate > alendronate > ibandronate > risedronate > etidronate > clodronate. The measurements of zeta potential show that the crystal
surface is modified by the adsorption of bisphosphonates in a manner best explained by molecular charges related to the protonation of their sidechain moieties, with risedronate showing substantial differences from alendronate, ibandronate, and zoledronate. The studies of the solid/liquid
interfacial properties show additional differences among the bisphosphonates that may influence their mechanisms for binding and inhibiting
crystal growth and dissolution. The observed differences in kinetic binding affinities, HAP zeta potentials, and interfacial tension are likely to
contribute to the biological properties of the various bisphosphonates. In particular, these binding properties may contribute to differences in
uptake and persistence in bone and the reversibility of effects. These properties, therefore, have potential clinical implications that may be
important in understanding differences among potent bisphosphonates, such as the apparently more prolonged duration of action of alendronate
and zoledronate compared with the more readily reversible effects of etidronate and risedronate.
2005 Elsevier Inc. All rights reserved.
Keywords: Bisphosphonates; Osteoporosis; Hydroxyapatite; Zeta potential; Bone binding affinity; Risedronate

Introduction
Bisphosphonates are now the most widely used drugs for
treating diseases associated with increased bone resorption,
such as osteoporosis. Following the demonstration that

Corresponding author. Fax: +1 513 622 5346.

E-mail address: ebetino.fh@pg.com (F.H. Ebetino).
8756-3282/$ - see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2005.05.003

inorganic pyrophosphate was a naturally occurring modulator

of bone metabolism, the bisphosphonates, which are metabolically stable analogs of pyrophosphate, were studied as
inhibitors of calcification and resorption for the treatment of
bone disorders [13]. The earliest studies showed that bisphosphonates could bind to hydroxyapatite (HAP) crystals, and
inhibit crystal growth and dissolution similar to pyrophosphate
[4,5]. The ability of bisphosphonates to inhibit bone resorption
was initially ascribed to these direct inhibitory effects on

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G.H. Nancollas et al. / Bone 38 (2006) 617627

mineral dissolution. However, with the development and study

of increasingly potent bisphosphonates, it became clear that
these physicochemical effects on bone mineral did not
completely explain the inhibition of bone resorption, and
cellular effects were likely to be involved [68]. It is now
known that bisphosphonates also act directly on osteoclasts and
interfere with specific intracellular biochemical processes such
as isoprenoid biosynthesis and subsequent protein prenylation
to inhibit cell activity [9,10].
The high binding affinities of bisphosphonates for bone may
affect many important biological properties of these compounds
including uptake and retention on the skeleton, diffusion of the
drug within bone, release of adsorbed drug from bone, potential
recycling of the desorbed drug back onto bone surfaces, and
effects on mineral dynamics and cellular function within bone.
The strong binding of bisphosphonates to bone mineral together
with the ability to be linked to a gamma-emitting technetium
isotope [11] was the basis for the use of bisphosphonates as
bone-scanning agents to detect metastases and other bone
lesions. Many studies have shown that bisphosphonates have
prolonged or persistent effects, both in experimental animals
and in humans, which is probably related to the rate and extent
of chemical desorption and cell-mediated release from bone
[1218].
Chemically, the bisphosphonates of medical interest are all
characterized by 2 phosphonate groups sharing a common
carbon atom (PCP). This PCP backbone is responsible for
the strong affinity for bone mineral and thus facilitates the
potent inhibitory effects on bone metabolism in vitro and in
vivo. Both phosphonate groups are required, as modifications to
one or both reduce affinity for bone mineral. It has been well
established that R1 substituents (Table 1) with additional
capability to co-ordinate to calcium, such as a hydroxyl (OH)
or amino (NH2), can display enhanced chemisorption to
mineral, presumably via tridentate binding to calcium [4].

Table 1
Bisphosphonate structures

Varying the R2 substituents can result in differences in

antiresorptive potency of several orders of magnitude. Many
bisphosphonates with an R2 side chain containing a basic
primary nitrogen atom in an alkyl chain (e.g., pamidronate and
alendronate) are more potent antiresorptive agents than either
etidronate or clodronate, whereas compounds with more highly
substituted nitrogen moieties in R2 (e.g., ibandronate) can
display further increases in antiresorptive potency. The most
potent antiresorptive bisphosphonates include those containing
a nitrogen atom within a heterocyclic ring (e.g., risedronate and
zoledronate). These enhancements in antiresorptive potency
resulting from differences in the R2 groups appear to be linked
to the biochemical activities of these drugs, particularly
inhibition of the farnesyl diphosphate synthase enzyme within
the mevalonic acid pathway in osteoclasts [1921].
Although earlier studies showed differences in mineral
binding among bisphosphonates such as etidronate and
clodronate in which R1 was varied, the effects on mineral
binding of varying R2 has not been systematically studied with
many nitrogen-containing bisphosphonates. In a previous study,
differences were found in the HAP adsorption isotherms at high
equilibrium concentrations, even among bisphosphonates with
the same R1 (OH) [20,22]. The method used, however, did not
allow for calculation of binding affinities.
In this present study, using an HAP crystal growth method,
we measured the kinetic binding affinities of 6 bisphosphonates
in current clinical use at concentrations more similar to in vivo
conditions. We have also studied the effects of these bisphosphonates on other HAP surface properties that are likely to
influence the mineral binding of bisphosphonates in vivo,
including zeta potential and interfacial tension.
Methods
Materials
Six bisphosphonates (clodronate, etidronate, risedronate, ibandronate,
alendronate, and zoledronate) were used in this study; their chemical names
and structures are listed in Table 1. Clodronate has an R1 substituent of Cl and
the other bisphosphonates tested have OH as the R1 substituent. All 6
compounds were synthesized and characterized in the Procter & Gamble
Pharmaceuticals laboratories utilizing previously published methods [19]. HAP,
prepared as described previously, was characterized by X-ray diffraction,
chemical analysis, and infrared spectroscopy [23]. The specific surface area was
35.2 0.4 m2/g (BET nitrogen adsorption; 20/80 N2/He).

Constant composition kinetic studies of crystal growth

A constant composition potentiostatic method was used to determine the
kinetic binding affinities. This HAP crystal growth method was utilized to
maintain the thermodynamic driving forces which would otherwise change
during the growth or dissolution reactions due to changes in the concentrations
of crystal lattice ions; the resulting supersaturated/undersaturated solutions make
it impossible to obtain reliable kinetic data for individual reacting phases. We
avoided these problems by using the constant composition method as constant
thermodynamic driving forces are maintained during the experiments [24].
Kinetic experiments were performed at pH 7.4. They were initiated by the
introduction of known amounts of HAP crystallites (seed mass 10 mg/200
mL) in magnetically stirred (450 rpm) double-jacketed vessels thermostated at
37.0 0.1C. The reaction solutions were prepared by slowly mixing filtered
solutions of calcium chloride and potassium dihydrogen phosphate with

G.H. Nancollas et al. / Bone 38 (2006) 617627

sodium chloride for a final ionic strength of 0.15 M. For experiments in the
presence of bisphosphonates, calculated amounts of bisphosphonates were
added prior to pH adjustment. A potentiometer incorporating glass and
reference electrodes to maintain a constant thermodynamic driving force
triggered the addition of titrant solutions. The titrants contained calcium,
phosphate, sodium chloride, and sodium hydroxide (for growth) to
compensate for the changes of ionic concentrations during the reaction as
described by Koutsoukos et al. [24].
The growth rates at any instant were determined from the recorded plots of
titrant volume added as a function of time. The overall rates, J, can be defined by
Eq. (1):
J

Ceff dV
AT dt

where dV/dt is the rate of titrant addition. Ceff is the effective titrant
concentration with respect to HAP, indicating the molar amount of grown
HAP per liter of the added titrant (in the current studies, Ceff = 2.0 104 M).
The value of the surface area during formation, AT, was estimated assuming a
uniform three-dimensional crystal growth.
The adsorption of inhibitor molecules on crystal surfaces may be interpreted
in terms of a Langmuir equilibrium adsorption isotherm. In terms of reaction
rate, a pseudo Langmuir adsorption isotherm may be written as Eq. (2),
RO
1
1
KL C
RO  Ri

where the RO, Ri are the rates in the absence and presence of additive,
respectively. C is the concentration of additive and KL is the adsorption affinity
constant.

Zeta potential
The zeta potential (), or the electrical potential at the shear plane at the
crystal surfaces, can be derived from measurements of the electrophoretic
mobilities of the suspended crystallites [25,26].
Zeta-potential measurements of HAP crystallites were made at a constant
ionic strength of 0.15 M (adjusted with sodium chloride) in the absence and
presence of bisphosphonates. The electrophoretic mobilities of suspended HAP
particles were measured with a laser-Doppler velocimetric instrument (Zetasizer
IIc, Malvern Instruments) [25].

619

the surface are replaced by water molecules to form units that escape into the
bulk solution. Higher values of interfacial tension, SL, indicate a greater
difficulty in forming such an interface between the solid and the aqueous
phase. A thin layer wicking capillary rise method (at pH 7.4) was used to
determine the contact angle formed between a liquid and the finely divided
seed crystal particles [30]. In this method, the measured rate of capillary rise
(i.e., the capillary rise h, in a time t) of a liquid L, through a layer of the
powder, coated on a glass microscope slide, was substituted into the
Washburn equation (Eq. (3)) [31].
h2

tReff gL cosh
2g

where Reff is the effective interstitial pore radius of the powder column, L is
the interfacial tension of the test liquid, is the viscosity of the liquid, and
the contact angle between the liquid and the solid.
The thin HAP crystallite layers (seed crystals) were prepared by uniformly
dropping 2 mL of a 23% HAP suspension on clean, horizontal, glass
microscope slides (7.5 2.5 cm). These dried coated glass slides were placed in
containers fitted with gas-tight ground glass stoppers and filled to a height of
about 5 mm with one of several test liquids. The rates of vertical movement of
the liquid front were recorded.

Statistics
Crystal growth experiments were performed 4 times for each compound
tested. Langmuir isotherm plots were generated and separate binding affinity
constants (KL) were calculated for each individual study. Data are presented as
mean SD. The data were normally distributed with equal group variances
(Bartlett's test accepted), and so across group comparisons were made with a
parametric one-way analysis of variance, followed by pair-wise comparisons
with Tukey's test (Sigmastat). Differences were considered statistically
significant at P < 0.05 on a two-tailed test.
Zeta-potential values were calculated using the Smoluchowski equation [26]
and are presented as the mean SD from 3 determinations. Statistical analysis to
compare the bisphosphonates tested was not conducted because common
concentrations were not always used due to the experimental conditions. The
values reported are interpolations for a 105 M concentration. Interfacial tension
and interfacial tension component values were calculated by means of Young's
equations [32] and are the averages from 3 separate experiments. A standard
deviation was calculated for the interfacial tension data.

Results
HAP growth

Interfacial tension
It has been recognized that there is a close relationship between solubility
and interfacial tension [2729]. During dissolution, some neighboring ions on

All 6 bisphosphonates studied produced concentrationdependent inhibition of HAP crystal growth (Table 2, Figs. 1

Table 2
Bisphosphonates and inhibition of HAP growth rates and kinetic binding affinities
Inhibitory concentrations of bisphosphonates/107 mol L1

KL/106 L
mol1

Inhibition of HAP growth rates

Clodronate
Etidronate
Risedronate
Ibandronate
Alendronate
Zoledronate

20%

50%

80%

100%

3.3
2.0
1.3
1.1
0.8
0.7

13.4
6.5
3.8
3.4
2.4
2.2

>>25
13.2
9.8
7.4
5.2
4.8

a
a
a
a

7.6
6.5

Concentrations inhibiting HAP growth rate calculated from HAP growth curves (at time 1020 min).
Kinetic affinity constants calculated from slopes of the Langmuir adsorption isotherm plots (values are means SD for n = 4).
a
Does not completely suppress the growth reaction.
Significantly different from risedronate K (P < 0.05).
L

0.72 0.12
1.19 0.10
2.19 0.17
2.36 0.32
2.94 0.24
3.47 0.18

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G.H. Nancollas et al. / Bone 38 (2006) 617627

Fig. 1. Typical constant composition growth curves (titrant volume vs. time) for HAP growth in the presence of varying concentrations of risedronate and alendronate
at pH 7.4. Seed mass = 11.0 mg and Ceff = 2.0 104 M.

and 2). The rank order of inhibitory potency at these concentrations was zoledronate (most potent) alendronate >
ibandronate risedronate > etidronate > clodronate (least
potent). Fig. 1 shows representative plots of titrant volume
versus HAP growth over time for risedronate and alendronate.
The initial non-linear portion of the titrant curves represents
expected titrant surges. These surges are observed during the
first 10 min of the reaction and are due to conditioning of the
seed surfaces to the metastable solutions when the slurry is
introduced, causing a small change in pH in the supersaturated
solutions.
The control growth rate for HAP in the absence of
bisphosphonate at pH 7.4 and = 7.40 was 1.28 107 mol
m2 min1, which was consistent with previous kinetic studies
[3337]. Clodronate was the weakest inhibitor of HAP growth
rate; a concentration of 1.34 106 mol L1 was required to
inhibit growth rate by 50% (IC50). The same rank order of
inhibitory potency was seen at 20% and 80% inhibition of HAP
growth (Table 2). Only zoledronate and alendronate completely

Fig. 2. HAP growth rate versus concentration for 6 different bisphosphonates at

pH 7.4.

inhibited (100%) the HAP growth, and this complete inhibition

required relatively high concentrations.
Fig. 3 shows the corresponding Langmuir plots from the
constant composition experiments for HAP growth in the
presence of the bisphosponates. The satisfactory linearity
suggests this application of the Langmuir model can be used
to compare the effects of bisphosphonates on crystal growth
over a range of bisphosphonate concentrations to yield valid
kinetic affinity constants, KL.
Clodronate had a KL of 0.72 106 L mol1, which is almost
50% lower than the KL for etidronate (1.19 106 L mol1)
(Table 2, Fig. 4), and may be explained by the lack of an OH
group at R1. In the bisphosphonate structure in which R1 is
OH, and the R2 side chain differs, it is clear the various R2
functional groups further influence binding to HAP. For
example, the KL for risedronate (2.19 106 L mol1) showed
a statistically significant difference (P < 0.05) from the KL for

Fig. 3. Langmuir adsorption isotherm plot for the kinetic growth results.

G.H. Nancollas et al. / Bone 38 (2006) 617627

Fig. 4. HAP adsorption affinity constants for the bisphosphonates under

experimental condition of growth.

clodronate, etidronate, alendronate, and zoledronate. Overall,

these values indicate bisphosphonates are able to bind with
more than 10 times greater affinity than most other inhibitors of
crystal growth [3845] and explain why the bisphosphonates
are so effective at targeting bone mineral and thus exerting their
overall biological effects.
Zeta potentials
Plots of HAP as a function of bisphosphonate concentration are shown in Fig. 5. At pH = 7.4, the HAP of 4.2 mV
became less negative in the presence of alendronate and
ibandronate while the addition of clodronate, etidronate, and
risedronate resulted in more negative HAP . In the presence of
zoledronate, even at increasing concentrations, the value was
almost unchanged. The effects on HAP were influenced by
pH. At a pH of 5.0 and in the absence of any bisphosphonate,
the HAP was positive, 9.6 mV, reflecting the increased
protonation of surface phosphate ions [26]. The zeta potentials
became increasingly more positive with increasing concentra-

621

tion for all the bisphosphonates with the exception of clodronate

which continued to induce a more negative HAP .
Effects on HAP are likely to result from charges on the
phosphonate groups and on the R2 functional groups, which will
be determined by the corresponding pKa values. However, the
current data support that the differences in effects on HAP
among the nitrogen-containing bisphosphonates at the 2 pHs
tested are likely the result of differences in the pKa values of the
functional groups (Table 3, Figs. 5 and 6).
The experimental phosphonate proton dissociation constants
of the different bisphosphonates in Table 3 show no difference
for pKa1 and pKa2 among the compounds. The considerably
lower pKa values of clodronate compared to the other bisphosphonates can be explained by the electron-withdrawing
influence of Cl, which facilitates dissociation of the phosphonate protons [4648]. At the lower pH, this influence of Cl
results in a higher percentage of monoprotonated species of
clodronate compared to the other bisphosphonates, which in
turn results in the marked decrease in HAP upon clodronate
adsorption (Fig. 5). At the higher pH (7.4), more phosphonate
protons dissociate leading to increased concentrations of
monoprotonated species of all the bisphosphonates. The
adsorption of these more negative species on HAP surfaces at
pH 7.4 accounted for the observed decreases in HAP with
etidronate and risedronate.
While the R2 side chain would be expected to influence
proton dissociation of the phosphonate groups, they appear to
have a greater differentiation effect on HAP due to their
degree of nitrogen protonation. Comparing risedronate and
ibandronate, the R2 side chain of the former contains a
pyridine, while the latter contains a tertiary amine. The pKa
values of these nitrogen substituents are 5.25 and 9.80,
respectively. At pH 7.4, the R2 nitrogen side chain of
risedronate is almost completely deprotonated, while the R2
nitrogen side chain in ibandronate remains protonated with its
higher pKa value of 9.80 (Fig. 6). Therefore, at pH 7.4, the
adsorption of risedronate resulted in more negative zeta
potential while the adsorption of ibandronate resulted in a

Fig. 5. HAP zeta potential in the presence of bisphosphonates. (a) pH = 7.4 and (b) pH = 5.0. Error bars indicate the standard deviation.

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G.H. Nancollas et al. / Bone 38 (2006) 617627

Table 3
Dissociation constants of the phosphoric acids, functional groups of bisphosphonates, and their effects on zeta potential ()
Sample

pKa1

pKa2

pKa3

pKa4

Clodronate
Etidronate
Risedronate
Ibandronate
Alendronate
Zoledronate

1.70
1.87

2.13
2.76
2.77
2.84
2.35
2.89

5.66
6.78
6.79
6.08
6.55
6.63

8.30
10.20
10.45
10.43
10.09
10.99

Functional group

/mV a at pH 5.0

/mV a at pH 7.4

Pyridine, pKa = 5.25

Trimethylamine, pKa = 9.80
Methylamine, pKa = 10.63
1-H imidazole, pKa = 6.953

9.1
+1.6
+0.6
+1.9
+2.9
+5.5

4.9
9.3
6.9
+5.4
>> +10
+0.9

The values of change of , , are differences between treated (interpolated concentration of 1.0 105 M bisphosphonates) and untreated HAP. Zeta potentials for
untreated HAP were 4.2 mV at pH 7.4, and +9.6 mV at pH 5.0.
a

less negative HAP . Alendronate, with an even higher pKa for

its functional R2 side chain group, showed an even greater
change in the positive direction for zeta potential under these
experimental conditions.
A related prediction based on protonation of R2 functional
groups explaining effects on HAP can also be applied to
zoledronate, which has an R2 group similar to 1-H-imidazole
and a pKa of approximately 7.0. At pH 5.0, the compounds with
a nitrogen ring (risedronate and zoledronate) remain protonated
and display a positive zeta potential, as do alendronate and
ibandronate. At pH 7.4, the H-imidazole of zoledronate is in
equilibrium with its deprotonated form reducing the positive
charge but yielding a more neutral effect on HAP than

risedronate. Etidronate and clodronate do not have pH-sensitive

side chains and remain net negative molecules so that their
adsorption always results in a decrease of the surface charge at
physiological pH. This may also account for the reduced pH
sensitivity of their affinity constants.
Interfacial tension
The measured interfacial tension (SL) of HAP surfaces in
contact with pure saturated solution was 8.05 mJ m2. This
value is consistent with the value previously estimated from
kinetic data (810 mJ m2) [49]. The thin-layer wicking
experiments show that the adsorption of the bisphosphonates

Fig. 6. Differing nitrogen charges/species among bisphosphonates at pH 7.4.

G.H. Nancollas et al. / Bone 38 (2006) 617627

623

Table 4
Interfacial tension and interfacial tension components of HAP after treatment with bisphosphonates
Sample

SL/mJ m2

LW/mJ m2

+/mJ m2

/mJ m2

Pore size/108 m

Control
Clodronate
Etidronate
Risedronate
Ibandronate
Alendronate
Zoledronate

8.05
5.33
2.83
3.80
2.46
2.03
1.46

37.46
37.72
37.22
36.78
37.85
38.19
38.87

1.08
1.20
1.75
1.64
1.93
2.07
2.56

18.57
21.62
24.45
23.08
25.14
25.85
26.97

1.707
1.698
1.585
1.686
1.574
1.536
1.512

SL is interfacial tension, and LW, +, and are the Liftshitz-van der Waals, Lewis acid, and Lewis base components of the interfacial energy, respectively. The values
have a standard deviation of approximately 6%.

on HAP surfaces changed the surface properties (Table 4).

The HAP interfacial tension decreased with increasing
bisphosphonate binding. The HAP slurry for the thin-layer
wicking experiments was prepared in NaCl solutions in a pH
range of 7.98.4, which is close to that of the growth
experiments. It can be seen that the order of decreasing
interfacial tension (Table 4) was similar to that of the affinity
constants determined in the HAP growth experiments,
namely, zoledronate > ibandronate > alendronate >
etidronate > risedronate > clodronate. The only difference
versus the affinity rankings reported earlier is the interchanged positions of etidronate and risedronate. For the thinlayer-wicking studies, the experimental interfacial energy
values have a standard deviation of approximately 6%.
Discussion
The results of this study show that even bisphosphonates that
share a common PCP structure, with OH at R1, can have
significantly different kinetic binding affinities for HAP with a
rank order of highest to lowest for the bisphosphonates studied
of zoledronate > alendronate > ibandronate = risedronate
> etidronate. These differences must be attributed to differences
in the R2 side chain. This study also showed that the nature of
the R2 side chain influences other surface properties, including
zeta potential and interfacial tension. Differences in binding
affinities and effects on mineral surface properties are likely to
be reflected in the apparent clinical differences among these
bisphosphonates, including differences in potency, pharmacokinetics, and persistence of effect. These clinical differences
may result from differences in uptake and retention on the
skeleton, diffusion of the drug within bone, release of adsorbed
drug from bone, potential recycling of the desorbed drug back
onto bone surfaces, effects on mineral dynamics, and effects on
cellular functions (Fig. 7).
Previous studies have shown significant differences in
binding affinities among bisphosphonates with different R1
groups, but have shown only small differences [50,51] or no
significant differences [52,53] in affinities due to different R2
groups. In our studies, we derived kinetic binding affinities at
low micromolar concentrations. This technique is more
sensitive than competitive binding approaches that determine
equilibrium/steady-state binding affinities, and is probably
more likely to mimic the in vivo bone:bisphosphonate

interaction, given the rapid clearance of bisphosphonates from

the blood. Previous competitive binding studies of radiolabeled
compounds to fetal mouse bones or to bone powder, determined
equilibrium/steady-state binding affinities at high micromolar
concentrations [5052]. The conditions of these other studies,
therefore, are more comparable to the equilibrium binding data
of Ebrahimpour and Ebetino [20,22] rather than to the kinetic
binding results determined here. Equilibrium binding studies
showed [52] differences of approximately 20% between these
two compounds, but these differences were not statistically
significant. We report a significant difference of approximately
35% in kinetic binding affinities for HAP for risedronate and
alendronate.
It is well known that mineral crystal growth and dissolution
takes place at active kink sites and that both processes are
retarded when these sites are occupied by additive molecules.
Physical chemical studies on the mineralization of surfaces have
demonstrated that domain electronic density between the
substrate and the mineralizing phase plays an essential role on
this inhibitory process [5456]. Bisphosphonates have different
molecular charges due to the different proton dissociations
(pKs), on both the phosphonate moieties and the R2 groups.
This study is the first to describe the effects of nitrogencontaining bisphosphonates on HAP zeta potential and
interfacial tension. For solidliquid systems, the zeta potential
is the electrical potential at the shear plane at the crystal surfaces
(or around the mineral surface contact point). It will change

Fig. 7. Bisphosphonate interaction with bone.

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G.H. Nancollas et al. / Bone 38 (2006) 617627

following the adsorption of highly charged anionic species such

as bisphosphonates [5760], and may influence the subsequent
binding of charged molecules. The observed changes in zeta
potential in the presence of different bisphosphonates are
consistent with expected charges on the R2 sidechain (e.g., the
degree of protonation of the nitrogen moiety). These charges on
crystals may affect several properties; for example, their ability
to attract other ions, and charged molecules and macromolecules. It may also affect the local binding of additional
bisphosphonate molecules by attracting or repelling further
binding on the basis of their electrical charge. In other words,
the capacity of any given surface region of bone mineral to
adsorb different bisphosphonates may vary as a result of zeta
potential.
Interfacial tension may also be important in the changes in
HAP surfaces due to the adsorption of bisphosphonate.
Interfacial tension plays an important role not only in the
stability of colloidal suspensions and the adsorption of
molecules at solid/solution interfaces but also in the classical
description of the shape and kinetics of growth of crystals
from solution [49,61,62]. A greater reduction in interfacial
tension reflects an easier ability in forming an interface between
the solid and the aqueous phase. In addition, the Lewis base
interfacial tension component, is an indicator of the ability
of a surface to induce heterogeneous nucleation [6366].
Higher values of reflect more favorable conditions for
surface nucleation important in crystal growth. The highest
Lewis base component of zoledronate in Table 4 is consistent
with this bisphosphonate having the highest binding affinity,
and with it being the strongest inhibitor of in vitro mineral
deposition and dissolution. These data suggest that the rank
order for promotion of surface nucleation is zoledronate >
alendronate > ibandronate > risedronate. Thus, the differences
among bisphosphonates in their effects on zeta potential and
interfacial tension have relevance in terms of bisphosphonate
interaction with the bone matrix, which may manifest clinically.
The binding of bisphosphonates to mineral is the basis for
the selective uptake of these compounds by bone. Accumulation
of these drugs on bone may also influence crystal behavior in a
way that contributes to their pharmacologic actions. The earliest
concept of how bisphosphonates retarded bone resorption was
that they inhibited crystal dissolution via a physical chemical
effect. This effect may contribute to the pharmacological actions
of bisphosphonates where larger molar doses are given (e.g.,
etidronate, clodronate, and pamidronate). In contrast, it is less
likely that more potent bisphosphonates, such as zoledronate,
risedronate, and alendronate, inhibit bone resorption through
physicochemical effects, since lower amounts are needed in
bone to inhibit resorption indicating the effects are likely to be
predominantly on cellular mechanisms.
The biological potency of individual bisphosphonates is
related to the important parameters of skeletal uptake/
retention and osteoclast inhibition. The results of these in
vitro binding experiments are consistent with what is known
about the relative skeletal uptake and retention of these
bisphosphonates in human subjects. There are few studies in
which skeletal uptake of different bisphosphonates has been

compared head to head in the same patient population.

Despite this, data derived from clinical studies suggest the
differences in initial skeletal uptake follow the same pattern
with a decreasing rank order of skeletal retention
(zoledronate > alendronate > risedronate > clodronate). For
example, the 24-h urinary excretion of one dose of
risedronate is 65%, while the corresponding excretion of
alendronate and zoledronate is 44% and 38%, respectively
[17,67,68]. In a recent pharmacokinetic study in humans, 14Clabeled risedronate and alendronate were compared. At 24 h,
there was a trend for increased retention for alendronate
compared to risedronate and there was a significantly higher
skeletal retention of alendronate at 72 h, consistent with its
higher mineral binding affinity [69].
The thermodynamically related phenomenon of desorption
or detachment of bisphosphonates from mineral is also an
important property in terms of how these drugs are delivered
to bone cells and exert their prolonged actions (Fig. 7).
Diffusion of bisphosphonates throughout the skeleton was
demonstrated by Francis et al. [70]. The adsorption and
desorption process may involve precipitation of bisphosphonate onto bone mineral and the solubility of the relevant
calcium-bisphosphonate salts may contribute to the resulting
equilibrium concentration of the bisphosphonate in solution.
The bisphosphonates with lower affinity would then be
predicted to produce the higher solution concentration
through desorption in the vicinity of the bone mineral, and
around bone cells such as osteoclasts. Thus, it is likely the
skeleton acts as a reservoir of bisphosphonate that produces
concentrations in solution locally in the vicinity of bone cells
that are related to the adsorption and desorption properties of
bisphosphonates on and off bone surfaces. This may explain
why the effects of bisphosphonates persist long after systemic
dosing is discontinued, and the differences among bisphosphonates appear to be in accordance with the physical
chemical affinity measurements reported here.
From the clinical point of view, the differences in mineral
binding affinity might be expected to influence how long it
takes for the inhibition of bone resorption to recover after the
bisphosphonate therapy is stopped. The speed of reversal of
effect after giving any bisphosphonate will depend not only on
the compound itself but also on the dose given and for how
long, as well as on the patient population studied. Thus, women
studied soon after menopause seem to recover more rapidly than
older women in whom bone turnover is lower, as has been
demonstrated with alendronate [71]. Unfortunately, there are no
head-to-head studies that allow direct comparisons to be made
among bisphosphonates. However, published data suggest the
effects of etidronate given cyclically wear off within a year [72],
and recent data indicate this is also true for risedronate [73].
These data appear to be in marked contrast to zoledronate which
displays a sustained inhibition of resorption for at least a year
after a single intravenous dose of 4 mg [14]. Reported studies
with alendronate also show an apparently long persistence of
effect after dosing; Khan et al. demonstrated bone resorption
remained suppressed for at least a year after short-term (3 days)
intravenous dosing with high doses. After oral dosing with

G.H. Nancollas et al. / Bone 38 (2006) 617627

alendronate at 10 mg/day for 5 years, there appears to be

continuing suppression of resorption for up to a further 5 years
after stopping [17], although interpretation of these results is
difficult because a placebo control group was not studied
alongside over the same time period. In general, these clinical
differences among bisphosphonates in terms of persistence of
effects mirror their affinities for mineral binding.
The long-term retention of the bisphosphonate may have
some disadvantages for treatment of certain populations such as
children and women of childbearing age, or when combination
therapies are needed (e.g., PTH) [74]. Long-term retention is
less likely to be a problem with lower-affinity compounds
whose effects are more rapidly reversed when therapy stops.
There also continues to be some concern about the long-term
use of bisphosphonates, particularly when there is marked
suppression of bone turnover such as that seen with some of the
higher-affinity drugs. Indeed, it may be unnecessary to suppress
to more than premenopausal levels in order to achieve maximal
fracture reduction [75].
The differences in reduction of bone turnover that have been
reported from clinical trials among the bisphosphonates given at
similar dose levels may be related to the differences in binding
affinity and binding capacity. The differences in the saturation
adsorption isotherms for risedronate and alendronate support
this concept with alendronate having a greater binding capacity,
possibly compensating for its inherent lower biochemical
potency [20,22].
In conclusion, the data described here reveal the differing
abilities of bisphosphonates to inhibit mineral growth in vitro,
and from this, kinetic affinity constants were derived. The
observed differences in binding affinities and in effects on
mineral surface properties relate to differences in the Ncontaining R2 functional groups, and will influence the rate and
extent of binding and release of the bisphosphonate with bone
mineral. The differences in kinetic affinity, zeta potential, and
interfacial tension are consistent with observed clinical
differences in potency, pharmacokinetics, and persistence of
effect among these bisphosphonates; thus, they likely contribute
to the clinical differences among the bisphosphonates, particularly when long-term dosing regimens are used.
Acknowledgments
We are grateful to Elaine B. Taylor for her assistance in the
preparation and editing of the manuscript. We thank the
National Institutes of Health (NIDCR) for a grant (DE03223)
in partial support of this work.
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