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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
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
618
Table 1
Bisphosphonate structures
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
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
620
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. 3. Langmuir adsorption isotherm plot for the kinetic growth results.
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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.
622
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
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
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%.
624
625
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