Journal of Equine Veterinary Science 34 (2014) 727–737

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Journal of Equine Veterinary Science

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Review Article

Bisphosphonates: Pharmacology and Clinical Approach to

Their Use in Equine Osteoarticular Diseases
Silvina Andrea Soto DVM *, Angelina Chiappe Barbará DVM, PhD
Department of Animal Physiology, School of Veterinary Science, Buenos Aires University, Argentina

a r t i c l e i n f o a b s t r a c t

Article history: Bisphosphonates (BPs) are drugs widely used in various bone diseases, mainly for their
Received 31 July 2013 skeletal antiresorptive effect. Nowadays, they are also used to treat degenerative pathol-
Received in revised form 30 December 2013 ogies such as arthritis because of their particular anti-inflammatory and analgesic prop-
Accepted 8 January 2014
erties. Since the sixties, BPs with different characteristics and biological activity profiles
Available online 17 January 2014
have been studied, although most of the pharmacologic and clinical trials have been car-
ried out in humans. By 2002, however, tiludronate was granted a marketing license in
Keywords:
France for use in horses. The objective of this article is to review the literature available on
Equine
Orthopedic disease BPs and apply this knowledge, either experimentally or clinically, to equine osteoarticular
Bisphosphonate disease therapy.
Osteoarticular disease Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction and geometric properties are inversely interrelated and

The effects of bisphosphonates (BPs) can be analyzed
from different points of view and different levels of bio-
logical complexity, that is, cells, tissues, and organs. But the
main pharmacologic effect of BPs is their whole bone
strengthening ability, which plays an important role on the
pathophysiology and evolution of osteoarticular ailments
in horses.
Bone strength is a very complex physiological property.
The ultimate strength of a bone before fracture is a com-
bination of its structural “stiffness” (resistance to defor-
mation to avoid crack generation) and “toughness”
(resistance to crack generation and progression). Both
stiffness and toughness of whole bone are exclusively
determined by tissue characteristics (or material proper-
ties) and by design features (or geometric properties) [1–3].
According to Frost “mechanostat” theory, bone material
* Corresponding author at: Silvina Andrea Soto, DVM, Department of
Animal Physiology, School of Veterinary Science, Buenos Aires University,
Chorroarin 280, C1427CWO Ciudad Autónoma de Buenos Aires,
Argentina.
E-mail address: ssoto@fvet.uba.ar (S.A. Soto).
biomechanically controlled in all vertebrates, apparently to
optimize the peak strains produced by maximal effort
under physiological conditions [4].
Furthermore, bones are elastic–plastic structures, and
their “elastic” (linear) pre-yield and “plastic” (nonlinear)
post-yield behavior is determined by different factors [5–7].
The pre-yield behavior of a bone depends chiefly on its
structural stiffness, which usually varies linearly with the
degree of mineralization [8–10] and the spatial distribution
of the mineralized tissue [11,12]. The post-yield behavior of
bones depends chiefly on bone structural toughness and is
determined by many additional factors, which can vary
regardless of tissue mineralization such as noncollagen
proteins, crystal quality and distribution, lamellar struc-
ture, density of lacunae, and microfracture density or
microdamage [13,14].
It is important to remark that at least part of the
inconsistency frequently observed in clinical studies
between positive effects of BPs on dual-energy X-ray
absorptiometry–bone mineral density (DEXA–BMD) and
bone strength, can be reasonable explained by the disso-
ciation that exists between strength, stiffness, and tough-
ness of cortical bone. No information is provided by DEXA

0737-0806/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jevs.2014.01.009
728 S.A. Soto, A. Chiappe Barbará / Journal of Equine Veterinary Science 34 (2014) 727–737
on the bone material or geometric properties that are the
true determinants of the bone structural properties.
Therefore, no DEXA data should be regarded as indicating
the state of or changes in bone strength or fragility.
Bisphosphonates effects on bone biomechanics are very
difficult to study primarily because of the complexity of
bone structure that differs from cortical or trabecular bone
[12] and also because there are many sources of variation
(type of compound, dose-time schedule, end points, and
individual variations: animal species, gender, age, meta-
bolic–endocrine status, and physical activity) in research
and clinical trials [15].
2. Pharmacochemistry and Pharmacokinetics of BPs
Bisphosphonates are synthetic organic compounds
characterized by C–P bonds in their chemical structure. Like
the germinal BPs used in the industry, BPs are analogs of
pyrophosphate with a carbon atom instead of an oxygen
atom. P–C–P bonds are resistant to high temperatures and
are chemically stable. They are also resistant to chemical
and enzymatic hydrolysis. Unlike pyrophosphate, this
chemical structure does not occur naturally, neither in
humans nor in animals. Besides, BPs are not biodegradable.
Bisphosphonates have a high affinity for metal ions and,
depending on the pH of the solution and on the metal, they
are capable of forming soluble and insoluble complexes and
aggregates. Their physicochemical properties are very
similar to those of pyrophosphate.
Bisphosphonates avidly bind to calcium phosphate
crystals and inhibit their growth, aggregation, and disso-
lution. Their bone mineral affinity is the basis of their use as
inhibitors of bone resorption [16]. The BP molecule features
necessary for biological activity have been clearly defined
in previous studies. The P–C–P bond is responsible for its
high binding affinity to hydroxyapatite (HAP) [17], and its
long permanence in bone crystals, but its half-life is still
unknown. The basic P–C–P structure can present different
changes, either by phosphate group esterification or by
changing the two side chains of carbon atoms, generically
named R1 and R2 [16].
Their mechanism of action differs according to
their biochemical structure. They can be “simple” or
nonnitrogen-containing BPs (etidronate, clodronate, and
tiludronate) and -amino or nitrogen-containing BPs, with a
nitrogen molecule that binds to its side chain R2. And in the
latter group, it is even possible to distinguish “alkyl-amino
BPs” (alendronate, pamidronate, ibandronate, and olpadr-
onate) and “heterocyclic nitrogen-containing BPs” (risedr-
onate and zoledronate). After absorption, BPs are taken up
by bone tissue and return to the bloodstream by physico-
chemical desorption, or by osteoclastic resorption, and
finally adhere again to bone, a process called “reattach-
ment” [18].
Recent studies show that the difference between the
individual BP profile of bone retention and effect duration
may be partially explained by particular BP HAP affinities.
After their administration, BPs are avidly taken up by bone.
Those compounds that bind more strongly to bone mineral
have a faster and more complete removal from blood-
stream. They also have a lower desorption and a higher
bone reattachment rate compared with compounds with
lower mineral affinity.
It has been suggested that BPs that display higher HAP
affinity, such as alendronate and zoledronate, undergo
continuous “bone recycling”, which could explain why
these drugs can be found in plasma and urine for long
periods after a single application [18]. On the other hand,
the difference in bone mineral affinity also causes a dif-
ferential diffusion of the drug into the bone compartments
[19]. Another important pharmacologic feature of BPs is
that the total dose administered determines their effect
(“total dose” principle). This phenomenon has been
described for several compounds, such as ibandronate and
zoledronate [20–22], and it is the basis for the design of
intermittent dosing regimens currently used in humans,
which can be on a monthly or even on a yearly basis.
Nevertheless, the use of intermittent dosing regimen that
allows a less frequent administration, but at higher doses, is
still under study because some side effects have not been
well characterized yet [17,23].
3. Pharmacodynamics and General Effects
Different BP effects must be considered: antiresorptive,
condroprotective, analgesic, anti-inflammatory, anti-
angiogenic, and adverse. However, the main effect of BPs is
the decrease in bone resorption. There are currently three
generations of BPs. The newer synthesized analogs show
greater antiresorptive activity and fewer side effects than
the older ones (Table 1).
The classical criterion for BPs comparison is based on
their antiresorptive effect [24], mainly mediated by osteo-
clasts (Table 2). The antiresorptive capacity of BPs is
dependent on both their binding affinity to bone mineral
and on their inhibitory action on osteoclasts [25].
However, it is important to note that for some com-
pounds such as alendronate, its higher mineral binding rate
can compensate its lower inhibition of osteoclast resorp-
tion activity, to achieve a determinant pharmacologic
potency. The molecular charge affecting bone surface
loading capacity [15] is another chemical feature that
should be considered.
The BPs scale in terms of their mineral affinity is
tiludronate > ibandronate > alendronate > pamidronate >
zoledronate, but considering farnesyl diphosphate synte-
thase (FPPS) enzyme inhibition, the scale is tiludronate >
pamidronate > alendronate > ibandronate > zoledronate
[15].
3.1. BPs Effects on Bone Cells
Bisphosphonates effects on bone cells are highly rele-
vant in determining bone mechanical properties at greater
Table 1
Bisphosphonates generation compounds
Generation Bisphosphonate Compounds
First Etidronate and clodronate
Second Tiludronate, pamidronate, and alendronate
Third Risedronate, ibandronate, and Zoledronate
 S.A. Soto, A. Chiappe Barbará / Journal of Equine Veterinary Science 34 (2014) 727–737
Table 2
Relative potency of the major bisphosphonates to inhibit bone resorption in the rat
1 10 100
Etidronate Clodronate and tiludronate Pamidronate
Adapted from Fleisch 1998 [24].
levels of complexity, tissue and organs, affecting bone
modeling and remodeling. The biochemical process
involved in the induction of osteoclast proapoptosis and
osteoblasts and osteocytes antiapoptosis will be described.
3.1.1. Osteoclasts
There is general consensus regarding the inhibitory effect
of BPs on osteoclast activity and principally on mature os-
teoclasts [15,16]. However, given that mature, multinucle-
ated osteoclasts result from the fusion of mononuclear
precursors of hematopoietic origin, BPs might also inhibit
bone resorption by preventing osteoclast formation [26–29].
Once BPs enter the osteoclastsdprobably by endocyto-
sisdthey have two main intracellular mechanisms of
action according to their molecular structure as follows:
Nonnitrogen-containing BPs
This group of BPs (etidronate, clodronate, and tiludro-
nate) are metabolized in the osteoclast and incorporated
into adenosine triphosphate (ATP) molecules, creating a
non-hydrolyzable analog known as “cytotoxic ATP” [30].
This beta, gamma-methylene ATP (AppCp)-type nucleotide
analog accumulates at higher concentrations inside the
osteoclast and is thus able to inhibit numerous intracellular
metabolic enzymes with detrimental effects on cell func-
tion leading to apoptosis [15,31].
In summary, the main mechanism of action of
nonamino-BPs is to induce apoptosis through the accu-
mulation of its metabolites, thus acting as prodrugs.
Nitrogen-containing BPs
Also known as amino-BPs, this subset of compounds
has greater antiresorptive potency than nonnitrogen-
containing BPs [31,32]. They alter the mevalonate pathway
responsible for the production of cholesterol, other sterols
and isoprenoid lipids such as isoprenyldiphosphate (iso-
prenyl pyrophosphate [IPP]), farnesyl diphosphate, and
geranylgeranyl diphosphate, affecting cell activity and sur-
vival by interfering with a posttranslational modification
process (prenylation) of key regulatory proteins necessary
for signaling [33].
Prenylation is necessary for the correct functioning
of small GTPases (Ras, Rab, Rho, and Rac), a large family
of hydrolase enzymes that can bind and hydrolyze guano-
sine triphosphate (GTP) and thereby regulate a variety
of important processes in osteoclasts, including cell
morphology, the arrangement of the cytoskeleton, brush
border formation, vesicular trafficking, and apoptosis [34].
The main mechanism of action by which nitrogen-
containing BPs prevent prenylation of GTPases is the
inhibition of FPPS, a peroxisomal enzyme, by acting as a
substrate analog.
729
>100 > 1,000 >1,000 > 10,000 >10,000
Alendronate Ibandronate Zoledronate
Because osteoclasts are unable to adequately replace the
abnormal proteins, the non-prenylated form accumulates.
The loss of osteoclast prenylation capacity may result in
the blockade of intracellular signaling processes, required
for osteoclast function. However, recent studies have sug-
gested that inhibited resorption is because of the accu-
mulation of non-prenylated GTPases in an “active” form,
rather than to the loss of protein prenylation capacity
[31,35]. The active form will generate an inappropriate
activation of downstream signaling pathways and exert a
dominant negative effect on signaling by sequestering ef-
fectors in non-productive cytoplasmic complexes.
On the other hand, the accumulation of IPP, as a result of
inhibiting FPPS, leads to the production of another
metabolite, triphosphoric acid 1-adenosin-50 -yl ester 3-(3-
methylbut-3-enyl) ester (ApppI), which, such as the
AppCp-type metabolites generated by “simple” BPs, also
leads to apoptosis [15]
Eventually, some nitrogen-containing BPs may cause
apoptosis not only by inhibiting protein prenylation but
also by accumulating the ApppI metabolite. Nevertheless,
apoptosis does not appear to be a requirement for inhibi-
tion by this type of BPs.
The FPPS–BP bond is also associated to the induction of a
FPPS enzyme conformational change, which is followed by
the binding of a second substrate IPP that confers greater
stability to the BP–FPPS inhibitory complex. This stabiliza-
tion is more evident in heterocyclic nitrogen-containing
BPs (risedronate and zoledronate) that have a higher
inhibitory effect as a result of the process when compared
with alkyl-amino-BPs such as alendronate, pamidronate,
ibandronate, and olpadronate [36].
Amino-BPs are also taken up by peripheral blood cells,
most likely monocytes, causing intracellular accumulation
of IPP by inhibiting FPPS. Although the mechanism by
which IPP diffuses is unclear, it is known that this metab-
olite is a ligand for a receptor present in a small subset of
T cells that possess a distinct T cell receptor (TCRg/d bearing
T cells).
The activation of these cells causes the release of tumor
necrosis factor alpha that triggers a proinflammatory, acute
phase reaction, leading to transient mild fever generally
after first exposure to the drug, especially after IV admin-
istration [37,38].
3.1.2. Osteocytes and Osteoblasts
The in vitro production of an osteoclast inhibitor by
osteoblasts previously exposed to BPs has been demon-
strated long ago [29,39] suggesting that part of the osteo-
clast inhibiting effect of BPs is mediated via a previous
effect on osteoblasts. However, such an observation
remains to be confirmed. Moreover, it has been shown that
BPs inhibit the apoptosis of osteocytes and osteoblasts and
induce the proliferation of osteoblasts both in vitro and
730 S.A. Soto, A. Chiappe Barbará / Journal of Equine Veterinary Science 34 (2014) 727–737
in vivo [40–46]. The antiapoptotic effect of BPs is inde-
pendent of the effect that these drugs have on osteoclasts.
Besides, analogs that have less or no antiresorptive activity
are still able to inhibit osteoblast and osteocyte apoptosis
in vitro [47,48].
In contrast to their effect on osteoclasts, the prosurvival
effect of BPs on cells of the osteoblastic lineage is exerted at
concentrations several orders of magnitude lower than the
ones required to inhibit osteoclast activity [43,49] indi-
cating that these effects are mediated by different
pathways.
Bisphosphonates have a concentration-dependent
effect on osteoblasts, and this effect is related to the rate
of proliferation and differentiation of cells of osteoblastic
lineage. Anabolic effects have been observed at lower
concentrations, ranging from 109 to 106, whereas an
inhibitory effect on growth and pro-differentiation activity
of these cells is exerted at values >105 [40,45,48,50,51]. It
was also shown that BPs exert a prosurvival effect on os-
teoblasts and osteocytes at similar doses and indepen-
dently of their biochemical structure [43,48–52].
The in vitro and in vivo inhibition of osteocyte
and osteoblast apoptosis caused by all BPs requires the
opening of connexin 43 (Cx43) hemichannels and the
subsequent activation of extracellular signal-regulated
kinases [43,44,53]. However, a recent study carried out
in vitro with alendronate suggests that Cx43 is dispensable
for BP binding and uptake, as well as to accomplish the
osteoblastic cell proliferative effects [46].
It has been hypothesized that BPs bind to another pro-
tein that, in turn, induces Cx43 hemichannel transduction
of antiapoptotic signaling and then after opening the
channel, BPs enter the cell by an independent mechanism.
3.2. BP Effects on Bone Mechanical Homeostasis
According to the bone mechanostat theory [4], bone
stiffness (and, indirectly, bone strength) is regulated by
osteocytes sensing the strain derived from the mechanical
usage of bones throughout the skeleton. This information is
transmitted to bone surface particularly to the lining cells
activating remodeling [54]. Thus the effect of BPs on bone
cells in terms of biomechanical connections between sen-
sors, osteocytes, and effectors, osteoblasts, and osteoclasts,
can be very important to understand their effect on bone
toughness. Parfitt et al [55] have confirmed that, in
humans, the density of live osteocytes per field surface unit
is largely the variable that best describes bone tissue stiff-
ness and strength and even fracture incidence.
However, because biopsies are not so easy to perform in
veterinary medicine, peripheral quantitative computed
tomography (pQCT) studies on bone geometry changes
would probably prove to be a simple practice to evaluate
biomechanical activity of long bones during horse training
[56,57]. Moreover, BPs’ effects can be extremely important
to the mechanical environment, optimizing bone geometry
in horses. Bisphosphonates can induce changes on long
bone geometry, mainly on the spatial distribution of
cortical bone. Cortical bone design, particularly the cross-
sectional moments of inertia, is the bone structural vari-
able best related to bone strength at the organ level [14,58].
3.3. BP Effects on Bone Toughness. Antiresorptive Effects
When a bone is subjected to an increasing load, there is
an initial resistance to deformation (stiffness) until the first
crack is produced. And then the whole bone structure re-
sists the progression of subsequent cracks (toughness) until
fracture is completed. Both properties depend inversely on
the degree of tissue mineralization (BMD). Instead, bone
structure stiffness and toughness are positively associated
to the architectural quality of bone design. The effects of
BPs on bone design will always improve bone structural
toughness.
However, the effects of BPs on bone material stiffness
and toughness are virtually unpredictable; they can
improve or impair bone tissue toughness. On one hand, by
blocking bone remodeling, not modeling, BPs can increase
bone collagen age, degree of mineralization, and crystal-
linity. This effect is known to greatly increase bone tissue
stiffness and hence reduce bone tissue toughness [59,60].
On the other hand, by stimulating osteoblasts, BPs can
also improve not only bone mass but also some aspects of
the microstructure of bone material [48] as circumferential
lamellar bone replacement by cylindrically shaped osteons
increasing its toughness [61].
The possible occurrence of both positive and negative
effects of BPs on bone material properties has been pro-
posed and experimentally supported by Kashii et al [62].
3.4. The Integrated BP Effects on Whole Bone as an Organ
Bisphosphonates are able to decrease bone resorption
not only through their direct osteoclast apoptotic effect but
also by preventing osteoblast and osteocyte apoptosis
[43,44]. Sometimes, especially in young patients, for the
treatment of bone fragility when a decrease in bone
remodeling is not desirable, it may be necessary to preserve
the viability of osteoblasts and osteocytes without inducing
osteoclast apoptosis. Novel analogs that have no anti-
resorptive effects but still retain antiapoptotic activity on
cells of the osteoblastic lineage may be of great value in
treating such osteopenic conditions in early skeletal
development period [43,48]. However, at normal thera-
peutic dosages of currently available BPs, the antiresorptive
osteoclastic effect predominates over the beneficial effect
on osteoblasts and osteocytes survival.
The ability of BPs to access the osteocytes network is
inversely related to their mineral binding affinity. There-
fore, BPs with lower affinity are able to penetrate deeper
into the bone and bind to osteoblasts and osteocytes
[19]. Because the overall effect of BPs depends on their
biochemical, pharmacokinetic, and pharmacodynamics
properties, unique to each compound, extrapolation from
one specific BP to another should always be done with
caution.
The prolonged and strong inhibition of bone resorption
caused by BPs, mainly by the first generation compounds,
may lead to increased bone fragility because of the inability
of bone to replace old tissue by new tissue in answer to a
biomechanical stimulus, with the consequent difficulty to
repair microfractures [16]. Several studies performed on
animal models have demonstrated that BPs increase
 S.A. Soto, A. Chiappe Barbará / Journal of Equine Veterinary Science 34 (2014) 727–737
microdamage in a dose-dependent manner, although no
apparent adverse consequences on whole bone mechanical
properties have been reported [23,63]. Hence, the impor-
tance of studying the interaction of these drugs and the
relationship between the geometric properties and bone
mineralization were governed by mechanical forces or
bone mechanostat [4,5]. Although the general effects of BPs
on bone structure and biomechanics have been studied in
detail in our laboratory in different ovariectomized osteo-
penic rat models [1,64–67].
The mechanical properties of long bones in bending are
known to depend on (1) the mechanical quality of cortical
bone as expressed by its intrinsic stiffness or elastic
modulus (E) and (2) the cross-sectional diaphyseal archi-
tecture, as indicated by axial cross-sectional moment of
inertia (xCSMI) [68,69]. The xCSMI and volumetric BMD of
cortical bone can be evaluated by pQCT, and the last term
has been regarded as one of the chief biological
determinants of E of solid bone tissue [2]. This is why it is
important to perform studies in horses applying BP therapy
in concordance with pQCT evaluation of large bone geom-
etry. Some of this data can be extracted from pQCT studies
carried out by Firth [56,57] in healthy horses.
The real existence of potentially microdamage induced
by BPs in humans and in animals has not been established
so far. Results obtained in different studies are controver-
sial, mainly with regard to the long-lasting anti-remodeling
effects of these compounds [70,71].
Like any structure bearing repetitive loads, bone accu-
mulates microdamage. However, unlike inert materials,
biologically active bone is able to sense microdamage and
repair it to maintain its mechanical competence[72]. The
use of BPs at low doses has been considered safe in bone
fractures inducing the formation of normal size bone callus,
and when the repaired process is completed, greater me-
chanical strength and higher bone calcium content is ach-
ieved [16,73,74].
3.5. Chondroprotective Effects
There are many opinions regarding the usefulness of BPs
in the treatment of osteoarthritis (OA) [75,76]. In recent
articles, the use of BPs as metalloproteinase inhibitors has
been suggested [77–79], and it has been demonstrated that
in vitro tiludronate can inhibit the activity of matrix met-
alloproteinases (MMP-1 and MMP-3), thus supporting the
idea that one of the mechanisms by which BPs accomplish
their effect is by chelating cations that are used as enzyme
cofactors [80].
In another in vitro study [81], tiludronate was able to
inhibit the interleukin (IL) 1–mediated secretion of carti-
lage matrix–degrading enzymes by chondrocytes and
synovial cells.
In a dog anterior cruciate ligament transaction model
follow by reconstructive surgery, the cartilage volume loss
was greatly prevented and concomitant treatment with
tiludronic acid provided an additional benefit reducing
experimental OA lesions [82].
Other articles also support the chondroprotective effects
of alendronate in OA by inhibiting tumor necrosis factor
731
beta, probably through the inhibition of MMP-13 and
MMP-9, and the reduction of cartilage neovascularization.
The alendronate inhibitory effect on MMP-9 could be
explained by its action on osteoclasts; however, its effect on
the MMP-13 expression in chondrocytes is less understood
and requires further research [75].
In an established adjuvant arthritis rat model, other
authors have suggested that the cartilage protective effect
of incadronate is possibly because of BPs acting on a specific
phagocytic cell, the chondroclast [83]. The literature also
supports the hypothesis that subchondral bone pathology
plays an important role in the development of chronic
cartilage lesions [84–87]. Thus, by regulating bone turn-
over, BPs might prevent the onset and progression of sub-
chondral bone–associated injuries.
3.6. Analgesic, Anti-Inflammatory, and Anti-Angiogenic
Effects of BPs
Excessive bone resorption and cartilage degradation, as
well as the inflammation of those tissues, are often painful.
It has also been found that angiogenesis is closely related to
bone inflammation [88,89]. Over the past 5 years, there has
been increasing interest in assessing the effects of BPs in
inflammatory and degenerative disease and in cancer
therapy [90–94]. In a trial conducted on knee OA in
humans, clodronate proved to relieve pain and improve
joint extension and mobility [95]. In addition, the analgesic
properties of zoledronate in a rat model with degenerative
joint disease have also been demonstrated [96].
The analgesic properties of BPs may be explained by
several mechanisms. In a rat model of persistent inflam-
matory pain, the pain relief caused by ibandronate was
attributed to its modulatory effect on the secretion of pain
soluble mediators, such as substance P, among others [97].
Anti-inflammatory properties have also been demon-
strated in vitro for tiludronate. This BP seems to inhibit
vascular endothelial growth factor synthesis induced by
prostaglandin F2a in osteoblasts [98], and cytokine and
nitric oxide secretion from activated macrophages in a
dose-dependent manner [99]. The synthesis of osteoblastic
IL-6 with resorptive and anti-osteogenic effects has also
been found to decrease [100,101].
Prospective, randomized, controlled, double-blind
in vitro studies to assess the effects of tiludronate have
shown a positive effect on gait impairment and joint
symptoms in a dog model with OA. The latter effect im-
proves OA structural changes and reduces the synthesis of
catabolic and inflammatory mediators, such as prosta-
glandin E2 (PGE2) in synovial fluid, MMP-13 and a dis-
integrin and metalloproteinase with thrombospondin
motifs 5 in cartilage, and cathepsin K in subchondral bone
[102].
Another mechanism proposed to explain the analgesic
properties of BPs is the inhibition of the osteoclastic
vacuolar-type (Hþ)-ATPase (V-ATPase), which creates an
acidic microenvironment during bone resorption by
secreting protons. Compounds such as zoledronate can
prevent acidosis by inhibiting the osteoclast V-ATPase thus
preventing pain caused by the activation of acid sensitive
732 S.A. Soto, A. Chiappe Barbará / Journal of Equine Veterinary Science 34 (2014) 727–737
ion channels in sensory nerve fibers [103]. This mechanism
may also explain the additional analgesic effect of tiludro-
nate [104].
The anti-angiogenic effect of zoledronate, alendronate,
pamidronate, and other BPs has been demonstrated both
in vitro and in vivo. That effect could be of clinical use not
only to treat bone inflammatory disorders as arthritis [1]
but also to treat some malignancies, which involve an
angiogenic process [105–107]. The strongest angiogenesis
inhibiting activity has been observed in nitrogen-
containing BPs. However, results obtained in a recent
study suggest that such inhibition was exerted in a FPPS
independent manner [108].
3.7. Side and Adverse Effects
In human medicine, some adverse effects have been
reported for BPs, even when administered at therapeutic
doses. These effects are generally mild and transient and
include hypocalcemia, especially after IV administration at
a relatively high dose.
Gastrointestinal irritation and kidney damage have
also been reported after oral administration, when
combined with aminoglycosides. Other less frequent ef-
fects, such as bone, joint, or muscle pain, atrial fibrillation,
ocular inflammation, esophageal cancer, and atypical frac-
tures, have also been associated to long time use of BPs
[16,71,109].
In recent years, a serious rare effect known as jaw
osteonecrosis (ONJ) has been observed and associated to IV
administration of amino-BPs, such as zoledronate and
ibandronate. However, that effect was only observed after
high doses and long-term administration, such as those
used in the treatment of cancer. Nevertheless, the latter
adverse effect cannot be directly attributed to BPs, because
ONJ is a multifactorial pathology [71,109–111].
4. Application of BPs in Orthopedics and
Osteoarticular Pathologies in Horses
In horses, as in humans, many orthopedic disorders are
characterized by intense bone remodeling, which can lead
to osteolysis because of excessive resorption and pain [112].
The use of BPs is an interesting alternative in the treatment
of these pathologies with high bone turnover. Because
certain BPs have been shown to have a protective effect on
cartilage to prevent damage [74,112], these active drugs
could also be an attractive treatment option for inflam-
matory, degenerative joint disease. To date, three BPs have
been used in equine medicine: pamidronate, tiludronate,
and zoledronate [113–119].
Pamidronate was the first BP studied in horses, [113].
This compound has proven to be safe when administered
IV at a dose of 0.4 mg/kg/d or 0.8 mg/kg/d for 15
consecutive days. In another study, this BP was shown to
be safe at a total dose of 0.6–0.8 mg/kg administered two
or three times every 7–10 days, depending on horse age
[120]. And in vitro study [121] to measure horse osteoclast
resorptive activity was carried out using different con-
centrations of pamidronate over a 7-day period. The study
demonstrated that high doses of pamidronate reduce bone
resorption, whereas low doses induce a transient increase
in bone resorption. This effect, attributed to an acute
phase reaction of pamidronate, also caused a dose-
dependent decrease in osteoclast counts, after a culture
of 7 days. This finding was probably because of increased
cell death rather than to a decrease in osteoclast forma-
tion. Paradoxically, pamidronate seems to produce a mild
and transient increase in osteoclast formation, but this
effect is reverted after the seventh day of culture. Data
from this in vitro study support the potential use of
pamidronate in equine orthopedic diseases, characterized
by increasing bone remodeling and provide useful infor-
mation for future pharmacokinetic studies and clinical
trials which should be aimed at achieving effective local
concentrations of the drug.
Most of the studies on osteoarticular treatments per-
formed in equines have been carried out with tiludronate
or pamidronate. Tiludronate was the only BP that obtained
a marketing license for equine medicine use as vials con-
taining 50 mg in several European countries. However,
license for vial containing 500 mg was suspended by the
European Commission on July 2010 [122]. Tildren has not
yet been approved by the Food and Drug Administration
(FDA). The importation of Tildren in to the United States is
not permitted unless authorization has been granted by the
FDA for compassionate use [123].
Tiludronate is the BP most studied in horses in terms of
pharmacodynamic side effects and efficacy [124,125]. In
equines, the plasma protein binding to tiludronate is about
80–85%, and this compound does not undergo biotrans-
formation; thus, the intact molecule is eliminated almost
exclusively in urine. After 10 daily IV doses of 0.1 mg/kg,
30–50% of the administered dose is fixed to bone. The latter
phenomenon also occurs with other BPs.
Once inside the bone, the distribution of the drug is not
homogenous, with the highest concentrations being found
at sites of high remodeling and cancelous bone, rather than
in bone cortex [126]. The tolerance and the effects of this BP
during the short-term IV administration were evaluated in
one study [127]. The authors concluded that the slow IV
infusion of 1 mg/kg of tiludronate in horses was well
tolerated with no significant adverse effects. The drug was
also found to induce a rapid and marked decrease in serum
C terminal telopeptides of type I collagen (CTX-1) thus
evidencing their antiresorptive effect without affecting
bone formation.
Another study compared the bioavailability of two
different schedules of IV administration of tiludronate and
their effect on the resorption marker CTX-1. The results of
this study suggested that both dosages produced similar
plasma exposure and pharmacologic effects in healthy
adult horses. In both systems, a decrease of CTX-1 plasma
was demonstrated. Nevertheless, the response of bone
resorption marker CTX-1 to the drug seemed to be less
reproducible when the total dose of 1 mg/kg was admin-
istered as 10 daily doses, as opposed to a single dose [125].
Further, the CTX-1 difference in the latter group was still
significant on day 3.
This finding could have practical implications, because
it appears that the administration of 10 consecutive doses
could be replaced by a single one. This effect has also
 S.A. Soto, A. Chiappe Barbará / Journal of Equine Veterinary Science 34 (2014) 727–737
been described in human medicine as the “total dose
principle”.
As in the case of humans, the most frequently described
side effect of this drug in horses is gastrointestinal pain
when administered as an IV infusion [125,127,128]. On the
other hand, it has been found that the intra-articular in-
jection of tiludronate in the equine distal inter-phalangeal
and metacarpal-phalangeal joints quickly diffuses from
the joint into the bloodstream inducing synovial mem-
brane inflammation, as confirmed by histologic examina-
tion [129].
As to the safety of zoledronic acid in horses, there is only
one recent study where the pharmacokinetics and phar-
macodynamics of the drug have been determined. Venous
blood was collected at several time points and the zole-
dronic acid concentration in plasma was measured in eight
healthy animals, after administration of a single IV dose.
Mean total calcium concentrations in plasma were found to
be lower than the reference range 7 days after zoledronic
acid administration. Besides, severe hypocalcemia without
clinical signs was observed in one horse 11 days after the
zoledronate infusion. Neither fever nor colic was observed
in any of the horses [130].
Results of these studies suggest that zoledronic acid can
be safely used in horses as a continuous IV infusion over
30 minutes at a dose (0.057 mg/kg) similar to that recom-
mended for humans (4 mg, label dose for the treatment of
osteoporosis and Paget disease). In horses, zoledronate
induced a longer effect on bone turnover than tiludronate.
However, authors recommend evaluating kidney function
and limiting dental procedures before and after adminis-
tration. They also suggest measuring calcium concentration
before and after treatment and offering horses either a diet
rich in calcium (alfalfa) or oral calcium supplementation
beginning 1 week before infusion until at least 3 weeks after
treatment. Pretreatment of horses with flunixinmeglumine
is also advisable to minimize the risk of colic and the
adverse effects associated with acute phase reaction [130].
4.1. Potential Indications for BP Therapy
4.1.1. Navicular Syndrome (Navicular Disease)
Navicular syndrome is primarily considered chronic
intermittent forelimb lameness, associated with an aseptic
and chronic inflammation of any of the anatomical ele-
ments that form the foot trochlear, including the navicular
bone and closely related structures as suspensor ligaments,
distal sesamoidean impair ligaments, deep digital flexor
plantar aponeurosis, and podotrochlear bursa [131]. It is a
disease with a complex, multifactorial etiology. Conditions
affecting any of the previously mentioned structures may
cause pain and result in lameness. Concurrent soft tissue
and navicular bone abnormalities are also common.
There is increasing evidence that abnormally high loads,
that is, nonphysiological biomechanical forces leading to
tissue degeneration are the most likely cause of navicular
disease [131]. Abnormal forces on the navicular bone could
arise from either excessive physiological loads applied to a
foot with normal conformation or normal loads applied to a
foot with abnormal conformation [132]. Those excessive
733
mechanical stresses increase navicular bone remodeling,
leading to lameness.
Areas of increased bone resorption and formation
(increased remodeling and high bone turnover) are often
typical of lesions within a diseased navicular bone [131],
supporting the potential of BPs to treat this condition.
In a preliminary clinical trial [114], pamidronate at a
monthly dose of 1 mg/kg, or placebo, were administered by
three times to a group of 11 horses with navicular syn-
drome after 3 months of corrective shoeing. The evaluation
was conducted 1 month after the last administration of
pamidronate. Although the dose was well tolerated by
horses with no side effects, no clear-cut conclusions could
be drawn from this study.
A more recent double-blind study compared treatment
with two different doses of tiludronate administered over
10 days to animals with signs of navicular disease [116].
That study demonstrated that horses treated with the
higher total dose (1 mg/kg bwt) showed optimal
improvement of lameness and returned to normal level of
activity after 2–6 months of treatment, whereas in the
group that received the lower dose (0.5 mg/kg bwt), results
were similar to placebo. In chronic cases, no significant
treatment efficacy was observed at any dose.
4.1.2. Distal Tarsal OA (Bone Spavin)
Bone spavin is a type of OA and synovitis that involves
distal intertarsal, tarsometatarsal, and occasionally prox-
imal intertarsal joints. Distal tarsal OA is considered to be
the most common cause of tarsal lameness [131]. Like other
osteoarthritic processes, bone spavin is a common end-
stage multifactorial condition involving conformational
defects and traumatic lesions among many others.
It is common to find radiographic changes associated to
the tarsal joint as osteophytes, subchondral bone lyses, and
narrowing of the interarticular space. However, these
findings do not always correlate with clinical outcomes,
and horses that present such radiological signs may be
lame or not. Conversely, the absence of radiographic signs
does not rule out the condition (occult or blind spavin) and
intra-articular anesthesia may be required to confirm
diagnosis, because at the early stage of the disease there
can only be synovitis [131]. Two different clinical trials
studied the efficacy of tiludronate to treat lame horses with
bone spavin clinical diagnosis associated to radiological
bony signs. The improvement in lameness clinical score
was optimal after 2 months of treatment [115,118].
4.1.3. Fetlock Suspensor Ligament Enthesopathy
Enthesopathy is a condition that occurs at the site of
insertion of tendons or ligaments to bone. Unusual and
excessive strain to the fetlock region may produce an injury
where the suspensory ligament attaches to the sesamoidean
bones. In sesamoiditis, the typical signs are osteolysis along
the vascular channels and bone proliferation at the abaxial
edge of the sesamoid. Enlarged channels in sesamoiditis
indicate increased bone resorption [131]. This may repre-
sent the initiation of the remodeling response to bone stress
by training or it may reflect an increase in blood flow
because of inflammation and injury to the suspensor liga-
ment, or both [133].
734 S.A. Soto, A. Chiappe Barbará / Journal of Equine Veterinary Science 34 (2014) 727–737
The condition is usually associated with pain causing
lameness as a result of inflammation at the ligament–bone
interface. Pamidronate and tiludronate may be effective for
treating the bony component of this condition and other
enthesopathies [78,112].
4.1.4. Axial Skeleton Osteoarthritic Disease
Back pain has been identified as a cause of poor per-
formance or locomotion failure in horses. Orthopedic
lesions are considered to be the primary cause of pain
associated with the thoracic–lumbar vertebral column. To
date, there is only one study concerning the use of tiludr-
onate in this equine axial skeleton disease [117]. All horses
included in that study had clinical manifestations of pain
associated with the thoracic–lumbar vertebral column and
obvious osteoarthritic changes most of which were in the
lumbar region. In that study, authors reported a higher rate
of clinical improvement in dorsal flexibility between days
0 and 60 in the treated group (12/15) as compared with
placebo (7/14), and this improvement was evident even
after 120 days. The highest positive response was recorded
after 60 days posttreatment. The authors concluded that
tiludronate might be an effective alternative in the man-
agement of horses with this condition and potentially in
the treatment of other intervertebral lesions and associated
pain. It has been proposed that tiludronate regulates the
deleterious effect of abnormal bone remodeling associated
to OA of articular processes of the synovial intervertebral
joints and to other pathological bone conditions in the
dorsal region. Tiludronate not only stabilizes bone lesions
but also may relieve pain by restoring the balance between
bone resorption and formation in pathological remodeling
process.
4.1.5. Subchondral Bone Cysts
Subchondral bone cysts are signs of osteochondrosis
and can also be found in arthritic processes. They represent
areas of subchondral bone osteolysis. One study showed
that the fibrous content of equine subchondral bone cysts
can produce prostaglandin E2 (PGE2) as well as nitric oxide
(NO), and the neutral metalloproteinases (NMPs) and that
the concentration of PGE2 was higher in cystic fibrous
tissue than in compared with joint synovial membrane
and articular cartilage. Further, conditioned media of ex-
plants of subchondral bone cyst fibrous tissue were able to
recruit osteoclasts and increase their activity in a bone
resorption assay [134]. This active bone resorption may
play a role in the pathogenesis of these lesions and is
responsible for their expansion and difficult treatment.
Bisphosphonates not only have antiresorptive effects but
also anti-inflammatory effects, inhibiting mediators and
metaloproteases. It has been suggested that the anti-
inflammatory effect is probably because of their ability to
chelate zinc, which is needed for these enzymes as a
cofactor [74,76,85].
4.1.6. Bone fragility disorder
Bone fragility disorder (BFD) is a progressive, debili-
tating condition, involving primarily axial skeleton and
proximal aspects of the appendicular skeleton, which has
been described in horses from the coastal region of
Northern California. A recent study provides histophato-
logic evidence that BFD involves abnormal osteoclastic
mediated bone resorption [135] BPs could therefore aid in
the treatment of horses with this disorder. Based on their
own previous findings using tiludronate in this disease,
Katzman et al. tested zoledronate at 0.075 mg/kg via a
slow, 30-minute IV infusion. Ten horses, diagnosed with
BFD by clinical examination and nuclear scintigraphy, were
treated with this drug. Six months later, clinical improve-
ment, defined as improvement in the lameness score,
resolution of signs of musculoskeletal pain, or both, was
detected in 9 of 10 horses. Moreover, no adverse effects
were detected [119].
5. Conclusions
In human medicine, the treatment with BPs has been
demonstrated to be effective in many bone diseases asso-
ciated to excessive bone turnover, and studies are being
performed to test their use in degenerative and inflam-
matory joint disease, such as OA and rheumatoid arthritis.
The final BP effect on whole bone strength and osteo-
articular diseases in horses can be unpredictable because
the true clinical outcome of the treatment depends on the
particular BP used, the dose-dependent effect, and the
administration schedule. Although BPs share many prop-
erties, it is clear that, as in humans, the outcome after
treatment of a certain bone disease differs between indi-
vidual compounds, and therefore, each BP must be studied
independently. To extrapolate the results obtained with
one BP to another BP is almost impossible mainly because
of the fact that the mechanisms of action may differ from
one to another, for example, pamidronate or tiludronate.
The comparison between studies is also hindered by the
heterogeneity of conditions used as described previously.
Moreover, some noninvasive determinations of the true
indicators of both bone material and geometric properties,
as those provided by quantitative computed tomography or
pQCT scans, are necessary in the study of horse skeletal
effects of BPs. Furthermore, any effect on bone mechanical
properties must exclusively come from effects exerted on
bone material quality, bone design, or both.
Because there are potential pharmacologic species-
specific differences, it is impossible to extrapolate data,
and further equine orthopedic research is necessary to
determine BP effectiveness in OA.
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