Effects of the bisphosphonate ibandronate on hyperalgesia, substance P, and cytokine levels in a rat...

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European Journal of Pain 12 (2008) 284–292

Effects of the bisphosphonate ibandronate on hyperalgesia,substance P, and cytokine levels in a rat model

of persistent inflammatory pain

Mauro Bianchi a,*, Silvia Franchi a, Paolo Ferrario a,Maria Luisa Sotgiu b, Paola Sacerdote a

a Department of Pharmacology, University of Milano, Via Vanvitelli 32, 20129 Milano, Italyb Institute of Bioimages and Molecular Physiology, CNR, Via Fratelli Cervi 93, 20090 Segrate, Italy

Received 27 March 2007; received in revised form 29 May 2007; accepted 14 June 2007Available online 30 July 2007

Abstract

The anti-inflammatory and analgesic properties of different bisphosphonates have been demonstrated in both animal and humanstudies. Ibandronate is a third-generation bisphosphonate effective in managing different types of bone pain. In this study we inves-tigated its effects in a standard pre-clinical model of inflammatory pain. We evaluated the effects of a single injection of differentdoses (0.5, 1.0, and 2.0 mg/kg i.p.) of ibandronate on inflammatory oedema and cutaneous hyperalgesia produced by the intrapl-antar injection of complete Freund’s adjuvant (CFA) in the rat hind-paw. In addition, we measured the effects of this drug (1.0 mg/kg i.p.) on hind-paw levels of different pro-inflammatory mediators (PGE-2, SP, TNF-a, and IL-1b). We also measured the levels ofSP protein and of its mRNA in the ipsilateral dorsal root ganglia (DRG). Ibandronate proved able to reduce the inflammatoryoedema, the hyperalgesia to mechanical stimulation, and the levels of SP in the inflamed tissue as measured 3 and 7 days followingCFA-injection. This drug significantly reduced the levels of TNF-a and IL-1b only on day 7. On the other hand, the levels of PGE-2in the inflamed hind-paw were unaffected by the administration of this bisphosphonate. Finally, ibandronate blocked the overex-pression of SP mRNA in DRG induced by CFA-injection in the hind-paw.

These data help to complete the pharmacodynamic profile of ibandronate, while also suggesting an involvement of severalinflammatory mediators, with special reference to substance P, in the analgesic action of this bisphosphonate.� 2007 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. Allrights reserved.

Keywords: Hyperalgesia; Ibandronate; Interleukin-1; Prostaglandin E2; Substance P; Tumor necrosis factor-a

1. Introduction

In the last few years bisphosphonates have becomeimportant in the management of pain in patients withmetastatic cancer or affected by postmenopausal osteo-porosis (Emkey et al., 2005; Body, 2006). Ibandronate

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doi:10.1016/j.ejpain.2007.06.005

* Corresponding author. Tel.: +39 02 50316930; fax: +39 0250316949.

E-mail address: [email protected] (M. Bianchi).

is a third-generation, nitrogen-containing, bisphospho-nate which has been shown to inhibit osteoclast-medi-ated bone resorption in women with postmenopausalosteoporosis, and to prevent skeletal-related events,improve quality of life, and reduce pain in patients withmetastatic bone disease (Body et al., 2004; Heidenreichand Ohlmann, 2004; Cameron et al., 2006; Croom andScott, 2006). Several studies have shown that bisphos-phonates of the first and the second-generation such asalendronate, clodronate and pamidronate, can exert

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M. Bianchi et al. / European Journal of Pain 12 (2008) 284–292 285

analgesic effects in experimental animals (Goicoecheaet al., 1999; Bonabello et al., 2001; Bonabello et al.,2003). Some data are available on the anti-nociceptiveeffects of the third-generation bisphosphonate zoled-ronic acid in rodents (Walker et al., 2002). The effectsof ibandronate in animal models of persistent pain,however, have yet to be investigated in detail.

The mechanism by which bisphosphonates maydecrease pain is largely unknown. In fact, the main effectof all the drugs belonging to this pharmacological fam-ily, including ibandronate, is to reduce bone resorptionby inhibiting osteoclast function (Fleisch, 1991; Russelland Rogers, 1999; McCormack and Plosker, 2006). Thisbiological action, however, does not fully explain theiranalgesic efficacy. In particular, the rapid appearanceof pain relief after the administration of ibandronatein patients with bone disease from breast cancer(Heidenreich et al., 2004) suggests a possible dissocia-tion between the analgesic and the metabolic effects ofthis compound.

Some anti-inflammatory effects of different bisphos-phonates, including ibandronate, have been investigatedin a few number of studies (Santini et al., 2004; Baussand Body, 2005; Toussirot and Wendling, 2005;Yamamoto et al., 2006). Nevertheless, the possibleanti-inflammatory action of ibandronate is still contro-versial (Zysk et al., 2003).

For all these reasons, we thought it would be of inter-est to explore in the present study the effects of ibandr-onate using an animal model of prolonged noxiousstimulation in which a single injection of complete Fre-und’s adjuvant (CFA) in the hind-paw caused inflamma-tory hyperalgesia (Stein et al., 1988 ). In addition toinvestigating the effects of this drug on inflammatoryoedema and mechanical hyperalgesia, we measured thechanges in the hind-paw levels of prostaglandin E-2(PGE-2), substance P (SP), tumor necrosis factor-a(TNF) and interleukin-1b (IL-1). Finally, we evaluatedthe changes in SP production in primary afferent sensoryneurons following the administration of ibandronate innormal and inflamed animals.

2. Methods

2.1. Animals

Male Sprague Dawley albino rats (Charles River,Calco, Italy) weighing between 200 and 250 g were used.The animals were housed 4 to a cage, at 22 ± 2 �C with alight-dark cycle of 12/12-h and free access to water andfood. The rats were allowed to habituate to the housingfacilities for 1 week before the experiments began.Behavioural studies were carried out in a quiet roombetween 10.00 and 12.00. Eight rats were used in eachexperimental group.

All procedures were approved by the Department ofPharmacology of the University of Milan Animal Careand Use Committee and followed the ethical guidelinesfor the treatment of animals of the International Associ-ation for the Study of Pain (Zimmermann, 1983). Allefforts were made to minimize the number of animalsand their suffering.

2.2. Induction of inflammation and drug treatments

Peripheral inflammation was induced by the injectionof a suspension of 0.1 mg/0.1 ml complete Freund’sadjuvant (CFA) containing heat-killed and dried myco-bacterium tuberculosis (H37Ra, ATCC 25177) in 85%paraffin oil and 15% mannide monooleate into the plan-tar surface of the left hind-paw. Control animals wereinjected with 0.1 ml of saline in the left hind-paw.

Ibandronate ([1-hydroxy-3-(methylpentylamino)pro-pylidene]bis-phosphonic acid) was supplied by RocheDiagnostics GmbH, Mannheim, Germany. The drugwas dissolved in saline and administered by intraperito-neal route (i.p.) in a volume of 0.2 ml/100 g bw. Thedrug injection was performed 1 h after the paw injectionof CFA. Control animals were treated i.p. with the samevolume of saline.

In the first set of experiments (evaluation of the effectson inflammatory oedema and hyperalgesia), ibandronatewas administered at the doses of 0.5, 1.0 and 2.0 mg/kg.For the further studies (evaluation of the effects on tissuelevels of different inflammatory mediators), the dose of1.0 mg/kg of ibandronate was chosen.

2.3. Evaluation of inflammatory oedema and hyperalgesia

The intensity of inflammatory oedema and hyperalge-sia was measured on day 1, 2, 3 and 7 after the intraplan-tar injection of CFA or saline. The paw swelling(oedema) was assessed by measuring the volume of bothhind-paws by a plethysmometer (7150 Plethysmometer,Basile, Comerio, Italy). The results are expressed as thealgebraic difference between the volume (ml) of inflamed(CFA-injected) and normal (saline-injected) hind-paw.

The Randall-Selitto paw-withdrawal test, which usesmechanical force as nociceptive stimulus, was used tomeasure inflammatory hyperalgesia. The stimulus wasapplied with an analgesymeter (Basile, Comerio, Italy)which generates a linearly increasing mechanical force,applied by a conical piece of plastic with a dome-shapedtip on the dorsal surface of the rat’s hind-paw. The ani-mals were gently held and incremental pressure (maxi-mum 250 g) was applied onto the dorsal surface of thehind-paw. The thresholds represent the pressure(expressed in grams) at which the animal withdrew itshind-paw. The results are expressed as the algebraic dif-ference between the thresholds measured on the rightand left (inflamed) hind-paw.

286 M. Bianchi et al. / European Journal of Pain 12 (2008) 284–292

The observer was blind to treatment allocation of theanimals.

2.4. Measurement of PGE -2, SP, TNF-a and IL-1b in

paw skin

On days 3 and 7 following the intraplantar injectionof CFA or saline the animals were killed by decapitationand the entire hind-paw skin was removed. The tissuesamples were weighed, frozen on dry ice and stored at�70 �C until further processing for PGE-2, SP, TNF,and IL-1 measurements (Bianchi et al., 2004).

Alter homogenization of skin tissue in phosphate buf-fered saline (PBS), PGE-2 extraction and purificationwas performed using a solid phase method utilizing100 mg Amprep C18 minicolumns (GE Healthcare, Col-ogno Monzese, Italy). The samples were eluted in ethylacetate, and evaporated to dryness in a Savant VacuumCentrifuge apparatus.

Quantitative determination of PGE-2 was performedby the enzyme immunoassay using a commerciallyavailable EIA kit (GE Healthcare, Cologno Monz-ese, Italy). The sensitivity of the PGE-2 EIA kit was50 pg/ml.

For the measurements of SP skin samples werehomogenized in 2 ml of 0.1 N acetic acid, centrifugedat 10000g and stored at �70 �C. SP was measured byradioimmunoassay (RIA) using antiserum and methodspreviously described and validated (Bianchi et al., 2004).The antibody was raised in rabbit against synthetic SP,and it is directed towards the C terminal of the peptide.I125–SP was purchased from GE Healthcare (ColognoMonzese, Italy). Sensitivity of the RIA was 10 pg/tubeand intra-assay and inter-assay variation coefficientwere 8% and 11%, respectively.

For TNF and IL-1 evaluation skin samples werehomogenized in 2 ml of phosphate buffered saline pH7.4 (PBS) containing 10 mM EDTA and 20 KIU/mlaprotinin (Sigma, Milano, Italy). After centrifugationat 10000g the supernatants were frozen at �70 �C .

TNF and IL-1 were measured by mean of ELISA kitspecific for rat TNF (Bender Medsystem, ProdottiGianni, Italy) and for the mature form of rat IL-1b(eBioscience, Societa Italiana Chimici, Italy). All theELISA procedures were performed according to themanufacturer’s instructions. The standards were recom-binant cytokine curves generated in doubling dilutionsfrom 2500 to 39 pg/ml.

2.5. Removal of dorsal root ganglia (DRG)

Three and 7 days after CFA-injection animals wereanaesthetized with sodium pentobarbital (60 mg/kg,i.p., 0.2 ml/100 g bw). L4, L5 and L6 DRG ipsilateralto the CFA-injected hind-paw were removed under dis-secting microscope, immediately frozen in liquid nitro-

gen and stored at �80�C until the SP protein contentand preprotachykinin gene expression measurement.

For the evaluation of SP levels, DRG were homoge-nized in 0.25 ml 0.1 N acetic acid, and the peptide mea-sured as described above for paw skin.

2.6. RNA isolation and real-time RT-PCR

Total RNA from DRG was purified using TRIzolreagent (Invitrogen, Life Technologies, San GiulianoMilanese, Italy) according to the manufacturer’s instruc-tions and resuspended in 6 ll of formamide. After puri-fication, total RNA concentrations were determinedfrom the sample absorbance value at 260 nm. 3000 ngof total RNA were treated with DNase (DNA-free-Ambion) to avoid false-positive results due to amplifica-tion of contaminating genomic DNA. First strandcDNA was synthesized from 1000 ng of total RNA ina final volume of 20 ll using M-MLV RT (MoloneyMurine Leukemia Virus Reverse Transcriptase; Invitro-gen, San Giuliano Milanese, Italy).

cDNA (2 ll) was subjected to real-time quantitativePCR using ABI PRISM 7000 (Applied Biosystems, For-ster City, CA). TaqMan PCR was performed in 25 llvolumes using Real Master Mix Probe ROX (Eppen-dorf, Hamburg, Germany). Custom probes were pre-pared by Applied Biosystem. The probes weredesigned to span an intron in order to avoid potentialamplification of contaminated DNA in the analyzedsamples (Lu et al., 2005) The probes were labelled atthe 5 0 end with 6-carboxy fluorescicein (FAM) and atthe 3 0 end with 6-carboxy-tetramethyl rhodamine(TAMRA). Table 1 shows the primers and probesequence for preprotachykinin (PPT, Genbank acces-sion number M15191) and GAPDH (Genbank acces-sion number AF106860). All PCR assays wereperformed in triplicate. Before using the DDCT methodfor relative quantification, we performed a validationexperiment to demonstrate that the efficiencies of targetand reference are equal. The reaction conditions were asfollows: 95�C for 2 min, followed by 40 cycles at 95 �Cfor 15 s (denaturation) and 60 �C for 1 min (annealingand elongation). As controls, we used the reaction mix-ture without the cDNA. Threshold cycle numbers (CT)were determined with an ABI PRISM 7000 SequenceDetection System (version 1.1 software) and trans-formed using the DCTð2�DDCTÞ comparative method.Gene-specific expression values were normalized toexpression values of GAPDH (endogenous control)within each sample. The levels of preprotachykinin wereexpressed relative to the calibrator value control group.Relative quantification was performed using the com-parative method. The amount of target, normalized toan endogenous reference and relative to a calibrator, isgiven by 2�DDCT . Briefly, the DCT value is determinedby subtracting the average GAPDH CT value from the

Table 1Primers/probe sequence used in the real-time quantitative RT-PCR to analyse transcription levels of SP

mRNA Primers Probes (50FAM-30TAMRA)

Substance P Sense 50-AAGTTTGCCAGCGATGCAA-3 0 ACAGGAGTTTCTCTGCCTCCAGCAGCAAntisense 5 0-AACCAAGGGAAGCGAAAGA-30

GAPDH Sense 50-AATGTATCCGTTGTGGATCTGACA-3 0 TCGGCCGCCTGCTTCACCAAntisense 5-AGCCCAGGATGCCCTTTAGT-30


average cytokines CT in the same sample. The calcula-tion of DDCT involves subtraction of the DCT calibratorvalue.

2.7. Statistical analysis

Data were analyzed by one way analysis of variance(ANOVA), followed by Bonferroni’s t-test for multiplecomparison. An effect was determined to be significantif the P-value was less than 0.05.

3. Results

3.1. Effects of ibandronate on inflammatory oedema and

mechanical hyperalgesia

The injection of CFA into hind-paw caused a markedincrease in paw volume (oedema), associated with adecrease in the paw-withdrawal latency to noxiousmechanical stimulation Both the inflammatory oedemaand hyperalgesia were evident at 1, 2, 3 and 7 days afterCFA-injection. Fig. 1, left panel, shows that the oedemaformation was significantly reduced by ibandronate 1.0

Fig. 1. Left panel: Effect of ibandronate (0.5, 1.0, and 2.0 mg/kg i.p.) on tIbandronate (IBN) or saline were administered 1 h after CFA-injection. Datathe volume of inflamed (CFA-injected) and uninflamed (saline-injected) hind-the inflammatory hyperalgesia induced by the hind-paw injection of CFA. IbThe evaluation was performed by Randall-Selitto Test. Data are expressed inmeasured in saline-injected and CFA-injected (inflamed) hind-paw. * = P <

and 2.0 mg/kg, while the dose of 0.5 mg/kg was noteffective. This drug effect was evident starting from thethird day after treatment and was similarly maintainedat the 7th day after ibandronate administration. Theeffect of ibandronate on inflammatory hyperalgesia isshown in the right panel of Fig. 1. The CFA-inducedmechanical hyperalgesia was significantly reduced byibandronate 1.0 and 2.0 mg/kg, while no significant dif-ference was identified between the values measured inanimals treated with ibandronate 0.5 mg/kg and thosemeasured in rats treated with saline. The anti-hyperalge-sic effect of ibandronate was evident starting from thethird day after treatment and was already present 7 daysafter treatment. It is important to note that we previ-ously established that, at these doses (0.5, 1.0, and2.0 mg/kg), ibandronate did not affect nociceptivethresholds to mechanical stimulation in the normal(uninflamed) hind-paws (data not shown).

3.2. Effects of ibandronate on inflammatory mediators in

the paw

In the first set of experiments we demonstrated thatthe doses of 1.0 and 2.0 mg/kg were similarly effective

he inflammatory oedema induced by the hind-paw injection of CFA.are expressed in ml, as mean ± SEM of the algebraic difference betweenpaw. Right panel: Effect of ibandronate (0.5, 1.0, and 2.0 mg/kg i.p.) onandronate (IBN) or saline were administered 1 h after CFA-injection.grams, as mean ± SEM of the algebraic difference between thresholds

0.05 vs CFA + saline.

Fig. 3. Effect of ibandronate (1.0 mg/kg i.p.) on the increase of SPtissue levels produced by CFA-injection into the hind-paw of rats.Control animals were treated intraplantarly and i.p. with saline.Ibandronate or saline were administered 1 h after intraplantar injec-tion of CFA or saline. The measurements were performed 3 and 7 daysafter CFA-injection. Values are means ± SEM of eight rats.* = P < 0.05 vs controls; # = P < 0.05 vs CFA + saline.


in reducing inflammatory oedema and hyperalgesia. Inthe light of this observation and of the information thatclinically relevant renal damage in rat was not notedafter the i.v. administration of ibandronate at a doseof 1.0 mg/kg (Pfister et al., 2003), we decided to performthe following studies on inflammatory mediators withthe lowest effective dose, i.e. 1.0 mg/kg.

At this dose, ibandronate did not affect the PGE-2levels in the skin of saline-injected hind-paws. Asexpected, CFA-injection caused a significant increasein PGE-2tissue concentrations. The administration ofibandronate to animals with inflamed paw did not mod-ify PGE-2 levels increased by CFA-injection (Fig. 2).

The injection of CFA caused a clear increase in SPlevels in the paw both at 3 and 7 days after the inductionof the inflammation (Fig. 3). This change in SP levelswas completely prevented at both time points by thetreatment with ibandronate. No significant effect of thisdrug on SP was observed in the paw of non-inflamedanimals.

Fig. 4 shows the results regarding the paw concentra-tions of TNF and IL-1. When administered to animalsintraplantarly injected with saline, ibandronate had noeffect either on the TNF or IL-1 levels. The injectionof CFA produced a significant increase in the skin con-tent of both cytokines in the ipsilateral paw. This effectof CFA was evident at 3 and 7 days post-injection. Theadministration of ibandronate to CFA-treated animalsdid not to modify cytokine concentrations as measured3 days after CFA-injection. On the other hand, at 7 daysafter the induction of the inflammation in animals trea-ted with ibandronate the paw skin levels of TNF and IL-1 resulted significantly lower than those measured in theinflamed paw of rats treated with saline.

Fig. 2. Effect of ibandronate (1.0 mg/kg i.p.) on the increase of PGE-2tissue levels produced by CFA-injection into the hind-paw of rats.Control animals were treated intraplantarly and i.p. with saline.Ibandronate or saline were administered 1 h after intraplantar injec-tion of CFA or saline. The measurements were performed 3 and 7 daysafter CFA-injection. Values are means ± SEM of eight rats.

3.3. Ibandronate effect on SP and preprotachykinin

mRNA levels in DRG

To assess the possible effect of ibandronate on SPproduction in primary afferent sensory neurons, ipsilat-eral L3-5 DRG were collected 3 and 7 days after CFA-injection in the hind-paw. Both SP protein and itsmRNA were evaluated. No significant differences werepresent in SP protein content in DRG obtained fromrats intraplantarly treated with saline or CFA, and theadministration of ibandronate did not modify SP eitherin control or in inflamed animals (Table 2).

Fig. 5 shows the fold increase in preprotachykiningene expression in DRG compared with control animals(i.e. not inflamed animals treated with saline). A slightbut significant increase in preprotachykinin expressionwas observed in DRG obtained from CFA-treated ani-mal; this effect was present both at 3 and 7 days afterthe induction of the inflammation. The administrationof ibandronate completely prevented the overexpressionof SP mRNA induced by CFA, while did not affect SPmRNA levels in animals injected intraplantarly withsaline.

4. Discussion

Ibandronate is a potent and long acting bisphosho-nate, highly effective in the treatment of osteoporosisand pain associated with metastatic bone disease (Bodyet al., 2004; Heidenreich and Ohlmann, 2004; Cameronet al., 2006; Croom and Scott, 2006). In the presentstudy we have shown that this drug is able to reduceinflammatory oedema and hyperalgesia in a rat model

Table 2SP protein content in DRG

SP (pg/lg protein)

3 days 7 daysControls 0.152± 0.04 0.161 ± 0.02Ibandronate 0.162 ± 0.06 0.154 ± 0.05CFA + saline 0.210± 0.05 0.220 ± 0.06CFA + ibandronate 0.200 ± 0.05 0.190 ± 0.013

Control animals were treated intraplantarly and i.p. with saline.Ibandronate or saline were administered 1 h after the intraplantarinjection of CFA or saline. Three and 7 days after hind-paw CFAadministration, L4, L5 and L6 DRG ipsilateral to the CFA-injectedhind-paw were removed, and pooled.Values are mean ± SEM of eight rats.

Fig. 5. Effect of ibandronate (1.0 mg/kg i.p.) on the increase ofpreprotachykinin mRNA expression in ipsilateral DRG produced byCFA-injection into the hind-paw of rats. Control animals were treatedintraplantarly and i.p. with saline. Ibandronate or saline wereadministered 1 h after intraplantar injection of CFA or saline. Themeasurements were performed 3 and 7 days after CFA-injection.Results are expressed as preprotachykinin mRNA expression inrelation to GADPH, and are presented as a fold increase relative tocontrol animals. Values are means ± SEM of four rats. * = P < 0.05 vscontrols; # = P < 0.05 vs CFA + Ibandronate.

Fig. 4. Effect of ibandronate (1.0 mg/kg i.p.) on the increase of TNF (left panel) and IL-1 (right panel) tissue levels produced by CFA-injection intothe hind-paw of rats. Control animals were treated intraplantarly and i.p. with saline. Ibandronate or saline were administered 1 h after intraplantarinjection of CFA or saline. The measurements were performed 3 and 7 days after CFA-injection. Values are means ± SEM of eight rats. * = P < 0.05vs controls; # = P < 0.05 vs CFA + saline.


of persistent pain. These effects became evident on thethird day after the administration of a single dose ofibandronate on the same day as the induction of inflam-

mation, and proved long lasting; in fact, they were stillevident one week after drug administration.

It is well known that a major problem associated withthe clinical use of bisphosphonates is related to theirrenal toxicity (Adami and Zamperlan, 1996). The renalsafety of these drugs depends on the dose and the dosinginterval. It is therefore important to point out that wehave demonstrated significant anti-inflammatory effectsafter the treatment with a dose of ibandronate(1.0 mg/kg) which has been reported to not induce clin-ically relevant nephrotoxicity after i.v. administration inthe rat (Pfister et al., 2003). As previously noted, thisfact also determined our choice to perform all the bio-chemical studies with this dose of ibandronate.Although there is no clear relation of this dose to thedoses used in humans, it is important to stress thatibandronate accumulates in bone after repeated dosing(Bauss et al., 2004). Thus, in the clinical setting, repeateddoses cause high concentrations in bone. Part of thebone-bound drug is released during bone turnover,and in consequence results in a high concentration inthe bone environment. In order to simulate such highconcentrations in our short-term animal model, weadministered a single high dose. Bone concentration ofibandronate is more relevant for the actions of this drugsince serum levels decrease fast due to the short half-lifeof ibandronate in both rats and humans (Barrett et al.,2004).

Peripheral inflammatory pain is associated with acomplex pattern of local changes. Following tissueinjury many pro-nociceptive and pro-inflammatorymediators are activated; they lower nociceptive thresh-olds and increase neuronal membrane excitability, lead-ing to hypernociception (Woolf, 1991; Cunha et al.,2005). Among these, the neuropeptide SP is present inC-fibres, is synthesized in DRG, and is transported toboth central and peripheral endings of primary afferentneurons (Maggi, 1995). In the spinal cord, it has excit-atory effects on dorsal horn neurons producing an


increase in nociception. Moreover, SP is released anti-dromically in the inflamed tissue, where it sustains theso-called neurogenic inflammation (Maggi, 1995; White,1997). In periphery, SP is able to sensitise afferent fibresand increase the sensitivity to nociceptive stimuli. Inaddition, SP can directly activate immune cells inducingthe chemotaxis of monocytes/macrophages, and theproduction of different pro-inflammatory cytokines(Bianchi et al., 2003; Delgado et al., 2003; Bianchiet al., 2004). It has been reported that more than 80%of SP synthesized by DRG neurons is being exportedtowards the terminal endings in periphery rather thanto the central nervous system (Maggi, 1995; White,1997). In this study, we observed a clear increase in SPrelease in the CFA-inflamed paw, associated with a sig-nificant increase of the expression of PPT I gene, thatencodes for SP mRNA (Krause et al., 1984; Lu et al.,2005), in the L4-6 DRG corresponding to the afferentsfrom the inflamed hind-paw. Rather surprisingly, wedid not find any increase in SP peptide levels in DRGof rats injected with CFA in the hind-paw. This mayreflect a dynamic interplay between the production ofthe peptide and its transport from the soma; one possi-ble explanation is that following an intense nerve stimu-lation, as happens during persistent inflammation, thenewly synthesized peptide is rapidly transported alongthe axon and rapidly released in periphery.

Our present data suggest an important involvementof SP in the anti-hyperalgesic effect of ibandronate. Infact, this bisphosphonate completely abolished theincrease in SP synthesis and release induced by CFA-injection in the hind-paw. It this context, it is importantto stress that antidromically released SP is able to recruitneutrophils and monocytes into inflamed tissue, and tostimulate lymphocytes, mast cells, and macrophages toproduce various cytokines, including IL-1 and TNF(Delgado et al., 2003; Hernanz et al., 2003). These cyto-kines can either directly sensitise nociceptors, or inducethe release of other pro-inflammatory and pro-nocicep-tive mediators (Safieh-Garabedian et al., 1995; Marc-hand et al., 2005). Interestingly, IL-1 has beenreported to regulate SP production in DRG (Moriokaet al., 2002); therefore a positive loop between SP andcytokines is active in the maintenance and perpetrationof inflammatory hyperalgesia. After CFA-injection weobserved a significant increase in paw IL-1 and TNF lev-els that was prevented by the administration of ibandr-onate. However, the cytokine increase was alreadyevident 3 days after CFA, whereas ibandronate was ableto reduce it only on day 7. Therefore, an overall analysisof our findings appears to suggest that the ibandronate-induced decrease in SP may have an impact on the pro-duction of IL-1 and TNF in the hind-paw.

It has been suggested that some bisphosphonates maydirectly modulate the production of cytokines frommonocyte/macrophages, either increasing (Pioli et al.,

1990; Takagi et al., 2005) or decreasing them (Dehghaniet al., 2004; Selander et al., 1996). Although we cannotrule out a direct effect of ibandronate on cytokine pro-duction, it is important to note that we did not observeany effect of ibandronate on cytokine production in theabsence of an inflammatory state.

In this study, the increase in PGE-2 hind-paw levelsinduced by CFA-injection was not affected by the treat-ment with ibandronate. This observation suggests thatthis drug does not interact with the cyclooxygenase(COX) enzymes, and is in accordance with findingsrecently published showing that several bisphosphonatesdo not interfere either with COX-1 or COX-2 activity(Tuominen et al., 2006).

With regard to the effects of ibandronate on SP, themechanisms by which this bisphosphonate may producea decrease in SP production remains to be clarified. Ithas recently been suggested that the activation of osteo-clasts in CFA-induced paw inflammation plays a role innociceptor sensitisation (Nagae et al., 2006). Osteoclastslocalized in the inflamed paw, indeed, may secrete pro-tons and make the microenvironment acidic. It is wellknown that two classes of acid-sensing nociceptors arepresent in sensory neurons: the acid-sensing ion chan-nels (ASICS) and the transient receptor potential chan-nel vanilloid member (TRPV1) (Rhee and Kress, 2001).TRPV-1 can be activated directly by hydrogen ions.TRPV-1 activation promotes inflammation mediatedby SP in several animal models (Tognetto et al., 2001;Dinh et al., 2004; Hutter et al., 2005; Kanai et al.,2005). It can therefore be hypothesised that ibandro-nate, by inhibiting osteoclastic activity, might preventproton production by these cells, and reduce the activa-tion of specific ion channels and the consequent produc-tion of SP by primary afferents.

The role of the nervous system in regulating bonebiology is now emerging, and a number of anatomicaland physiological evidences demonstrate the presenceof sensory SP containing nerve in the bone (Bjurholmet al., 1988; Hukkanen et al., 1992; Fras et al., 2003;Goto and Tanaka, 2002). The rich innervation of perios-teum by SP positive fibres further supports the notion ofa role for this peptide in bone pain. A body of dataexists showing that bone malignancies induce peripheraland central sensitisation of the nervous system. A criti-cal link between inflammation and cancer has beendescribed, and several pro-inflammatory mediators,including TNF and IL-1, have been identified to exerta crucial role in the development of hyperalgesia (Clo-hisy and Mantyh, 2003; Aggarwal et al., 2006). More-over, it has been demonstrated that osteoclastic boneresorption is associated with an inflammatory state adja-cent to bone (Nagae et al., 2006). In view of all thesefindings, the ability of ibandronate to reduce inflamma-tory hyperalgesia, and to inhibit the mechanisms acti-vated by tachykinins and cytokines in inflammatory


conditions may contribute to explain the reduction ofpain observed after treatment with this bisphosphonatein patients with osteoporosis and cancer pain associatedwith metastatic bone disease.

References

Adami S, Zamperlan S. Adverse effects of bisphosphonates. Drug Saf1996;14:158–70.

Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G.Inflammation and cancer: How hot is the link? Biochem Pharmacol2006;72:1605–21.

Barrett J, Worth E, Bauss F, Epstein S. Ibandronate: a clinicalpharmacological and pharmacokinetic update. J Clin Pharmacol2004;44:951–65.

Bauss F, Body J. Ibandronate in metastatic bone disease: a review ofpreclinical data. Anti-cancer Drugs 2005;16:107–18.

Bauss F, Graham R, Russell G. Ibandronate in osteoporosis:preclinical data and rationale for intermittent dosing. OsteoporosInt 2004;15:423–33.

Bianchi M, Broggini M, Balzarini P, Baratelli P, Ferrario P, PaneraiAE, et al. Effects of tramadol on synovial fluid concentrations ofsubstance P and interleukin-6 in patients with knee osteoarthritis:comparison with paracetamol. Int Immunopharmacol2003;3:1901–8.

Bianchi M, Martucci C, Biella G, Ferrario P, Sacerdote P. Increasedsubstance P and tumor necrosis factor alpha in the paws followingformalin injection in rat tail. Brain Res 2004;1019:255–8.

Bjurholm A, Kreicbergs A, Brodin E, Schultzberg M. Substance P-and CGRP-immunoreactive nerves in bone. Peptides1988;9:165–71.

Body JJ. Breast cancer: bisphosphonates therapy for metastatic bonedisease. Clin Cancer Res 2006;12:6258s–63s.

Body JJ, Diel IJ, Lichinitzer M, Lazarev A, Pecherstorfer M, Bell R,et al. Oral ibandronate reduces the risk of sletal complications inbreast cancer patients with metastatic bone disease: results fromtwo randomizes, placebo-controlled phase III studies. Br J Cancer2004;90:1133–7.

Bonabello A, Galmozzi MR, Bruzzese T, Zara GP. Analgesic effect ofbisphosphonates in mice. Pain 2001;91:269–75.

Bonabello A, Galmozzi MR, Canaparo R, Serpe L, Zara GP. Long-term analgesic effect of clodronate in rodents. Bone 2003:567–574.

Cameron D, Fallon, Diel I. Ibandronate: its role in metastatic breastcancer. Oncologist 2006;11(Suppl. 1):27–33.

Clohisy DR, Mantyh PW. Bone cancer pain. Cancer 2003;97:866–73.Croom KF, Scott LJ. Intravenous ibandronate in the treatment od

osteoporosis. Drugs 2006;66:1593–601.Cunha TM, Verri WA, Silva JS, Poole S, Cunha FQ, Ferreira SH. A

cascade of cytokines mediates mechanical inflammatory hyperno-ciception in mice. Proc Natl Acad Sci 2005;102:1755–60.

Dehghani F, Conrad A, Kohl A, Korf HW, Hailer NP. Clodronateinhibits the secretion of proinflammatory cytokines and NO byisolated microglial cells and reduces the number of proliferatingglial cells in excitotoxically injured hippocampal slice cultures. ExpNeurol 2004;186:241–51.

Delgado AV, Mc Manus AT, Chambers JP. Production of tumornecrosis factor – alpha, interleukin-1b, interleukin-2 and interleu-kin-6 by rat leukocytes subpopulation after exposure to substanceP. Neuropeptides 2003;37:355–61.

Dinh QT, Groneberg DA, Peiser C, Mingomataj E, Joachim RA, WittC, et al. Substance P expression in TRPV-1 and trkA-positivedorsal root ganglion neurons innervating the mouse lung. RespirPhysiol Neurobiol 2004;144:15–24.

Emkey R, Koltun W, Beusterien K, Seidman L, Kivitz A, Devas V,et al. Patient preference for once-monthly ibandronate versusonce-weekly alendronate in a randomized, open-label, cross-overtrial: the Boniva Alendronate Trial in Osteoporosis (BALTO).Curr Med Res Opin 2005;21:1895–903.

Fleisch H. Bisphosphonates: pharmacology and use in the treatment oftumor induced hypercalcaemic and metastatic bone disease. Drugs1991;42:919–44.

Fras C, Kravetz P, Mody DR, Heggeness MH. Substance P containingnerve within the human vertebral body: an immunohistochemicalstudy of the basivertebral nerve. Spine 2003;3:63–7.

Goicoechea C, Porras E, Alfaro MJ, Martin MI. Alendronate inducesantinociception in mice, not related with its effects in bone. Jpn JPharmacol 1999;79:433–7.

Goto T, Tanaka TM. Tachykinin and tachykinin receptors in bone.Microsc Res Tech 2002;58:91–7.

Heidenreich A, Ohlmann CH. Ibandronate: its pharmacology andclinical efficacy in the management of tumor-induced hypercalce-mia and metastatic bone disease. Expert Rev Anticancer Ther2004;4:991–1005.

Heidenreich A, Ohlmann C, Body JJ. Ibandronate in metastatic bonepain. Semin Oncol 2004;31(Suppl. 10):67–72.

Hernanz A, Medina S, de Miguel E, Martin-Mola E. Effect ofCGRP, neuropeptide Y, substance P and vasoactive intestinalpeptide on Interleukin-1 beta, interleukin-6 and tumor necrosisfactor-alpha production by peripheral whole blood cells fromrheumatoid arthritis and osteoarthritis patients. Regul Pept2003;115:19–24.

Hukkanen M, Konttinen YT, Rees RG, Santavirta S, Terenghi G,Polak JM. Distribution of nerve ending and sensory neuropeptidesin rat synovium, meniscus and bone. Int J Tissue React1992;14:1–10.

Hutter MM, Wick EC, Day AL, Maa J, Zerega EC, Richmond AC,et al. Transient receptor potential vanilloid (TRPV-1) promotesneurogenic inflammation in the pancreas via activation of theneurokinin-1 receptor. Pancreas 2005;30:260–5.

Kanai Y, Nakazato E, Fujiuchi A, Hara T, Imai A. Invovement of anincreased spinal TRPV-1 sensitization throughout its upregulationin mechanical allodynia of CCI rats. Neuropharmacology2005;49:977–84.

Krause JE, Chirgwin JM, Carter MS, Xu ZS, Hershey AD. Three ratpreprotachikinin mRNAs encoded the neuropeptide SP andneurokinin A. Proc Natl Acad Sci USA 1984;84:881–5.

Lu CL, Pasricha PJ, Hsieh JC, Lu RH, Lai CR, Wu LL, et al.Changes of the neuropeptides content and gene expression in spinalcord and dorsal root ganglion after noxious colorectal distension.Reg Pept 2005;131:66–73.

Maggi CA. Tachykinins and calcitonin gene related peptide (CGRP)as co-trasmitters released from peripheral endings of sensorynerves. Prog Neurobiol 1995;45:1–98.

Marchand F, Perretti M, Mc Mahon SB. Role of the immune systemin chronic pain. Nat Rev Neurosci 2005;6:521–31.

McCormack PL, Plosker GL. Ibandronic acid: a review of its use inthe treatment of bone metastases of breast cancer. Drugs2006;66:711–28.

Morioka N, Takeda K, Kumagai K, Hanada T, Ikoma K, Hide I,et al. Interleukin-1b-induced substance P release from rat culturedprimary afferents neurons driven by two phospholipase A2enzymes: secretory type IIA and cytosolic type IV. J Neurochem2002;80:989–97.

Nagae M, Hiraga T, Wakabayashi H, Wang L, Iwara K, Yoneda T.Osteoclasts play a part in pain due to inflammation adjacent tobone. Bone 2006;39:1107–15.

Pfister T, Atzpodien E, Bauss F. The renal effects of minimallynephrotoxic doses of ibandronate and zoledronate following singleand intermittent intravenous administration in rats. Toxicology2003;191:159–67.


Pioli G, Hughes DE, Milan A, Aarden L, Meager A, Chapman K.Bisphosphonates stimulate the production of cytokines by humanperipheral blood mononuclear cells in vitro. Calcif Tissue Int1990;46:A54.

Rhee PW, Kress M. Molecular physiology of proton transduction innociceptors. Curr Opin Pharmacol 2001;1:45–51.

Russell RG, Rogers MJ. Bisphosphonates: from the laboratory to theclinic and back again. Bone 1999;25:97–106.

Safieh-Garabedian B, Poole S, Allchorn A, Winter J, Woolf C.Contribution of IL-1 beta to the inflammation-induced increase innerve growth factor levels and inflammatory hyperalgesia. Br JPharmacol 1995;115:1265–75.

Santini D, Fratto ME, Vincenzi B, La Cesa A, Dianzani C, Tonini G.Bisphosphonate effects in cancer and inflammatory diseases. Invitro and in vivo modulation of cytokine activities. Biodrugs2004;18:269–78.

Selander KS, Monkkonnen J, Karhukorpi EK, Harkonen P, Hann-uniemi R, Vaananen HK. Characteristics of clodronate-inducedapoptosis in osteoclasts and macrophages. Mol Pharmacol1996;50:1127–38.

Stein C, Millan MJ, Herz A. Unilateral inflammation of the hindpawin rats as a model of prolonged noxious stimulation: alterations inbehavior and nociceptive thresholds. Pharmacol Biochem Behav1988;31:445–51.

Takagi K, Takagi M, Kanangat S, Warrington KC, Shigemitsu H,Postlewaite A. Modulation of TNF-alpha gene expression by IFN-gamma and pamidronate in murine macrophages: regulation bySTAT1 dependent pathways. J Immunol 2005;174:1801–10.

Tognetto M, Amadesi S, Harrison S, Creminon C, Trevisani M,Carreras M, et al. Anandamide excites central terminals of dorsalroot ganglion neurons via vanilloid receptor-1 activation. JNeurosci 2001;21:1104–9.

Toussirot E, Wendling D. Bisphosphonates as anti-inflammatoryagents in ankylosing spondylitis and spondylarthropathies. ExpertOpin Pharmacother 2005;6:35–43.

Tuominen OM, Ylitalo-heikkala R, Vehmas TI, Mucha I, Ylitalo P,Riutta A. Effect of bisphosphonates on prostaglandin E2 andtromboxane B2 production in human whole blood and monocytesstimulated by lipopolysaccaride and A23187. Methods Find ExpClin Pharmacol 2006;28:361–7.

Walker K, Medhurst SJ, Kidd BL, Glatt M, Bowes M, Patel S, et al.Disease modifying and anti-nociceptive effects of the bisphospho-nate, zoledronic acid in a model of bone cancer pain. Pain2002;100:219–29.

White DM. Release of substance P from peripheral sensory nerveterminals. J Peripher Nerv Syst 1997;2:191–201.

Woolf CJ. Generation of acute pain. Br Med Bull 1991;47:523–33.Yamamoto K, Yoshino S, Shue G, Nagashima M. Inhibitory effect of

bone resorption and inflammation with etidronate therapy inpatients with rheumatoid arthritis for 3 years and in vitro assay inarthritis models. Rheumatol Int 2006;26:627–32.

Zimmermann M. Ethical guidelines for investigation of experimentalpain in conscious animals. Pain 1983;16:109–10.

Zysk SP, Durr HR, Gebhard HH, Schmitt-Sody M, Refior HJ,Messmer K, et al. Effects of ibandronate on inflammation inmouse antigen-induced arthritis. Inflamm Res 2003;52:221–6.

Effects of the bisphosphonate ibandronate on hyperalgesia, substance P, and cytokine levels in a rat...

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