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  RESEARCH PAPER bph_1408 681..693 Anti-nociceptive effect of kinin B 1  and B 2  receptorantagonists on peripheralneuropathy induced bypaclitaxel in mice Robson Costa 1 , Emerson M Motta 1 , Rafael C Dutra 1 ,Marianne N Manjavachi 1 , Allisson F Bento 1 , Fernanda R Malinsky 2 , João B Pesquero 2 and João B Calixto 1 1  Department of Pharmacology, Centre of Biological Sciences, Universidade Federal de SantaCatarina, Florianópolis, SC, Brazil, and   2  Department of Biophysics, Universidade Federal de São Paulo, São Paulo, SP, Brazil Correspondence  João B. Calixto, Departmentof Pharmacology, Centre of Biological Sciences, UniversidadeFederal de Santa Catarina,Campus Universitário,88049-900, Florianópolis, SC,Brazil. E-mail:calixto@farmaco.ufsc.br orcalixto3@terra.com.br ---------------------------------------------------------------- Keywords neuropathic pain; chemotherapy;paclitaxel; kinin; B 1  receptor; B 2 receptor ---------------------------------------------------------------- Received 28 November 2010 Revised 14 March 2011 Accepted 23 March 2011 BACKGROUND AND PURPOSE In the current study, we investigated the role of both kinin B 1  and B 2  receptors in peripheral neuropathy induced by thechronic treatment of mice with paclitaxel a widely used chemotherapeutic agent. EXPERIMENTAL APPROACH Chemotherapy-evoked hyperalgesia was induced by i.p. injections of paclitaxel (2 mg·kg - 1 ) over 5 consecutive days.Mechanical and thermal hyperalgesia were evaluated between 7 and 21 days after the first paclitaxel treatment. KEY RESULTS Treatment with paclitaxel increased both mechanical and thermal hyperalgesia in mice (C57BL/6 and CD1 strains). Kininreceptor deficient mice (B 1 ,or B 2  receptor knock-out and B 1 B 2  receptor, double knock-out) presented a significant reduction inpaclitaxel-induced hypernociceptive responses in comparison to wild-type animals. Treatment of CD1 mice with kinin receptor antagonists (DALBK for B 1  or Hoe 140 for B 2  receptors) significantly inhibited both mechanical and thermal hyperalgesia whentested at 7 and 14 days after the first paclitaxel injection. DALBK and Hoe 140 were also effective against paclitaxel-inducedperipheral neuropathy when given intrathecally or i.c.v.. A marked increase in B 1  receptor mRNA was observed in the mousethalamus, parietal and pre-frontal cortex from 7 days after the first paclitaxel treatment. CONCLUSIONS AND IMPLICATIONS Kinins acting on both B 1  and B 2  receptors, expressed in spinal and supra-spinal sites, played a crucial role in controlling thehypernociceptive state caused by chronic treatment with paclitaxel. Abbreviations ABP ,  brachial plexus avulsion ;  B 1 R - / - ,  B 1  receptor deficient ;  B 2 R - / - ,  B 2  receptor deficient ;  B 1 B 2 R - / - ,  B 1  and B 2  receptordeficient ;  BBB ,  blood-brain barrier ;  DABK ,  des-Arg 9 -BK ;  DALBK ,  des-Arg 9 -Leu 8 -BK ;  DRG ,  dorsal root ganglia Introduction Kinins are potent endogenous algogenic peptides, and theirrole in pain transmission has been extensively reviewed(Couture  et al ., 2001; Calixto  et al ., 2004; Huang and Player,2010). Once formed from their precursors, the kininogens, bythe action of kallikrein enzymes, kinins are released and exerttheir actions via the activation of two subtypes of GPCRs,named B 1  and B 2  receptors (nomenclature follows Alexander et al ., 2009). The B 2  receptor displays higher affinity for BJP  British Journal of Pharmacology DOI:10.1111/j.1476-5381.2011.01408.xwww.brjpharmacol.org British Journal of Pharmacology (2011)  164  681–693 681 © 2011 The AuthorsBritish Journal of Pharmacology © 2011 The British Pharmacological Society  bradykinin (BK) and kallidin peptides, while the B 1  receptorpresents high affinity for the kinin metabolites, des-Arg 9 -BK(DABK) and Lys-des-Arg 9 -BK. B 2  receptors are usuallyexpressed in a constitutive manner throughout peripheraland central tissues, mediating most of the physiologicaleffects of the kinins and the acute phase of inflammatory andnociceptive responses. In contrast, B 1  receptors are generallyabsent under physiological conditions, being quicklyup-regulated after tissue injury or during certain inflamma-tory process. Therefore, they might represent importantplayers in the chronic phase of pain and inflammation(Calixto  et al ., 2004; Marceau and Regoli, 2004; Huang andPlayer, 2010). Nevertheless, the constitutive expression of B 1 receptors in sensory neurons has been reported (Ma andHeavens, 2000; Wothersponn and Winter, 2000; Ma, 2001).Many groups have reported that both kinin receptors areinvolved in the onset and/or maintenance of neuropathicpain (Petersen  et al ., 1998; Levy and Zochodne, 2000;Yamaguchi-Sase  et al ., 2003; Rashid  et al ., 2004; Ferreira  et al .,2005; Lai  et al ., 2006; Quintão  et al ., 2008), a chronic condi-tion characterized by spontaneous pain, allodynia and hype-ralgesia, which remains without satisfactory treatment andcompromises the quality of life (Woolf and Mannion, 1999; Jensen and Baron, 2003). Nerve injuries (caused by surgery ortrauma), some pathological states (e.g. diabetes mellitus,herpes zoster or HIV infection) and chemotherapy are themain causes of peripheral neuropathy in humans (Woolf andMannion, 1999). Chemotherapy-induced peripheral neur-opathy is a common side effect of several anticancer drugs,including vincristine, oxaliplatin and paclitaxel (Wolf   et al .,2008). Paclitaxel, derived from  Taxus brevifolia  and commer-cially known as Taxol, is one of the most effective and com-monly used anti-neoplastic drugs. Its major dose-limiting sideeffect is the appearance of peripheral sensory neuropathycharacterized by painful paresthesias of the hands and feet(Polomano and Bennett, 2001; Dougherty  et al ., 2004). Inaccordance with these clinical findings, chronic treatmentwith paclitaxel in rodents induced mechanical and thermalhyperalgesia, and has been used as a reproducible model toevaluate chemotherapy-induced peripheral neuropathy(Cliffer  et al ., 1998; Dina  et al ., 2001; Polomano  et al ., 2001).Recent evidence has suggested that kinins, and theirreceptors, might play a critical role in peripheral neuropathyinduced by chemotherapy (Bujalska  et al ., 2008; Bujalska andMakulska-Nowak, 2009a,b). However, the mechanismsunderlying these actions still remain unclear. Hence, in thepresent study, in order to provide new evidence on the rel-evance of both kinin B 1  and B 2  receptors in chemotherapy-induced neuropathy, we sought to analyse, by the use of kinin receptor knock-out mice in combination with selectivekinin receptor antagonists and molecular analysis, the con-tribution of these receptors to the thermal and mechanicalhyperalgesia induced by paclitaxel. Methods  Animals All animal care and experimental procedures complied withthe National Institutes of Health Animal Care Guidelines(NIH publications 80-23), and were approved by the EthicsCommittee of the Universidade Federal de Santa Catarina(protocol number PP00032). The animals were housed in aroom with controlled temperature (22    2°C) and humidity(around 60–80%) under a 12:12 h light–dark cycle (lights on0600 h). Food and water were provided  ad libitum . Adult maleCD1 mice (8–10 weeks) were used in this study. In someexperiments, male C57BL/6 wild-type mice, C57BL/6 kininB 1 - or B 2  receptor-deficient mice (B 1 R - / - and B 2 R - / - , respec-tively) and mice lacking the genes encoding both kininreceptors (double knock-out mice, B 1 B 2 R - / - ) were also used.Wild-type and knock-out mice were srcinally obtained fromthe Centro de Desenvolvimento de Modelos Experimentaispara Medicina e Biologia, from the Universidade Federal deSão Paulo (São Paulo, Brazil). Deletion of the entire codingsequence for kinin B 1  and B 2  receptors was achieved accord-ing to the methodology previously described by Pesquero et al . (2000) and Rupniak  et al . (1997) respectively. Micelacking both kinin receptors (B 1 B 2 R - / - ) were generated accord-ing to the methodology described by Cayla  et al . (2007). Theanimals were randomly distributed between the experimentalgroups (six animals per group), and all behavioural experi-ments were conducted without knowledge of the treatmentsin order to reduce experimental bias. The number of animalsand the intensity of noxious stimuli used were the minimumnecessary to demonstrate consistent effects. There were nowithdrawals or exclusions in this study.  Peripheral neuropathy induced by paclitaxel The neuropathy induced by paclitaxel was induced accordingto the methodology described previously by Polomano  et al .(2001) and adapted for use in mice. Briefly, mice wereinjected i.p. with paclitaxel (2 mg·kg - 1 per injection) for 5consecutive days (days 1–5), using an injection volume of 10 mL·kg - 1 . The cumulative paclitaxel dose was 10 mg·kg - 1 .Control animals received only the vehicle (0.9% NaCl). Inorder to assess general toxicity, mouse body weight and rectaltemperature were measured at regular intervals of time, for 21days after the first paclitaxel administration. Tests for alteredpain sensitivity began on day 7 and continued until day 14 or21.  Mechanical hyperalgesia in hind paws To assess the mechanical hypernociceptive response, micewere placed individually in clear Plexiglas boxes (9  ¥  7  ¥ 11 cm)onelevatedwire-meshplatformstoallowaccesstotheventral surface of the right hind paw (Ugo Basile, Comerio,VA, Italy). The animals were acclimatized for 1 h beforebehavioural testing. The withdrawal response frequency (in%) was measured following 10 applications (with a durationof   ~ 3 s each, and an interval of   ~ 20 s among each) of von Freyhairs (VFHs, Stoelting, Chicago, IL, USA). Stimuli were deliv-ered from below to the plantar surface of the right hind paw.The 0.6 g VFH filament produces a mean withdrawal fre-quency of about 20%, which is considered to be an adequatevalue for the measurement of mechanical hyperalgesia(Quintão  et al ., 2008). Hence, the 0.6 g VFH was usedthroughout this study. All the groups were evaluated beforevehicle or paclitaxel injections, in order to determine basal BJP  R Costa et al. 682 British Journal of Pharmacology (2011)  164  681–693  mechanical thresholds. The incidence of mechanical hyper-algesia was  ~ 90% in paclitaxel-treated animals.  Hind paw thermal hyperalgesia (paw flick) A radiant heat analgesiometer (Tail-Flick Analgesia Meter,Albarsch, Porto Alegre, Brazil) was used to measure latenciesfor paw withdrawal according to the method described byMenéndez  et al . (2002). All the animals were evaluated todetermine the basal thermal threshold (I.R. intensity of 15),and then they were submitted to paclitaxel injections, asdescribed earlier. Thermal hyperalgesia was evaluated atseveral time intervals after the initiation of vehicle or pacli-taxel treatment. Twenty seconds was adopted as the maximaltime of reaction to avoid possible tissue damage. The devel-opment of thermal hyperalgesia by paclitaxel treatments wasnot reproduced in all experiments conducted in CD1animals, and its incidence was variable among experiments(from 10 to 80%). The effect of drug treatments on thisparameter was assessed only when the incidence of thermalhyperalgesia reached  ~ 80%. Overt nociception The procedure used was similar to that described previously(Ferreira  et al ., 2005). Twenty microlitres of BK (10 nmol perpaw) or DABK solution (20 nmol per paw) was injected intra-plantarly (i.pl.) under the surface of the right hindpaw 7 daysafter the first treatment with paclitaxel or vehicle in CD1mice. Separate groups of animals received an i.pl. injection of saline (0.9% NaCl). The animals were placed individually inchambers (transparent glass cylinders of 20 cm diameter) andwere allowed to adapt to the chambers for 20 min beforealgogen or saline injection. After challenge, the mice wereobserved individually for 10 min. The amount of time spentlicking the injected paw was measured with a chronometerand was considered as indicative of overt nociception.  Mechanical and thermal hyperalgesia after  paclitaxel treatment in kinin receptor knock-out mice The relevance of kinin B 1  or B 2  receptors for the mechanicaland thermal hyperalgesia induced by paclitaxel was analysedusing kinin B 1  and B 2  receptor knock-out mice (B 1 R - / - andB 2 R - / - ), and the corresponding wild-type mice (C57BL/6strains). The full functional contribution of the kallikrein–kinin system was checked using C57BL/6 double knock-outmice lacking the two kinin receptors (B 1 B 2 R - / - ). Briefly, theanimals were submitted to five paclitaxel injections asdescribed earlier, and the mechanical and thermal hyperalge-sia were evaluated at several times after paclitaxel injections.Each set of experiments used four groups: wild-typevehicle- and paclitaxel-injected mice, B 1 R - / - , B 2 R - / - or B 1 B 2 R - / - paclitaxel-treated mice.  Intrathecal (i.t.) and i.c.v. drug injections The i.t. drug injections were performed in accordance withthe method described by Hylden and Wilcox (1980), withminor modifications (Ferreira  et al ., 2002a). The animals werelightly anaesthetized with isoflurane, and a needle connectedto a microsyringe by polyethylene tubing was introducedthrough the skin. Subsequently, 5  m L of saline solution (0.9%NaCl) alone (control) or containing the drugs was injectedbetween the L5 and L6 vertebral spaces. For i.c.v. injections,the animals were lightly anaesthetized with isoflurane, and5  m L of sterile saline containing the drugs was injecteddirectly into the lateral ventricle (coordinates from bregma:1 mm lateral; 1 mm rostral; 3 mm vertical), as described pre-viously by Laursen and Belknap (1986). The control animalsreceived the same volume of saline.  Effect of selective kinin receptor antagonistson the hypernociceptive responses induced by paclitaxel To assess the contribution of kinin B 1  and B 2  receptors to thedevelopment of mechanical and thermal hyperalgesiainduced by paclitaxel, different groups of CD1 mice weretreated with the selective peptide kinin B 1  or B 2  receptorantagonists, des-Arg 9 -Leu 8 -BK (DALBK; 100 nmol·kg - 1 ) andHoe 140 (50 nmol·kg - 1 ), respectively, administered by the i.p.route twice a day (each 12 h) for 6 days (days 1–6), starting atthe time of the first (day 1) paclitaxel treatment. Mechanicaland thermal hyperalgesia were evaluated between days 7 and9 after the first paclitaxel injection.To analyse the involvement of kinin B 1  or B 2  receptors onthe established mechanical and thermal hyperalgesia inducedby paclitaxel, CD1 mice were treated with the selectivepeptide B 1  or B 2  receptor antagonists, DALBK or Hoe 140,respectively, 7 or 14 days after the first paclitaxel treatment bydifferent pathways of administration. First, DALBK (100–300 nmol·kg - 1 ) or Hoe 140 (30–100 nmol·kg - 1 ) was given bythe i.p. route in order to evaluate its systemic effect. In otherexperimental groups, to evaluate the peripheral effect of theantagonists, DALBK (3 nmol per paw) or Hoe 140 (3 nmol perpaw) was injected by the i.pl. pathway. Finally, the centraleffect of single injections of DALBK (10 pmol) or Hoe 140(100 pmol) was tested by the i.t. or i.c.v. route. Mechanicaland/or thermal hyperalgesia were evaluated, as described pre-viously, between 1 and 6 h after drug treatment.To check the effect of repeated administration of kininreceptor antagonists on the established mechanical hyperal-gesia induced by paclitaxel, CD1 mice were treated withDALBK (100 nmol·kg - 1 , i.p.) or Hoe 140 (50 nmol·kg - 1 , i.p.)twice a day (every 12 h) for 2 days (days 7 and 8). Mechanicalhyperalgesia was evaluated between days 7 and 11 after thefirst paclitaxel injection.The protocols of all tested drugs (doses and time of injec-tions) were chosen in accordance with previous publicationsof our group (Ferreira  et al ., 2002a,b; 2004; 2008; Costa  et al .,2006; 2010; Quintão  et al ., 2008). Quantitative real-time PCR The expression of B 1  receptor mRNA was measured usingquantitative real-time PCR according to the methoddescribed previously (Ferreira  et al ., 2005). Seven and four-teen days after the first injection of vehicle or paclitaxel, mice(four to six in each group) were killed, and the plantar skin of the right hind paw, lumbar dorsal root ganglia (DRG)(between the L 4  and L 6  segments), lumbar spinal cord seg-ments (L 4 –L 6 ), thalamus, hypothalamus, parietal cortex and BJP Paclitaxel-induced neuropathy and kinin receptors British Journal of Pharmacology (2011)  164  681–693 683  pre-frontal cortex were isolated, dissected, frozen in liquidnitrogen and stored at  - 80°C until use. Thawed tissues werehomogenized in 0.3–1 mL of TRIzol reagent (Invitrogen,Carlsbad, CA, USA), and total RNA was isolated according tothe instructions of the manufacturer. RNA concentration inthe samples was determined by a NanoDrop 1100 (NanodropTechnologies, Wilmington, DE, USA). Reverse transcriptionassay was carried out using M-MLV Reverse Transcriptase(Invitrogen) according to the manufacturer’s instructions.cDNA was amplified in duplicate using a TaqMan UniversalPCR Master Mix Kit (Applied Biosystems, Foster City, CA,USA) with specific TaqMan Gene Expression target genes(Applied Biosystems): the 3 ′  quencher FAM-labelled probe formouse B 1 R (Mm00432059_s1) and the 3 ′  quencher VIC-labelled probe for mouse GAPDH (Mm03302249_g1), thelatter being used as an endogenous control for normalization.PCR was performed in a 96-well Optical Reaction Plate(Applied Biosystems). The thermocycler parameters were asfollows: 50°C for 2 min, 95°C for 10 min, 60 cycles of 95°Cfor 15 s and 60°C for 1 min. Both FAM and VIC correspon-dent fluorescence were acquired at the end of each extensionphase. The PCR cycle (when a given fluorescence threshold iscrossed by the amplification curve) was considered our firstparameter to analyse mRNA expression and named  C t .  D C t values were calculated by subtracting GAPDH  C t  from kininB 1 R  C t  to obtain the 2 -DD Ct  parameter, which represents relativeB 1 R/GAPDH expression.  Data analysis Results are presented as the mean    SEM of six to eightanimals for each experimental group. The percentages of inhibition are reported as the difference (in percentage)between the areas under the time–response curve of the testgroup in relation to the corresponding control group. Statis-tical comparisons of the data were performed by two-way ANOVA  followed by Bonferroni’s post-test, one-way  ANOVA  fol-lowed by the Newman–Keuls post-test or Student’s  t  -test,using GraphPad Prism software version 5.01 (GraphPad Soft-ware Inc., La Jolla, CA, USA).  P   values  < 0.05 were consideredsignificant.  Materials The following drugs were used: Cremophor EL, DABK, BK andDALBK were purchased from Sigma Chemical Company (St.Louis, MO, USA). Hoe 140 was kindly donated by Sanofi-Aventis (Bridgewater, NJ, USA). Paclitaxel (6 mg·mL - 1 in Cre-mophor EL) was obtained from Dosa S.A. Laboratory (BuenosAires, Argentina). All drugs were diluted in saline (0.9%NaCl). The paclitaxel stock solution (6 mg·mL - 1 in Cremo-phor EL) was diluted in saline to a concentration of 0.2 mg·mL - 1 (solution for injection). Results  Mechanical and thermal hyperalgesiafollowing paclitaxel treatment in kininreceptor-deficient mice As illustrated in Figures 1A,B and 2, the 5 day treatmentwith daily i.p. injections of paclitaxel (2 mg·kg - 1 ) induced asignificant decrease in both mechanical and thermal (heat)withdrawal threshold in C57BL/6 and CD1 mice strains com-pared with vehicle-treated groups. Mechanical and thermalhyperalgesia was significant 7 days after the initial injectionof paclitaxel, and persisted for up to 21 and 14 days, respec-tively (Figure 1). When B 1 R - / - , B 2 R - / - or B 1 B 2 R - / - mice weretreated with paclitaxel, both mechanical and thermal hyper-nociceptive responses were notably reduced during almostthe entire period of evaluation in comparison to wild-typemice (Figure 1A,B). As expected, the inhibition of paclitaxel-induced hyperalgesia by concomitant deficiency of bothkinin B 1  and B 2  receptors (B 1 B 2 R - / - , double knock-out mice)was greater than that caused by the single ablation of B 1  or B 2 receptors (Figure 1C,D). Of note, the inhibition of paclitaxel-induced mechanical hyperalgesia by double deletion of bothkinin receptors persisted up to 21 days, while single ablations(B 1  or B 2  receptors) were effective only for the period of 14days (Figure 1A). On the other hand, there was no significantdifference in the percentage of weight gain and rectal tem-perature between vehicle- and paclitaxel-treated animals inboth CD1 and C57BL/6 mice during 21 days of testing (datanot shown).  Effect of selective kinin B 1  R or B 2  Rantagonists on the genesis of hypernociceptiveresponses induced by paclitaxel treatment  The involvement of kinin receptors in the onset of mechani-cal and thermal hyperalgesia induced by paclitaxel treatmentwas assessed by treating CD1 mice with selective kinin B 1  orB 2  receptor antagonists, DALBK (100 nmol·kg - 1 ) or Hoe 140(50 nmol·kg - 1 ) respectively. The antagonists were given bythe i.p. route twice a day (every 12 h) for 6 days (betweendays 1 and 6), starting at the time of the first paclitaxeltreatment. As can be seen in Figure 2A, DALBK (B 1  receptorantagonist) or Hoe 140 (B 2  receptor antagonist) treatmentswere able to prevent the mechanical hyperalgesia only at theinitial time point (day 7) after paclitaxel injection (77  15%and 61  4% of inhibition respectively). In addition, Hoe 140treatment prevented the paclitaxel-induced thermal hyper-nociceptive response at the initial stage (69  13% of inhibi-tion), while the B 1  receptor antagonist (DALBK) wasineffective on this parameter (Figure 2B).  Effect of systemic treatment with selectivekinin B 1  or B 2  receptor antagonists on theestablished mechanical and thermalhyperalgesia induced by paclitaxel treatment  In an attempt to further evaluate the participation of kininreceptors in the maintenance of mechanical and thermalhyperalgesia induced by paclitaxel, CD1 mice were intraperi-toneally treated with the selective kinin B 1  (DALBK) or B 2 receptor (Hoe 140) antagonists, 7 and 14 days after the firstpaclitaxel injection. The results depicted in Figure 3 demon-strate that systemic treatment with DALBK (150 and300 nmol·kg - 1 , i.p.) or Hoe 140 (50 and 100 nmol·kg - 1 , i.p.)was effective in inhibiting the mechanical hyperalgesiainduced by paclitaxel for up to 2–3 h after drug administra-tion when assessed at days 7 (Figure 3A,B) and 14 (Figure 3C).The inhibition values obtained for mechanical hyperalgesiaare shown in Table 1 and are expressed as the area under the BJP  R Costa et al. 684 British Journal of Pharmacology (2011)  164  681–693

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