Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice

UNCORRECTED PROOF 1Analgesic and an i-inflamma o y e ec i eness o si aglip in and 2 ildaglip in in mice 3Q1 Judi Újhelyi a , Zol án Újhelyi b , And ea Szalai a , János F. László c,d , Mayella Cayasso a , 4Miklós Vecse nyés b , Róbe Pó szász a, ⁎ 5 a Depa men o Pha macology and Pha maco he apy, Uni e si y o Deb ecen, Deb ecen, Nagye dei k . 98, 4032 Hunga y 6 b Depa men o Pha maceu ical Technology, Uni e si y o Deb ecen, Deb ecen, Nagye dei k . 98, 4032 Hunga y 7 c Depa men o Compu e Science, Uni e si y o Deb ecen, Deb ecen, Kassai ú 26, 4028 Hunga y 8 d e-Come s LLC, Budapes , Csej ei u. 1-3 1025 Hunga y abs ac 9a icle in o 10 A icle his o y: 11 Recei ed 31 Janua y 2014 12 Recei ed in e ised o m 5 Sep embe 2014 13 Accep ed 11 Sep embe 2014 14 A ailable online xxxx 15 Keywo ds: 16 A h i is 17 Mechanical and he mal ouch sensi i i y 18 Mice 19 Mus a d oil 20 Si aglip in 21 Vildaglip in 22To alida e hepo en ial an i-inflamma o yandanalgesic oleo si a-and ildaglip in,fi e di e en expe imen al 23models we e usedin mice: i) mus a d oil-induced ea edema, ii) neu ophil accumula ion,iii) mechanicaland i ) 24 he mal ouch sensi i i y in comple e F eund's adju an -induced a h i is and ) capsaicin-induced plasma 25ex a asa ion in he u ina y bladde . Fo he comple e examina ion pe iod in i) he dose o 10 mg si aglip in as 26well as 1–10 mg ildaglip in was ound o significan ly dec ease ea edema as compa ed o posi i e con ol 27(pb0.05, n= 8/g oup). All doses o si aglip in p o ided an an i-inflamma o y e ec pb0.005 (n= 10/ 28g oup) in es ii) and an analgesic e ec in iii) excep 3 mg. Vildaglip in was simila ly e ec i e in es ii) 29(pb0.005, n= 10/g oup) as si aglip in, bu i ailed o a ec mechanical ouch sensi i i y. Unlike mechanical 30 ouch sensi i i y, bo h glip ins could beneficially ac on he he mal h eshold (pb0.05, n= 10/g oup). And 31only in es s ) could bo h glip ins e e se inflamma ion. Fu he s udies a e needed o suppo he sugges ion 32 ha he u iliza ion o hese beneficial e ec s o glip ins may be conside ed in he ea men o Type 2 diabe ic 33pa ien s. 34 © 2014 Published by Else ie B.V. 3536 37 38 39 1. In oduc ion 40 Ch onic inflamma ion and pain can be highly debili a ing. To educe 41 he inflamma ion i sel o o elie e he ela ed pain is a jus ifiable 42 expec a ion o he pa ien s. An i-inflamma o y and analgesic d ugs a e 43 commonly p esc ibed o he symp oma ic ea men o di e en 44 diseases and he ange o chemical classes o a ailable d ugs is qui e 45 b oad. The mos equen ly used d ugs a e he non-s e oidal an i- 46 inflamma o y d ugs, al hough he applica ion o s e oid compounds in 47 se ious cases is also widely accep ed. The condi ions when hese d ugs 48 a e applied a e mos ly immune-d i en diseases like mul iple scle osis, 49 inflamma o y bowel disease, o heuma oid a h i is. Mo eo e , diabe- 50 es ela ed pain such as diabe ic neu opa hy o pain ul diabe ic neu i is 51 a flic s a majo i y o diabe ic pa ien s especially, i he diabe es is no 52 ea ed adequa ely. 53 Since diabe es (especially ype-2 diabe es) has a g owingp e alence 54 wo ldwide, no el ea men s o he disease a e in he ocus o scien ific 55 in e es . The wo mos ecen ly accep ed inc e in mechanisms in ol ing 56d ug ca ego ies a e he deg ada ion- esis an glucagon-like pep ide-1 57(GLP-1) ecep o agonis s (inc e in mime ics) and he inhibi o s o 58dipep idyl pep idase-4 (DPP-4) ac i i y (inc e in enhance s) [1]. The 59pha macological ac ions o GLP-1 analogues and DPP-4 inhibi o s ha e 60been e iewed ecen ly [2]. 61The e a e in es inal ho mones eleased a e he o al adminis a ion 62o glucose. These ho mones a e eleased in a glucose-dependen man- 63ne and a e esponsible o augmen ing insulin sec e ion, p omo ing ß 64cell p oli e a ion and educing apop osis. This is defined as he inc e in 65e ec . The wo mos impo an ho mones in ol ed in he inc e in 66mechanism a e he glucose-dependen insulino opic polypep ide 67(GIP) and GLP-1 [1,3]. Bo h GIP and GLP-1 a e apidly inac i a ed a e 68 hei elease; he hal -li e o ac i e GLP-1 being less han 2 minu es. 69The inac i a ion is caused by a unca ion o he pep ides by he emo al 70o he N- e minal pep ide end. This p ocess is execu ed by he enzyme 71dipep idyl pep idase-4 (DPP-4) [4]. DPP-4 is a 110-kDa ype-II in eg al 72memb ane glycop o ein wi h ubiqui ous exp ession and whose enzyme 73ac i i y has been eco ded in a s, mice and humans. I is p esen in he 74epi helial cells o he in es ine, kidney, li e , lung, hymus, lymph node, 75spleen, p os a e and in adipocy es, as well as on ac i a ed lymphocy es 76and monocy es [5]. Besides he inc e in ho mones, a numbe o 77bioac i e pep ides a e po en ial subs a es o DPP-4. These include 78neu opep ide Y, pep ide YY, gas in- eleasing polypep ide, pi ui a y Regula o y Pep ides xxx (2014) xxx–xxx ⁎Co esponding au ho a : Dep . o Pha macology and Pha maco he apy, Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Deb ecen, Nagye dei k . 98, 4032 Hunga y. Tel.: +36 52 411717x55304. E-mail add ess: obe .po [email protected] (R. Pó szász). REGPEP-04531; No o Pages 7 h p://dx.doi.o g/10.1016/j. egpep.2014.09.006 0167-0115/© 2014 Published by Else ie B.V. Con en s lis s a ailable a ScienceDi ec Regula o y Pep ides jou nal homepage: www.else ie .com/loca e/ egpep Please ci e his a icle as: Újhelyi J, e al, Analgesic and an i-inflamma o y e ec i eness o si aglip in and ildaglip in in mice, Regul Pep (2014), h p://dx.doi.o g/10.1016/j. egpep.2014.09.006 UNCORRECTED PROOF 79 adenyla e-cyclase-ac i a ing polypep ide, insulin-like g ow h ac o -1, 80 subs ance P and a ious chemokines [6]. DPP-4 is also known as he 81 cell su ace an igen CD26 and i can associa e wi h he lymphocy e 82 cell-su ace molecules CD45 and adenosine deaminase (ADA) o ha e 83 a co-s imula o y unc ion in he immune esponse [5]. An in e es ing 84 obse a ion is he inc ease in he plasma concen a ion o DPP-4 as a 85 soluble p o ein du ing con inuous ea men o humans by si aglip in 86 (100 mg/day). This migh o igina e om shedding o CD26 p o eins 87 om mononuclea cells e oked by si aglip in [8]. 88 Dipep idyl pep idase-4 inhibi o s, like si aglip in and ildaglip in, 89 ha e been al eady in oduced o he ma ke since 2006 and a e used 90 o he ea men o ype-2 diabe es. Glip ins a e ound o imp o e he 91 ascula endo helial unc ion, hus pe o ming pleio opic ca dio ascu- 92 la ac ions [7]. The sa e y o he glip in amily was ques ioned ecen ly, 93 bu in wo long- e m ca dio ascula ou come ials, Saxaglip in Assess- 94 men o Vascula Ou comes Reco ded in Pa ien s wi h Diabe es 95 Melli us-TH ombolysis in Myoca dial In a c ion 53 (SAVOR-TIMI 53), 96 i has been p o en ha saxaglip in is sa e om he ca dio ascula 97 poin o iew. I was shown ha he p ima y endpoin s o he s udy (a 98 composi e o ca dio ascula dea h, non- a al myoca dial in a c ion o 99 non- a al ischemic s oke) occu ed in 7.3% o he saxaglip in g oup 100 compa ed wi h 7.2% o he placebo g oup (ClinicalT ials.go Iden ifie : 101 NCT01107886). The conclusion o Ca dio ascula Ou comes S udy o 102 Aloglip in in Pa ien s Wi h Type 2 Diabe es and Acu e Co ona y Syn- 103 d ome (EXAMINE) s udy (ClinicalT ials.go Iden ifie : NCT00968708) 104 was ha in ype-2 diabe ic pa ien s wi h ecen acu e co ona y 105 synd ome, majo ca dio ascula e en a es o aloglip in we e no 106 inc eased compa ed o placebo. In his ial acu e panc ea i is de elop- 107 men as a se ious ad e se e en was only 0.07% compa ed o placebo 108 (0.15 %), hus i is alid o s a e ha aloglip in is ee om his side e ec . 109 Bo h inc e ins, GIP andGLP-1 s imula e insulin sec e ion in a glucose 110 dependen manne and consequen ly, DPP-4 inhibi o ea men does 111 no inc ease he isk o hypoglycaemia. No only was he occu ence o 112 hypoglycaemic e en s inciden ally simila o lowe when compa ing 113 g oups ea ed wi h DPP-4 inhibi o (ei he mono he apy o in combi- 114 na ion)wi h placebo ea ed g oups in di e en s udies, bu henumbe 115 o epo ed ad e se e en s did no di e om he ac i ely ea ed 116 g oups. [4]. I has been demons a ed in animal s udies ha oxici y 117 may be caused by he inhibi ion o o he enzymes in his amily, like 118 DPP-8 and DPP-9 [9], so he selec i i y o inhibi o s o DPP-4 is c ucially 119 impo an o ensu e an op imal sa e y p ofile. Since bo h si aglip in and 120 ildaglip in show a highe ela i e selec i i y o DPP-4, he isk o 121 de elopmen o ad e se e ec s due o inhibi ion o o he enzymes is 122 minimized [4,10]. Howe e , i did u n ou ha du ing he pos ma ke - 123 ing pe iod o glip ins hese DPP-4 inhibi o s inc eased he a e o in ec- 124 ions such as nasopha yngi is and u ina y ac in ec ions [11].In 125 addi ion, panc ea i is was epo ed mainly associa ed wi h he use o 126 si aglip in and linaglip in [12], al hough a ecen me a-analysis could 127 no find di e ences be ween DPP-4 inhibi o s [13]. In spi e o he in- 128 c eased isk o in ec ions, si aglip in and ildaglip in a e well ole a ed 129 in gene al. Besides he p ima y a ge ed he apeu ic a ea, in i o and 130 in i o s udies showed an i-inflamma o y p ope ies o DPP-4 inhibi o s 131 ha could lead o a no el d ug class o an i-inflamma o y diso de s 132 [14]. Al e ed ci cula ing pep idase ac i i y and memb ane DPP-4 133 exp ession ha e been demons a ed in a numbe o human inflamma o- 134 ydiseases[15]. DPP-4 is esponsible o hemodifica ion o a numbe o 135 egula o y ac o s, such as pep ides o chemokines and a ec s he 136 signaling unc ions. This sugges s ha DPP-4 is in ol ed in de e mining 137 immune esponse and p ocession o inflamma o y diso de s as well. As 138 men ioned p e iously, DPP-4 is also known as he cell su ace an igen 139 CD26, which signals T-cells o p oli e a e. Howe e , his mechanism 140 canno be a ibu ed o he DPP-4 inhibi ion [16] because he T-cell 141 ac i a ion seems o be independen o he DPP-4 enzyme ac i i y and 142 he ADA-binding capabili y [16,17]. Mo eo e , e e sible DPP-4 inhibi- 143 o Lys[Z(NO 2 )]-py olidide was shown o supp ess au oimmune 144 encephalomyeli is and up egula ed TGF-β1sec e ionin i o [18]. 145The possible an i-inflamma o y p ope y o he glip in g oup can 146be conside ed as an addi ional alue o hese d ugs in diabe ic 147pa ien s wi h neu i is o diabe ic neu opa hy, o pa ien s wi h 148a he oscle osis conside ing ha hese diseases a e d i en by inflam- 149ma o y p ocesses [19]. Mo eo e , he educ ion in plasma C- eac i e 150p o ein concen a ion and sys olic blood p essu e ha e been 151desc ibed o exena ide [20].Thean i-inflamma o y ac ion o 152si aglip in [8] and exena ide [19] a e p o en biochemically in 153humans, hus in he p esen se ies o expe imen s we aimed o 154examine he possible an i-inflamma o y e ec o wo po en DPP-4 155inhibi o s, si aglip in and ildaglip in. They we e applied in in i o 156inflamma ion and analgesic models in mice. 1572. Ma e ials and me hods 1582.1. Animals and e hics 159Expe imen s we e pe o med on 25–35 g CD1 male mice (Cha les 160Ri e ,Gödöllő, Hunga y),kep unde s anda dpa hogen- eecondi ions 161a 24–25 °C and p o ided wi h s anda d oden chow and wa e 162ad libi um. The ligh /da k cycle was 12 h/12 h. Animal p ocedu es 163we e app o ed by he local animal e hics commi ee and Na ional 164Food Chain Sa e y O fice Animal Heal h and Animal Wel a e Di ec o a e 165unde he numbe 26/2007/DE MÁB in acco dance wi h he Eu opean 166Communi ies Council Di ec i es (86/609/ECC) and he Hunga ian Ac 167 o he P o ec ion o Animals in Resea ch (XXVIII . 32§) and complied 168wi h he ecommenda ions o he In e na ional Associa ion o he 169S udy o Pain [21] and he Helsinki Decla a ion. The design o he 170s udy was ca ied ou in a manne in which o minimize he numbe 171o animals used and hei su e ing. 1722.2. Subs ances and hei applica ion 173Mice we e dosed wi h 1, 3 o 10 mg/kg si aglip in o ildaglip in 174(Nanjing Ange Pha maceu icals, Nanjing, Jiangsu, China) dissol ed in 175saline by o al ga age (1 ml/100 g). Con ol g oups we e gi en he 176 ehicle in he same amoun and way. A single applica ion was used in 177 he case o one-day expe imen s, while daily applica ion was used in 178 he 21 day long expe imen s, as sugges ed by Thomas e al. [22]. 179T ea men s and measu emen s we e implemen ed 30 min a e he 180o al ga age in e e y case. 1812.3. Allyl-iso hiocyana e (AITC)-induced inflamma ion model 182Anes hesia was induced by hiopen al (T apanal, Sandoz, Basle, 183Swi ze land) in an amoun o 50 mg/kg in ape i oneally (i.p.), epea - 184ed as equi ed. The inne and ou e su ace o he igh ea was hen 185smea ed wi h 1% allyl-iso hiocyana e (AITC) (Sigma-Ald ich, Budapes , 186Hunga y) dissol ed in pa a fin oil, using a co on-wool s ick. This ea - 187men was applied 30 min a e he o al ga age (subs ances dissol ed in 188saline o ehicle in he con ol g oup) and he p ocedu e was epea ed 18945 min a e he fi s applica ion ollowing he ins uc ions o Bán ölgyi 190[23] and Inoue e al. [24]. Thus he o al adminis a ion o glip ins was 191pe o med fi s ly and he induc ion o inflamma ion was ca ied ou 192secondly. 193A he end o he expe imen he animals we e sac ificed by ce ical 194disloca ion and ea s we e s o ed on -20 °C o he neu ophil accumula- 195 ion assay. 1962.4. Measu emen o ea edema 197Ea hickness was measu ed by a mic ome e calipe (Ox o d P eci- 198sion, Leices e , England) wi h 0.1 mm accu acy be o e he AITC ea - 199men , 15 min a e he fi s AITC applica ion, hen by each hou du ing 200a 6 hou pe iod a e each AITC ea men acco ding o Inoue e al. [24] 201wi h sligh modifica ions. Glip in ea men was pe o med 30 minu es 2J. Újhelyi e al. / Regula o y Pep ides xxx (2014) xxx–xxx Please ci e his a icle as: Újhelyi J, e al, Analgesic and an i-inflamma o y e ec i eness o si aglip in and ildaglip in in mice, Regul Pep (2014), h p://dx.doi.o g/10.1016/j. egpep.2014.09.006 UNCORRECTED PROOF 202 be o e he commencemen o ea edema induc ion. Da a we e exp essed 203 in mic ome e s. 204 2.5. Measu emen o neu ophil accumula ion 205 F ozen ea samples we e hawed a oom empe a u e, chopped in o 206 small pieces, and homogenized in 0.05 M po assium phospha e bu e 207 con aining 0.5% HTAB (hexadecyl ime hylammonium b omide, 208 Sigma-Ald ich, Budapes , Hunga y), 1 ml bu e /ea . The homogena e 209 was cen i uged a 11000 g a 4 °C o 10 min and 200 μlo hesupe na- 210 an was placed in o Eppendo ubes. 211 Myelope oxidase ac i i y was assayed by measu ing he H 2 O 2 - 212 dependen oxida ion o 3,3′,5,5′- e ame hylbenzidine (TMB, Sigma- 213 Ald ich, Budapes , Hunga y) as sugges ed by Suzuki e al. [25].Ini s 214 oxidized o m, TMB has a blue colo , which was measu ed spec opho- 215 ome ically a 620 nm. The eac ion was pe o med in 96-well mic o i- 216 e pla es a oom empe a u e. The eac ion mix u e consis ed o 25 μl 217 o he issue sample, 25 μlo TMB(final concen a ion 0.16 mM) 218 dissol ed in dime hylsul oxide (DMSO) and 200 μlH 2 O 2 (final concen- 219 a ion 0.24 mM, Sigma-Ald ich, Budapes , Hunga y) dilu ed in 0.08 M 220 phospha e bu e pH 5.4 a e Schie wagen e al. [26]. The op ical densi- 221 y (OD) was measu ed a 5 min in e als o 30 min using a mic opla e 222 eade (FLUOs a OPTIMA, BMG Lab ech, O enbe g, Ge many). Da a 223 was exp essed in a bi a y uni s o abso bance. 224 2.6. Induc ion o a h i is 225 Ch onic a h i is o he igh ibio a sal join o mice was induced by 226 he subcu aneous injec ion o 0.1 ml o F eund's comple e adju an 227 (CFA, killed Mycobac e ia suspended in pa a fin oil, 1 mg/ml as p o id- 228 ed by Sigma-Ald ich, Budapes , Hunga y) in o he plan a su ace o he 229 igh hind paw and oo o he ail. To enhance sys emic e ec s, an addi- 230 ional injec ion in o he ail was gi en he ollowing day as desc ibed by 231 Helyes e al. [27]. In o de o minimize he su e ing o mice, sho - e m 232 gene al anes hesia was induced by 1% isoflu ane (Abbo Labo a o ies, 233 Budapes , Hunga y) deli e ed in 1:2 oxygen/ni ous oxide mix u e. 234 2.7. Measu emen o plasmaex a asa ion in he u ina y bladde o mice 235 Mice we e anaes he ized by i.p. adminis a ion o hiopen al 236 (50 mg/kg). A la e al ail ein was cannula ed o in a enous adminis- 237 a ion. 1 o 3 mg o ildaglip in o si aglip in was adminis e ed by o al 238 ga age 30 minu es be o e he commencemen o he capsaicin chal- 239 lenge. E ans blue (30 mg/kg) and 1 minu e la e capsaicin (1 mg/kg) 240was injec ed h ough he enous cannula. Each animal was sac ificed 241by ansca diac pe usion wi h 50 ml o 0.9% w/ saline in o he le ca - 242diac en icle 10 min a e in a enous injec ion o E ans blue a 37 °C. 243The u ina y bladde was hen emo ed and weighed. Excised issues 244we e incuba ed in 1 ml o o mamide o 48 h and E ans blue con en 245was measu ed spec opho ome ically a 620 nm and exp essed as 246μg/g we mass o he issue. 2472.8. Measu emen o mechano-nocicep i e h eshold 248Touch sensi i i y on he plan a su ace was measu ed wi h on F ey 249filamen s (Bioseb, Cha ille, F ance) be o e he expe imen , 3, 7, 10, 14, 25017, and 21 days ollowing he fi s CFA adminis a ion. The se o 20 251monofilamen s p o ided an app oxima e loga i hmic scale o ac ual 252 o ce and a linea scale o pe cei ed in ensi y. Mice we e placed in o a 253Plexiglas cage wi h a pi ed floo . Following animal acclima iza ion he 254ope a o placed he monofilamen unde he animal's paw and p essed 255agains he su ace ill he animal indica ed he p essu e sensa ion by 256pulling back o shaking i s paw, o he monofilamen cu ed wi hou 257any kind o eac ion s a ing wi h 0.008 g and anging up o 300 g. 2582.9. Inc easing- empe a u e ho pla e es 259The pla e (Supe ech, Pécs, Hunga y) in con ac wi h he paws has 260been slowly wa med up om oom empe a u e and he h eshold 261 empe a u e p oducing he fi s noci ensi e beha io (e.g., paw licking) 262was eco ded. Since he empe a u e was inc eased g adually in o he 263noxious ange, s ess associa ed wi h he es ing p ocedu e was mini- 264mized. The hea ed su ace dimensions we e 110 × 80 mm su ounded 265by 350 mm high anspa en Plexiglas walls. The commanding compu - 266e p og am was se o p oduce a 3 °C/min empe a u e inc ease o he 267pla e as p oposed by László e al. [28]. When he hind paw licking o 268flinching was obse ed he h eshold empe a u e was eco ded. The 269measu emen was e mina ed a he h eshold le el o when he pla e 270 empe a u e eached 50 °C o a oid issue damage [29]. Da a we e 271exp essed in °C. 2722.10. S a is ics 273Since baseline alues o di e se g oups we e significan lydi e en in 274all measu emen s, a baseline co ec ion was ca ied ou on aw alues. 275Baseline co ec ed alues we e ega ded as p ima y ou comemeasu es. 276Two-way ANOVA wi h eplica ion was used o mul iple g oup analysis 277wi h he ime poin o obse a ion and he ea men op ion as ac o s Fig. 1. Time e olu ion o baseline co ec ed ea hickness (μm) in allyl-iso hiocyana e (AITC)-induced ea edema model in mice as a unc ion o he amoun o A) si aglip in and B) ildaglip in adminis e ed by o al ga age. E o ba s deno e s anda d e o o he mean. Lines be ween ma ke s guide he eye only. Fo he comple e ime pe iod × (pb0.001), #(pb0.005), and $ (pb0.001) showed significan di e ences o posi i e con ol, 1 mg, and 3 mg si aglip in, and × (pb0.001), # (pb0.01) o posi i e con ol and 1 mg ildaglip in, espec i ely as assessed by Games-Howell pos hoc es . 3J. Újhelyi e al. / Regula o y Pep ides xxx (2014) xxx–xxx Please ci e his a icle as: Újhelyi J, e al, Analgesic and an i-inflamma o y e ec i eness o si aglip in and ildaglip in in mice, Regul Pep (2014), h p://dx.doi.o g/10.1016/j. egpep.2014.09.006 UNCORRECTED PROOF 278 o he comple e du a ion o he expe imen s. Games-Howell es s we e 279 used as pos hoc analysis o bina y compa ison o g oup a e ages. 280 Significan di e ences a he 95% confidence in e al we e ecognized, 281 i pb0.05. Below 0.001 no nume ic alues o pa e p o ided in he ex . 282 3. Resul s 283 3.1. AITC-induced ea edema 284 Bo h o ally adminis e ed glip ins significan ly dec eased ea hick- 285 ness in he comple e ime pe iod compa ed o posi i e con ol (AITC 286 only) in a dose-dependen manne as seen in Fig. 1.Themaximum 287 e ec o AITC was measu ed a 2 hou pos -challenge ime in ei he 288 case. Fo he comple e examina ion pe iod 10 mg si aglip in as well as 289 1–10 mg ildaglip in was ound o significan ly dec ease ea edema as 290 compa ed o posi i e con ol. 291 3.2. AITC-induced neu ophil accumula ion 292 The e ol ed inflamma ion was shown by he high le el o 293 myelope oxidase enzyme in he posi i e con ol g oup (AITC only), see 294Fig. 2. The model is sui able o measu ing he ex en o inflamma ion, 295since hese da a defini ely di e ge om he nega i e con ol g oup 296 esul s. Si aglip in ea men was ound significan ly e ec i e in blocking 297 he e olu ion o inflamma ion; e e y examined dose could e e se in- 298flamma ion. (Blind samples we e no included in he hypo hesis es ing.) 299The e ec o ildaglip in ea men was simila o ha o si aglip in, 300al hough he dose o 3 mg/kg had only an insignifican impac . 3013.3. Measu emen o plasmaex a asa ion in he u ina y bladde o mice 302The capsaicin-induced plasma ex a asa ion in u ina y bladde s o 303mice was inhibi ed by si aglip in (1 mg p= 0.025 and 3 mg pb0.001) 304and ildaglip in (bo h 1 mg and 3 mg pb0.001) significan ly (Fig. 4). 305Di e ence in ac ion was seen be ween he highe doses (3 mg/kg) o 306 ilda- and si aglip in. The lowe dose o si aglip in (1 mg/kg) p oduced 307 he leas significan inhibi ion compa ed o he con ol. 3083.4. Touch sensi i i y in CFA-induced a h i is 309Resul s show ha he mechano-nocicep i e h eshold o he un- 310 ea ed g oup was significan ly highe han in he CFA ea ed (posi i e Fig. 2. Time e olu ion o he baseline co ec ed abso bance o myelope oxidase in an allyl-iso hiocyana e (AITC)-induced ea edema model in mice as a unc ion o he amoun o A) si aglip in o B) ildaglip in adminis e ed by o al ga age. E o ba s deno e s anda d e o o he mean. Lines be ween ma ke s guide he eye only. Fo he comple e ime pe iod *(pb0.05) and × (pb0.01) showed significan di e ences o nega i e and posi i e con ol and * (pb0.05), × (pb0.005), # (pb0.01), and $ (pb0.005) o nega i e, posi i e con ol, 1 mg, and 3 mg ildaglip in, espec i ely as assessed by Games-Howell pos hoc es . Fig. 3. Time e olu ion o he mechano-nocicep i e h eshold in F eund's comple e adju an (CFA)-induced a h i is model in mice as a unc ion o he amoun o A) si aglip in o B) ildaglip in adminis e ed by o al ga age. E o ba s deno e s anda d e o o he mean. Lines be ween ma ke s guide he eye only. Fo he comple e ime pe iod * (pb0.001), ×(pb0.01), and # (pb0.05) showed significan di e ences o nega i e, posi i e con ol and 1 mg si aglip in, and * (pb0.001) o nega i e con ol, espec i ely as assessed by Games-Howell pos hoc es . 4J. Újhelyi e al. / Regula o y Pep ides xxx (2014) xxx–xxx Please ci e his a icle as: Újhelyi J, e al, Analgesic and an i-inflamma o y e ec i eness o si aglip in and ildaglip in in mice, Regul Pep (2014), h p://dx.doi.o g/10.1016/j. egpep.2014.09.006 UNCORRECTED PROOF 311 con ol) g oup du ing he whole 21 day expe imen al pe iod (Fig. 3). In 312 he glip in ea ed g oups e e y h eshold was significan ly lowe 313 han in he nega i e con ol g oup; consequen ly nei he si a-, no 314 ildaglip in ea men was able o hinde he e olu ion o allodynia. 315 Si aglip in was significan ly ha m ul in an amoun o 3 and 10 mg/kg 316 doses as compa ed o he posi i e con ol, meanwhile ildaglip in 317 emained ine ec i e in all doses. 318 3.5. Inc easing- empe a u e ho pla e es in CFA-induced a h i is 319 Th eshold empe a u e o he un ea ed g oup was significan ly 320 highe han in he CFA- ea ed g oup du ing he whole 21 day expe i- 321 men al pe iod, ollowing he fi s day as shown in Fig. 5. E e y dose o 322 ei he si aglip in o ildaglip in significan ly inc eased he h eshold 323 empe a u e, compa ed o heposi i e con ol g oup. Nei he si aglip in 324 no ildaglip in could inhibi inflamma ion; he h eshold in all glip in 325 ea ed g oups emained significan ly lowe han in he nega i e con ol 326 g oups. 3274. Discussion 328Acco ding o ou p esen da a, we can conclude ha he s udied 329glip ins had a dose-dependen an i-inflamma o y e ec in in i o 330mouse models. The applied me hods we e sensi i e enough o de ec 331 he ac ion o glip ins. Dipep idyl pep idase inhibi o s we e e iewed 332as an eme ging d ug class o a ious inflamma o y diseases [7]. The 333an i-inflamma o y ac iono hese d ugs we e desc ibed in human s ud- 334ies [8] and o exena ide [19]. Si aglip in significan ly imp o es endo- 335 helial unc ion and inflamma o y s a e in pa ien s wi h co ona y 336a e y disease and uncon olled diabe es melli us [30], o ming a mile- 337s one in he way owa ds widening he spec um o glip ins' indica ion. 338Mo eo e , he GLP-1 ecep o (GLP-1R) is exp essed in lymphoid issue 339and he numbe s o CD4+ and CD8+ T-cells in lymph nodes was 340shown o inc ease a e exena ide (a GLP-1R agonis ) ea men . I 341could also educe he numbe o CD4+ CD25+ Foxp3 + egula o yT- 342cells in he hymus, bu no in he spleen [31], hus playing a egula o y 343 ole in he immune sys em and can influence inflamma o y p ocesses 344[32]. Howe e , Kim e al. [33] we e unable o de ec he e ec o ei he 345GIP o GLP-1 on splenic o hymic CD4+ T-cell mig a ion in i o [33]. 346Eosinophil cell ec ui men (in alle gic as hma o in a opic de ma i is) 347is desc ibed o be media ed by CCL11(eosinophil chemo ac ic p o ein) 348and he ec ui men p o ed o be mo e e ec i e a e pha macological 349inhibi ion o DPP-4 enzyme o in DPP-4-deficien F344 a s [34].The 350ac i a ion o ansien ecep o po en ial anky in 1 (TRPA1) e okes 351nocicep ion h ough subs ance P elease om he p ima y senso yneu- 352 ons; p38 mi ogen-ac i a ed p o ein kinase (p38 MAPK) inhibi o 353SB203580 significan ly a enua ed AITC-e oked subs ance P elease 354[35]. Allyl-iso hiocyana e is capable o inducing ea edemain he p ope 355dose as desc ibed ea lie [23]; he maximum au icle swelling was 356measu ed in he second hou . Bo h examined chemicals, si aglip in 357and ildaglip in we e able o dec ease he AITC-induced inflamma ion 358in a dose-dependen manne howe e , si aglip in had a highe impac . 359This e ec canno be explained by he egula o y ole o GLP-1 on p38 360MAPK, as i was desc ibed as an induce [36]; nei he can i bea ibu ed 361 o he e ec o glip ins on subs ance P me abolism [37]. Mo eo e , he 362physiological ole o GLP-1 is so dominan ha i s inhibi ion can s ill 363o e ide he p38 MAPK-induce p ope y and he algogenic e ec o 364ele a ed subs ance P. T ea men by DPP4 inhibi o I40 significan ly 365 educed he se e i y o expe imen al alle gic encephalomyeli is (EAE), 366in mice concei ably h ough up- egula ing TGF-be a 1 [18].Fu he mo e, 367a dose-dependen inhibi ion o he sec e ion o he p o-inflamma o y 368cy okine TNF-alpha was measu ed in i o [18]. The abili y o glip ins o Fig. 4. Inhibi ion o capsaicin-induced plasmaex a asa ion in u ina y bladde s o mice by Vildaglip in and Si aglip in. Glip ins we e adminis e ed by o al ga age in 1 o 3 mg/kg dose 30 minu es be o e he capsaicin (1 mg/kg) in a enous challange. E ans blue dye was adminis e ed in 30 mg/kg i. . and he plasmaex a asa ion was de e mined spec o- pho ome ically a 620 nm wa e leng h. E o ba s deno e s anda d e o o he mean. * and × deno e significan di e ences o nega i e con ol and o ildaglip in 1 mg, espec i ely as assessed by Games-Howell pos hoc es . Fig. 5. Time e olu ion o he he mo-nocicep i e h eshold in F eund's comple e adju an (CFA)-induced a h i is model in mice as a unc ion o he amoun o A) si aglip in o B) ildaglip in adminis e ed by o al ga age. E o ba s deno e s anda d e o o he mean. Lines be ween ma ke s guide he eye only. Fo he comple e ime pe iod * (pb0.001), ×(pb0.05), and # (pb0.05) showed significan di e ences o nega i e, posi i e con ol, and 1 mg si aglip in, and * (pb0.001), × (pb0.05), # (pb0.05), and $ (pb0.01) o nega i e, posi i e con ol, 1 mg, and 3 mg ildaglip in, espec i ely as assessed by Games-Howell pos hoc es . 5J. Újhelyi e al. / Regula o y Pep ides xxx (2014) xxx–xxx Please ci e his a icle as: Újhelyi J, e al, Analgesic and an i-inflamma o y e ec i eness o si aglip in and ildaglip in in mice, Regul Pep (2014), h p://dx.doi.o g/10.1016/j. egpep.2014.09.006 UNCORRECTED PROOF 369 egula e TNF-alpha, INF-gamma, and a a ie y o in e leukins can be a - 370 ibu ed only o he DPP4 inhibi o y ac i i y, because he compounds 371 used in he p esen se ies o expe imen s ha e a high specifici y o 372 DPP4 and p obably do no ha e any inhibi o y e ec on DPP8 o 9 in 373 he applied doses [38]. Howe e , ildaglip in ine ec i eness in wo 374 models (myelope oxidase measu emen and ouch sensi i i y in CFA- 375 induced a h i is) a 3 mg/kg can be he esul o DPP-9 ac i i y a enua- 376 ion ha ing 66 nM IC50 alue in i o compa ed o 130 nM IC50 o 377 si aglip in [39]. Inhibi ion o DPP-8/9 can lead o he de elopmen o 378 ad e se e ec s in oden s [9,40], bu o he s udies s a e ha he inhibi ion 379 o DPP-8/9 do no ha e any clinical consequence [41]. A educed exp es- 380 sion o ni osa i e s ess and inflamma ion hallma ks wi hin he b ain o 381 ch onically adminis e ed si aglip in was desc ibed ea lie in a mouse 382 model o Alzheime 's disease [42]. An explana ion could easily ise 383 conside ing he ac ha GLP-1 can ha e g ow h- ac o -like p ope ies 384 simila o insulin and he an i-inflamma o y ac i i y is a seconda y ac ion 385 [43]. In ou expe imen s, he an i-inflamma o y ac ion seems o be di ec 386 as demons a ed by he accumula ed numbe o neu ophil cells 387 (measu ed by myelope oxidase enzyme ac i i y) in he inflamed ea ; 388 his accumula ion could be inhibi ed by he glip in p e- ea men . 389 Simila ly o he abo e men ioned es s, si aglip in ea men had a 390 highe impac in he compensa ion o he CFA-induced a h i is, 391 whe e ildaglip in showed no e ec i eness. In case o measu ing he 392 high empe a u e sensi i i y, bo h subs ances showed equal e ec i e- 393 ness. Ou esul s lead o he conclusion ha si aglip in has a s onge 394 influence on he e olu ion o inflamma ion; howe e , ildaglip in 395 showed highe e ec i eness in he inhibi ion o capsaicin-induced 396 plasma ex a asa ion in he u ina y bladde . Al hough he in es iga ed 397 molecules ha e he same e ec i eness in he ea men o ype-2 398 diabe es, i seems ha hey do no ac in he same way, in he immune 399 esponse. 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