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

UNCORRECTED PROOF 1Analgesic and anti-inflammatory effectiveness of sitagliptin and 2vildagliptin in mice 3Q1 Judit Újhelyi a , Zoltán Újhelyi b , Andrea Szalai a , János F. László c,d , Mayella Cayasso a , 4Miklós Vecsernyés b , Róbert Pórszász a, ⁎ 5 a Department of Pharmacology and Pharmacotherapy, University of Debrecen, Debrecen, Nagyerdei krt. 98, 4032 Hungary 6 b Department of Pharmaceutical Technology, University of Debrecen, Debrecen, Nagyerdei krt. 98, 4032 Hungary 7 c Department of Computer Science, University of Debrecen, Debrecen, Kassai út 26, 4028 Hungary 8 d e-Comers LLC, Budapest, Csejtei u. 1-3 1025 Hungary abstract9article info 10 Article history: 11 Received 31 January 2014 12 Received in revised form 5 September 2014 13 Accepted 11 September 2014 14 Available online xxxx 15 Keywords: 16 Arthritis 17 Mechanical and thermal touch sensitivity 18 Mice 19 Mustard oil 20 Sitagliptin 21 Vildagliptin 22To validate thepotential anti-inflammatoryandanalgesicroleofsita-and vildagliptin,five different experimental 23models were usedin mice: i) mustard oil-induced ear edema, ii) neutrophil accumulation,iii) mechanicaland iv) 24thermal touch sensitivity in complete Freund's adjuvant-induced arthritis and v) capsaicin-induced plasma 25extravasation in the urinary bladder. For the complete examination period in i) the dose of 10 mg sitagliptin as 26well as 1–10 mg vildagliptin was found to significantly decrease ear edema as compared to positive control 27(pb0.05, n= 8/group). All doses of sitagliptin provided an anti-inflammatory effect pb0.005 (n= 10/ 28group) in test ii) and an analgesic effect in iii) except 3 mg. Vildagliptin was similarly effective in test ii) 29(pb0.005, n= 10/group) as sitagliptin, but it failed to affect mechanical touch sensitivity. Unlike mechanical 30touch sensitivity, both gliptins could beneficially act on the thermal threshold (pb0.05, n= 10/group). And 31only in tests v) could both gliptins reverse inflammation. Further studies are needed to support the suggestion 32that the utilization of these beneficial effects of gliptins may be considered in the treatment of Type 2 diabetic 33patients. 34 © 2014 Published by Elsevier B.V. 3536 37 38 39 1. Introduction 40 Chronic inflammation and pain can be highly debilitating. To reduce 41 the inflammation itself or to relieve the related pain is a justifiable 42 expectation of the patients. Anti-inflammatory and analgesic drugs are 43 commonly prescribed for the symptomatic treatment of different 44 diseases and the range of chemical classes of available drugs is quite 45 broad. The most frequently used drugs are the non-steroidal anti46 inflammatory drugs, although the application of steroid compounds in 47 serious cases is also widely accepted. The conditions when these drugs 48 are applied are mostly immune-driven diseases like multiple sclerosis, 49 inflammatory bowel disease, or rheumatoid arthritis. Moreover, diabe50 tes related pain such as diabetic neuropathy or painful diabetic neuritis 51 afflicts a majority of diabetic patients especially, if the diabetes is not 52 treated adequately. 53 Since diabetes (especially type-2 diabetes) has a growingprevalence 54 worldwide, novel treatments of the disease are in the focus of scientific 55 interest. The two most recently accepted incretin mechanisms involving 56drug categories are the degradation-resistant glucagon-like peptide-1 57(GLP-1) receptor agonists (incretin mimetics) and the inhibitors of 58dipeptidyl peptidase-4 (DPP-4) activity (incretin enhancers) [1]. The 59pharmacological actions of GLP-1 analogues and DPP-4 inhibitors have 60been reviewed recently [2]. 61There are intestinal hormones released after the oral administration 62of glucose. These hormones are released in a glucose-dependent man63ner and are responsible for augmenting insulin secretion, promoting ß 64cell proliferation and reducing apoptosis. This is defined as the incretin 65effect. The two most important hormones involved in the incretin 66mechanism are the glucose-dependent insulinotropic polypeptide 67(GIP) and GLP-1 [1,3]. Both GIP and GLP-1 are rapidly inactivated after 68their release; the half-life of active GLP-1 being less than 2 minutes. 69The inactivation is caused by a truncation of the peptides by the removal 70of the N-terminal peptide end. This process is executed by the enzyme 71dipeptidyl peptidase-4 (DPP-4) [4]. DPP-4 is a 110-kDa type-II integral 72membrane glycoprotein with ubiquitous expression and whose enzyme 73activity has been recorded in rats, mice and humans. It is present in the 74epithelial cells of the intestine, kidney, liver, lung, thymus, lymph node, 75spleen, prostate and in adipocytes, as well as on activated lymphocytes 76and monocytes [5]. Besides the incretin hormones, a number of 77bioactive peptides are potential substrates for DPP-4. These include 78neuropeptide Y, peptide YY, gastrin-releasing polypeptide, pituitary Regulatory Peptides xxx (2014) xxx–xxx ⁎Corresponding author at: Dept. of Pharmacology and Pharmacotherapy, Medical and Health Science Centre, University of Debrecen, Debrecen, Nagyerdei krt. 98, 4032 Hungary. Tel.: +36 52 411717x55304. E-mail address: [email protected] (R. Pórszász). REGPEP-04531; No of Pages 7 http://dx.doi.org/10.1016/j.regpep.2014.09.006 0167-0115/© 2014 Published by Elsevier B.V. Contents lists available at ScienceDirect Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep Please cite this article as: Újhelyi J, et al, Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice, Regul Pept (2014), http://dx.doi.org/10.1016/j.regpep.2014.09.006 UNCORRECTED PROOF 79 adenylate-cyclase-activating polypeptide, insulin-like growth factor-1, 80 substance P and various chemokines [6]. DPP-4 is also known as the 81 cell surface antigen CD26 and it can associate with the lymphocyte 82 cell-surface molecules CD45 and adenosine deaminase (ADA) to have 83 a co-stimulatory function in the immune response [5]. An interesting 84 observation is the increase in the plasma concentration of DPP-4 as a 85 soluble protein during continuous treatment of humans by sitagliptin 86 (100 mg/day). This might originate from shedding of CD26 proteins 87 from mononuclear cells evoked by sitagliptin [8]. 88 Dipeptidyl peptidase-4 inhibitors, like sitagliptin and vildagliptin, 89 have been already introduced to the market since 2006 and are used 90 for the treatment of type-2 diabetes. Gliptins are found to improve the 91 vascular endothelial function, thus performing pleiotropic cardiovascu92 lar actions [7]. The safety of the gliptin family was questioned recently, 93 but in two long-term cardiovascular outcome trials, Saxagliptin Assess94 ment of Vascular Outcomes Recorded in Patients with Diabetes 95 Mellitus-THrombolysis in Myocardial Infarction 53 (SAVOR-TIMI 53), 96 it has been proven that saxagliptin is safe from the cardiovascular 97 point of view. It was shown that the primary endpoints of the study (a 98 composite of cardiovascular death, non-fatal myocardial infarction or 99 non-fatal ischemic stroke) occurred in 7.3% of the saxagliptin group 100 compared with 7.2% of the placebo group (ClinicalTrials.gov Identifier: 101 NCT01107886). The conclusion of Cardiovascular Outcomes Study of 102 Alogliptin in Patients With Type 2 Diabetes and Acute Coronary Syn103 drome (EXAMINE) study (ClinicalTrials.gov Identifier: NCT00968708) 104 was that in type-2 diabetic patients with recent acute coronary 105 syndrome, major cardiovascular event rates for alogliptin were not 106 increased compared to placebo. In this trial acute pancreatitis develop107 ment as a serious adverse event was only 0.07% compared to placebo 108 (0.15 %), thus it is valid to state that alogliptin is free from this side effect. 109 Both incretins, GIP andGLP-1 stimulate insulin secretion in a glucose 110 dependent manner and consequently, DPP-4 inhibitor treatment does 111 not increase the risk of hypoglycaemia. Not only was the occurrence of 112 hypoglycaemic events incidentally similar or lower when comparing 113 groups treated with DPP-4 inhibitor (either monotherapy or in combi114 nation)with placebotreated groups in different studies, but thenumber 115 of reported adverse events did not differ from the actively treated 116 groups. [4]. It has been demonstrated in animal studies that toxicity 117 may be caused by the inhibition of other enzymes in this family, like 118 DPP-8 and DPP-9 [9], so the selectivity of inhibitors to DPP-4 is crucially 119 important to ensure an optimal safety profile. Since both sitagliptin and 120 vildagliptin show a higher relative selectivity for DPP-4, the risk of 121 development of adverse effects due to inhibition of other enzymes is 122 minimized [4,10]. However, it did turn out that during the post market123 ing period of gliptins these DPP-4 inhibitors increased the rate of infec124 tions such as nasopharyngitis and urinary tract infections [11].In 125 addition, pancreatitis was reported mainly associated with the use of 126 sitagliptin and linagliptin [12], although a recent meta-analysis could 127 not find differences between DPP-4 inhibitors [13]. In spite of the in128 creased risk of infections, sitagliptin and vildagliptin are well tolerated 129 in general. Besides the primary targeted therapeutic area, in vitro and 130 in vivo studies showed anti-inflammatory properties of DPP-4 inhibitors 131 that could lead to a novel drug class for anti-inflammatory disorders 132 [14]. Altered circulating peptidase activity and membrane DPP-4 133 expression have been demonstrated in a number of human inflammato134 rydiseases[15]. DPP-4 is responsible for themodification of a numberof 135 regulatory factors, such as peptides or chemokines and affects the 136 signaling functions. This suggests that DPP-4 is involved in determining 137 immune response and procession of inflammatory disorders as well. As 138 mentioned previously, DPP-4 is also known as the cell surface antigen 139 CD26, which signals T-cells to proliferate. However, this mechanism 140 cannot be attributed to the DPP-4 inhibition [16] because the T-cell 141 activation seems to be independent of the DPP-4 enzyme activity and 142 the ADA-binding capability [16,17]. Moreover, reversible DPP-4 inhibi143 tor Lys[Z(NO 2 )]-pyrrolidide was shown to suppress autoimmune 144 encephalomyelitis and upregulated TGF-β1secretionin vivo [18]. 145The possible anti-inflammatory property of the gliptin group can 146be considered as an additional value of these drugs in diabetic 147patients with neuritis or diabetic neuropathy, or patients with 148atherosclerosis considering that these diseases are driven by inflam149matory processes [19]. Moreover, the reduction in plasma C-reactive 150protein concentration and systolic blood pressure have been 151described for exenatide [20].Theanti-inflammatory action of 152sitagliptin [8] and exenatide [19] are proven biochemically in 153humans, thus in the present series of experiments we aimed to 154examine the possible anti-inflammatory effect of two potent DPP-4 155inhibitors, sitagliptin and vildagliptin. They were applied in in vivo 156inflammation and analgesic models in mice. 1572. Materials and methods 1582.1. Animals and ethics 159Experiments were performed on 25–35 g CD1 male mice (Charles 160River,Gödöllő, Hungary),keptunderstandardpathogen-freeconditions 161at 24–25 °C and provided with standard rodent chow and water 162ad libitum. The light/dark cycle was 12 h/12 h. Animal procedures 163were approved by the local animal ethics committee and National 164Food Chain Safety Office Animal Health and Animal Welfare Directorate 165under the number 26/2007/DE MÁB in accordance with the European 166Communities Council Directives (86/609/ECC) and the Hungarian Act 167for the Protection of Animals in Research (XXVIII tv. 32§) and complied 168with the recommendations of the International Association for the 169Study of Pain [21] and the Helsinki Declaration. The design of the 170study was carried out in a manner in which to minimize the number 171of animals used and their suffering. 1722.2. Substances and their application 173Mice were dosed with 1, 3 or 10 mg/kg sitagliptin or vildagliptin 174(Nanjing Ange Pharmaceuticals, Nanjing, Jiangsu, China) dissolved in 175saline by oral gavage (1 ml/100 g). Control groups were given the 176vehicle in the same amount and way. A single application was used in 177the case of one-day experiments, while daily application was used in 178the 21 day long experiments, as suggested by Thomas et al. [22]. 179Treatments and measurements were implemented 30 min after the 180oral gavage in every case. 1812.3. Allyl-isothiocyanate (AITC)-induced inflammation model 182Anesthesia was induced by thiopental (Trapanal, Sandoz, Basle, 183Switzerland) in an amount of 50 mg/kg intraperitoneally (i.p.), repeat184ed as required. The inner and outer surface of the right ear was then 185smeared with 1% allyl-isothiocyanate (AITC) (Sigma-Aldrich, Budapest, 186Hungary) dissolved in paraffin oil, using a cotton-wool stick. This treat187ment was applied 30 min after the oral gavage (substances dissolved in 188saline or vehicle in the control group) and the procedure was repeated 18945 min after the first application following the instructions of Bánvölgyi 190[23] and Inoue et al. [24]. Thus the oral administration of gliptins was 191performed firstly and the induction of inflammation was carried out 192secondly. 193At the end of the experiment the animals were sacrificed by cervical 194dislocation and ears were stored on -20 °C for the neutrophil accumula195tion assay. 1962.4. Measurement of ear edema 197Ear thickness was measured by a micrometer caliper (Oxford Preci198sion, Leicester, England) with 0.1 mm accuracy before the AITC treat199ment, 15 min after the first AITC application, then by each hour during 200a 6 hour period after each AITC treatment according to Inoue et al. [24] 201with slight modifications. Gliptin treatment was performed 30 minutes 2J. Újhelyi et al. / Regulatory Peptides xxx (2014) xxx–xxx Please cite this article as: Újhelyi J, et al, Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice, Regul Pept (2014), http://dx.doi.org/10.1016/j.regpep.2014.09.006 UNCORRECTED PROOF 202 before the commencement of ear edema induction. Data were expressed 203 in micrometers. 204 2.5. Measurement of neutrophil accumulation 205 Frozen ear samples were thawed at room temperature, chopped into 206 small pieces, and homogenized in 0.05 M potassium phosphate buffer 207 containing 0.5% HTAB (hexadecyltrimethylammonium bromide, 208 Sigma-Aldrich, Budapest, Hungary), 1 ml buffer/ear. The homogenate 209 was centrifuged at 11000 g at 4 °C for 10 min and 200 μlofthesuperna210 tant was placed into Eppendorf tubes. 211 Myeloperoxidase activity was assayed by measuring the H 2 O 2 - 212 dependent oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma213 Aldrich, Budapest, Hungary) as suggested by Suzuki et al. [25].Inits 214 oxidized form, TMB has a blue color, which was measured spectropho215 tometrically at 620 nm. The reaction was performed in 96-well microti216 ter plates at room temperature. The reaction mixture consisted of 25 μl 217 of the tissue sample, 25 μlofTMB(final concentration 0.16 mM) 218 dissolved in dimethylsulfoxide (DMSO) and 200 μlH 2 O 2 (final concen219 tration 0.24 mM, Sigma-Aldrich, Budapest, Hungary) diluted in 0.08 M 220 phosphate buffer pH 5.4 after Schierwagen et al. [26]. The optical densi221 ty (OD) was measured at 5 min intervals for 30 min using a microplate 222 reader (FLUOstar OPTIMA, BMG Labtech, Ortenberg, Germany). Data 223 was expressed in arbitrary units of absorbance. 224 2.6. Induction of arthritis 225 Chronic arthritis of the right tibiotarsal joint of mice was induced by 226 the subcutaneous injection of 0.1 ml of Freund's complete adjuvant 227 (CFA, killed Mycobacteria suspended in paraffin oil, 1 mg/ml as provid228 ed by Sigma-Aldrich, Budapest, Hungary) into the plantar surface of the 229 right hind paw and root of the tail. To enhance systemic effects, an addi230 tional injection into the tail was given the following day as described by 231 Helyes et al. [27]. In order to minimize the suffering of mice, short-term 232 general anesthesia was induced by 1% isoflurane (Abbott Laboratories, 233 Budapest, Hungary) delivered in 1:2 oxygen/nitrous oxide mixture. 234 2.7. Measurement of plasmaextravasation in the urinary bladder of mice 235 Mice were anaesthetized by i.p. administration of thiopental 236 (50 mg/kg). A lateral tail vein was cannulated for intravenous adminis237 tration. 1 or 3 mg of vildagliptin or sitagliptin was administered by oral 238 gavage 30 minutes before the commencement of the capsaicin chal239 lenge. Evans blue (30 mg/kg) and 1 minute later capsaicin (1 mg/kg) 240was injected through the venous cannula. Each animal was sacrificed 241by transcardiac perfusion with 50 ml of 0.9% w/v saline into the left car242diac ventricle 10 min after intravenous injection of Evans blue at 37 °C. 243The urinary bladder was then removed and weighed. Excised tissues 244were incubated in 1 ml of formamide for 48 h and Evans blue content 245was measured spectrophotometrically at 620 nm and expressed as 246μg/g wet mass of the tissue. 2472.8. Measurement of mechano-nociceptive threshold 248Touch sensitivity on the plantarsurface was measured with von Frey 249filaments (Bioseb, Chaville, France) before the experiment, 3, 7, 10, 14, 25017, and 21 days following the first CFA administration. The set of 20 251monofilaments provided an approximate logarithmic scale of actual 252force and a linear scale of perceived intensity. Mice were placed into a 253Plexiglas cage with a pitted floor. Following animal acclimatization the 254operator placed the monofilament under the animal's paw and pressed 255against the surface till the animal indicated the pressure sensation by 256pulling back or shaking its paw, or the monofilament curved without 257any kind of reaction starting with 0.008 g and ranging up to 300 g. 2582.9. Increasing-temperature hot plate test 259The plate (Supertech, Pécs, Hungary) in contact with the paws has 260been slowly warmed up from room temperature and the threshold 261temperature producing the first nocifensive behavior (e.g., paw licking) 262was recorded. Since the temperature was increased gradually into the 263noxious range, stress associated with the testing procedure was mini264mized. The heated surface dimensions were 110 × 80 mm surrounded 265by 350 mm high transparent Plexiglas walls. The commanding comput266er program was set to produce a 3 °C/min temperature increase of the 267plate as proposed by László et al. [28]. When the hind paw licking or 268flinching was observed the threshold temperature was recorded. The 269measurement was terminated at the threshold level or when the plate 270temperature reached 50 °C to avoid tissue damage [29]. Data were 271expressed in °C. 2722.10. Statistics 273Since baselinevalues ofdiverse groups were significantlydifferentin 274all measurements, a baseline correction was carried out on raw values. 275Baseline corrected values were regarded as primary outcomemeasures. 276Two-way ANOVA with replication was used for multiple group analysis 277with the time point of observation and the treatment option as factors Fig. 1. Time evolution of baseline corrected ear thickness (μm) in allyl-isothiocyanate (AITC)-induced ear edema model in mice as a function of the amount of A) sitagliptin and B) vildagliptin administered by oral gavage. Error bars denote standard error of the mean. Lines between markers guide the eye only. For the complete time period × (pb0.001), #(pb0.005), and $ (pb0.001) showed significant differences to positive control, 1 mg, and 3 mg sitagliptin, and × (pb0.001), # (pb0.01) to positive control and 1 mg vildagliptin, respectively as assessed by Games-Howell post hoc test. 3J. Újhelyi et al. / Regulatory Peptides xxx (2014) xxx–xxx Please cite this article as: Újhelyi J, et al, Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice, Regul Pept (2014), http://dx.doi.org/10.1016/j.regpep.2014.09.006 UNCORRECTED PROOF 278 for the complete duration of the experiments. Games-Howell tests were 279 used as post hoc analysis for binary comparison of group averages. 280 Significant differences at the 95% confidence interval were recognized, 281 if pb0.05. Below 0.001 no numeric values of pare provided in the text. 282 3. Results 283 3.1. AITC-induced ear edema 284 Both orally administered gliptins significantly decreased ear thick285 ness in the complete time period compared to positive control (AITC 286 only) in a dose-dependent manner as seen in Fig. 1.Themaximum 287 effect of AITC was measured at 2 hour post-challenge time in either 288 case. For the complete examination period 10 mg sitagliptin as well as 289 1–10 mg vildagliptin was found to significantly decrease ear edema as 290 compared to positive control. 291 3.2. AITC-induced neutrophil accumulation 292 The evolved inflammation was shown by the high level of 293 myeloperoxidase enzyme in the positive control group (AITC only), see 294Fig. 2. The model is suitable for measuring the extent of inflammation, 295since these data definitely diverge from the negative control group 296results. Sitagliptin treatment was found significantly effective in blocking 297the evolution of inflammation; every examined dose could reverse in298flammation. (Blind samples were not included in the hypothesis testing.) 299The effect of vildagliptin treatment was similar to that of sitagliptin, 300although the dose of 3 mg/kg had only an insignificant impact. 3013.3. Measurement of plasmaextravasation in the urinary bladder of mice 302The capsaicin-induced plasma extravasation in urinary bladders of 303mice was inhibited by sitagliptin (1 mg p= 0.025 and 3 mg pb0.001) 304and vildagliptin (both 1 mg and 3 mg pb0.001) significantly (Fig. 4). 305Difference in action was seen between the higher doses (3 mg/kg) of 306vildaand sitagliptin. The lower dose of sitagliptin (1 mg/kg) produced 307the least significant inhibition compared to the control. 3083.4. Touch sensitivity in CFA-induced arthritis 309Results show that the mechano-nociceptive threshold of the un310treated group was significantly higher than in the CFA treated (positive Fig. 2. Time evolution of the baseline corrected absorbance of myeloperoxidase in an allyl-isothiocyanate (AITC)-induced ear edema model in mice as a function of the amount of A) sitagliptin or B) vildagliptin administered by oral gavage. Error bars denote standard error of the mean. Lines between markers guide the eye only. For the complete time period *(pb0.05) and × (pb0.01) showed significant differences to negative and positive control and * (pb0.05), × (pb0.005), # (pb0.01), and $ (pb0.005) to negative, positive control, 1 mg, and 3 mg vildagliptin, respectively as assessed by Games-Howell post hoc test. Fig. 3. Time evolution of the mechano-nociceptive threshold in Freund's complete adjuvant (CFA)-induced arthritis model in mice as a function of the amount of A) sitagliptin or B) vildagliptin administered by oral gavage. Error bars denote standard error of the mean. Lines between markers guide the eye only. For the complete time period * (pb0.001), ×(pb0.01), and # (pb0.05) showed significant differences to negative, positive control and 1 mg sitagliptin, and * (pb0.001) to negative control, respectively as assessed by Games-Howell post hoc test. 4J. Újhelyi et al. / Regulatory Peptides xxx (2014) xxx–xxx Please cite this article as: Újhelyi J, et al, Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice, Regul Pept (2014), http://dx.doi.org/10.1016/j.regpep.2014.09.006 UNCORRECTED PROOF 311 control) group during the whole 21 day experimental period (Fig. 3). In 312 the gliptin treated groups every threshold was significantly lower 313 than in the negative control group; consequently neither sita-, nor 314 vildagliptin treatment was able to hinder the evolution of allodynia. 315 Sitagliptin was significantly harmful in an amount of 3 and 10 mg/kg 316 doses as compared to the positive control, meanwhile vildagliptin 317 remained ineffective in all doses. 318 3.5. Increasing-temperature hot plate test in CFA-induced arthritis 319 Threshold temperature of the untreated group was significantly 320 higher than in the CFA-treated group during the whole 21 day experi321 mental period, following the first day as shown in Fig. 5. Every dose of 322 either sitagliptin or vildagliptin significantly increased the threshold 323 temperature, compared to thepositive control group. Neither sitagliptin 324 nor vildagliptin could inhibit inflammation; the threshold in all gliptin 325 treated groups remained significantly lowerthan in the negative control 326 groups. 3274. Discussion 328According to our present data, we can conclude that the studied 329gliptins had a dose-dependent anti-inflammatory effect in in vivo 330mouse models. The applied methods were sensitive enough to detect 331the action of gliptins. Dipeptidyl peptidase inhibitors were reviewed 332as an emerging drug class for various inflammatory diseases [7]. The 333anti-inflammatory actionof these drugs were described in human stud334ies [8] and for exenatide [19]. Sitagliptin significantly improves endo335thelial function and inflammatory state in patients with coronary 336artery disease and uncontrolled diabetes mellitus [30], forming a mile337stone in the way towards widening the spectrum of gliptins' indication. 338Moreover, the GLP-1 receptor (GLP-1R) is expressed in lymphoid tissue 339and the numbers of CD4+ and CD8+ T-cells in lymph nodes was 340shown to increase after exenatide (a GLP-1R agonist) treatment. It 341could also reduce the number of CD4+ CD25+ Foxp3 + regulatoryT342cells in the thymus, but not in the spleen [31], thus playing a regulatory 343role in the immune system and can influence inflammatory processes 344[32]. However, Kim et al. [33] were unable to detect the effect of either 345GIP or GLP-1 on splenic or thymic CD4+ T-cell migration in vitro [33]. 346Eosinophil cell recruitment (in allergic asthma or in atopic dermatitis) 347is described to be mediated by CCL11(eosinophil chemotactic protein) 348and the recruitment proved to be more effective after pharmacological 349inhibition of DPP-4 enzyme or in DPP-4-deficient F344 rats [34].The 350activation of transient receptor potential ankyrin 1 (TRPA1) evokes 351nociception through substance P release from the primary sensoryneu352rons; p38 mitogen-activated protein kinase (p38 MAPK) inhibitor 353SB203580 significantly attenuated AITC-evoked substance P release 354[35]. Allyl-isothiocyanate is capable of inducing earedemain the proper 355dose as described earlier [23]; the maximum auricle swelling was 356measured in the second hour. Both examined chemicals, sitagliptin 357and vildagliptin were able to decrease the AITC-induced inflammation 358in a dose-dependent manner however, sitagliptin had a higher impact. 359This effect cannot be explained by the regulatory role of GLP-1 on p38 360MAPK, as it was described as an inducer [36]; neither can it beattributed 361to the effect of gliptins on substance P metabolism [37]. Moreover, the 362physiological role of GLP-1 is so dominant that its inhibition can still 363override the p38 MAPK-inducer property and the algogenic effect of 364elevated substance P. Treatment by DPP4 inhibitor I40 significantly 365reduced the severity of experimental allergic encephalomyelitis (EAE), 366in mice conceivably through up-regulating TGF-beta 1 [18].Furthermore, 367a dose-dependent inhibition of the secretion of the pro-inflammatory 368cytokine TNF-alpha was measured in vitro [18]. The ability of gliptins to Fig. 4. Inhibition of capsaicin-induced plasmaextravasation in urinary bladders of mice by Vildagliptin and Sitagliptin. Gliptins were administered by oral gavage in 1 or 3 mg/kg dose 30 minutes before the capsaicin (1 mg/kg) intravenous challange. Evans blue dye was administered in 30 mg/kg i.v. and the plasmaextravasation was determined spectrophotometrically at 620 nm wave length. Error bars denote standard error of the mean. * and × denote significant differences to negative control and to vildagliptin 1 mg, respectively as assessed by Games-Howell post hoc test. Fig. 5. Time evolution of the thermo-nociceptive threshold in Freund's complete adjuvant (CFA)-induced arthritis model in mice as a function of the amount of A) sitagliptin or B) vildagliptin administered by oral gavage. Error bars denote standard error of the mean. Lines between markers guide the eye only. For the complete time period * (pb0.001), ×(pb0.05), and # (pb0.05) showed significant differences to negative, positive control, and 1 mg sitagliptin, and * (pb0.001), × (pb0.05), # (pb0.05), and $ (pb0.01) to negative, positive control, 1 mg, and 3 mg vildagliptin, respectively as assessed by Games-Howell post hoc test. 5J. Újhelyi et al. / Regulatory Peptides xxx (2014) xxx–xxx Please cite this article as: Újhelyi J, et al, Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice, Regul Pept (2014), http://dx.doi.org/10.1016/j.regpep.2014.09.006 UNCORRECTED PROOF 369 regulate TNF-alpha, INF-gamma, and a variety of interleukins can be at370 tributed only to the DPP4 inhibitory activity, because the compounds 371 used in the present series of experiments have a high specificity to 372 DPP4 and probably do not have any inhibitory effect on DPP8 or 9 in 373 the applied doses [38]. However, vildagliptin ineffectiveness in two 374 models (myeloperoxidase measurement and touch sensitivity in CFA375 induced arthritis) at 3 mg/kg can be the result of DPP-9 activity attenua376 tion having 66 nM IC50 value in vitro compared to 130 nM IC50 for 377 sitagliptin [39]. Inhibition of DPP-8/9 can lead to the development of 378 adverse effects in rodents [9,40], but other studies state that the inhibition 379 of DPP-8/9 do not have any clinical consequence [41]. A reduced expres380 sion of nitrosative stress and inflammation hallmarks within the brain of 381 chronically administered sitagliptin was described earlier in a mouse 382 model of Alzheimer's disease [42]. An explanation could easily rise 383 considering the fact that GLP-1 can have growth-factor-like properties 384 similar to insulin and the anti-inflammatory activity is a secondary action 385 [43]. In our experiments, the anti-inflammatory action seems to be direct 386 as demonstrated by the accumulated number of neutrophil cells 387 (measured by myeloperoxidase enzyme activity) in the inflamed ear; 388 this accumulation could be inhibited by the gliptin pre-treatment. 389 Similarly to the above mentioned tests, sitagliptin treatment had a 390 higher impact in the compensation of the CFA-induced arthritis, 391 where vildagliptin showed no effectiveness. In case of measuring the 392 high temperature sensitivity, both substances showed equal effective393 ness. Our results lead to the conclusion that sitagliptin has a stronger 394 influence on the evolution of inflammation; however, vildagliptin 395 showed higher effectiveness in the inhibition of capsaicin-induced 396 plasma extravasation in the urinary bladder. Although the investigated 397 molecules have the same effectiveness in the treatment of type-2 398 diabetes, it seems that they do not act in the same way, in the immune 399 response. Both substances represent promising options for the therapy 400 of inflammatory disorders. 401 Conflict of interest 402Q2 The authors declare that there are no conflicts of interest. 403 References 404 [1] Drucker DJ. The biology of incretin hormones. Cell Metab 2006;3:153–65. 405 [2] Juillerat-Jeanneret L. Dipeptidyl peptidase IV and its inhibitors: therapeutics for type 406 2 diabetes and what else? J Med Chem 2014;57:2197–212. 407 [3] Elrick H, Stimmler L, Hlad Jr CJ, Arai Y. Plasma insulin response to oral and 408 intravenous glucose administration. J Clin Endocrinol Metab 1964;24:1076–82. 409 [4] Ahren B, Schmitz O. GLP-1 receptor agonists and DPP-4 inhibitors in the treatment 410 of type 2 diabetes. Horm Metab Res 2004;36:867–76. 411 [5] Gorrell MD. Dipeptidyl peptidase IV and related enzymes in cell biology and liver 412 disorders. Clin Sci (Lond) 2005;108:277–92. 413 [6] Mentlein R. Dipeptidyl-peptidase IV, (CD26)–role in the inactivation of regulatory 414 peptides. Regul Pept 1999;85:9–24. 415 [7] Balakumar P, Dhanaraj SA. Cardiovascular pleiotropic actions of DPP-4 inhibitors: a 416 step at the cutting edge in understanding their additional therapeutic potentials. 417 Cell Signal 2013;25:1799–803. 418 [8] Makdissi A, Ghanim H, Vora M, Green K, Abuaysheh S, Chaudhuri A, Dhindsa S, 419 Dandona P. Sitagliptin exerts an antiinflammatory action. J Clin Endocrinol Metab 420 2012;97:3333–41. 421 [9] Lankas GR, Leiting B,Roy RS,Eiermann GJ, BeconiMG, Biftu T, Chan CC, EdmondsonS, 422 Feeney WP, He H, Ippolito DE, Kim D, Lyons KA, OkHO, Patel RA, Petrov AN, Pryor KA, 423 Qian X, Reigle L, Woods A, et al. Dipeptidyl peptidase IV inhibition for the treatment 424 of type 2 diabetes: potential importance of selectivity over dipeptidyl peptidases 8 425 and 9. Diabetes 2005;54:2988–94. 426 [10] Barnett A. DPP-4 inhibitors and their potential role in the management of type 2 427 diabetes. Int J Clin Pract 2006;60:1454–70. 428 [11] Amori RE, Lau J, Pittas AG. Efficacy and safety of incretin therapy in type 2 diabetes: 429 systematic review and meta-analysis. JAMA 2007;298:194–206. 430 [12] Mikhail N. Safety of dipeptidyl peptidase 4 inhibitors for treatment of type 2 431 diabetes. Curr Drug Saf 2011;6:304–9. 432 [13] Monami M, Ahren B, Dicembrini I, Mannucci E. Dipeptidyl peptidase-4 inhibitors 433 and cardiovascular risk: a meta-analysis of randomized clinical trials. Diabetes 434 Obes Metab 2013;15:112–20. 435 [14] Yazbeck R, Howarth GS, Abbott CA. Dipeptidyl peptidase inhibitors, an emerg436 ing drug class for inflammatory disease? Trends Pharmacol Sci 2009;30: 437 600–7. 438[15] Reinhold D, Bank U, Tager M, Ansorge S, Wrenger S, Thielitz A, Lendeckel U, Faust J, 439Neubert K, Brocke S. DP IV/CD26, APN/CD13 and related enzymes as regulators of T 440cell immunity: implications for experimental encephalomyelitis and multiple 441sclerosis. Front Biosci 2008;13:2356–63. 442[16] Ohnuma K, Dang NH, Morimoto C. Revisiting an old acquaintance: CD26 and its 443molecular mechanisms in T cell function. Trends Immunol 2008;29:295–301. 444[17] Yu DM, Slaitini L, Gysbers V, Riekhoff AG, Kahne T, Knott HM, De Meester I, Abbott 445CA, McCaughan GW, Gorrell MD. Soluble CD26/dipeptidyl peptidase IV enhances 446human lymphocyte proliferation in vitro independent of dipeptidyl peptidase 447enzyme activity and adenosine deaminase binding. Scand J Immunol 2011;73: 448102–11. 449[18] Steinbrecher A, Reinhold D, Quigley L, Gado A, Tresser N, Izikson L, Born I, Faust J, 450Neubert K, Martin R, Ansorge S, Brocke S. Targeting dipeptidyl peptidase IV 451(CD26) suppresses autoimmune encephalomyelitis and up-regulates TGF-beta 1 452secretion in vivo. J Immunol 2001;166:2041–8. 453[19] Chaudhuri A, Dandona P, Fonseca V. Cardiovascular benefits of exogenous insulin. J 454Clin Endocrinol Metab 2012;97:3079–91. 455[20] Viswanathan P, Chaudhuri A, Bhatia R, Al-Atrash F, Mohanty P, Dandona P. 456Exenatide therapy in obese patients with type 2 diabetes mellitus treated with 457insulin. Endocr Pract 2007;13:444–50. 458[21] Zimmermann M. Ethical guidelines for investigations of experimental pain in 459conscious animals. Pain 1983;16:109–10. 460[22] Thomas L, Eckhardt M, Langkopf E, Tadayyon M, Himmelsbach F, Mark M. (R)-8461(3-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylm 462ethyl)-3,7-dihydro-purine-2,6-dione (BI 1356), a novel xanthine-based dipeptidyl 463peptidase 4 inhibitor, has a superior potency and longer duration of action 464compared with other dipeptidyl peptidase-4 inhibitors. J Pharmacol Exp Ther 4652008;325:175–82. 466[23] Banvolgyi A, Pozsgai G, Brain SD, Helyes ZS, Szolcsanyi J, Ghosh M, Melegh B, Pinter 467E. Mustard oil induces a transient receptor potential vanilloid 1 receptor468independent neurogenic inflammation and a non-neurogenic cellular inflammatory 469component in mice. Neuroscience 2004;125:449–59. 470[24] Inoue H, Asaka T, Nagata N, Koshihara Y. Mechanism of mustard oil-induced skin 471inflammation in mice. Eur J Pharmacol 1997;333:231–40. 472[25] Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T. Assay method for 473myeloperoxidase in human polymorphonuclear leukocytes. Anal Biochem 1983; 474132:345–52. 475[26] Schierwagen C, Bylund-Fellenius AC, Lundberg C. Improved method for quantification 476of tissue PMN accumulation measured by myeloperoxidase activity. J Pharmacol 477Methods 1990;23:179–86. 478[27] Helyes Z, Szabo A, Nemeth J, Jakab B, Pinter E, Banvolgyi A, Kereskai L, Keri G, 479Szolcsanyi J. Antiinflammatory and analgesic effects of somatostatin released from 480capsaicin-sensitive sensory nerve terminals in a Freund's adjuvant-induced chronic 481arthritis model in the rat. Arthritis Rheum 2004;50:1677–85. 482[28] László J, Szilvási J, Fényi A, Szalai A, Gyires K, Pórszász R. Daily exposure to 483inhomogeneous static magnetic field significantly reduces blood glucose level 484in diabetic mice. Int J Radiat Biol 2011;87:36–45. 485[29] Tjolsen A, Rosland JH, Berge OG, Hole K. The increasing-temperature hot-plate test: 486an improved test of nociception in mice and rats. J Pharmacol Methods 1991;25: 487241–50. 488[30] Matsubara J, Sugiyama S, Akiyama E, Iwashita S, Kurokawa H, Ohba K, Maeda H, 489Fujisue K, Yamamoto E, Kaikita K, Hokimoto S, Jinnouchi H, Ogawa H. Dipeptidyl 490peptidase-4 inhibitor, sitagliptin, improves endothelial dysfunction in association 491with its anti-inflammatory effects in patients with coronary artery disease and 492uncontrolled diabetes. Circ J 2013;77:1337–44. 493[31] Hadjiyanni I, Baggio LL, Poussier P, Drucker DJ. Exendin-4 modulates diabetes onset 494in nonobese diabetic mice. Endocrinology 2008;149:1338–49. 495[32] Okeke EB, Okwor I, Mou Z, Jia P, Uzonna JE. CD4 + CD25+ regulatory T cells 496attenuate lipopolysaccharide-induced systemic inflammatory responses and 497promotes survival in murine Escherichia coli infection. Shock 2013;40:65–73. 498[33] Kim SJ, Nian C, McIntosh CH. Sitagliptin (MK0431) inhibition of dipeptidyl peptidase 499IV decreases nonobese diabetic mouse CD4+ T-cell migration through incretin500dependent and -independent pathways. Diabetes 2010;59:1739–50. 501[34] Forssmann U, Stoetzer C, Stephan M, Kruschinski C, Skripuletz T, Schade J, Schmiedl 502A, Pabst R, Wagner L, Hoffmann T, Kehlen A, Escher SE, Forssmann WG, Elsner J, von 503Horsten S. Inhibition of CD26/dipeptidyl peptidase IV enhances CCL11/eotaxin504mediated recruitment of eosinophils in vivo. J Immunol 2008;181:1120–7. 505[35] Nakamura Y, Une Y, Miyano K, Abe H, Hisaoka K, Morioka N, Nakata Y. Activation of 506transient receptor potential ankyrin 1 evokes nociception through substance P 507release from primary sensory neurons. J Neurochem 2012;120:1036–47. 508[36] Kahne T, Reinhold D, Neubert K, Born I, Faust J, Ansorge S. Signal transduction events 509induced or affected by inhibition of the catalytic activity of dipeptidyl peptidase IV 510(DP IV, CD26). Adv Exp Med Biol 2000;477:131–7. 511[37] Guieu R, Fenouillet E, Devaux C, Fajloun Z, Carrega L, Sabatier JM, Sauze N, Marguet 512D. CD26 modulates nociception in mice via its dipeptidyl-peptidase IV activity. 513Behav Brain Res 2006;166:230–5. 514[38] Thornberry NA, Weber AE. Discovery of JANUVIA (Sitagliptin), a selective dipeptidyl 515peptidase IV inhibitor for the treatment of type 2 diabetes. Curr Top Med Chem 5162007;7:557–68. 517[39] LiuY,SiM,TangL,ShangguanS,WuH,LiJ,WuP,MaX,LiuT,HuY.Synthesis 518and biological evaluation of novel benzyl-substituted (S)-phenylalanine 519derivatives as potent dipeptidyl peptidase 4 inhibitors. Bioorg Med Chem 5202013;21:5679–87. 521[40] Burkey BF, Hoffmann PK, Hassiepen U, Trappe J, Juedes M, Foley JE. Adverse effects of 522dipeptidyl peptidases 8 and 9 inhibition in rodents revisited. Diabetes Obes Metab 5232008;10:1057–61. 6J. Újhelyi et al. / Regulatory Peptides xxx (2014) xxx–xxx Please cite this article as: Újhelyi J, et al, Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice, Regul Pept (2014), http://dx.doi.org/10.1016/j.regpep.2014.09.006 UNCORRECTED PROOF 524 [41] Kirby M, Yu DM, O'Connor S, Gorrell MD. Inhibitor selectivity in the clinical 525 application of dipeptidyl peptidase-4 inhibition. Clin Sci (Lond) 2010;118:31–41. 526 [42] D'Amico M, Di Filippo C, Marfella R, Abbatecola AM, Ferraraccio F, Rossi F, Paolisso G. 527 Long-term inhibition of dipeptidyl peptidase-4 in Alzheimer's prone mice. Exp 528 Gerontol 2010;45:202–7. 529[43] Bak AM, Egefjord L, Gejl M, Steffensen C, Stecher CW, Smidt K, Brock B, Rungby J. 530Targeting amyloid-beta by glucagon-like peptide -1 (GLP-1) in Alzheimer's disease 531and diabetes. Expert Opin Ther Targets 2011;15:1153–62. 7J. Újhelyi et al. / Regulatory Peptides xxx (2014) xxx–xxx Please cite this article as: Újhelyi J, et al, Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice, Regul Pept (2014), http://dx.doi.org/10.1016/j.regpep.2014.09.006