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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

New Peptide Inhibitor of Dipeptidyl Peptidase IV, EMDB-1 Extends the Half-Life of GLP-2 and Attenuates Colitis in Mice after Topical Administration

Maciej Salaga, Anna Mokrowiecka, Marta Zielinska, Ewa Malecka-Panas, Radzislaw Kordek, Elzbieta Kamysz and Jakub Fichna
Journal of Pharmacology and Experimental Therapeutics October 2017, 363 (1) 92-103; DOI: https://doi.org/10.1124/jpet.117.242586

Abstract

Protease inhibition has become a possible new approach in inflammatory bowel disease (IBD) therapy. A serine exopeptidase, dipeptidyl peptidase IV (DPP IV), is responsible for the inactivation of incretin hormone, glucagon-like peptide 2 (GLP-2), a potent stimulator of intestinal epithelium regeneration and growth. Recently, we showed that the novel peptide analog of endomorphin-2, Tyr-Pro-D-ClPhe-Phe-NH2 (EMDB-1) is a potent blocker of DPP IV and has an inhibitory effect on gastrointestinal (GI) smooth muscle contractility. The aim of this study was to characterize the anti-inflammatory effect and mechanism of action of EMDB-1 in the mouse GI tract. We used two models of experimental colitis (induced by TNBS and DSS). The anti-inflammatory effect of EMDB-1 was assessed by the determination of macroscopic score, ulcer score, colonic wall thickness, as well as myeloperoxidase activity. Additionally, we measured the expression of GLP-2, GLP2R, and DPP IV in the colon of control and colitic animals treated with the test compound. The expression of GLP-2 and GLP2R in the serum and colon of IBD patients and healthy control subjects has been assessed. We showed that EMDB-1 elevates the half-life of GLP-2 in vitro and attenuates acute, semichronic, and relapsing TNBS as well as DSS-induced colitis in mice after topical administration. The anti-inflammatory action of EMDB-1 is associated with changes in the level of colonic GLP-2 but not DPP IV expression. Our results validate DPP IV as a pharmacological target for the anti-IBD drugs, and its inhibitors based on natural substrates, such as EMDB-1, have the potential to become valuable anti-inflammatory therapeutic agents.

Introduction

Inflammatory bowel disease (IBD) is a group of chronic inflammatory bowel disorders mainly represented by Crohn’s disease (CD) and ulcerative colitis (UC). The main symptoms of IBD include abdominal pain and other clinical symptoms, such as diarrhea and rectal bleeding. Attenuation of these ailments can be achieved through the use of nonsteroidal anti-inflammatory drugs, corticosteroids, or biologic agents, such as anti–tumor necrosis factor α (TNFα) or anti–α4β7 integrin antibodies, depending on the stage and the severity of the disease (Raine, 2014; Rogler, 2015). However, all available treatment options have debatable efficiency (a significant fraction of nonresponders), whereas their use, especially in prolonged administration, may bring major side effects.

The etiology of IBD is not fully understood, and there is substantial evidence that immunologic, genetic, and environmental factors are the main contributors in its pathogenesis. Both CD and UC are characterized by an excessive activation of common inflammatory pathways, which results in an enhanced secretion of proinflammatory cytokines, such as interleukins (ILs; IL-1β, IL-2, IL-6, IL-8, IL-12, and IL-17), TNFα, and an imbalance in the levels of proinflammatory and anti-inflammatory factors in the tissue (Jump and Levine, 2004). Recent studies (Baumgart and Carding, 2007; Geuking et al., 2014) suggest that the etiology of IBD also involves factors (both genetic and environmental) that cause dysfunction of the epithelial barrier with consequent aberrations in the mucosal responses to gut microbiota. Intestinal epithelial cells constitute a specific form of a physical, chemical, and immune barrier between the external and internal environment. Any damage to these cells may lead to an increased inflammatory process. Disturbance in the epithelial barrier seems to be the key element in the onset of IBD and, subsequently, in the frequent relapses. One of the factors responsible for the regeneration and growth of the epithelium are incretin hormones, such as glucagon-like peptide 2 (GLP-2), which are produced by the enteroendocrine L cells of the small intestine and the colon. GLP-2, a 33-amino acid peptide released by the neuroendocrine convertase 1 from the proglucagon is one of the most potent modulators of the intestinal function. GLP-2 signals through a G protein–coupled receptor (GLP2R), expressed predominantly in the small intestine and colon (Yazbeck et al., 2008). It is a potent intestinotrophic growth factor that stimulates crypt cell proliferation and inhibits crypt cell apoptosis (Hartmann et al., 2000). GLP-2 has also been shown to improve epithelial barrier function and to increase mucosal hexose and glucose transport. It is also known to alleviate the symptoms in animal models of small intestinal and colonic injury, such as a nonsteroidal anti-inflammatory drug–induced enteritis, necrotic colitis, postoperative ileus, and dextran sulfate sodium (DSS)–induced colitis (Hartmann et al., 2000; Bank et al., 2006; Yazbeck et al., 2008; Moore et al., 2010; Mimura et al., 2013; Salaga et al., 2013; Nakame et al., 2016).

GLP-2 undergoes swift enzymatic degradation and thus its potential therapeutic action rapidly ceases after administration [half-life (t1/2) = 5–7 minutes in humans]. The enzyme mainly responsible for decomposition of GLP-2 is dipeptidyl peptidase IV (DPP IV), which cleaves the protein from its bioactive form, GLP-2(1-33), to inactive GLP-2(3-33) (Salaga et al., 2013). DPP IV is ubiquitously expressed on the surface of epithelial cells, and the highest levels in humans have been reported to occur in the intestine, bone marrow, and kidney, although there are also soluble DPP IV forms in plasma and other body fluids (Salaga et al., 2013).

There have been some attempts to target proteases, including DPP IV, by synthetic inhibitors as a novel form of treatment of gut inflammation. One of the strategies commonly used to design a novel enzyme inhibitor is to modify the structure of its natural substrate to obtain a compound that actively binds to the enzyme active site and blocks its catalytic activity. Using this approach, our group designed and synthesized a series of peptide DPP IV inhibitors based on the structure of endomorphin (EM)-2, which is a potent endogenous µ-opioid receptor (MOR) agonist and DPP IV substrate. We found that one of the compounds [Tyr-Pro-D-ClPhe-Phe-NH2 (EMDB-1)] significantly extends the t1/2 of EM-2 and does not exhibit affinity toward MOR in vitro (Fichna et al., 2006b). The results of the experiments with isolated proteolytic enzymes suggest that EMDB-1 is a competitive and selective inhibitor of DPP IV (Fichna et al., 2010). Cravezic et al. (2011) demonstrated that EMDB-1 significantly prolonged the analgesic and antidepressant-like effects, induced by exogenous EMs, by blocking EM-degrading enzymes. Fichna et al. (2010) reported that EMDB-1 has a significant influence on the intestinal tissue, showing inhibitory effects on the smooth muscle contractility of the rat ileum. Encouraged by the previously reported results, here we aimed at testing the hypothesis that EMDB-1 attenuates experimental colitis in mice by elevation of the intestinal GLP-2 level. The nonselective opioid antagonist naloxone (NLX) was employed to exclude the role of opioid receptors in the action of EMDB-1. Moreover, we characterized the expression of GLP-2, GLP2R, and DPP IV in the control and colitic EMDB-1–treated animals. To translate our results to clinical conditions, we measured the expression of GLP-2 and GLP2R in the serum and colon of IBD patients and healthy control subjects.

Materials and Methods

Determination of EMDB-1 Inhibitory Activity In Vitro

The potency of EMDB-1 was determined using the DPP IV Inhibitor Screening Assay Kit (Cayman Chemical, Ann Arbor, MI), which uses the fluorogenic substrate Gly-Pro-Aminomethylcoumarin (AMC) to measure DPP IV activity. Cleavage of the peptide bond by the enzyme releases the free AMC group, whose fluorescence can be measured using an excitation wavelength of 350–360 nm and emission wavelength of 450–465 nm (Victor 3 Multilabel Microplate Reader; PerkinElmer, Waltham, MA). Multiple concentrations of EMDB-1 ranging from 10−10 to 10−4 M were tested to obtain a concentration-response curve. Curve-fitting analysis has been performed to obtain the IC50 value. A well-known DPP IV inhibitor, sitagliptin, was used as a positive control for the assay.

GLP-2 Degradation Studies and High-Performance Liquid Chromatography Analysis

The degradation studies were performed using purified DPP IV isolated from porcine kidney, according to the modified method described previously (Fichna et al., 2006b). Briefly, the lyophilized enzyme was reconstituted in 50 mM Tris-HCl (pH 7.4). The aliquots (100 µl, 0.2 mg protein/ml) were incubated with 50 µl of either GLP-2 (0.042 mM) and 50 µl of EMDB-1 (0.21 mM) or Tris, over 0, 7.5, 15, 22.5, and 30 minutes at 37°C in a final volume of 200 µl. The reaction was stopped at the required time by placing the tube on ice and acidifying with 20 µl of a 1 M aqueous HCl solution. The aliquots were centrifuged at 20,000g for 10 minutes at 4°C. The obtained supernatants were filtered through Millex-GV Syringe Filters (EMD Millipore, Billerica, MA) and analyzed by high-performance liquid chromatography on a Gemini-NX C18 (4.6 × 150 mm; particle size 5µm; pore size 110 Å; Phenomenex, Torrance, CA), using the solvent system of 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B) and a linear gradient of 3–80% of B over 20 minutes with a flow rate of 2.5 ml/min. Three independent experiments for each assay were carried out in duplicate. The rate constants of degradation (k) were obtained by a least-squares linear regression analysis of logarithmic EM peak areas [ln(A/A0)], where A is the amount of peptide remaining and A0 is the initial amount of peptide, versus time. Degradation t1/2 values were calculated from the rate constants as ln2/k.

Animals

Experimentally naive male C57BL/6 mice were obtained from the vivarium at the University of Lodz, Poland. All animals used in the experiments weighed 22–26 g (6–8 weeks of age). Mice were housed at a constant temperature (22°C) and maintained under a 12-hour light/dark cycle (lights on at 6:00 AM) in sawdust-lined plastic cages. Chow pellets (Agropol S.J., Motycz, Poland) and tap water were available ad libitum. All animal protocols were approved by the Medical University of Lodz Animal Care Committee (Protocol 36/ŁB670/2015) and complied with the European Communities Council Directive of September 22, 2010 of the EU (2010/63/EU). All efforts were made to minimize animal suffering and to reduce the number of animals used. Groups of 6–10 animals were used in all in vivo experiments.

Induction of Colitis

TNBS Model.

Colitis was induced by intracolonic (i.c.) administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS), as described previously (Salaga et al., 2014b). Briefly, mice were weighed and lightly anesthetized with 1% isoflurane (Baxter, Deerfield, IL) and TNBS (0.1 ml of 30% ethanol in saline; acute and semichronic models: 4 mg TNBS/animal; chronic relapsing model: first dose, 150 mg TNBS/kg; second dose, 75 mg TNBS/kg) was injected into the colon through a catheter inserted 3 cm proximally from the anus. Mice were then maintained in an inclined position for 1 minute to ensure the proper distribution of the inductor in the colon. Next, recovery was allowed with food and water supplied. Control animals received vehicle alone (30% ethanol in saline; TNBS was replaced with an equivolume of water). Preliminary experiments demonstrated that the dose of TNBS used in this study resulted in reproducible colitis manifested by body weight decrease as well as macroscopic damage and biochemical alterations that are characteristic for the disease.

DSS Model.

Colitis was induced by the addition of DSS to drinking water from day 0 to day 5 (3% w/v; mol. wt., 40,000; Lot No. 5237K; MP Biomedicals, Aurora, OH). On days 6 and 7, the animals received water without DSS. Control animals received tap water. Animal body weight as well as general health and disease symptoms were monitored daily.

Pharmacological Treatments

In this study, we used three different therapeutic regimens in the TNBS model. In the acute paradigm, the effect of EMDB-1 was evaluated as follows: colitis was induced on day 0, and EMDB-1 was administered twice daily from day 0 to day 2 at doses ranging from 0.1 to 3 mg/kg, i.c., with the first treatment 30 minutes before the TNBS instillation. Animals were euthanized on day 3 by rapid cervical dislocation, and the evaluation of disease parameters was performed (Fig. 2A). Nonselective opioid receptor antagonist NLX was administered i.p. 30 minutes before EMDB-1 (3 mg/kg) at a dose of 1 mg/kg, which was selected based on the preliminary studies and previously published data (Salaga et al., 2015).

In the semichronic TNBS model, a curative treatment mode was tested. Inflammation was induced on day 0, and animals received EMDB-1 (1 mg/kg, i.c., twice daily) from day 3 to day 6 (Fig. 3A). On day 7, mice were euthanized by rapid cervical dislocation, and the evaluation of colonic damage was performed.

The therapeutic activity of EMDB-1 in chronic, relapsing colitis was evaluated in the model described earlier by Martin et al. (2014) with minor modifications. Briefly, colitis was induced on day 0 by i.c. administration of TNBS at a dose of 150 mg/kg. Between days 7 and 13, mice were injected with EMDB-1 at a dose of 1 mg/kg, i.c., twice daily. The relapse of colitis was induced on day 11 by the second administration of the TNBS solution at a dose of 75 mg/kg. Three days later, mice were sacrificed (day 14, endpoint) and the evaluation of colonic damage was performed (Fig. 4A). The doses of TNBS used in this experiment were selected based on the preliminary experiments involving titration and evaluation of disease parameters as well as the survival rate of the mice. In all experiments, control animals received vehicle (100 µl, i.p.) alone.

In the DSS model, animals were treated with EMDB-1 (1 mg/kg, i.c., twice daily) from day 3 to day 6 (Fig. 6A). On day 7, mice were sacrificed and the evaluation of colonic damage was performed. In all experiments, control animals received vehicle (100 µl, i.c.) alone.

Evaluation of Colonic Damage

TNBS-Induced Colitis.

After euthanasia, the colon was rapidly removed, opened longitudinally, rinsed with phosphate-buffered saline (PBS), and immediately examined. Macroscopic colonic damage was determined by an established semiquantitative scoring system by adding individual scores for ulcer, colonic shortening, wall thickness, and the presence of hemorrhage, fecal blood, and diarrhea, as described previously (Salaga et al., 2014a). For scoring ulcer and colonic shortening, the following scale was used: ulcer, 0.5 points for each 0.5 cm; shortening of the colon, 1 point for >15% and 2 points for >25% (based on a mean length of the colon in untreated mice of 7.87 ± 0.12 cm; n = 8). The wall thickness was measured in millimeters, a thickness of n mm corresponded to n scoring points. The presence of hemorrhage, fecal blood, or diarrhea increased the score by 1 point for each additional feature.

DSS-Induced Colitis.

After euthanasia on day 7 after the addition of DSS to the drinking water, the entire colon was isolated and weighed with fecal content. Then, the colon was opened along the mesenteric border and cleaned of fecal material. A total macroscopic damage score was calculated for each animal based on the 1) stool consistency (where 0 means normal well-shaped fecal pellets and 3 means diarrhea), 2) colon epithelial damage considered as a number of ulcers (0–3), and 3) colon length and weight scores considered as a percentage of loss of each parameter in relation to the control group (0 points, ≤5% weight/length loss; 1 point, 5–14% weight/length loss; 2 points, 15–24% weight/length loss; 3 points, 25–35% weight/length loss; and 4 points, ≥35% weight/length loss). A total score of 0 means no inflammation (Salaga et al., 2014a). The presence (score = 1) or absence (score = 0) of fecal blood was also recorded.

Determination of Tissue Myeloperoxidase Activity

The method described by Salaga et al. (2014a) was used to quantify the myeloperoxidase (MPO) activity. Briefly, 0.5 cm segments of colon were weighed and homogenized in hexadecyltrimethylammonium bromide buffer (0.5% hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer, pH 6.0; 50 mg of tissue/ml) immediately after isolation. Next, the homogenate was centrifuged for 15 minutes (13,200g, 4°C), and the supernatant was used in the subsequent steps. On a 96-well plate, 200 µl of 50 mM potassium phosphate buffer (pH 6.0), containing 0.167 mg/ml O-dianisidine hydrochloride and 0.05 µl of 1% hydrogen peroxide was added to 7 µl of the supernatant. Absorbance was measured at 450 nm (iMARK Microplate Reader; BIO-RAD, Watford, UK) at 0, 30, and 60 seconds after initiation of the reaction. All measurements were performed in triplicate. MPO was expressed in milliunits per gram of wet tissue, 1 unit being the quantity of enzyme able to convert 1 µmol hydrogen peroxide to water in 1 minute at room temperature. Units of MPO activity per 1 minute were calculated from a standard curve using purified peroxidase enzyme.

Histology

After the macroscopic scoring, segments of the distal colon were stapled flat, mucosal side up, onto cardboard and fixed in 10% neutral-buffered formalin for 24 hours at 4°C. After subsequent dehydration samples were embedded in paraffin, sectioned at 5 μm, and mounted onto slides. Next, sections were stained with hematoxylin and eosin and examined using (Motic AE31 Microscope; Ted Pella, Stockholm, Sweden). Photographs were taken using a digital imaging system consisting of a digital camera (Moticam 2300; Ted Pella, Redding, CA) and image analysis software (Motic Images Plus 2.0; Motic Deutschland, Wetzlar, Germany). A microscopic total damage score was determined in a blinded fashion based on the presence (score = 1) or absence (score = 0) of goblet cell depletion, the presence (score = 1) or absence (score = 0) of crypt abscesses, the destruction of mucosal architecture (normal = 1, moderate = 2, extensive = 3), the extent of muscle thickening (normal = 1, moderate = 2, extensive = 3), and the presence and degree of cellular infiltration (normal = 1, moderate = 2, transmural = 3).

Study Population for the Evaluation of Human GLP-2 and GLP2R Expression

To quantify the relative expression of human GLP-2 and GLP2R, forceps biopsy samples and serum were analyzed. In total, 13 specimens prepared from human colon biopsy samples and 29 serum samples were used for the study. The study population comprised 9 patients with CD (21–53 years of age), 12 patients with UC (25–73 years of age), and 8 healthy, unrelated control subjects (25–65 years of age) recruited from January 2014 to January 2015 (for more detailed information, see Supplemental Table 1). The specimens were frozen shortly after isolation and kept at −80°C until further analysis. The diagnosis of CD and UC in patients was assessed according to established clinical criteria using endoscopic, radiologic, and histopathologic criteria. This human part of the study was approved by the Ethics Committee of the Medical University of Lodz. All patients gave written, informed consent prior to the analysis.

Determination of GLP-2 Protein Level by Enzyme-Linked Immunosorbent Assay

For determination of GLP-2 in the mouse colon and human serum the competitive inhibition enzyme immunoassay kit was used (MyBioSource, San Diego, CA). Briefly, mouse tissue segments were rinsed in ice-cold PBS to remove excess blood and weighed before the homogenization. Next, tissues were minced using a motorized cordless tissue grinder (Fisher Scientific, Goteborg, Sweden) in the 20 volumes of mammalian cell lysis buffer (50 mM Tris-HCl, pH 7.5; 1 mM EDTA, 150 mM NaCl; 0.1% SDS; 0.5% deoxycholic acid; 1% Igepal CA-630; Sigma-Aldrich, Poznan, Poland) containing protease inhibitor cocktail (AEBSF, pepstatin A, bestatin, leupeptin, and aprotinin). Then, the homogenates were centrifuged for 12 minutes at 10,000g and at 4°C. The supernatant was used for the procedure following manufacturer instructions. Human serum was diluted five times with PBS before assaying. The amount of GLP-2 in the colonic samples was calculated from the standard curve prepared with the purified GLP-2 standard provided in the kit.

Western Blot

Fragments of tissues were mixed with the mammalian cell lysis buffer (50 mM Tris-HCl, pH 7.5; 1 mM EDTA, 150 mM NaCl; 0.1% SDS; 0.5% deoxycholic acid; 1% Igepal CA-630; Sigma-Aldrich) containing protease inhibitor cocktail (4-benzenesulfonyl fluoride, pepstatin A, bestatin, leupeptin, aprotinin). Then, samples were homogenized using a motorized cordless tissue grinder (Fisher Scientific, Goteborg, Sweden). The homogenate was cleared by centrifugation at 10,000 g for 12 minutes. The concentration of total protein pool was evaluated in each sample (in triplicate) using the Pierce 660 nm Protein Assay (Thermo Scientific, Rockford, IL). Electrophoresis of the samples (15 µg of protein/well) was performed on a precast 4–20% SDS-PAGE gel (BIO-RAD SA, Warsaw, Poland) in electrophoretic buffer (0.1% SDS, 192 mM glycine, and 25 mM Tris, pH 8.3). Separated proteins were transferred using a semi-dry system onto polyvinylidene difluoride membranes (pore size, 0.45 µm; Life Technologies, Carlsbad, CA) in transfer buffer containing 15% (v/v) methanol, 192 mM glycine, and 25 mM Tris, pH 8.3. The polyvinylidene difluoride membranes were incubated at room temperature for 1 hour in 5% nonfat dry milk in PBS with Tween 20 (PBST; PBS, 0.1% Tween 20) to saturate nonspecific protein binding sites. Subsequently, the membranes were incubated with specific primary antibodies diluted in 1% nonfat dry milk in PBST for 80 minutes at room temperature for immunodetection of the studied proteins. The primary rabbit anti-mouse GLP2R polyclonal antibody (H-57; dilution, 1:1000; catalog #sc-99092; Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-mouse DPP IV monoclonal antibody (dilution, 1:1000; catalog #MBS690035; MyBioSource), and mouse anti-glycerylaldehyde-3-phosphate dehydrogenase (dilution, 1:15,000; MAB374; Merck, Warsaw, Poland) were used. After the wash steps (six times for 2 minutes) using PBST, membranes were incubated with appropriate horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature and then the bands were visualized using SuperSignal West Pico Western Blotting Substrate (Thermo Scientific) as a substrate for the localization of horseradish peroxidase activity. Qualitative and quantitative analysis was performed by measuring integrated optical density by ImageLab version 5.2.1 for Windows program (BIO-RAD SA). For the determination of protein weight, we have used 5 µl/lane Precision Plus Protein Standards (BIO-RAD SA).

Statistics

Statistical analysis was performed using Prism version 5.0 (GraphPad Software, La Jolla, CA). The data are expressed as the mean ± SEM. A Student’s t test or one-way ANOVA followed by Newman-Keuls post hoc test were used for analysis. P values <0.05 were considered to be statistically significant.

Drugs

All drugs and reagents, unless otherwise stated, were purchased from Sigma-Aldrich. DSS (mol. wt., 40,000) was purchased from MP Biomedicals (Solon, OH). NLX was purchased from Tocris Bioscience (Bristol, UK). EMDB-1 was synthesized by the solid-phase method using fluorenylmethyloxycarbonyl chemistry. Particulars concerning the synthesis, purification, and physicochemical characteristics of this peptide are as described previously (Fichna et al., 2006a). All drugs were dissolved in 5% dimethylsulfoxide in saline, which was used as a vehicle. The vehicles in the concentrations used had no effects on the observed parameters.

Results

EMDB-1 Is a Potent Inhibitor of DPP IV and Protects GLP-2 against Enzymatic Degradation In Vitro

In vitro experiments employing the Gly-Pro-AMC demonstrated that, in line with a previous report (Fichna et al., 2006b), EMDB-1 exhibits a potent inhibitory activity toward DPP IV (IC50 = 89.7 ± 3 nM) (Fig. 1A). Furthermore, the degradation of t1/2 values for GLP-2 incubated with EMDB-1 was increased approximately 4-fold compared with GLP-2 incubated alone (Fig. 1B). However, the difference between the t1/2 values did not reach statistical significance (P = 0.11).

Fig. 1.
Fig. 1.

EMDB-1 inhibited DPP IV activity in a dose-dependent manner and extended the t1/2 of GLP-2 in vitro. (A) Concentration-response curve showing the inhibitory effect of EMDB-1 on DPP IV activity. (B) Effect of EMDB-1 on the t1/2 of GLP-2 in the presence of purified DPP IV. Data represent the mean ± SEM of three independent experiments performed in triplicate.

EMDB-1 Exhibits Anti-Inflammatory Effect in TNBS- and DSS-Induced Colitis

To evaluate the anti-inflammatory activity of EMDB-1 in the mouse GI tract, we used a well-established mouse model of acute colitis induced by TNBS. The i.c. administration of TNBS resulted in reproducible colitis in mice, as indicated by elevated macroscopic colon damage scores and MPO activity. EMDB-1 administered twice daily (i.c.) at doses of 0.1, 1, and 3 mg/kg significantly improved colitis in a dose-dependent manner, as shown by decreased macroscopic and ulcer scores, colonic wall thickness, and MPO activity (Fig. 2, B–E).

Fig. 2.
Fig. 2.

EMDB-1 injected i.c., twice daily over 3 days at doses ranging from 0.1 to 3 mg/kg attenuated TNBS-induced colitis in mice. The effect of EMDB-1 (3 mg/kg, i.c.) was not blocked by the nonselective opioid receptor antagonist NLX (1 mg/kg, i.p.). (A) Scheme illustrating the protocol and treatment regimen used in this experiment. The figure shows data for macroscopic score (B), ulcer score (C), colonic wall thickness (D), and MPO activity (E). &P < 0.05, &&P < 0.01, &&&P < 0.001, compared with control mice. *P < 0.05, **P < 0.01 ***P < 0.001, compared with TNBS-treated mice. Data represent the mean ± SEM of 6–10 mice/group. D, day.

To examine the possible involvement of opioid receptors in the effect of EMDB-1, we used a nonselective opioid antagonist, NLX (1 mg/kg, i.p.). As shown in Fig. 2, B–E, NLX did not block the anti-inflammatory effect of EMDB-1. NLX administered alone did not affect the disease parameters, besides the ulcer score, which was significantly reduced in this experimental group. EMDB-1 at a dose of 1 mg/kg, i.c. (twice daily), was used in all subsequent experiments.

Our previous observations as well as data from the literature show that the severity of colitis in mice manifested by its clinical symptoms, such as body weight loss, reaches its maximum at day 3 after TNBS injury (Sans et al., 2001; Monteleone et al., 2012). Hence, to investigate the healing effect of EMDB-1 on established colitis, we administered the compound twice daily from day 3 after TNBS administration. Treatment with EMDB-1 resulted in the significant improvement of colitis as indicated by reduced macroscopic score, ulcer score, colonic wall thickness, and MPO activity (Fig. 3, B–E). To mimic the pattern of symptoms that occurs in humans (including the acute phase of colitis, and the recovery and relapse periods), we evaluated the effect of EMDB-1 in the chronic, relapsing colitis paradigm. EMDB-1 (1 mg/kg, i.c.) administered twice daily from day 7 to day 13 significantly attenuated the clinical and molecular parameters of the disease, as shown by reduced macroscopic and ulcer scores, colonic wall thickness, MPO activity, as well as decreased body weight loss and expression of TNFα and IL-1β (Fig. 4, B–G). Histologic evaluation of the mouse colon specimens supported the macroscopic observations. Pathologic changes induced by TNBS were reversed after treatment with EMDB-1 (Fig. 5). Analysis of sections of distal colon from untreated animals showed intact epithelium, the absence of edema, and normal muscle architecture (Fig. 5A). Severe microscopic damage, characterized by the loss of mucosal architecture, the thickening of smooth muscle, the presence of crypt abscesses, and extensive cellular infiltration was observed in TNBS-treated colon specimens (Fig. 5B). The histologic damage was reduced after i.c. EMDB-1 administration (1 mg/kg twice daily from day 7 to day 13) (Fig. 5C).

Fig. 3.
Fig. 3.

The i.c. administration of EMDB-1 (1 mg/kg, twice daily) alleviates established TNBS-induced colitis in mice. (A) A schematic illustration of the protocol and treatment regimen used in this experiment. The figure shows data for microscopic score (B), ulcer score (C), colonic wall thickness (D), and MPO activity (E). &&&P < 0.001, compared with control mice. **P < 0.01, ***P < 0.001, compared with TNBS-treated mice. Data represent the mean ± SEM of 6–10 mice/group. D, day.

Fig. 4.
Fig. 4.

The effect of EMDB-1 (1 mg/kg, i.c.) injected twice daily over 7 days on chronic, relapsing colitis in mice. (A) A scheme illustrating the experimental protocol and treatment regimen used in this model. Figure shows data for macroscopic score (B), ulcer score (C), colonic wall thickness (D), MPO activity (E), body weight changes (F), as well as TNFα and IL-1β expression (G). &P < 0.05, &&P < 0.01, &&&P < 0.001, compared with control mice. **P < 0.01, ***P < 0.001, compared with TNBS-treated mice. Data represent the mean ± SEM of 6–10 mice/group. D, day.

Fig. 5.
Fig. 5.

Microscopic total damage score and representative micrographs of hematoxylin and eosin–stained sections of distal colon obtained from the chronic, relapsing colitis model. Figure shows pictures of control (A), TNBS (B), TNBS + EMDB-1 (1 mg/kg, i.c., twice daily from day 7 to day 13)–treated mice (C). Scale bar = 100 μm. &&&P < 0.001, compared with control mice, ***P < 0.001, compared with TNBS-treated mice. Data represent the mean ± SEM of 6 mice/group.

To test the anti-inflammatory activity of EMDB-1 in the mouse model mimicking UC, a DSS-induced colitis was used. Animals treated with 3% DSS in drinking water developed severe colonic injury (Fig. 6). The i.c. administration of EMDB-1 (1 mg/kg, twice daily) between days 3 and 6 alleviated the disease, as demonstrated by the macroscopic colon damage score (Fig. 6B), colon weight (Fig. 6C), colon length (Fig. 6D), MPO activity (Fig. 6E), and body weight loss (Fig. 6F).

Fig. 6.
Fig. 6.

EMDB-1 (1 mg/kg, i.c.) injected twice daily over 3 days (i.e., D3 to D6) exhibits a healing effect on DSS-induced colitis in mice. (A) A scheme illustrating experimental protocol and treatment used in this model. The figure shows data for macroscopic score (B), colon weight (C), colon length (D), MPO activity (E), and body weight changes (F). &&P < 0.01, &&&P < 0.001, compared with control mice. *P < 0.05, **P < 0.01, ***P < 0.001, compared with DSS-treated mice. Data represent the mean ± SEM of 6–10 mice/group. D, day.

EMDB-1 Alters the Expression of GLP-2 and GLP2R but Not DPP IV in Control and TNBS-Treated Mice

For further characterization of possible mechanisms of the anti-inflammatory action of EMDB-1, changes in the colonic levels of GLP-2, GLP2R, and DPP IV were characterized in naive (“healthy”) and TNBS-treated animals. In naive animals, the level of GLP-2 was significantly elevated (approximately 9-fold increase) after treatment with EMDB-1 (1 mg/kg, i.c.). However, in the TNBS-treated mice this effect was substantially stronger (approximately 19-fold increase) (Fig. 7A).

Fig. 7.
Fig. 7.

Effect of EMDB-1 (1 mg/kg, i.p.) administered twice daily on the expression of GLP-2, GLP2R, and DPP IV in the distal colon of control and TNBS-treated mice. The figure shows quantitative analysis by enzyme-linked immunosorbent assay and Western blotting of GLP-2 (A), GLP2R (B), and DPP IV (C) expression. Western blot data were normalized to glyceraldehyde-3-phosphate dehydrogenase, which was used as an internal control for protein loading. &P < 0.05, compared with control mice. ***P < 0.001, compared with TNBS-treated mice. Data represent the mean ± SEM of 6–7 mice/group.

The expression of GLP2R in the naive animals treated with EMDB-1 has significantly decreased compared with the vehicle-treated mice. A similar pattern was observed in the TNBS-treated animals; however, the reduction in expression was weaker and did not reach statistical significance (Fig. 7B).

Treatment with EMDB-1 did not alter the expression of DPP IV in both control and TNBS-treated mice (Fig. 7C).

Expression of GLP-2 and GLP2R Is Altered during CD and UC

To translate our observations from animals to human conditions, we examined GLP-2 and GLP2R protein expression levels in serum and colon specimens from patients with CD and UC and healthy control subjects. We found that the expression of GLP-2 in human serum is significantly decreased in patients with CD (Fig. 8A). Western blot analysis of GLP2R protein expression in the colon showed a significant decrease in the expression of this receptor in UC patients (Fig. 7C).

Fig. 8.
Fig. 8.

Determination of GLP-2 expression in serum and colon specimens obtained from patients with diagnosed UC and CD versus healthy control subjects. The figure shows data for the concentration of GLP-2 (A), determined by enzyme-linked immunosorbent assay, in the serum and the relative expression of GLP2R (B) measured by Western blotting in the colonic specimens. &P < 0.05, &&P < 0.01, compared with the healthy control subjects. HC, healthy control subject.

Discussion

IBD is a chronic, relapsing inflammatory disorder of the GI tract affecting several million patients worldwide. A high incidence of IBD was reported in developed countries; however, the number of cases of IBD in the regions formerly absent from IBD incidence maps is rapidly increasing (Molodecky et al., 2012). The lack of a fully effective and safe treatment strategy against IBD forces the pursuit of novel compounds targeting proteins that modulate the function of the gut in pathophysiological conditions. In this study, we aimed at testing the concept that the inhibition of DPP IV attenuates colonic inflammation via increased levels of GLP-2.

We showed that a new, highly potent peptide inhibitor of DPP IV, EMDB-1, displays anti-inflammatory activity in TNBS- and DSS-induced mouse models of experimental colitis. Moreover, we demonstrated that this effect is associated with the striking elevation of colonic GLP-2 levels. We also showed that EMDB-1 is effective in the model of chronic, relapsing colitis that recapitulates the course of IBD in humans, a finding of high clinical relevance. Finally, we quantified the expression of GLP-2 as well as GLP2R in human serum and colonic specimens to evaluate the potential involvement of the incretin signaling in the pathophysiology of IBD.

The significant anti-inflammatory action of EMDB-1 validates the therapeutic use of DPP IV inhibitors in the treatment of IBD and corroborates the concept of protease inhibition as a way to combat diseases characterized by immunologic imbalance. Proteases are widely distributed in the human body (to date, 500–600 different proteases have been identified), including the GI tract, where they serve not only for digestion purposes but also for maintenance of gut homeostasis (e.g., immune cells activation and interaction with gut microbiota) (Vergnolle, 2016). In pathophysiological conditions, such as inflammation, the content of proteases in the gut increases due to the infiltration of immune cells (e.g., neutrophils release significant amounts of elastase and proteinase-3), which use these enzymes to degrade tissues and absorbed biologic molecules, thereby increasing the phagocytic properties of these cells (Vergnolle, 2016). In line with this, the expression of a very large number of proteases is increased in IBD. Enzymatic activity of these upregulated proteases is not balanced by endogenous inhibitors since their expression often remains unchanged. Thus, re-equilibration of the protease-antiprotease balance with exogenous inhibitors may be a useful therapeutic strategy (Vergnolle, 2016).

Recently, a novel therapeutic strategy, referred to as peptidase-targeted immunoregulation, has been proposed that involves, for the purpose of restoration of immune balance, limiting the activation of immune cells and the induction of endogenous immunosuppressive mechanisms, such as transforming growth factor β and regulatory T cells, through the inhibition of peptidase-dependent pathways (Bank et al., 2006; Salaga et al., 2013). Experimental data indicate that peptidase-targeted immunoregulation resulted in the suppression of cell proliferation and reduced synthesis of proinflammatory cytokines without affecting cellular vitality (Bank et al., 2006). Moreover, protease inhibitors seem to be superior to exogenous receptor ligands since they allow for finer tuning of the signaling pathways, which are transduced through the elevation of endogenous mediators. Furthermore, their action may be limited in time and space, ensuring greater pharmacological control. Here we show that targeting DPP IV attenuates experimental colitis after topical administration.

Our results are in line with the report by Mimura et al. (2013), which showed that synthetic DPP IV inhibitor anagliptin improves body weight loss, disease activity index, as well as histologic score in the model of DSS-induced colitis. Moreover, in this study we show for the first time that a peptide compound targeting DPP IV potentially could be used as an anti-inflammatory agent in the GI tract. Of note, EMDB-1 was effective in two animal models that mimic both major types of IBD, namely CD recapitulated in TNBS-induced colitis and UC recapitulated in the DSS-induced colitis.

To investigate the mechanism of action of EMDB-1, we characterized its effect on the levels of GLP-2, GLP2R, and DPP IV in control and TNBS-treated mice. These experiments undoubtedly demonstrated that the increase in the levels of colonic GLP-2 is associated with the anti-inflammatory effect of EMDB-1. Moreover, no change in the expression of DPP IV has been observed; hence, the elevation of GLP-2 may be attributed solely to the inhibition of this protease. Particularly interesting is the fact that the protective effect of the DPP IV inhibitor on GLP-2 was substantially stronger in the TNBS-treated animals versus control animals, leading to the approximately 19-fold increase of this incretin hormone in the colon. This phenomenon may be explained by the increase in the number of L cells that secrete GLP-2 into the colonic mucosa exposed to the TNBS, as previously demonstrated by O’Hara et al. (2007). The presence of a large amount of GLP-2 in the colon likely leads to the improvement of colitis by the stimulation of cells that express GLP2R via various previously described mechanisms, such as the stimulation of subepithelial myofibroblasts to release growth factors and transforming growth factor β, the inhibition of proinflammatory cytokines (e.g., TNFα, IL-1β), and the enhancement of mesenteric blood flow (for comprehensive review, see Hornby and Moore, 2011).

Moreover, treatment with EMDB-1 significantly reduced the expression of GLP2R in control mice but not in TNBS-treated mice. It is probably caused by a common adaptive mechanism regulating G-protein–coupled receptor signaling that is based on a reduction of the number of receptors in response to the steep increase in the concentration of the ligand. Of note, this effect was weaker (insignificant) in the animals with colitis, suggesting that the expression of GLP2R is maintained at the higher level by some additional mechanism to sustain the restorative GLP-2–mediated signaling. This phenomenon together with the increased bioavailability and prolonged t1/2 of GLP-2 further support the concept of aiming at DPP IV in the IBD therapy.

Translational experiments in human tissues showed that levels of GLP-2 are strongly reduced in the serum of patients with CD but not in those with UC. Recently, Sigalet et al. (2013), in a pilot study, showed that the postprandial GLP-2 level is reduced in patients with CD in the acute phase of the disease but not in the remission phase. This observation points to the conclusion that an increase of GLP-2 level would restore the colonic damage in patients with CD. Furthermore, our results suggest that GLP-2 signaling is also impaired in patients with UC due to the low receptor expression, which further supports the concept of targeting the incretin system in the anti-IBD therapy. Here we tested the i.c. administration of EMDB-1 and obtained encouraging results in different models of colonic inflammation. Consequently, we envisage the use of an enema consisting of DPP IV inhibitors in patients with CD and UC, especially since our test compound ameliorated both TNBS- and DSS-induced colitis. At this point, topical administration of compounds modulating incretin signaling seems to be the most rational since the therapeutic potential of GLP-2 in GI diseases is based on the multiple indirect effects in the gut, where this compound is locally secreted. Of note, one of the natural substrates of DPP IV are endogenous opioid peptides, such as EMs that activate MOR-dependent pathways in the GI tract. Immune cells express opioid receptors, and thus opioids may be involved in the regulation of inflammatory processes. It has been demonstrated that the activation of MORs located on the peripheral immune and nerve cells exert anti-inflammatory effect (Borzsei et al., 2008; Sobczak et al., 2014a,b). Moreover, it was observed that MOR−/− mice are more susceptible to inflammation than their wild-type littermates (Philippe et al., 2003). Here, to exclude the possible involvement of opioid peptides in the anti-inflammatory activity of EMDB-1, we used a nonselective opioid antagonist, NLX, which did not alter any of the measured parameters. Based on this observation, together with the fact that EMDB-1 does not exhibit affinity toward opioid receptors, we conclude that MOR signaling cannot be taken into consideration regarding the EMDB-1 mechanism of action.

Conclusions and Future Perspectives.

In the present study, we showed that a novel peptide inhibitor of DPP IV, EMDB-1, exerts a potent anti-inflammatory effect in the mouse models of colitis, which is associated with the increase of colonic GLP-2 levels. These findings offer a new alternative in the pharmacological strategies of IBD treatment. EMDB-1 may now be used as a pattern for the design and synthesis of peptides displaying not only low binding to opioid receptors and high inhibitory activity toward DPP IV, but also resistance to degradation by proteases located in the upper GI tract. Such modification would allow oral administration of the compound, which is preferable in the context of clinical application. The design and synthesis of peptide DPP IV inhibitors is thus a promising direction in the search for innovative anti-IBD drugs. Such therapy could be particularly appealing for the group of patients for whom other therapies did not provide sufficient relief.

Concerns about the side effects of the GLP-2 that may hamper its therapeutic utility mainly relate to its potential carcinogenic effect. It has been raised that long-term stimulation of GLP2R may contribute to the increased proliferation and protection of the tumor cells. Indeed, it has been shown that exogenous GLP-2 elevates the number of aberrant crypt foci and leads to the formation of adenocarcinomas in the azoxymethane-treated mice (Iakoubov et al., 2009). On the other hand, it was reported that long-term GLP-2 treatment has no effect on the growth of human colon cancer cells in nude mice (neither on the number or size of polyps in ApcMin/+ mice) (Koehler et al., 2008). Given these contradictory data, it is difficult to provide a clear-cut answer on the potential threat of GLP2R agonists. Hence nowadays, one of the greatest challenges to the field is to clearly estimate the risk of carcinogenesis after treatment with GLP-2.

Authorship Contributions

Participated in research design: Salaga and Fichna.

Conducted experiments: Salaga, Zielinska, and Kamysz.

Contributed new reagents or analytic tools: Mokrowiecka, Malecka-Panas, and Kordek.

Performed data analysis: Salaga, Zielinska, and Kamysz.

Wrote or contributed to the writing of the manuscript: Salaga, Kamysz, and Fichna.

Footnotes

    • Received May 2, 2017.
    • Accepted July 12, 2017.

  • This work was supported by the Medical University of Lodz [Grant #502-03/1-156-04/502-14-298 to M.S. and Grant #503/1-156-04/503-11-001 to J.F.], the National Science Centre [Grant #UMO-2013/11/N/NZ7/02354 to M.S. and Grant #UMO-2013/11/B/NZ7/01301 to J.F.], and the Iuventus Plus Program of the Polish Ministry of Science and Higher Education (Grant IP2015 068774 to M.S.). No potential conflicts of interest relevant to this article are reported.

  • https://doi.org/10.1124/jpet.117.242586.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

AMC
aminomethylcoumarin
CD
Crohn’s disease
DPP IV
dipeptidyl peptidase IV
DSS
dextran sulfate sodium
EM
endomorphin
EMDB-1
Tyr-Pro-D-ClPhe-Phe-NH2
GI
gastrointestinal
GLP-2
glucagon-like peptide 2
GLP2R
glucagon-like peptide 2 receptor
IBD
inflammatory bowel disease
i.c.
intracolonic
IL
interleukin
MOR
µ-opioid receptor
MPO
myeloperoxidase
NLX
naloxone
PBS
phosphate-buffered saline
PBST
phosphate-buffered saline with Tween 20
t1/2
half-life
TNBS
2,4,6-trinitrobenzene sulfonic acid
TNFα
tumor necrosis factor α
UC
ulcerative colitis

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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

EMDB-1 in Colitis

Maciej Salaga, Anna Mokrowiecka, Marta Zielinska, Ewa Malecka-Panas, Radzislaw Kordek, Elzbieta Kamysz and Jakub Fichna
Journal of Pharmacology and Experimental Therapeutics October 1, 2017, 363 (1) 92-103; DOI: https://doi.org/10.1124/jpet.117.242586
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