CROHNS-00956; No of Pages 12 Journal of Crohn's and Colitis (2014) xx, xxx–xxx
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Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition of fatty acid amide hydrolase (FAAH) M. Sałaga a , A. Mokrowiecka b , P.K. Zakrzewski c , A. Cygankiewicz c , E. Leishman d , M. Sobczak a , H. Zatorski a , E. Małecka-Panas b , R. Kordek e , M. Storr f , W.M. Krajewska c , H.B. Bradshaw d , J. Fichna a,⁎ a
Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Lodz, Poland Department of Digestive Tract Diseases, Faculty of Medicine, Medical University of Lodz, Lodz, Poland c Department of Cytobiochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland d Department of Psychological and Brain Sciences, Gill Center for Biomolecular Science, Indiana University, Bloomington, IN, USA e Department of Pathology, Faculty of Medicine, Medical University of Lodz, Lodz, Poland f Department of Medicine, Division of Gastroenterology, Ludwig Maximilians University of Munich, Munich, Germany b
Received 12 November 2013; received in revised form 29 January 2014; accepted 29 January 2014 KEYWORDS Fatty acid amide hydrolase; Intestinal inflammation; Inflammatory bowel diseases; PF-3845; Experimental colitis
Abstract Background and aims: Pharmacological treatment and/or maintenance of remission in inflammatory bowel diseases (IBD) is currently one of the biggest challenge in the field of gastroenterology. Available therapies are mostly limited to overcoming the symptoms, but not the cause of the disease. Recently, the endocannabinoid system has been proposed as a novel target in the treatment of IBD. Here we aimed to assess the anti-inflammatory action of the novel fatty acid amide hydrolase (FAAH) inhibitor PF-3845 and its effect on the endocannabinoid and related lipid metabolism during the course of experimental colitis. Methods: We used two models of experimental colitis in mice (TNBS- and DSS-induced) and additionally, we employed LC/MS/MS spectrometry to determine the changes in biolipid levels in the mouse colon during inflammation.
Abbreviations: 2-AG, 2-arachidonyloglicerol; 2-OG, 2-oleoylglycerol; AA, arachidonic acid; AEA, anandamide; COX, cyclooxygenase; DMSO, dimethyl sulfoxide; DSS, dextran sulfate sodium; FAAH, fatty acid amide hydrolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HTAB, hexadecyltrimethylammonium bromide; i.c., intracolonic; i.p., intraperitoneal; MAGL, monoacylglycerol lipase; MO, allyl isothiocyanate; MPO, myeloperoxidase; MRM, multiple reaction monitoring; p.o., per os; PBS, phosphate buffered saline; PGE2, prostaglandin E2; TNBS, 2,4,6-trinitrobenzene sulfonic acid. ⁎ Corresponding author at: Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland. Tel.: + 48 42 272 57 07; fax: + 48 42 272 56 94. E-mail address: [email protected] (J. Fichna). 1873-9946/$ - see front matter © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.crohns.2014.01.025 Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
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M. Sałaga et al. Results: We showed that the FAAH inhibitor PF-3845 reduced experimental TNBS-induced colitis in mice and its anti-inflammatory action is associated with altering the levels of selected biolipids (arachidonic and oleic acid derivatives, prostaglandins and biolipids containing glycine in the mouse colon). Conclusions: We show that FAAH is a promising pharmacological target and the FAAH-dependent biolipids play a major role in colitis. Our results highlight and promote therapeutic strategy based on targeting FAAH-dependent metabolic pathways in order to alleviate intestinal inflammation. © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.
1. Introduction Ulcerative colitis (UC) and Crohn's disease (CD), which belong to the group of inflammatory bowel diseases (IBD), are chronic, progressive and relapsing inflammatory disorders that may cause disability over time (for review see Ref. 1). Both disorders are characterized by an excessive activation of common inflammatory pathways, which results in an enhanced secretion of pro-inflammatory cytokines, such as interleukins (IL-1α, -1β, -2, -6, -8, -12, and -17), TNF or INF, and an imbalance in the levels of pro- and anti-inflammatory factors in the tissue.2 There is also growing evidence that the neuronal system is significantly engaged in the pathophysiology of colitis, including signaling in the intrinsic enteric nervous system (ENS) and from ENS to the central nervous system (CNS).1,3 Although the impaired immune response and disrupted neural signaling could be the causes of IBD, the etiology of this disorder still remains unclear. The natural plant-derived cannabinoids have been known for centuries, but only relatively recently the endogenous cannabinoids, namely anandamide (AEA) and 2-arachidonyloglicerol (2-AG), have been discovered in the mammalian tissues.4,5 Endogenous and plant-derived cannabinoids share similar biological properties, which include antinociception and the regulation of GI motility, secretion, proliferation, and immune functions.6,7 The principal sites of action for cannabinoids are the “classical” CB receptors, i.e. CB1 and CB2. CB1 receptors are located primarily presynaptically on the surface of nerve cells of the central and the peripheral nervous system, and also in the peripheral tissues like cardiovascular tissue, urinary bladder and gastrointestinal (GI) tract (small intestine, spleen).8,9 CB2 receptors are located mainly on cells of the immune system (e.g. macrophages, neutrophils), but were also detected in the microglial cells and neurons of the CNS.8,9 Both types of receptors are coupled with the Gi-protein, which is responsible for the inhibition of adenylate cyclase activity; it was also shown that cannabinoids modulate the activity of protein kinases.10 The action of endocannabinoids in the living organism is strongly regulated by their metabolism; for example, the half-life of AEA can be measured in the order of minutes.11 Fatty acid amide hydrolase (FAAH) is primarily responsible for the intracellular degradation of AEA into arachidonic acid (AA) and ethanol amine.12 In addition, FAAH has been identified as the key enzyme in the metabolism of N-arachidonoyl glycine as well as its family of endogenous structural analogs.13,14 FAAH possesses a number of channels and cavities involved in substrate or inhibitor binding and by using specific FAAH inhibitors the level of the endogenous AEA and potentially other lipids can be
elevated and thus its action in the organism prolonged and intensified. Since the blockade of FAAH produces effects similar to those that can be achieved by the CB receptor agonists, it has been suggested as an attractive pharmacological strategy for the treatment of IBD in humans, possibly without the side effects typical for cannabinoids. The use of specific FAAH inhibitors to improve colitis in animal models has recently been reported.15,16 Moreover, it was observed that FAAH-deficient mice are more resistant to experimental DNBS-induced colitis than the wild type animals.17 The increased level of AEA in the colonic tissue of IBD patients, translated well the key findings from animal tissues to humans.15 Evaluating novel drugs for the treatment of IBD, we characterized the anti-inflammatory action of the selective FAAH inhibitor PF-3845 in two mouse models of intestinal inflammation. In particular, we examined the effects of PF-3845 employing different routes of administration and different dosing regimens. Moreover, we characterized the mechanism of action of PF-3845 by using specific CB1 and CB2 receptors antagonists. Since visceral pain is one of the most common symptoms in IBD, we also investigated the antinociceptive effect of PF-3845 in TNBS-treated animals. Finally, we quantified the levels of endocannabinoid and metabolically-related biolipids in the course of colitis to assess the biomolecular pathways responsible for the antiinflammatory action of the FAAH blocker.
2. Materials and methods 2.1. Animals Experimentally naive male C57B1/6 mice were obtained from the Animal House at the University of Lodz, Poland. All animals used in the experiments weighed 25–30 g. Mice were housed at a constant temperature (22 °C) and maintained under a 12-hour light/dark cycle 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 University of Lodz Animal Care Committee (Protocol #670/ 2013), following the guidelines of the Polish Council of Animal Care. All efforts were made to minimize animal suffering and to reduce the number of animals used. Groups of 6–8 animals were used in all in vivo experiments.
2.2. Induction of colitis 2.2.1. TNBS model Colitis was induced by intracolonic (i.c.) administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS), as described
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
Inhibition of FAAH in experimental colitis previously.18 Briefly, mice were lightly anesthetized with 1% isoflurane (Baxter Healthcare Corp., IL, USA) and TNBS (4 mg in 0.1 mL of 30% ethanol in saline) was administered into the colon through a catheter inserted to the anus (3 cm proximally). Control animals received vehicle alone (0.1 mL of 30% ethanol in saline). Preliminary experiments demonstrated that the dose of TNBS used in this study induced reproducible colitis. 2.2.2. DSS model Colitis was introduced by the addition of DSS to drinking water starting from day 0 to day 5 (4% wt/vol; molecular weight 40,000; MP Biomedicals, Aurora, OH, Lot No. 5237K). On days 6 and 7 the animals received water without DSS. Control animals received tap water. Animal body weight was monitored daily and mean water and food consumption was recorded.
2.3. Pharmacological treatments The selective FAAH inhibitor PF-3845 was administered once daily at the dose of 10 mg/kg (intraperitoneally, i.p., and i.c.) in TNBS and DSS models, and 10 mg/kg twice daily (orally, p.o.), in TNBS model, with the first treatment 30 min before the induction of colitis. Selective CB1 (AM 251, 1 mg/kg, i.p.), and CB2 (AM 630, 1 mg/kg, i.p.) receptor antagonists were administered 10 min before PF-3845. All drugs were dissolved in 5% dimethyl sulfoxide (DMSO) in saline, which was used as vehicle. Control animals received vehicle (100 μL i.p., 100 μL i.c. or 150 μL p.o.) alone. Neither the antagonists, nor vehicle influenced the observed parameters when given alone.
2.4. Evaluation of colonic damage 2.4.1. TNBS model Animals were sacrificed by cervical dislocation 3 days after TNBS infusion. The colon was rapidly removed, opened longitudinally, rinsed with phosphate buffered saline (PBS), and immediately examined. Macroscopic colonic damage was assessed by an established semiquantitative scoring system by adding individual scores for ulcer, colonic shortening, wall thickness, and presence of hemorrhage, fecal blood, and diarrhea, as described previously.18 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 N 15%, 2 points for N 25% (based on a mean length of the colon in untreated mice of 8.07 ± 0.20 cm, n = 6). The wall thickness was measured in mm. The presence of hemorrhage, fecal blood, or diarrhea increased the score by 1 point for each additional feature. 2.4.2. DSS model Mice were sacrificed by cervical dislocation on day 7 following addition of DSS to the drinking water. The entire colon was immediately isolated and weighed with fecal content. Colon was then opened along the mesenteric border and fecal material removed. A total macroscopic damage score was calculated for each animal consisting of stool consistence (0–3), colon epithelial damage considered as number of ulcers (0–3), colon length and weight scores (0–4), where score = 0 means no inflammation.18 The presence (score = 1) or absence (score = 0) of fecal blood was also recorded.
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2.5. Determination of tissue myeloperoxidase activity The method described by Fichna and collaborators18 was used to assess granulocyte infiltration and to quantify the myeloperoxidase (MPO) activity. Briefly, 1-cm segments of colon were weighed and homogenized in hexadecyltrimethylammonium bromide (HTAB) buffer (0.5% HTAB in 50 mM potassium phosphate buffer, pH 6.0; 50 mg of tissue/mL) immediately after isolation and the homogenate was centrifuged (15 min, 13,200 ×g, 4 °C). On a 96-well plate, 200 μL of 50 mM potassium phosphate buffer (pH 6.0), containing 0.167 mg/mL of O-dianisidine hydrochloride and 0.05 μL of 1% hydrogen peroxide was added to 7 μL of supernatant. Absorbance was measured at 450 nm (iMARK Microplate Reader, Biorad, United Kingdom). 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 of hydrogen peroxide to water in 1 min at room temperature. Units of MPO activity per 1 min were calculated from a standard curve using purified peroxidase enzyme.
2.6. 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 h at 4 °C. Samples were then dehydrated, embedded in paraffin, sectioned at 5 μm and mounted onto slides. Subsequently sections were stained with hematoxylin and eosin and examined using (Motic AE31 microscope, Ted Pella, Sweden). Photographs were taken using a digital imaging system consisting of a digital camera (Moticam 2300, Ted Pella, Sweden) and image analysis software (Motic Images Plus 2.0, Germany). A microscopic total damage score was determined 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).
2.7. Mustard oil model of visceral pain Behavioral responses to i.c. administration of mustard oil (MO, allyl isothiocyanate) were assessed as described previously.19 For the determination of pain behaviors, 50 μL of MO (1% in 70% ethanol) was injected i.c. under isoflurane anesthesia. To avoid stimulation of somatic nociceptors, perianal area was covered with Vaseline. After 5 min of recovery, spontaneous behaviors were recorded on a videotape for 20 min for later analysis by an observer blinded for experimental conditions. Postures defined as pain-related behaviors: 1) licking of the abdomen, 2) stretching the abdomen, 3) squashing of lower abdomen against the floor, and 4) abdominal retractions, were each counted as 1. PF-3845 was administered p.o. and i.c. 20 and 15 min before the MO instillation, respectively. All drugs were dissolved in 5% DMSO in saline, which was used as vehicle in control experiments.
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
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2.8. Determination of endocannabinoid levels in mouse tissue 2.8.1. Sample isolation and preparation Upon isolation, the colon samples were frozen at −80 °C and kept until further processed. For endocannabinoid level determination, they were removed from the freezer, shocked with liquid nitrogen, weighed and placed in centrifuge tubes on ice. Forty one volumes of methanol were added to each tube followed by 10 μL of 100 pM N-arachidonoyl glycine-d8, which was added to act as an internal standard to determine the recovery of the compounds of interest. The tubes were then covered with parafilm and left on ice and in darkness for approximately 2 h. The samples were then homogenized using a Polytron for approximately 1 min on each sample. The samples were then centrifuged at 19,000 ×g at 24 °C for 20 min. The supernatants were collected and placed in polypropylene tubes (15 or 50 mL) and HPLC-grade water was added making the final supernatant/water solution 25% organic. To isolate the compounds of interest partial purification of the 25% solution was performed on a Preppy apparatus assembled with 500 mg C18 solid-phase extraction columns. The columns were conditioned with 5 mL of HPLC-grade methanol immediately followed by 2.5 mL of HPLC-grade water. The supernatant/water solution was then loaded onto the C18 column, and then washed with 2.5 mL of HPLC grade water followed by 1.5 mL of 40% methanol. Elutions of 1.5 mL of 60, 75%, 85%, and 100% methanol were collected in individual autosampler vials and then stored in a − 80 °C freezer until mass spectrometer analysis.
2.8.2. LC/MS/MS analysis and quantification Samples were removed from the − 80 °C freezer and allowed to warm at room temperature then vortexed for approximately 1 min before being placed into the autosampler and held at 24 °C (Agilent 1100 series autosampler, Palo Alto, CA, USA) for LC/MS/MS analysis. Ten–twenty microliters of eluants were injected separately for each sample to be rapidly separated using a C18 Zorbax reversed-phase analytical column to scan for individual compounds. Gradient elution (200 μL/min) then occurred, under the pressure created by two Shimadzu 10AdVP pumps (Columbia, MD, USA). Next, electrospray ionization was accomplished using an Applied Biosystems/MDS Sciex (Foster City, CA, USA) API3000 triple quadrupole mass spectrometer. A multiple reaction monitoring (MRM) setting on the LC/MS/MS was then used to analyze levels of each compound present in the sample injection. Synthetic standards were used to generate optimized MRM methods and standard curves for analysis.
2.8.3. Data analysis The amount of analyte in each sample was calculated by using a combination of calibration curves of the synthetic standards obtained from the Analyst software and deuterium-labeled internal standards. The standards provided a reference for the retention times by which the analytes could be compared. They also helped to identify the specific precursor ion and fragment ion for each analyte which enabled their isolation. These processes provide confidence in the claim that the compounds measures were, in fact, the compound of interest. The amount
of each compound in each tissue was then converted to moles per gram tissue, which is how it was statistically analyzed.
2.9. Study population for the evaluation of human FAAH mRNA levels To quantify the relative expression of human FAAH, forceps biopsy samples were analyzed. In total 44 specimens prepared from human colon biopsies were used for the study. The study population comprised 15 patients with Crohn's disease (CD, ages 20–68), 15 patients with ulcerative colitis (UC, ages 24–59) and 14 healthy, unrelated controls (ages 27–81) recruited from January 2011 to January 2012 (for more detailed information see supplementary file, Table 1). The specimens were frozen shortly after isolation and kept at −70 °C until further analysis. The diagnosis of CD and UC in patients was assessed accordingly 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 genetic analysis.
2.10. Determination of FAAH mRNA levels The RNA was isolated according to manufacturer's protocol using the PureLink RNA Mini kit (Life Technologies, Carlsbad, CA, USA) as described before.20 Briefly, tissue samples were homogenized in lysis buffer (600 μL), complemented with 1% 2-mercaptoethanol (St. Louis, MO, USA). Subsequently, the homogenates were centrifuged to clear the solution. Supernatants were washed and finally the purified total RNA samples were eluted into collection tubes using diethyl pyrocarbonate (DEPC) treated water (50 μL). To measure the purity and quantity of isolated RNA, dedicated spectrophotometer (BioPhotometer; Eppendorf, Germany) was used. Total RNA (1 μg) was transcribed onto cDNA with First Strand cDNA synthesis kit (Fermentas, Canada). Quantitative analysis was performed using fluorescently labeled TaqMan probe Hs00155015_m1 for human FAAH and Hs01003267_m1 for mouse hypoxanthineguanine phosphoribosyltransferase (HPRT, endogenous control, Life Technologies, Carlsbad, CA, USA) on Mastercycler S realplex 4 apparatus (Eppendorf, Germany). All experiments were performed in triplicate. The Ct (threshold cycle) values for studied genes were normalized to Ct values obtained for a housekeeping gene HPRT. Relative amount of mRNA copies was calculated using following equation: 2−ΔCt × 1000. There was no alteration in the levels of mRNA of HPRT in samples from all patient groups regardless of the studied group.
2.11. Statistics Statistical analysis was performed using Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). The data are expressed as means ± SEM. Student t-test or one-way ANOVA followed by Newman–Keuls post-hoc test were used for analysis. P values b 0.05 were considered statistically significant.
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
Inhibition of FAAH in experimental colitis
2.12. Drugs All drugs and reagents, unless otherwise stated, were purchased from Sigma-Aldrich (Poznan, Poland). DSS (MW 40,000) was purchased from MP Biomedicals (Solon, OH, USA). N-3-pyridinyl4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]1-piperidine-carboxamide (PF-3845) was prepared by TriMen Chemicals (Lodz, Poland). N-(Piperidin-1-yl)-5-(4-iodophenyl)1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and [6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]1H-indol-3-yl](4-methoxyphenyl) methanone (AM630) were purchased from Tocris Bioscience (Bristol, UK).
3. Results 3.1. PF-3845 has anti-inflammatory effect in TNBS-induced colitis in mice The i.c. instillation of TNBS resulted in a reproducible colitis in mice, manifested by increased macro- and microscopic colon damage scores and elevated MPO activity. PF-3845 significantly attenuated TNBS-colitis in various routes of administration (i.p. — Fig. 1A; p.o. — Fig. 1B; i.c. — Fig. 1C), as demonstrated by decreased total macroscopic score (4.68 ± 0.36 for i.p. administration of PF-3845 at the dose of 10 mg/kg once daily vs. 7.35 ± 0.52 for TNBS-treated mice; 3.39 ± 0.33 for p.o. administration of PF-3845 at the dose of 10 mg/kg twice daily vs. 5.61 ± 0.64 for TNBS-treated mice; 3.49 ± 0.34 for i.c. administration of PF-3845 at the dose of 10 mg/kg once daily vs. 7.35 ± 0.52 for TNBS-treated mice). The MPO activity after i.p. injection of PF-3845 (10 mg/ kg, once daily) was non-significantly elevated compared to the TNBS-treated group (Fig. 1A). The p.o. administration of PF-3845 at the dose of 10 mg/kg (twice daily) caused a substantial reduction of the MPO activity compared to the TNBS-treated group, but the difference did not reach a statistical significance (Fig. 1B). Similarly, the i.c. injection of the FAAH blocker (10 mg/kg, once daily) non-significantly decreased the MPO activity (Fig. 1C). The oral gavage was used as the principal route of administration in subsequent experiments. Specific CB1 and CB2 receptor antagonists (AM251 and AM630, respectively) were used to characterize the mechanism of action by which PF-3845 attenuates inflammation in the TNBS model. As shown in Fig. 2, the anti-inflammatory effect of PF-3845 at the dose of 10 mg/kg (p.o.; twice daily) was blocked by the CB1 receptor antagonist AM251 (1 mg/kg i.p.), as demonstrated by the increase in the macroscopic score (Fig. 2A), ulcer score (Fig. 2C) and shortening of colon (Fig. 2D). AM251 did not influence the MPO activity in the inflamed tissue (Fig. 2B). The microscopic evaluation of colon sections stained with hematoxylin/eosin was in good agreement with observation of the macroscopic parameters (Fig. 3). Histological analysis of sections of distal colon from untreated animals showed intact epithelium, absence of edema, and normal muscle architecture (Fig. 3A). Severe microscopic damage, characterized by loss of mucosal architecture, thickening of smooth muscle, presence of crypt abscesses, and extensive cellular infiltrate was observed in colon specimens 3 days post-TNBS (Fig. 3B). The histological changes were normalized after p.o. PF-3845
5 treatment (Fig. 3C). The protective effect of PF-3845 was reversed by administration of CB1 antagonist AM251, but not CB2 antagonist AM630.
3.2. PF-3845 administered orally has no effect on DSS-induced colitis in mice In order to extend our observations on the anti-inflammatory action of PF-3845, a DSS-induced mouse model of UC was used. Animals treated with 4% DSS in drinking water developed severe colonic injury, which was not reversed by p.o. administration of PF-3845 (10 mg/kg, twice daily, Fig. 4). Treatment with PF-3845 did not reduce any of evaluated parameters: macroscopic colon damage score (Fig. 4A), MPO activity (Fig. 4B), colon weight (Fig. 4C) or colon length (Fig. 4D).
3.3. PF-3845 administered orally has no antinociceptive effect in TNBS-treated animals To evaluate the antinociceptive activity of PF-3845 in inflammatory conditions, we used a MO-induced model of abdominal pain. The i.c. instillation of MO in TNBStreated mice resulted in a significant increase in number of pain-related behaviors compared to control animals (Fig. S1). Neither p.o., nor i.c. administration of PF-3845 at the dose of 10 mg/kg 20 min before the instillation of MO produced a statistically significant antinociceptive effect (Fig. S1).
3.4. PF-3845 alters the levels of endocannabinoids in TNBS-induced colitis For further characterization of possible mechanisms of the anti-inflammatory action of PF-3845, changes in the levels of endocannabinoids and their metabolites in colonic tissues were characterized in naive (“healthy”) and TNBStreated animals. In naïve animals, the levels of 2-AG and 2-oleoylglycerol (2-OG) were significantly lower after treatment with PF-3845 (10 mg/kg, p.o., Fig. 5A and B, respectively). However, in the TNBS-treated mice, 2-AG and 2-OG levels were not significantly altered after treatment with PF-3845 compared to the animals receiving vehicle. As shown in Fig. 5C, the level of endogenous prostaglandin E2 (PGE2) was significantly elevated after treatment with PF-3845 in control animals, but it was significantly decreased in TNBS-treated mice. Quantification of the levels of biolipids containing glycine showed that O-Gly, L-Gly, A-Gly, and D-Gly were significantly lower in naive animals after the treatment with PF-3845, but not in TNBS-treated mice (Fig. 5D). Interestingly, there were no significant changes in the levels of N-acyl ethanolamines in both naive and TNBS-treated animals after treatment with PF-3845 (Fig. S2).
3.5. Expression of FAAH mRNA in the human colon is decreased during UC and CD To translate our observations from animals to human, we examined the FAAH mRNA expression levels in colon
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
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Figure 1 PF-3845 (10 mg/kg) injected twice daily over 3 days in various routes of administration attenuated TNBS-induced colitis in mice. Figure shows data for macroscopic scores and MPO activity after (A) i.p., (B) p.o. and (C) i.c. administration. *P b 0.05, **P b 0.01, and ***P b 0.001, as compared to control mice. &&P b 0.01 and &&&P b 0.001, vs. TNBS-treated animals. Data represent mean ± SEM of 6–8 mice per group.
specimens from patients with UC, CD and healthy controls. We found that the relative expression of FAAH mRNA is decreased in patients with UC and CD compared with healthy subjects; however, the difference did not reach statistical significance (Fig. 6). Moreover, the expression was lower in patients with UC than CD but the difference was not statistically significant.
4. Discussion In the present study we showed that the highly potent and selective FAAH inhibitor PF-3845 displayed anti-inflammatory activity in TNBS-induced mouse model of experimental colitis and this effect was mediated by CB1 receptors. We also showed that PF-3845 was effective after i.p., p.o., and i.c.
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
Inhibition of FAAH in experimental colitis
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Figure 2 The anti-inflammatory effect of PF-3845 (10 mg/kg, p.o.) injected twice daily over 3 days in TNBS-treated mice was reversed by the CB1 antagonist AM 251 (1 mg/kg, i.p.), but not the CB2 antagonist AM 630 (1 mg/kg, i.p.). Figure shows data for macroscopic score (A), MPO activity (B), ulcer score (C), and colon length (D). &P b 0.05, &&P b 0.01, and &&&P b 0.001, as compared to control mice, *P b 0.05 and **P b 0.01, as compared to TNBS-treated mice. #P b 0.05 and ##P b 0.01, vs. PF-3845-treated animals. Data represent mean ± SEM of 6–8 mice per group.
administration, a finding of high clinical relevance. The significant anti-inflammatory action of PF-3845 suggests a possible therapeutic use of FAAH inhibitors in the treatment of IBD. Previous studies demonstrating colitis-associated changes in CB receptor expression in animals and humans indicate that the endogenous cannabinoid system plays an important role in the course of intestinal inflammation.15,17,21–23 However, neither natural, nor synthetic CB receptor ligands have entered clinical trials in IBD, which is due to the vast spectrum of adverse effects caused by these compounds, ranging from increase in body temperature, seizures and hallucinogenic effects or physical dependence.24–27 Of note, a natural FAAH inhibitor derived from Cannabis sativa, cannabidiol, has been shown to reduce inflammation-induced intestinal hypermotility and to ameliorate murine colitis induced by TNBS.28–31 Our findings point out that elevating the level of endogenous cannabinoids by application of a selective FAAH inhibitor may become of greater importance than the use of classical CB agonists. This is mainly due to the time- and site-specificity of its action, which allows for finer tuning of the levels of endogenous cannabinoid agonists with concomitant potent anti-inflammatory effect. Moreover, it was shown that FAAH inhibitors do not substitute for THC in the drug discrimination paradigm, do not produce hypothermia,
hypomotility, catalepsy, or memory impairment.32 Several lines of evidence suggest that PF-3845 is a good template for the development of future anti-IBD therapies based on selective inhibitors of FAAH, which plays an important role in biolipid turnover. In our study we showed that in control (no TNBS) animals there was a significant decrease of 2-AG and 2-OG after PF-3845 administration. However, the levels of 2-AG and 2-OG in experimental colitis were similar or even higher than those in colitis after treatment with PF-3845. Of interest, the action of PF-3845 did not influence the levels of N-acyl ethanolamines including AEA. These observations suggest that FAAH is one of the major enzymes involved in the development of colitis and indicate protective and restorative actions of FAAH inhibitors, which is in good agreement with the data reported previously by D'Argenio et al., Storr et al. and Massa et al.15–17 Furthermore, our data suggests that the role of monoacylglycerol lipase (MAGL) in the degradation of 2-AG and 2-OG during colitis becomes of less importance in favor of FAAH, which takes over its role in this process. In our study, PF-3845 significantly improved macro- and microscopic damage scores, thus demonstrating excellent anti-inflammatory potential in various routes of administration, including oral an intracolonic. It is very likely that
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
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Figure 3 Microscopic total damage score and representative micrographs of hematoxylin and eosin-stained sections of distal colon from (A) control, (B) TNBS, (C) TNBS + PF-3845 (10 mg/kg, p.o., twice daily)-treated mice, (D) TNBS + PF3845 + AM 251, (E) TNBS + PF3845 + AM 630. Scale bar = 100 μm. &&&P b 0.001, as compared with control mice, **P b 0.01 and ***P b 0.001, as compared to TNBS-treated mice. ###P b 0.001, vs. PF-3845-treated animals. Data represent mean ± SEM of 6–8 mice per group.
PF-3845 simultaneously, indirectly interacts with both neuronal and immune systems via endogenous biolipids, which may affect neurons abundant in CB1 receptors, and also stimulate receptors localized on immune cells, such as mast cells and neutrophils.33 Interestingly, the PF-3845-mediated improvement of colitis was not entirely consistent with measurements of MPO activity in the mouse colon specimens, which may suggest the principal role of AEA in the anti-inflammatory action of PF-3845. Several studies have shown that AEA does
not affect neutrophil influx and/or accumulation in the inflamed tissue and therefore MPO activity did not significantly decrease after PF-3845 treatment.33 It needs to be underlined that changes in MPO activity after treatment with PF-3845 depend on the route of administration of the FAAH inhibitor. The i.p. injection of PF-3845 produced an increase in MPO activity, which is in opposition to both, p.o. and i.c. administration. This may be due to the fact that after p.o. and i.c. administration the compound could act directly on
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
Inhibition of FAAH in experimental colitis
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Figure 4 PF-3845 (10 mg/kg, p.o.) injected twice daily over 7 days did not attenuate the DSS-induced colitis. Figure shows data for macroscopic score (A), MPO activity (B), colon weight (C), and colon length (D). ***P b 0.001, as compared to control mice. Data represent mean ± SEM of 6–8 mice per group.
immune cells located in the gut tissues, as well as on the nerve endings of the ENS, which may be somehow hindered after i.p. administration. Of interest, the administration of PF-3845 caused a significant decrease in the level of PGE2 in colonic samples from mice with colitis, which was contrary to the effect observed in control (no TNBS) animals. These data provide new insight into the mechanism of action of PF-3845, which may involve an indirect interaction with cyclooxygenase (COX)-dependent pathways. It is not clear how PF-3845 blocked the action of COX; we suggest that this may be due to the decrease of intracellular supply of arachidonic acid (AA), which is the substrate for COX. AA is one of the products of FAAH and MAGL activity and thus the blockade of FAAH and the putative decreased activity of MAGL in the inflamed colon discussed earlier result in the local reduction of AA in cells and impaired action of COX. Another hypothesis concerns the issue that 2-AG is a good substrate for COX, thus it can compete with other compounds for precedence in the active site of this enzyme. Because PF-3845 elevates level of 2-AG, this competition is becoming stronger, which results in the impaired production of prostaglandins.34
This indirect interaction with COX-dependent pathways further explains the anti-inflammatory action of PF-3845 in the colon. Recently, it has been shown by Dey et al. that PGE2 and other agonists of prostaglandin EP2 receptors stimulate production of IL-8 in the colonic cells. Decrease in the level of PGE2 after the treatment with PF-3845 contributes to the reduced secretion of IL-8, which is a potent chemotactic agent for immune cells and can trigger an acute host inflammatory response.35 The anti-inflammatory action of PF-3845 was additionally assessed in the DSS model of colitis. In contrast to the TNBS-induced model, we found that the selective FAAH blocker has no anti-inflammatory potential. We may postulate that this is closely associated with the difference between the mechanisms underlying the pathogenesis of DSS- and TNBSinduced colitis at the molecular and cellular level. The inflammatory pathways activated in DSS and TNBS models of colitis are related, but not identical (for review see Ref 36). The main difference concerns the profile of pro-inflammatory cytokines secreted by immune cells in each model. DSS elicits the secretion of large amounts of TNF-α and IL-4, -5, and -6, which are mainly responsible for
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
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Figure 5 Effect of PF-3845 on biolipid levels in control and TNBS-treated mice colon specimens. Figure shows changes in levels of A) 2-AG, B) 2-OG, C) PGE2, and D) biolipids containing glycine. *P b 0.05, **P b 0.01, and ***P b 0.001 vs. control animals. #P b 0.05, vs. TNBS-treated animals. Data represent mean ± SEM of 6–8 colon samples per group.
the tissue damage. In TNBS model, the role of TNF-α is limited to the stimulation of IL-12 and IL-18 secretion by T cells.36 Furthermore, although the resulting inflammation is almost always channeled into a final common pathway of inflammation, mediated by either an excessive Th1 or Th2 response, there are significant, model-based differences. In the TNBS model, the immunological response is a result of reaction to a specific antigen (a hapten) and it is mainly
Figure 6 Determination of FAAH mRNA levels in colon specimens from patients with diagnosed UC and CD vs. healthy subjects. There is an observable difference between control and UC as well as CD groups, but it did not reach statistical significance.
mediated by Th1 cells, whereas DSS leads to the activation of lymphocytes and the induction of Th1 and/or Th2 responses. Moreover in DSS–induced colitis a T cell–mediated inflammation can be superimposed on macrophage-induced inflammation.36 We postulate that these differences in mechanisms of pathogenesis underlie the lack of anti-inflammatory action of PF-3845 in the DSS colitis and are not affected by the changes in biolipid metabolism caused by the FAAH inhibition. Finally, we found that the expression of FAAH at the mRNA level in the human colon is decreased in UC and CD patients. Previously, Marquez et al.37 observed no significant difference in FAAH protein levels in the colon between UC patients and healthy subjects. However, it was also demonstrated that the FAAH protein level is strongly associated with severity of the disease expressed by clinical score and the samples from patients with moderate clinical score contained significantly higher amount of FAAH compared to controls, while in patients with severe clinical score the levels of FAAH were lower compared to healthy subjects.37 In our study, almost 20% of the patients with diagnosed UC were characterized by severe clinical score (supporting information), what could be revealed by our final outcome. It is also possible that there are other, yet undiscovered, regulating mechanisms, which affect translation of FAAH mRNA and thus alter the outcomes of FAAH expression at mRNA and protein levels. Interestingly, Storr et al. have shown that expression of FAAH mRNA fluctuates during different stages of colonic
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
Inhibition of FAAH in experimental colitis inflammation in the murine TNBS model of intestinal inflammation.16 FAAH mRNA expression was reduced in the early stage of colonic inflammation (1 day after TNBS instillation), suggesting that FAAH is suppressed to protect against inflammation. However, there is a significant tendency for recovery in FAAH mRNA levels during the progression of the disease and when the damage is maximal (3 days after TNBS), FAAH mRNA was increased in the colon samples. This supports the hypothesis that FAAH levels alter during the course of colonic inflammation and contribute to the restorative or protective properties of this enzyme in colitis.16 In conclusion, in the present study we showed that PF-3845 exerts potent anti-inflammatory effect in the mouse model of TNBS-induced colitis, which is principally mediated by the CB1 receptors. Additionally, PF-3845 strongly, multidirectionally affects biolipid metabolism in the colon, in particular during colonic inflammation, what underlies its anti-inflammatory potential. Our observations promote a novel strategy in IBD treatment, based on the regulation of biolipid turnover. The novel therapy may be particularly appealing for the group of patients, in case of which other therapies did not provide sufficient relief.
Conflict of interest The authors declare no conflict of interest.
Author contributions Study concept and design: JF, MStorr. Acquisition and analysis of data, statistical analysis: MSałaga, AM, PKZ, AC, EL, MSobczak, HZ, RK, JF. Drafting of the manuscript: MSałaga, JF, WMK, MStorr. Obtained funding: JF, MSałaga, EMP, MStorr, WMK, HBB. Study supervision and final approval of the version to be submitted: JF.
Acknowledgments Supported by the Iuventus Plus Program of the Polish Ministry of Science and Higher Education (0119/IP1/2011/71 and 0107/IP1/2013/72 to JF), the Medical University of Lodz (502-03/1-156-02/502-14-140 to MSałaga), the Deutsche Forschungsgemeinschaft (STO 645/6-1 to MS) and NIH DA032150 (to HBB).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.crohns.2014.01.025.
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31. Capasso R, Borrelli F, Aviello G, Romano B, Scalisi C, Capasso F, et al. Cannabidiol, extracted from Cannabis sativa, selectively inhibits inflammatory hypermotility in mice. Br J Pharmacol 2008;154:1001–8. 32. Ahn K, McKinney MK, Cravatt BF. Enzymatic pathways that regulate endocannabinoid signaling in the nervous system. Chem Rev 2008;108:1687–707. 33. Farquhar-Smith WP, Rice AS. A novel neuroimmune mechanism in cannabinoid-mediated attenuation of nerve growth factor-induced hyperalgesia. Anesthesiology 2003;99:1391–401. 34. Ligresti A, Bisogno T, Matias I, De Petrocellis L, Cascio MG, Cosenza V, et al. Possible endocannabinoid control of colorectal cancer growth. Gastroenterology 2003;125:677–87. 35. Dey I, Chadee K. Prostaglandin E2 produced by Entamoeba histolytica binds to EP4 receptors and stimulates interleukin-8 production in human colonic cells. Infect Immun 2008;76:5158–63. 36. Strober W, Fuss IJ, Blumberg RS. The immunology of mucosal models of inflammation. Annu Rev Immunol 2002;20:495–549. 37. Marquez L, Suarez J, Iglesias M, Bermudez-Silva FJ, Rodriguez de Fonseca F, Andreu M. Ulcerative colitis induces changes on the expression of the endocannabinoid system in the human colonic tissue. PLoS One 2009;4:e6893.
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Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition of fatty acid amide hydrolase (FAAH) M. Sałaga a , A. Mokrowiecka b , P.K. Zakrzewski c , A. Cygankiewicz c , E. Leishman d , M. Sobczak a , H. Zatorski a , E. Małecka-Panas b , R. Kordek e , M. Storr f , W.M. Krajewska c , H.B. Bradshaw d , J. Fichna a,⁎ a
Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Lodz, Poland Department of Digestive Tract Diseases, Faculty of Medicine, Medical University of Lodz, Lodz, Poland c Department of Cytobiochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland d Department of Psychological and Brain Sciences, Gill Center for Biomolecular Science, Indiana University, Bloomington, IN, USA e Department of Pathology, Faculty of Medicine, Medical University of Lodz, Lodz, Poland f Department of Medicine, Division of Gastroenterology, Ludwig Maximilians University of Munich, Munich, Germany b
Received 12 November 2013; received in revised form 29 January 2014; accepted 29 January 2014 KEYWORDS Fatty acid amide hydrolase; Intestinal inflammation; Inflammatory bowel diseases; PF-3845; Experimental colitis
Abstract Background and aims: Pharmacological treatment and/or maintenance of remission in inflammatory bowel diseases (IBD) is currently one of the biggest challenge in the field of gastroenterology. Available therapies are mostly limited to overcoming the symptoms, but not the cause of the disease. Recently, the endocannabinoid system has been proposed as a novel target in the treatment of IBD. Here we aimed to assess the anti-inflammatory action of the novel fatty acid amide hydrolase (FAAH) inhibitor PF-3845 and its effect on the endocannabinoid and related lipid metabolism during the course of experimental colitis. Methods: We used two models of experimental colitis in mice (TNBS- and DSS-induced) and additionally, we employed LC/MS/MS spectrometry to determine the changes in biolipid levels in the mouse colon during inflammation.
Abbreviations: 2-AG, 2-arachidonyloglicerol; 2-OG, 2-oleoylglycerol; AA, arachidonic acid; AEA, anandamide; COX, cyclooxygenase; DMSO, dimethyl sulfoxide; DSS, dextran sulfate sodium; FAAH, fatty acid amide hydrolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HTAB, hexadecyltrimethylammonium bromide; i.c., intracolonic; i.p., intraperitoneal; MAGL, monoacylglycerol lipase; MO, allyl isothiocyanate; MPO, myeloperoxidase; MRM, multiple reaction monitoring; p.o., per os; PBS, phosphate buffered saline; PGE2, prostaglandin E2; TNBS, 2,4,6-trinitrobenzene sulfonic acid. ⁎ Corresponding author at: Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland. Tel.: + 48 42 272 57 07; fax: + 48 42 272 56 94. E-mail address: [email protected] (J. Fichna). 1873-9946/$ - see front matter © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.crohns.2014.01.025 Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
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M. Sałaga et al. Results: We showed that the FAAH inhibitor PF-3845 reduced experimental TNBS-induced colitis in mice and its anti-inflammatory action is associated with altering the levels of selected biolipids (arachidonic and oleic acid derivatives, prostaglandins and biolipids containing glycine in the mouse colon). Conclusions: We show that FAAH is a promising pharmacological target and the FAAH-dependent biolipids play a major role in colitis. Our results highlight and promote therapeutic strategy based on targeting FAAH-dependent metabolic pathways in order to alleviate intestinal inflammation. © 2014 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.
1. Introduction Ulcerative colitis (UC) and Crohn's disease (CD), which belong to the group of inflammatory bowel diseases (IBD), are chronic, progressive and relapsing inflammatory disorders that may cause disability over time (for review see Ref. 1). Both disorders are characterized by an excessive activation of common inflammatory pathways, which results in an enhanced secretion of pro-inflammatory cytokines, such as interleukins (IL-1α, -1β, -2, -6, -8, -12, and -17), TNF or INF, and an imbalance in the levels of pro- and anti-inflammatory factors in the tissue.2 There is also growing evidence that the neuronal system is significantly engaged in the pathophysiology of colitis, including signaling in the intrinsic enteric nervous system (ENS) and from ENS to the central nervous system (CNS).1,3 Although the impaired immune response and disrupted neural signaling could be the causes of IBD, the etiology of this disorder still remains unclear. The natural plant-derived cannabinoids have been known for centuries, but only relatively recently the endogenous cannabinoids, namely anandamide (AEA) and 2-arachidonyloglicerol (2-AG), have been discovered in the mammalian tissues.4,5 Endogenous and plant-derived cannabinoids share similar biological properties, which include antinociception and the regulation of GI motility, secretion, proliferation, and immune functions.6,7 The principal sites of action for cannabinoids are the “classical” CB receptors, i.e. CB1 and CB2. CB1 receptors are located primarily presynaptically on the surface of nerve cells of the central and the peripheral nervous system, and also in the peripheral tissues like cardiovascular tissue, urinary bladder and gastrointestinal (GI) tract (small intestine, spleen).8,9 CB2 receptors are located mainly on cells of the immune system (e.g. macrophages, neutrophils), but were also detected in the microglial cells and neurons of the CNS.8,9 Both types of receptors are coupled with the Gi-protein, which is responsible for the inhibition of adenylate cyclase activity; it was also shown that cannabinoids modulate the activity of protein kinases.10 The action of endocannabinoids in the living organism is strongly regulated by their metabolism; for example, the half-life of AEA can be measured in the order of minutes.11 Fatty acid amide hydrolase (FAAH) is primarily responsible for the intracellular degradation of AEA into arachidonic acid (AA) and ethanol amine.12 In addition, FAAH has been identified as the key enzyme in the metabolism of N-arachidonoyl glycine as well as its family of endogenous structural analogs.13,14 FAAH possesses a number of channels and cavities involved in substrate or inhibitor binding and by using specific FAAH inhibitors the level of the endogenous AEA and potentially other lipids can be
elevated and thus its action in the organism prolonged and intensified. Since the blockade of FAAH produces effects similar to those that can be achieved by the CB receptor agonists, it has been suggested as an attractive pharmacological strategy for the treatment of IBD in humans, possibly without the side effects typical for cannabinoids. The use of specific FAAH inhibitors to improve colitis in animal models has recently been reported.15,16 Moreover, it was observed that FAAH-deficient mice are more resistant to experimental DNBS-induced colitis than the wild type animals.17 The increased level of AEA in the colonic tissue of IBD patients, translated well the key findings from animal tissues to humans.15 Evaluating novel drugs for the treatment of IBD, we characterized the anti-inflammatory action of the selective FAAH inhibitor PF-3845 in two mouse models of intestinal inflammation. In particular, we examined the effects of PF-3845 employing different routes of administration and different dosing regimens. Moreover, we characterized the mechanism of action of PF-3845 by using specific CB1 and CB2 receptors antagonists. Since visceral pain is one of the most common symptoms in IBD, we also investigated the antinociceptive effect of PF-3845 in TNBS-treated animals. Finally, we quantified the levels of endocannabinoid and metabolically-related biolipids in the course of colitis to assess the biomolecular pathways responsible for the antiinflammatory action of the FAAH blocker.
2. Materials and methods 2.1. Animals Experimentally naive male C57B1/6 mice were obtained from the Animal House at the University of Lodz, Poland. All animals used in the experiments weighed 25–30 g. Mice were housed at a constant temperature (22 °C) and maintained under a 12-hour light/dark cycle 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 University of Lodz Animal Care Committee (Protocol #670/ 2013), following the guidelines of the Polish Council of Animal Care. All efforts were made to minimize animal suffering and to reduce the number of animals used. Groups of 6–8 animals were used in all in vivo experiments.
2.2. Induction of colitis 2.2.1. TNBS model Colitis was induced by intracolonic (i.c.) administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS), as described
Please cite this article as: Sałaga M, et al, Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition..., J Crohns Colitis (2014), http://dx.doi.org/10.1016/j.crohns.2014.01.025
Inhibition of FAAH in experimental colitis previously.18 Briefly, mice were lightly anesthetized with 1% isoflurane (Baxter Healthcare Corp., IL, USA) and TNBS (4 mg in 0.1 mL of 30% ethanol in saline) was administered into the colon through a catheter inserted to the anus (3 cm proximally). Control animals received vehicle alone (0.1 mL of 30% ethanol in saline). Preliminary experiments demonstrated that the dose of TNBS used in this study induced reproducible colitis. 2.2.2. DSS model Colitis was introduced by the addition of DSS to drinking water starting from day 0 to day 5 (4% wt/vol; molecular weight 40,000; MP Biomedicals, Aurora, OH, Lot No. 5237K). On days 6 and 7 the animals received water without DSS. Control animals received tap water. Animal body weight was monitored daily and mean water and food consumption was recorded.
2.3. Pharmacological treatments The selective FAAH inhibitor PF-3845 was administered once daily at the dose of 10 mg/kg (intraperitoneally, i.p., and i.c.) in TNBS and DSS models, and 10 mg/kg twice daily (orally, p.o.), in TNBS model, with the first treatment 30 min before the induction of colitis. Selective CB1 (AM 251, 1 mg/kg, i.p.), and CB2 (AM 630, 1 mg/kg, i.p.) receptor antagonists were administered 10 min before PF-3845. All drugs were dissolved in 5% dimethyl sulfoxide (DMSO) in saline, which was used as vehicle. Control animals received vehicle (100 μL i.p., 100 μL i.c. or 150 μL p.o.) alone. Neither the antagonists, nor vehicle influenced the observed parameters when given alone.
2.4. Evaluation of colonic damage 2.4.1. TNBS model Animals were sacrificed by cervical dislocation 3 days after TNBS infusion. The colon was rapidly removed, opened longitudinally, rinsed with phosphate buffered saline (PBS), and immediately examined. Macroscopic colonic damage was assessed by an established semiquantitative scoring system by adding individual scores for ulcer, colonic shortening, wall thickness, and presence of hemorrhage, fecal blood, and diarrhea, as described previously.18 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 N 15%, 2 points for N 25% (based on a mean length of the colon in untreated mice of 8.07 ± 0.20 cm, n = 6). The wall thickness was measured in mm. The presence of hemorrhage, fecal blood, or diarrhea increased the score by 1 point for each additional feature. 2.4.2. DSS model Mice were sacrificed by cervical dislocation on day 7 following addition of DSS to the drinking water. The entire colon was immediately isolated and weighed with fecal content. Colon was then opened along the mesenteric border and fecal material removed. A total macroscopic damage score was calculated for each animal consisting of stool consistence (0–3), colon epithelial damage considered as number of ulcers (0–3), colon length and weight scores (0–4), where score = 0 means no inflammation.18 The presence (score = 1) or absence (score = 0) of fecal blood was also recorded.
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2.5. Determination of tissue myeloperoxidase activity The method described by Fichna and collaborators18 was used to assess granulocyte infiltration and to quantify the myeloperoxidase (MPO) activity. Briefly, 1-cm segments of colon were weighed and homogenized in hexadecyltrimethylammonium bromide (HTAB) buffer (0.5% HTAB in 50 mM potassium phosphate buffer, pH 6.0; 50 mg of tissue/mL) immediately after isolation and the homogenate was centrifuged (15 min, 13,200 ×g, 4 °C). On a 96-well plate, 200 μL of 50 mM potassium phosphate buffer (pH 6.0), containing 0.167 mg/mL of O-dianisidine hydrochloride and 0.05 μL of 1% hydrogen peroxide was added to 7 μL of supernatant. Absorbance was measured at 450 nm (iMARK Microplate Reader, Biorad, United Kingdom). 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 of hydrogen peroxide to water in 1 min at room temperature. Units of MPO activity per 1 min were calculated from a standard curve using purified peroxidase enzyme.
2.6. 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 h at 4 °C. Samples were then dehydrated, embedded in paraffin, sectioned at 5 μm and mounted onto slides. Subsequently sections were stained with hematoxylin and eosin and examined using (Motic AE31 microscope, Ted Pella, Sweden). Photographs were taken using a digital imaging system consisting of a digital camera (Moticam 2300, Ted Pella, Sweden) and image analysis software (Motic Images Plus 2.0, Germany). A microscopic total damage score was determined 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).
2.7. Mustard oil model of visceral pain Behavioral responses to i.c. administration of mustard oil (MO, allyl isothiocyanate) were assessed as described previously.19 For the determination of pain behaviors, 50 μL of MO (1% in 70% ethanol) was injected i.c. under isoflurane anesthesia. To avoid stimulation of somatic nociceptors, perianal area was covered with Vaseline. After 5 min of recovery, spontaneous behaviors were recorded on a videotape for 20 min for later analysis by an observer blinded for experimental conditions. Postures defined as pain-related behaviors: 1) licking of the abdomen, 2) stretching the abdomen, 3) squashing of lower abdomen against the floor, and 4) abdominal retractions, were each counted as 1. PF-3845 was administered p.o. and i.c. 20 and 15 min before the MO instillation, respectively. All drugs were dissolved in 5% DMSO in saline, which was used as vehicle in control experiments.
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2.8. Determination of endocannabinoid levels in mouse tissue 2.8.1. Sample isolation and preparation Upon isolation, the colon samples were frozen at −80 °C and kept until further processed. For endocannabinoid level determination, they were removed from the freezer, shocked with liquid nitrogen, weighed and placed in centrifuge tubes on ice. Forty one volumes of methanol were added to each tube followed by 10 μL of 100 pM N-arachidonoyl glycine-d8, which was added to act as an internal standard to determine the recovery of the compounds of interest. The tubes were then covered with parafilm and left on ice and in darkness for approximately 2 h. The samples were then homogenized using a Polytron for approximately 1 min on each sample. The samples were then centrifuged at 19,000 ×g at 24 °C for 20 min. The supernatants were collected and placed in polypropylene tubes (15 or 50 mL) and HPLC-grade water was added making the final supernatant/water solution 25% organic. To isolate the compounds of interest partial purification of the 25% solution was performed on a Preppy apparatus assembled with 500 mg C18 solid-phase extraction columns. The columns were conditioned with 5 mL of HPLC-grade methanol immediately followed by 2.5 mL of HPLC-grade water. The supernatant/water solution was then loaded onto the C18 column, and then washed with 2.5 mL of HPLC grade water followed by 1.5 mL of 40% methanol. Elutions of 1.5 mL of 60, 75%, 85%, and 100% methanol were collected in individual autosampler vials and then stored in a − 80 °C freezer until mass spectrometer analysis.
2.8.2. LC/MS/MS analysis and quantification Samples were removed from the − 80 °C freezer and allowed to warm at room temperature then vortexed for approximately 1 min before being placed into the autosampler and held at 24 °C (Agilent 1100 series autosampler, Palo Alto, CA, USA) for LC/MS/MS analysis. Ten–twenty microliters of eluants were injected separately for each sample to be rapidly separated using a C18 Zorbax reversed-phase analytical column to scan for individual compounds. Gradient elution (200 μL/min) then occurred, under the pressure created by two Shimadzu 10AdVP pumps (Columbia, MD, USA). Next, electrospray ionization was accomplished using an Applied Biosystems/MDS Sciex (Foster City, CA, USA) API3000 triple quadrupole mass spectrometer. A multiple reaction monitoring (MRM) setting on the LC/MS/MS was then used to analyze levels of each compound present in the sample injection. Synthetic standards were used to generate optimized MRM methods and standard curves for analysis.
2.8.3. Data analysis The amount of analyte in each sample was calculated by using a combination of calibration curves of the synthetic standards obtained from the Analyst software and deuterium-labeled internal standards. The standards provided a reference for the retention times by which the analytes could be compared. They also helped to identify the specific precursor ion and fragment ion for each analyte which enabled their isolation. These processes provide confidence in the claim that the compounds measures were, in fact, the compound of interest. The amount
of each compound in each tissue was then converted to moles per gram tissue, which is how it was statistically analyzed.
2.9. Study population for the evaluation of human FAAH mRNA levels To quantify the relative expression of human FAAH, forceps biopsy samples were analyzed. In total 44 specimens prepared from human colon biopsies were used for the study. The study population comprised 15 patients with Crohn's disease (CD, ages 20–68), 15 patients with ulcerative colitis (UC, ages 24–59) and 14 healthy, unrelated controls (ages 27–81) recruited from January 2011 to January 2012 (for more detailed information see supplementary file, Table 1). The specimens were frozen shortly after isolation and kept at −70 °C until further analysis. The diagnosis of CD and UC in patients was assessed accordingly 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 genetic analysis.
2.10. Determination of FAAH mRNA levels The RNA was isolated according to manufacturer's protocol using the PureLink RNA Mini kit (Life Technologies, Carlsbad, CA, USA) as described before.20 Briefly, tissue samples were homogenized in lysis buffer (600 μL), complemented with 1% 2-mercaptoethanol (St. Louis, MO, USA). Subsequently, the homogenates were centrifuged to clear the solution. Supernatants were washed and finally the purified total RNA samples were eluted into collection tubes using diethyl pyrocarbonate (DEPC) treated water (50 μL). To measure the purity and quantity of isolated RNA, dedicated spectrophotometer (BioPhotometer; Eppendorf, Germany) was used. Total RNA (1 μg) was transcribed onto cDNA with First Strand cDNA synthesis kit (Fermentas, Canada). Quantitative analysis was performed using fluorescently labeled TaqMan probe Hs00155015_m1 for human FAAH and Hs01003267_m1 for mouse hypoxanthineguanine phosphoribosyltransferase (HPRT, endogenous control, Life Technologies, Carlsbad, CA, USA) on Mastercycler S realplex 4 apparatus (Eppendorf, Germany). All experiments were performed in triplicate. The Ct (threshold cycle) values for studied genes were normalized to Ct values obtained for a housekeeping gene HPRT. Relative amount of mRNA copies was calculated using following equation: 2−ΔCt × 1000. There was no alteration in the levels of mRNA of HPRT in samples from all patient groups regardless of the studied group.
2.11. Statistics Statistical analysis was performed using Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). The data are expressed as means ± SEM. Student t-test or one-way ANOVA followed by Newman–Keuls post-hoc test were used for analysis. P values b 0.05 were considered statistically significant.
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2.12. Drugs All drugs and reagents, unless otherwise stated, were purchased from Sigma-Aldrich (Poznan, Poland). DSS (MW 40,000) was purchased from MP Biomedicals (Solon, OH, USA). N-3-pyridinyl4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]1-piperidine-carboxamide (PF-3845) was prepared by TriMen Chemicals (Lodz, Poland). N-(Piperidin-1-yl)-5-(4-iodophenyl)1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and [6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]1H-indol-3-yl](4-methoxyphenyl) methanone (AM630) were purchased from Tocris Bioscience (Bristol, UK).
3. Results 3.1. PF-3845 has anti-inflammatory effect in TNBS-induced colitis in mice The i.c. instillation of TNBS resulted in a reproducible colitis in mice, manifested by increased macro- and microscopic colon damage scores and elevated MPO activity. PF-3845 significantly attenuated TNBS-colitis in various routes of administration (i.p. — Fig. 1A; p.o. — Fig. 1B; i.c. — Fig. 1C), as demonstrated by decreased total macroscopic score (4.68 ± 0.36 for i.p. administration of PF-3845 at the dose of 10 mg/kg once daily vs. 7.35 ± 0.52 for TNBS-treated mice; 3.39 ± 0.33 for p.o. administration of PF-3845 at the dose of 10 mg/kg twice daily vs. 5.61 ± 0.64 for TNBS-treated mice; 3.49 ± 0.34 for i.c. administration of PF-3845 at the dose of 10 mg/kg once daily vs. 7.35 ± 0.52 for TNBS-treated mice). The MPO activity after i.p. injection of PF-3845 (10 mg/ kg, once daily) was non-significantly elevated compared to the TNBS-treated group (Fig. 1A). The p.o. administration of PF-3845 at the dose of 10 mg/kg (twice daily) caused a substantial reduction of the MPO activity compared to the TNBS-treated group, but the difference did not reach a statistical significance (Fig. 1B). Similarly, the i.c. injection of the FAAH blocker (10 mg/kg, once daily) non-significantly decreased the MPO activity (Fig. 1C). The oral gavage was used as the principal route of administration in subsequent experiments. Specific CB1 and CB2 receptor antagonists (AM251 and AM630, respectively) were used to characterize the mechanism of action by which PF-3845 attenuates inflammation in the TNBS model. As shown in Fig. 2, the anti-inflammatory effect of PF-3845 at the dose of 10 mg/kg (p.o.; twice daily) was blocked by the CB1 receptor antagonist AM251 (1 mg/kg i.p.), as demonstrated by the increase in the macroscopic score (Fig. 2A), ulcer score (Fig. 2C) and shortening of colon (Fig. 2D). AM251 did not influence the MPO activity in the inflamed tissue (Fig. 2B). The microscopic evaluation of colon sections stained with hematoxylin/eosin was in good agreement with observation of the macroscopic parameters (Fig. 3). Histological analysis of sections of distal colon from untreated animals showed intact epithelium, absence of edema, and normal muscle architecture (Fig. 3A). Severe microscopic damage, characterized by loss of mucosal architecture, thickening of smooth muscle, presence of crypt abscesses, and extensive cellular infiltrate was observed in colon specimens 3 days post-TNBS (Fig. 3B). The histological changes were normalized after p.o. PF-3845
5 treatment (Fig. 3C). The protective effect of PF-3845 was reversed by administration of CB1 antagonist AM251, but not CB2 antagonist AM630.
3.2. PF-3845 administered orally has no effect on DSS-induced colitis in mice In order to extend our observations on the anti-inflammatory action of PF-3845, a DSS-induced mouse model of UC was used. Animals treated with 4% DSS in drinking water developed severe colonic injury, which was not reversed by p.o. administration of PF-3845 (10 mg/kg, twice daily, Fig. 4). Treatment with PF-3845 did not reduce any of evaluated parameters: macroscopic colon damage score (Fig. 4A), MPO activity (Fig. 4B), colon weight (Fig. 4C) or colon length (Fig. 4D).
3.3. PF-3845 administered orally has no antinociceptive effect in TNBS-treated animals To evaluate the antinociceptive activity of PF-3845 in inflammatory conditions, we used a MO-induced model of abdominal pain. The i.c. instillation of MO in TNBStreated mice resulted in a significant increase in number of pain-related behaviors compared to control animals (Fig. S1). Neither p.o., nor i.c. administration of PF-3845 at the dose of 10 mg/kg 20 min before the instillation of MO produced a statistically significant antinociceptive effect (Fig. S1).
3.4. PF-3845 alters the levels of endocannabinoids in TNBS-induced colitis For further characterization of possible mechanisms of the anti-inflammatory action of PF-3845, changes in the levels of endocannabinoids and their metabolites in colonic tissues were characterized in naive (“healthy”) and TNBStreated animals. In naïve animals, the levels of 2-AG and 2-oleoylglycerol (2-OG) were significantly lower after treatment with PF-3845 (10 mg/kg, p.o., Fig. 5A and B, respectively). However, in the TNBS-treated mice, 2-AG and 2-OG levels were not significantly altered after treatment with PF-3845 compared to the animals receiving vehicle. As shown in Fig. 5C, the level of endogenous prostaglandin E2 (PGE2) was significantly elevated after treatment with PF-3845 in control animals, but it was significantly decreased in TNBS-treated mice. Quantification of the levels of biolipids containing glycine showed that O-Gly, L-Gly, A-Gly, and D-Gly were significantly lower in naive animals after the treatment with PF-3845, but not in TNBS-treated mice (Fig. 5D). Interestingly, there were no significant changes in the levels of N-acyl ethanolamines in both naive and TNBS-treated animals after treatment with PF-3845 (Fig. S2).
3.5. Expression of FAAH mRNA in the human colon is decreased during UC and CD To translate our observations from animals to human, we examined the FAAH mRNA expression levels in colon
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Figure 1 PF-3845 (10 mg/kg) injected twice daily over 3 days in various routes of administration attenuated TNBS-induced colitis in mice. Figure shows data for macroscopic scores and MPO activity after (A) i.p., (B) p.o. and (C) i.c. administration. *P b 0.05, **P b 0.01, and ***P b 0.001, as compared to control mice. &&P b 0.01 and &&&P b 0.001, vs. TNBS-treated animals. Data represent mean ± SEM of 6–8 mice per group.
specimens from patients with UC, CD and healthy controls. We found that the relative expression of FAAH mRNA is decreased in patients with UC and CD compared with healthy subjects; however, the difference did not reach statistical significance (Fig. 6). Moreover, the expression was lower in patients with UC than CD but the difference was not statistically significant.
4. Discussion In the present study we showed that the highly potent and selective FAAH inhibitor PF-3845 displayed anti-inflammatory activity in TNBS-induced mouse model of experimental colitis and this effect was mediated by CB1 receptors. We also showed that PF-3845 was effective after i.p., p.o., and i.c.
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Figure 2 The anti-inflammatory effect of PF-3845 (10 mg/kg, p.o.) injected twice daily over 3 days in TNBS-treated mice was reversed by the CB1 antagonist AM 251 (1 mg/kg, i.p.), but not the CB2 antagonist AM 630 (1 mg/kg, i.p.). Figure shows data for macroscopic score (A), MPO activity (B), ulcer score (C), and colon length (D). &P b 0.05, &&P b 0.01, and &&&P b 0.001, as compared to control mice, *P b 0.05 and **P b 0.01, as compared to TNBS-treated mice. #P b 0.05 and ##P b 0.01, vs. PF-3845-treated animals. Data represent mean ± SEM of 6–8 mice per group.
administration, a finding of high clinical relevance. The significant anti-inflammatory action of PF-3845 suggests a possible therapeutic use of FAAH inhibitors in the treatment of IBD. Previous studies demonstrating colitis-associated changes in CB receptor expression in animals and humans indicate that the endogenous cannabinoid system plays an important role in the course of intestinal inflammation.15,17,21–23 However, neither natural, nor synthetic CB receptor ligands have entered clinical trials in IBD, which is due to the vast spectrum of adverse effects caused by these compounds, ranging from increase in body temperature, seizures and hallucinogenic effects or physical dependence.24–27 Of note, a natural FAAH inhibitor derived from Cannabis sativa, cannabidiol, has been shown to reduce inflammation-induced intestinal hypermotility and to ameliorate murine colitis induced by TNBS.28–31 Our findings point out that elevating the level of endogenous cannabinoids by application of a selective FAAH inhibitor may become of greater importance than the use of classical CB agonists. This is mainly due to the time- and site-specificity of its action, which allows for finer tuning of the levels of endogenous cannabinoid agonists with concomitant potent anti-inflammatory effect. Moreover, it was shown that FAAH inhibitors do not substitute for THC in the drug discrimination paradigm, do not produce hypothermia,
hypomotility, catalepsy, or memory impairment.32 Several lines of evidence suggest that PF-3845 is a good template for the development of future anti-IBD therapies based on selective inhibitors of FAAH, which plays an important role in biolipid turnover. In our study we showed that in control (no TNBS) animals there was a significant decrease of 2-AG and 2-OG after PF-3845 administration. However, the levels of 2-AG and 2-OG in experimental colitis were similar or even higher than those in colitis after treatment with PF-3845. Of interest, the action of PF-3845 did not influence the levels of N-acyl ethanolamines including AEA. These observations suggest that FAAH is one of the major enzymes involved in the development of colitis and indicate protective and restorative actions of FAAH inhibitors, which is in good agreement with the data reported previously by D'Argenio et al., Storr et al. and Massa et al.15–17 Furthermore, our data suggests that the role of monoacylglycerol lipase (MAGL) in the degradation of 2-AG and 2-OG during colitis becomes of less importance in favor of FAAH, which takes over its role in this process. In our study, PF-3845 significantly improved macro- and microscopic damage scores, thus demonstrating excellent anti-inflammatory potential in various routes of administration, including oral an intracolonic. It is very likely that
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Figure 3 Microscopic total damage score and representative micrographs of hematoxylin and eosin-stained sections of distal colon from (A) control, (B) TNBS, (C) TNBS + PF-3845 (10 mg/kg, p.o., twice daily)-treated mice, (D) TNBS + PF3845 + AM 251, (E) TNBS + PF3845 + AM 630. Scale bar = 100 μm. &&&P b 0.001, as compared with control mice, **P b 0.01 and ***P b 0.001, as compared to TNBS-treated mice. ###P b 0.001, vs. PF-3845-treated animals. Data represent mean ± SEM of 6–8 mice per group.
PF-3845 simultaneously, indirectly interacts with both neuronal and immune systems via endogenous biolipids, which may affect neurons abundant in CB1 receptors, and also stimulate receptors localized on immune cells, such as mast cells and neutrophils.33 Interestingly, the PF-3845-mediated improvement of colitis was not entirely consistent with measurements of MPO activity in the mouse colon specimens, which may suggest the principal role of AEA in the anti-inflammatory action of PF-3845. Several studies have shown that AEA does
not affect neutrophil influx and/or accumulation in the inflamed tissue and therefore MPO activity did not significantly decrease after PF-3845 treatment.33 It needs to be underlined that changes in MPO activity after treatment with PF-3845 depend on the route of administration of the FAAH inhibitor. The i.p. injection of PF-3845 produced an increase in MPO activity, which is in opposition to both, p.o. and i.c. administration. This may be due to the fact that after p.o. and i.c. administration the compound could act directly on
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Figure 4 PF-3845 (10 mg/kg, p.o.) injected twice daily over 7 days did not attenuate the DSS-induced colitis. Figure shows data for macroscopic score (A), MPO activity (B), colon weight (C), and colon length (D). ***P b 0.001, as compared to control mice. Data represent mean ± SEM of 6–8 mice per group.
immune cells located in the gut tissues, as well as on the nerve endings of the ENS, which may be somehow hindered after i.p. administration. Of interest, the administration of PF-3845 caused a significant decrease in the level of PGE2 in colonic samples from mice with colitis, which was contrary to the effect observed in control (no TNBS) animals. These data provide new insight into the mechanism of action of PF-3845, which may involve an indirect interaction with cyclooxygenase (COX)-dependent pathways. It is not clear how PF-3845 blocked the action of COX; we suggest that this may be due to the decrease of intracellular supply of arachidonic acid (AA), which is the substrate for COX. AA is one of the products of FAAH and MAGL activity and thus the blockade of FAAH and the putative decreased activity of MAGL in the inflamed colon discussed earlier result in the local reduction of AA in cells and impaired action of COX. Another hypothesis concerns the issue that 2-AG is a good substrate for COX, thus it can compete with other compounds for precedence in the active site of this enzyme. Because PF-3845 elevates level of 2-AG, this competition is becoming stronger, which results in the impaired production of prostaglandins.34
This indirect interaction with COX-dependent pathways further explains the anti-inflammatory action of PF-3845 in the colon. Recently, it has been shown by Dey et al. that PGE2 and other agonists of prostaglandin EP2 receptors stimulate production of IL-8 in the colonic cells. Decrease in the level of PGE2 after the treatment with PF-3845 contributes to the reduced secretion of IL-8, which is a potent chemotactic agent for immune cells and can trigger an acute host inflammatory response.35 The anti-inflammatory action of PF-3845 was additionally assessed in the DSS model of colitis. In contrast to the TNBS-induced model, we found that the selective FAAH blocker has no anti-inflammatory potential. We may postulate that this is closely associated with the difference between the mechanisms underlying the pathogenesis of DSS- and TNBSinduced colitis at the molecular and cellular level. The inflammatory pathways activated in DSS and TNBS models of colitis are related, but not identical (for review see Ref 36). The main difference concerns the profile of pro-inflammatory cytokines secreted by immune cells in each model. DSS elicits the secretion of large amounts of TNF-α and IL-4, -5, and -6, which are mainly responsible for
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Figure 5 Effect of PF-3845 on biolipid levels in control and TNBS-treated mice colon specimens. Figure shows changes in levels of A) 2-AG, B) 2-OG, C) PGE2, and D) biolipids containing glycine. *P b 0.05, **P b 0.01, and ***P b 0.001 vs. control animals. #P b 0.05, vs. TNBS-treated animals. Data represent mean ± SEM of 6–8 colon samples per group.
the tissue damage. In TNBS model, the role of TNF-α is limited to the stimulation of IL-12 and IL-18 secretion by T cells.36 Furthermore, although the resulting inflammation is almost always channeled into a final common pathway of inflammation, mediated by either an excessive Th1 or Th2 response, there are significant, model-based differences. In the TNBS model, the immunological response is a result of reaction to a specific antigen (a hapten) and it is mainly
Figure 6 Determination of FAAH mRNA levels in colon specimens from patients with diagnosed UC and CD vs. healthy subjects. There is an observable difference between control and UC as well as CD groups, but it did not reach statistical significance.
mediated by Th1 cells, whereas DSS leads to the activation of lymphocytes and the induction of Th1 and/or Th2 responses. Moreover in DSS–induced colitis a T cell–mediated inflammation can be superimposed on macrophage-induced inflammation.36 We postulate that these differences in mechanisms of pathogenesis underlie the lack of anti-inflammatory action of PF-3845 in the DSS colitis and are not affected by the changes in biolipid metabolism caused by the FAAH inhibition. Finally, we found that the expression of FAAH at the mRNA level in the human colon is decreased in UC and CD patients. Previously, Marquez et al.37 observed no significant difference in FAAH protein levels in the colon between UC patients and healthy subjects. However, it was also demonstrated that the FAAH protein level is strongly associated with severity of the disease expressed by clinical score and the samples from patients with moderate clinical score contained significantly higher amount of FAAH compared to controls, while in patients with severe clinical score the levels of FAAH were lower compared to healthy subjects.37 In our study, almost 20% of the patients with diagnosed UC were characterized by severe clinical score (supporting information), what could be revealed by our final outcome. It is also possible that there are other, yet undiscovered, regulating mechanisms, which affect translation of FAAH mRNA and thus alter the outcomes of FAAH expression at mRNA and protein levels. Interestingly, Storr et al. have shown that expression of FAAH mRNA fluctuates during different stages of colonic
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Inhibition of FAAH in experimental colitis inflammation in the murine TNBS model of intestinal inflammation.16 FAAH mRNA expression was reduced in the early stage of colonic inflammation (1 day after TNBS instillation), suggesting that FAAH is suppressed to protect against inflammation. However, there is a significant tendency for recovery in FAAH mRNA levels during the progression of the disease and when the damage is maximal (3 days after TNBS), FAAH mRNA was increased in the colon samples. This supports the hypothesis that FAAH levels alter during the course of colonic inflammation and contribute to the restorative or protective properties of this enzyme in colitis.16 In conclusion, in the present study we showed that PF-3845 exerts potent anti-inflammatory effect in the mouse model of TNBS-induced colitis, which is principally mediated by the CB1 receptors. Additionally, PF-3845 strongly, multidirectionally affects biolipid metabolism in the colon, in particular during colonic inflammation, what underlies its anti-inflammatory potential. Our observations promote a novel strategy in IBD treatment, based on the regulation of biolipid turnover. The novel therapy may be particularly appealing for the group of patients, in case of which other therapies did not provide sufficient relief.
Conflict of interest The authors declare no conflict of interest.
Author contributions Study concept and design: JF, MStorr. Acquisition and analysis of data, statistical analysis: MSałaga, AM, PKZ, AC, EL, MSobczak, HZ, RK, JF. Drafting of the manuscript: MSałaga, JF, WMK, MStorr. Obtained funding: JF, MSałaga, EMP, MStorr, WMK, HBB. Study supervision and final approval of the version to be submitted: JF.
Acknowledgments Supported by the Iuventus Plus Program of the Polish Ministry of Science and Higher Education (0119/IP1/2011/71 and 0107/IP1/2013/72 to JF), the Medical University of Lodz (502-03/1-156-02/502-14-140 to MSałaga), the Deutsche Forschungsgemeinschaft (STO 645/6-1 to MS) and NIH DA032150 (to HBB).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.crohns.2014.01.025.
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