Peptides 69 (2015) 56–65

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Neuromedin U inhibits food intake partly by inhibiting gastric emptying Louise S. Dalbøge a,b , Søren L. Pedersen a , Thomas Secher a , Birgitte Holst b , Niels Vrang a , Jacob Jelsing a,∗ a b

Gubra ApS, Agern Alle 1, 2970 Hørsholm, Denmark University of Copenhagen, The Novo Nordisk Foundation Center for Basic Metabolic Research, Blegdamsvej 3B, 2200 Copenhagen N, Denmark

a r t i c l e

i n f o

Article history: Received 23 January 2015 Received in revised form 31 March 2015 Accepted 7 April 2015 Available online 18 April 2015 Keywords: Neuromedin U NMUR1 NMUR2 Gastric emptying In situ hybridization Lipidation Tachyphylaxis

a b s t r a c t Neuromedin U (NMU) is a gut-brain peptide, implicated in energy and glucose homeostasis via the peripherally expressed NMU receptor 1 (NMUR1) and the central NMUR2. We investigated the effects of a lipidated NMU analog on gastric emptying (GE), glucose homeostasis and food intake to evaluate the use of a NMU analog as drug candidate for treatment of obesity and diabetes. Finally mRNA expression of NMU and NMUR1 in the gut and NMUR2 in the hypothalamus was investigated using a novel chromogen-based in situ hybridization (ISH) assay. Effects on food intake (6 and 18 h post dosing) were addressed in both mice and rats. The effects on GE and glycaemic control were assessed in mice, immediately after the first dose and after seven days of bidaily (BID) dosing. The lipidated NMU analog exerted robust reductions in GE and food intake in mice and improved glycaemic control when measured immediately after the first dose. No effects were observed after seven days BID. In rats, the analog induced only a minor effect on food intake. NMU mRNA was detected in the enteric nervous system throughout the gut, whereas NMUR1 was confined to the lamina propria. NMUR2 was detected in the paraventricular (PVN) and arcuate nuclei (ARC) in mice, with a reduced expression in ARC in rats. In summary, the anorectic effect of the lipidated NMU is partly mediated by a decrease in gastric emptying which is subject to tachyphylaxis after continuous dosing. Susceptibility to NMU appears to be species specific. © 2015 Elsevier Inc. All rights reserved.

Introduction Neuromedin U (NMU) is a neuropeptide, which is expressed and secreted in both the brain and gut [1–3]. Two G-proteincoupled receptor subtypes for NMU have been identified: the neuromedin U receptor 1 (NMUR1) and neuromedin U receptor 2 (NMUR2) [2,4–7]. NMUR1 is mainly expressed peripherally with highest expression in the gastrointestinal tract [2,4,5,8]. In contrast, NMUR2 is predominantly expressed in the central nervous system where expression is mainly found in the hippocampus, spinal cord and hypothalamus [2,4–7]. The amino acid sequence of NMU is highly conserved across species indicating physiological importance of this peptide [6].

Abbreviations: ARC, arcuate nucleus; AUC, area under the curve; DIO, diet induced obesity; FFPE, formalin fixated paraffin embedded; GE, gastric emptying; GE-OGTT, gastric emptying and oral glucose tolerance test; GLP-1, gastrointestinal (GI) tract, glucagon-like peptide-1; GLP-2, glucagon-like peptide-2; NMU, neuromedin U; NMUR1, neuromedin U receptor 1; NMUR2, neuromedin U receptor 2; PVN, paraventricular nucleus; s.c., subcutaneous. ∗ Corresponding author. Tel.: +45 31 52 26 52. E-mail address: [email protected] (J. Jelsing). http://dx.doi.org/10.1016/j.peptides.2015.04.010 0196-9781/© 2015 Elsevier Inc. All rights reserved.

Despite this, the exact role of NMU and the NMU receptors still needs to be clarified. Studies have indicated that NMU could play a role in regulation of blood pressure, smooth muscle contraction, nociception, stress and inflammation [6]. Furthermore, both genetic and pharmacological data indicate that NMU could be involved in energy and appetite homeostasis. Mice lacking the gene encoding NMU become obese, and in humans, a genetic variation in the NMU gene has been associated with overweight and obesity [9,10]. Central administration of NMU has been shown to decrease food intake and body weight in rodents by increasing energy expenditure, locomotor activity, core body temperature, and oxygen consumption [3,11–14]. In addition, it has been shown that subcutaneous (s.c.) administration of NMU to diet induced obese (DIO) mice decreases food intake, body weight, and increases energy expenditure. These data support a role for peripherally administered NMU in regulation of appetite, body weight and glucose homeostasis [15]. Even though several studies involving NMU have been conducted, the mechanism of action of NMU on food intake and appetite modulation is still poorly defined and only a few studies focus specifically on unraveling the mechanisms behind the anorectic effect of peripheral administered NMU. The distribution of NMUR2 in key hypothalamic areas may indicate a direct central

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effect. However, many gut brain peptides have also been shown to reduce food intake, at least partly, due to an inhibitory effect on gastric emptying [16,17]. The present study was initiated to get a more in-depth knowledge about the pharmacological and physiological effects of NMU and its involvement in food intake. We used a long-acting lipidated NMU peptide analog (GUB07-007) to investigate effects on food intake, glucose homeostasis and gastric emptying immediately after the first dose and after seven days of bidaily (BID) dosing. The effects were compared to the long acting Glucagonlike peptide-1 (GLP-1) analog liraglutide to evaluate the use of a lipidated NMU analog as a potential drug candidate for treatment of obesity and diabetes. Finally, we investigated the mRNA expression of NMU and NMUR1 in the gastrointestinal tract, as well as the expression of NMUR2 in the hypothalamus, by means of a novel chromogen-based in situ hybridization technique. Materials and methods Peptide synthesis The lipidated NMU analog (GUB07-007) (H-FRVDEEFQK(Pam␥E-OH)PFASQSRGYFLFRPRN-NH2 ) was prepared by automated solid-phase peptide synthesis using the Fmoc/tBu strategy on Rink amide TentaGel resin (Rapp polymere GmbH, Tuebingen, Germany). The couplings were performed using Fmoc-N␣ protected amino acids, N,N -diisopropylcarbodiimide and ethyl cyanoglyoxylate-2-oxime (oxyma) in N,N-dimethylformamide (Iris Biotech GmbH, Marktredwitz, Germany) for 2 × 2 h. The N␣ -deprotections were performed using 40% piperidine in N-methyl-2-pyrrolidione (Iris Biotech GmbH, Marktredwitz, Germany) for 3 min followed by 20% piperidine in N-methyl-2-pyrrolidione for 22 min. The lipidation was performed selectively on-resin on the N␧ amine of a Lys-9 using the alloc protecting group. Following linear assembly of the peptide backbone, the alloc protecting group was removed using tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3 )4 ] (Sigma–Aldrich, Brøndby, Denmark) and borane dimethylamine complex (Sigma–Aldrich, Brøndby, Denmark) as scavenger in dichloromethane (Sigma-Aldrich, Brøndby, Denmark). The mixture reacted for 2.5 h at room temperature. Fmoc-Glu-OtBu (4 eq.) (Iris Biotech GmbH, Marktredwitz, Germany) was coupled to the free amine using N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (Iris Biotech GmbH, Marktredwitz, Germany) as coupling reagent and DIEA (Iris Biotech GmbH, Marktredwitz, Germany) as base for 2 h at room temperature. Palmitic acid (Sigma–Aldrich, Brøndby, Denmark) was incorporated using HATU and DIEA for 2 h at room temperature. Finally, the peptide was simultaneously side-chain deprotected and released from the solid support by a TFA cocktail containing trifluoro acetic acid (TFA) (Iris Biotech GmbH, Marktredwitz, Germany), triethylsilane (Sigma–Aldrich, Brøndby, Denmark) and H2 O (95/2.5/2.5) as scavengers for 2 h. The peptide was precipitated by the addition of diethylether (Sigma–Aldrich, Brøndby, Denmark). The peptide was purified by RP-HPLC and identified by LC-MS. The final products were obtained with >95% purity. For a detailed description see [18]. Animal studies All animal experiments were conducted in accordance with internationally accepted principles for the care and use of laboratory animals and were covered by a personal license issued for Jacob Jelsing (approved by the Danish Committee for Animal Research, permit number: 2013-15-2934-00784).

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In a recent paper, we have described the development and specific effects on food intake of a library of long-acting lipidated NMU analogs [18]. Based on these data, GUB07-007 (0.3 ␮mol/kg) was selected as one of the most efficacious for the present study. Effect of a single dose of GUB07-007 on cumulative food intake in male NMRI mice and SPD rats The effect on cumulative food intake was measured following a single dose of GUB07-007 using a fully automated food intake monitoring system (HM-2; MBRose ApS, Faaborg, Denmark), allowing for advanced synchronous real-time monitoring of food intake behavior of individual animals as previously described [18]. A total of 24 male NMRI mice, 6–7 weeks old (approx. 25–30 g body weight) at the time of arrival were obtained from Taconic (Denmark). Upon arrival, the animals were uniquely identified with s.c. implantable microchips (Pet ID Microchip, E-vet, Haderslev, Denmark), transferred to the HM-2 system and acclimatized to their new environment. The animals were housed in groups of 4 in a light-, temperature-, and humidity-controlled room (a 12/12 LD cycle, lights on at 02:00 AM; 22 ± 2 ◦ C; 50% relative humidity). The mice had ad libitum access to regular chow diet (Altromin 1324, Brogaarden A/S, Lynge, Denmark) and domestic quality tap water. Mice arrived at day-7, and a minimum of 5 days of habituation to the system was allowed prior to beginning of the study. During these days the animals were handled daily to accustom them to the experimental paradigm. On the day of dosing, the animals were randomized into three groups according to body weight (n = 8), which received vehicle (0.9% NaCl (Fresenius Kabi, Uppsala, Sweden) + 0.1% BSA (Roche Diagnostics, Mannheim, Germany) + 5% DMSO (Sigma-Aldrich, St. Louis, USA)), the positive control liraglutide (0.05 ␮mol/kg, (Liraglutide, Lot. AP52177, Maaloev, Denmark)) or the lipidated NMU analog GUB07-007 (0.3 ␮mol/kg), respectively. Animals were dosed s.c. in the lower back (dose volume 10 ml/kg) in the afternoon just prior to lights out, and food intake data were collected for 18 h post dosing using the HM-2 system with automatic food recordings every 5 min. The efficacy of GUB07-007 on food intake was tested in male Sprague Dawley rats (8 weeks of age, Taconic, Denmark) using a similar setup, except the rats were housed only 2 per cage, and the dose volume was 5 ml/kg. Effect of seven days BID of GUB07-007 in male NMRI mice The effect of seven days BID of GUB07-007, on food intake, body weight, oral glucose tolerance and gastric emptying was investigated in a total of 24 male NMRI mice. The mice were obtained from Taconic (Denmark) and were 8 weeks old (approx. 35–40 g body weight) at the time of arrival. They were acclimatized to their new environment for at least one week. Animals were single housed in a light-, temperature-, and humidity-controlled room (a 12/12 LD cycle, lights on at 06:00 AM; 22 ± 2 ◦ C; 50% relative humidity). All animals had free access to standard chow (Altromin 1324, Brogaarden A/S, Lynge, Denmark) and domestic quality tap water. Body weight, food and water intake were measured daily from day-5 to day-7 in the morning between 8 and 9 AM. One day before the experiment the mice were randomized according to body weight into three experimental groups (n = 8), which received vehicle s.c. bi-daily (BID) (0.9% NaCl (Fresenius Kabi, Uppsala, Sweden) + 0.1% BSA (Roche Diagnostics, Mannheim, Germany) + 5% DMSO (Sigma–Aldrich, St. Louis, USA)), the positive control liraglutide 0.05 ␮mol/kg s.c. BID or the lipidated NMU analog GUB07-007 0.3 ␮mol/kg s.c. BID, respectively. The test period was initiated with a gastric emptying and oral glucose tolerance test (GE-OGTT) performed in the morning on experimental day 0. Clean cages were provided for the mice on the day prior to the test, and the animals were semi-fasted (50% of their average 24-h intake was given in the afternoon the day before

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the test). The s.c. administration of compounds was performed at t = −2 h, and at t = 0 min the mice were gavaged with 100 mg/kg acetaminophen and 2 g/kg glucose (10 mg/ml acetaminophen (Sigma–Aldrich, St. Louis, USA) dissolved in 200 mg/ml glucose solution (B. Braun, Melsungen, Germany), dose volume: 10 ml/kg). Blood samples for estimation of blood glucose were collected at time points t = −120, −60, 0, 15, 30, 60, 120 and 240 min. Samples were collected into 10 ␮l heparinized glass capillary tubes, suspended in buffer (0.5 ml of glucose/lactate system solution (EKFdiagnostics, Germany)) and immediately analyzed for glucose using a BIOSEN c-Line glucose meter (EKF-diagnostics, Germany) according to manufacturer’s instructions. For analysis of acetaminophen, blood samples of 40 ␮l were taken in heparin tubes (Microvette 100LH, Sarstede, Nümbrecht, Germany) from the tail vein at time points 15, 30, 60, and 120 min and chilled on ice. Samples were centrifuged (at 5000 × g for 15 min at 4 ◦ C) and plasma was collected for acetaminophen determination and stored at −80 degrees Celsius until analysis. The plasma acetaminophen concentrations were determined in duplicate using a commercially available acetaminophen Kit (MULTIGENT, 2K9920, Abbot Laboratories, Abbott Park, IL 60064, USA). Following the last blood sample, food was re-introduced and all animals continued into a 7-day BID study. On day 7 the GE-OGTT test was repeated using the same procedures as described above, and animals were terminated using CO2 anesthesia and cervical dislocation. Chromogen-based in situ hybridization Tissue preparation The distribution of NMUR2 in mice and rat brains was investigated on fresh frozen cryo sections prepared as follows: After animal decapitation, the brains were removed as quickly as possible, snap frozen on dry ice and stored at −80 ◦ C until processing. The forebrain and midbrain were mounted with Tissue-Tek (Sakura finetek, Alpen ann den Rijn, Nederland) in a cryostat, trimmed and cut in the rostral-caudal direction throughout the entire hypothalamus. A 12 ␮m thick coronal section was cut for every 200 ␮m and collected on Super Frost Plus slides (Thermo Scientific, Walldorf, Germany). Slides were briefly air dried, and subsequently stored at −80 ◦ C until hybridizations were performed. The distribution of NMU and NMUR1 mRNA in the rat gastrointestinal tract (GI) was investigated using formalin-fixated paraffin embedded (FFPE) slides. Two male Sprague Dawley rats (8 weeks of age, Taconic, Denmark) were sacrificed using CO2 anesthesia and decapitation, and the entire gut was removed as quickly as possible and divided into stomach, duodenum, jejunum/ileum and colon. The feces were carefully removed from each segment by flushing with cold phosphate-buffered saline (PBS, Gibco 10010, Invitrogen Corporation, Invitrogen, Paisley, UK). Next, the segments were fixated in 10% neutral buffered formalin (4% formaldehyde, CellPath Ltd. Newton, Powys, UK) for 20 h at room temperature. After fixation, 4–8 transverse biopsies were sampled systematically using an equal sampling distance between each biopsy. In addition, a piece of the stomach and a piece of the junction between the stomach and duodenum were collected. After sampling, the biopsies were infiltrated and embedded in blocks of paraffin. The blocks were trimmed and 5 ␮m sections were cut from each block on a Microm HM340E (ThermoScientific, Walldorf, Germany) and put onto Superfrost Plus Slides (Thermo Scientific, Walldorf, Germany).

ViewRNA ISH Tissue 1-Plex Assay user manual. In brief, slides were de-paraffinated in xylene and ethanol series, a hydrophobic barrier was drawn around the sections with a hydrophobic barrier pen and sections were allowed to dry before being placed for 5 min in pre-treatment 1 solution at 95 ◦ C. Slides were then protease digested and fixated before the probe hybridization mixture was applied (40 ◦ C for 3 h). Signal was then amplified by applying PreAmp1 followed by an Amp1 Solution and a Label Probe-AP Solution. Signal was detected with Fast Red and sections were counterstained with Gill’s hematoxylin and mounted with Ultramount or pertex. Virtual images were obtained using an Aperio ScanScope® Scanner. The distribution of NMUR2 and NMUR1 mRNA was investigated using the RNAscope® 2.0 High Definition (HD) – BROWN and RED Assays (Cat#310036, and Cat#310035, Advanced Cell Diagnostics, Hayward, CA, USA) and a NMUR1 (NM 023100, Cat#410201, Advanced Cell Diagnostics, Hayward, CA, USA) and a NMUR2 (NM 153079.4, Cat#314111, Advanced Cell Diagnostics, Hayward, CA, USA) probe. Slides were treated according to the RNAscope® 2.0 High Definition (HD) – BROWN Assay user manual as described by Wang and colleagues [19]. Separate pre-treatment conditions were used for fresh frozen and FFPE slides. The FFPE slides were de-paraffinated in xylene and ethanol series and allowed to dry before incubation for 10 min with pre-treatment 1. Next, the slides were boiled for 10 min in pre-treatment 2, where after the sections were incubated with pre-treatment 3 for 30 min at 40 ◦ C. The fresh frozen slides were fixated in cold 10% neutral formalin buffer (4% formaldehyde, CellPath Ltd. Newton, Powys, UK) for 1 h at 4 ◦ C. Then, the slides were dehydrated and incubated for 10 min with pre-treatment 1 at room temperature followed by 10 min incubation with pre-treatment 3 at room temperature. Next, both FFPE and fresh frozen slides were incubated with probe hybridization mixture for 2 h at 40 ◦ C. Signal was then amplified by applying amplifier (AMP) 1 for 30 min at 40 ◦ C, AMP 2 for 15 min at 40 ◦ C, AMP 3 for 30 min at 40 ◦ C, AMP 4 for 15 min at 40 ◦ C, AMP 5 for 30 min at room temperature and AMP 6 for 15 min at room temperature. Finally, the signal was detected with DAB or fast RED, and the sections were counterstained with Gill’s hematoxylin (Sigma–Aldrich, St. Louis, USA) and mounted with pertex or EcoMount (Biocare Medical, Concord, CA, USA). Virtual images were obtained using a ScanScope® AT slide scanner (Aperio). Data, reporting and statistical evaluation Graphical presentations, calculations, and statistical analyses were carried out using GraphPad Prism for Windows 5.04 (Graphpad software, San Diego, CA, USA). Results are presented as mean ± standard error of mean (SEM). Area under the curve (AUC) was calculated using GraphPad Prism. Food intake 6 and 18 h post dosing, and AUC parameters were compared across groups using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. Body weight, food and water intake, as well as glucose and acetaminophen curves were analyzed using a two-way repeatedmeasures ANOVA. Between subject factors were treatment groups whereas time (sample fraction) was used as repeated measure. Significant effects were followed by Bonferroni’s post hoc test. P < 0.05 was considered statistical significant. Results

In situ hybridization Distribution of NMU mRNA was investigated using the QuantiGene® ViewRNA ISH Tissue 1-Plex Assay kit (QVT0051, Affymetrix/Panomics, Santa Clara, CA, USA) and a NMU (NM 022239, VC1-16059, Affymetrix/Panomics, Santa Clara, CA, USA) probe. Slides were treated according to QuantiGene®

Effect of a single dose of GUB07-007 on cumulative food intake in mice and rats The effect of a single s.c dose of the lipidated NMU analog GUB07-007 on food intake was assessed during the dark phase in ad

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Fig. 1. Effect of a single dose of GUB07-007 on cumulative food intake in mice and rats. The lipidated NMU analog, GUB07-007, was administered s.c. just prior to lights out and food intake was monitored for 18 h post dosing. (A) Time response curve, (B) cumulated food intake 6 h and (C) 18 h following s.c. administration of GUB07-007, and liraglutide to mice. (D) Time response curve, (E) cumulated food intake 6 h and (F) 18 h following s.c. administration of GUB07-007 and liraglutide to rats. Data are given as mean ± SEM with n = 6–8/group. Statistical analysis: time response curve were compared by two-way ANOVA with Bonferronis post hoc test: p < 0.05 versus vehicle control indicated by vertical lines. Cumulated food intake 6 and 18 h post dosing was compared with a one-way ANOVA with Newman–Keuls Multiple Comparison Test (*p < 0.05, **p < 0.01, ***p < 0.001 for significance).

libitum feed mice and rats (Fig. 1). As for mice, s.c. administration of liraglutide or GUB07-007 resulted in a robust reduction in cumulative food intake (Fig. 1A–C). GUB07-007 significantly reduced 6 h food intake as compared to both vehicle and liraglutide (p < 0.001 vs. vehicle and p < 0.05 vs. liraglutide) and 18 h food intake as compared to vehicle-treated mice (p < 0.001). In contrast, the suppressing effect on food intake of GUB07-007 was much lower in rats (Fig. 1D–F), showing significant reduction in cumulative food intake observed only 6 h post dosing (p < 0.01 vs. vehicle) and with no effect 18 h post dosing. Effect of seven days BID of GUB07-007 on food intake, body weight, gastric emptying and glycaemic control in mice The effect of seven days BID of GUB07-007 on food intake, body weight, oral glucose tolerance and gastric emptying was investigated in male NMRI mice. Both GUB07-007 and liraglutide significantly reduced 24 h food intake after the first dose (Fig. 2). However, whereas the effects of liraglutide on food intake lasted (although gradually faded) throughout the experiment, the effects of GUB07-007 almost vanished after the first dose, and no effect on food intake was evident on days 2–7. In agreement with this observation, a significant decrease in body weight was observed for the liraglutide-treated group only, while no clear effect was observed for the GUB07-007-treated group.

In agreement with the anorectic effects, administration of GUB07-007 potently inhibited gastric emptying rate, as shown by the ability to reduce plasma acetaminophen levels over time (Fig. 3A). This was also evident when expressed as area under the curve (p < 0.001 vs. vehicle and p < 0.001 vs. liraglutide) (Fig. 3B). An inhibitory effect on gastric emptying was also observed for the liraglutide-treated group, although not as potent as the group treated with the lipidated NMU analog. In contrast, neither liraglutide nor the lipidated NMU analog inhibited gastric emptying after seven days BID treatment (Fig. 3C and D). The effects of GUB07-007 and liraglutide on glucose homeostasis are shown in Fig. 4. Both compounds significantly reduced the peak glucose level 30 min after the glucose load (Fig. 4A). This resulted in an overall reduction in blood glucose level as calculated by area under the curve (AUC0–240 ) as compared to vehicle (Fig. 4B). After seven days BID only liraglutide significantly reduced peak glucose levels and reduced AUC0–240 , whereas no effect of GUB07-007 was observed (Fig. 4C and D). Distribution of NMUR2 in mice and rat hypothalamus The distribution of the central NMUR2 was investigated in both mice and rat hypothalami using chromogen-based in situ hybridization. In mice, NMUR2 mRNA was abundantly expressed in the arcuate nucleus (ARC) and in the cells surrounding the

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Fig. 2. Effects of seven days BID treatment with GUB07-007 on body weight and food intake. Effects of seven days BID administration of the lipidated NMU analog, GUB07-007, and liraglutide on (A) body weight change (in percent of day-1) and (B) daily food intake. Data are given as mean ± SEM with n = 8/group. Statistical analysis: two-way ANOVA with Bonferronis post hoc test against vehicle, (*p < 0.05, **p < 0.01, ***p < 0.001 for liraglutide versus vehicle and ˆˆˆp < 0.001 for NMU versus vehicle).

Fig. 3. Effect of GUB07-007 on gastric emptying after a single dose and after seven days BID treatment. Effect of liraglutide and GUB07-007 on (A) acetaminophen time–response curve (0–120) and (B) area under the curve (AUC0–120 ) measured immediately following the first dose. Effect of liraglutide and GUB07-007 on (C) acetaminophen time–response curve (0–120) and (D) area under the curve (AUC0–120 ) measured after seven days BID dosing. Data are given as mean ± SEM with n = 8/group. Compounds were administered at t = −120, and glucose acetaminophen solution (100 mg/kg Acetaminophen and 2 g/kg glucose) at t = 0, mice were semi fasted (clean cages were provided and 50% of the average 24-h intake was given in the morning the day before the test. Statistical analysis: time response curves were compared by two-way ANOVA with Bonferronis post hoc test: (***p < 0.001 for liraglutide versus vehicle and ˆˆˆp < 0.001 for NMU versus vehicle). AUC were compared with a one-way ANOVA with Newman–Keuls Multiple Comparison Test (*p < 0.05, ***p < 0.001 for significance).

third ventricle (Fig. 5B, C and E, F). In addition, sparse expression was observed in the paraventricular nucleus (PVN) (Fig. 5A and D), the dorsomedial hypothalamus and the cells surrounding the ventromedial hypothalamus. In rats, NMUR2 expression was seen in the PVN (Fig. 5G and J), and abundant expression was found in the ependymal layer of the third ventricle (Fig. 5I and L), particularly in the ventral portion of the 3rd ventricle harboring the tanycytes [20]. Expression of NMUR2 was also localized in the rat ARC. However, in contrary to the mouse the expression of NMUR2 in the rat ARC appeared markedly lower (Fig. 5H and K).

Distribution of NMU and NMUR1 mRNA in rat GI tract NMU mRNA was expressed in the myenteric plexus, located between the longitudinal and circular smooth muscle layers. This finding was evident throughout the gut, i.e. the duodenum, jejunum, ileum, caecum and colon. In addition, sparse expression was observed in the submucosal plexus in the duodenum, jejunum and ileum segments (Fig. 6). In contrast, NMUR1 expression was confined to the lamina propria in the mucosal villi in the duodenum, jejunum, ileum, caecum and colon (Fig. 7). NMUR1 expression was also detected in scattered cells of the gastric mucosa.

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Fig. 4. Effect of GUB07-007 on glycaemic control after a single dose and after seven days BID treatment. Effect of liraglutide and GUB07-007 on (A) glucose time–response curve (−60 to 240) and (B) area under the curve (AUC0–240 ) measured immediately after the first dose. Effect of liraglutide and GUB07-007 on (C) glucose time–response curve (−60 to 240) and (D) area under the curve (AUC0–240 ) measured after seven days BID dosing. Data are given as mean ± SEM with n = 8/group. Compounds were administered at t = −120, and glucose acetaminophen solution (100 mg/kg Acetaminophen and 2 g/kg glucose) at t = 0, mice were semi fasted (clean cages were provided and 50% of the average 24-h intake was given in the morning the day before the test. Statistical analysis: time response curves were compared by two-way ANOVA with Bonferronis post hoc test: (** p < 0.01, ***p < 0.001 for liraglutide versus vehicle and ˆˆp < 0.01, ˆˆˆp < 0.001 for NMU versus vehicle). AUC were compared with a one-way ANOVA with Newman–Keuls Multiple Comparison Test (*p < 0.05, **p < 0.01, ***p < 0.001 for significance).

Discussion In the present study we show that GUB07-007, a novel lipidated NMU analog, potently inhibits food intake and gastric emptying, and improves glycaemic control in mice, but also that these effects are short lasting and seem to be subject to rapid tachyphylaxis. Additionally, we describe the anatomical localization of NMU and NMUR1 mRNA expression throughout the gastrointestinal tract where NMU mRNA was detected in the enteric nervous system, whereas NMUR1 was confined to the lamina propria. Finally we show species-specific differences in the expression pattern of NMUR2 in the hypothalamus of rats and mice. We have previously described the development of novel lipidated NMU analogs with improved pharmacokinetic properties leading to potent reductions in 18 h cumulative food intake in mice [18]. In the present study we verified previous observations and extended the analyses to describe the effect of a single dose of GUB07-007 on cumulative food intake in rats. Surprisingly, the anorexigenic effect of the lipidated NMU analog was markedly lower in rats. The mechanism by which peripheral administered NMU reduces food intake is currently unknown, but accumulating evidence suggests that central mechanisms involved in satiety may play a role [3,11,13,21]. As NMUR2 is predominantly expressed in the CNS we hypothesized that this receptor could be important for the pharmacological effect of NMU and NMU analogs [4]. Accordingly, we set out to investigate whether the observed species-dependent differences in the anorectic properties of the lipidated NMU analog was in fact related to differences in NMUR2 distribution in mice and rat hypothalami. Interestingly, we found

that NMUR2 expression is highly abundant in mice ARC, whereas only weak expression was observed in the rat ARC. This finding concurs with previous reports showing no or very limited NMUR2 expression in the rat ARC [3,7,22]. It is well known that the ARC plays an essential role in the regulation of energy homeostasis and food intake, and that hormone receptors in the ARC are accessible from the periphery [23–25]. Accordingly it is very likely that NMUR2 expressed in the ARC contributes to the anorexigenic effects of the lipidated NMU analog. Hence, a differential expression of NMUR2 in the ARC may explain the attenuated effect of GUB07-007 in rats. Species-dependent differences in enzymatic degradation of the NMU analog may, however, also be involved. For example, it has previously been shown that discrepancies in the effect of glucagon-like peptide-2, which show intestinotrophic efficacy in mice, but not in rats, are attributed to differences in the activity of the enzyme dipeptidyl peptidase-4 [26]. In mice, GUB07-007 was tested in a seven days study with BID dosing to assess the long-term effect on food intake, body weight, gastric emptying and glycaemic control as compared to the GLP-1 analog liraglutide. In contrast to liraglutide, which led to a pronounced decrease in food intake accompanied by a sustained decrease in body weight and improved glycaemic control throughout the experiment, the effect of the lipidated NMU analog on food intake, body weight and glycaemic control diminished after the first dosing, with no effect following multiple dosing. An improved glycaemic control following a single peripheral dose of NMU is in line with previous data reported from Peier et al. demonstrating that peripheral NMU administration improves glucose tolerance following an oral glucose tolerance test and increases insulin secretion

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Fig. 5. Distribution of NMUR2 mRNA in mice and rat hypothalamus. Expression of NMUR2 in mouse hypothalamus is shown in A–F and rat hypothalamus in G–L. (A) Expression of NMUR2 mRNA in mouse PVN and (B + C) mouse ARC. (D), (E) and (F) are magnification of squared boxes in A, B and C respectively. (G) Expression NMUR2 mRNA in rat PVN, (F) rat ARC and (I) 3rd ventricle. (J), (K) and (L) are magnification of squared boxes in G, H and I respectively. Arrows denotes cell expressing NMUR2 mRNA.

[15]. However, other reports with diverging findings blur the role of NMU in controlling insulin release [27]. The tested lipidated NMU analog was also shown to exert a pronounced effect on gastric emptying. Delay of gastric emptying has been associated with an increased feeling of satiety and decreased food intake [28], and many gut brain peptides have been shown to reduce food intake, at least partly, due to an inhibitory effect on gastric emptying [16,17]. Accordingly, it was considered likely that the observed effect of NMU on gastric emptying together with the above-mentioned centrally induced effects on satiety were mainly responsible for the observed reductions in food intake.

The mechanism by which NMU inhibits gastric emptying is unknown. Other gut peptides such as GLP-1and CCK have has been ascribed to inhibit gastric emptying via the vagus nerve, and mRNA for both receptors is known to be expressed in the nodose ganglia [29–31]. Consequently, it can be speculated that NMU inhibits gastric emptying via a similar mechanism. However, NMU receptor expression has not been detected in the nodose ganglion, and the mechanism behind the NMU-induced reduction in gastric emptying is consequently not considered related to a direct activation of vagal afferents [15]. It is, however, possible that NMU activates the vagus nerve via secondary signaling pathways or by other

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Fig. 6. Distribution of NMU mRNA in the rat gastrointestinal tract. The expression of NMU mRNA was investigated using a chromogen-based in situ hybridization assay. NMU mRNA was expressed in the myenteric plexus and the submucosal plexus. (A) Expression of NMU mRNA in rat duodenum, (B) magnification of squared box in A, (C) magnification of squared box in B. (D) Expression of NMU mRNA in rat jejunum, (E) magnification of squared box in D, (F) magnification of squared box in E. (G) Expression of NMU mRNA in rat colon, (H) magnification of squared box in G, (I) magnification of squared box in G.

mechanisms. In agreement with the latter it has been shown that central administration of NMU decreases gastric emptying indicating that NMU may inhibits gastric emptying directly via centrally signaling pathways [32]. The inhibitory effect on gastric emptying by both NMU and liraglutide was, however, completely abolished following seven days of BID. We have previously reported a short-lived effect of liraglutide on gastric emptying [33]. However, notwithstanding the diminished effect of liraglutide on gastric emptying, the effect of liraglutide on bodyweight and glycaemic control is persistent [33]. In contrast to liraglutide, the anorectic effect of the NMU analog appeared to be short-lived and effects on food intake and body weight were only observed the first day after dosing. Consequently, it is likely that decreased gastric emptying contributed to the observed anorectic effects of the NMU analog. Similar short-lived effects on food intake and body weight have also been described for other gut-brain peptides such as neurotensin, xenin and cholecystokinin (CCK) [34,35]. Repeated in vitro administration of NMU8 to isolated porcine jejunum has been shown to lead to tachyphylaxis [36]. Similarly, the contractile response of human isolated ileum was also characterized by the development of tachyphylaxis following repeated application of NMU8 [37]. Collectively, these data suggest that rapid development of tachyphylaxis may explain the

lack of effect on food intake following repeated administration of the lipidated NMU analog. Neuner et al. and Ingallinella et al. have previously reported long-lasting effects of PEGylated and human serum albumin conjugated NMU analogs on food intake and body weight [21,38]. Based on these data it may be speculated that the development of tachyphylaxis is analog-specific. This is partly substantiated by Verbaeys and colleagues, who showed that PEGylated CCK analogs did not lead to development of tolerance tachyphylaxis [39]. NMU and NMUR1 mRNA have previously been investigated using qPCR in rats and humans in which they have been reported as highly expressed in the gastrointestinal tract [1,2,4,5,8]. However, little information is available on the tissue specific localization of NMU and NMUR1. Using a novel and highly sensitive chromogenbased in situ hybridization technique, which allows for a good visualization of mRNA distribution, we demonstrate that the peripheral NMUR1 is expressed in the mucosal lamina propria throughout the entire gut, as well as in scattered cells of the gastric mucosa. In contrast, NMU mRNA is restricted exclusively to the enteric ganglia from the proximal duodenum to the distal colon, with no expression in the stomach. In line with these observations, NMU immunoreactivity has previously been described as being located in the enteric ganglion and in the nerve fibers running

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Fig. 7. Distribution of NMUR1 mRNA in the rat gastrointestinal tract. The expression of NMUR1 mRNA was investigated using a chromogen-based in situ hybridization assay. NMUR1 mRNA was expressed in scattered cells of the gastric mucosa and in the lamina propria in the mucosal villi. (A) Expression of NMU mRNA in rat stomach, (B) magnification of squared box in A, (C) magnification of squared box in B. (D) Expression of NMUR 1 mRNA in rat duodenum, (E) magnification of squared box in D, (F) magnification of squared box in E. (G) Expression of NMUR1 mRNA in rat jejunum, (H) magnification of squared box in G, (I) magnification of squared box in G.

parallel to the axes of the villi in the rat intestine [40]. A similar expression pattern has also been seen in the intestine from pig and mice by application of immunohistochemical analyses of the NMU hormone [41,42]. Moreover, Ballesta et al., were not able to demonstrate any NMU immunoreactivity in rat stomach, thereby confirming our analyses of NMU mRNA expression [40]. Others have reported on NMU mRNA expression in the rat stomach measured using qPCR, although the expression was only found at low levels [2,8,43]. Thus, although not observed in the present study, a weak expression of NMU mRNA in the stomach cannot be ruled out. The exact physiological role of NMU and NMUR1 in the gastrointestinal tract remains unclear. The cellular location suggests that it may be involved in the regulation of mucosal functions such as intestinal motility, ion transport and absorptive/secretory functions [6,36,40]. This is supported by previous findings showing that NMU has prokinetic activity, increases intestinal transit rate in mice and is important for the induction of normal gastrointestinal motility via NMUR1-dependent pathways [41,44]. Our data implies that NMU elicits these effects via paracrine/neurocrine rather than endocrine signaling pathways. In line with this, circulating levels of endogenous NMU have not been detected in plasma [15].

In summary, GUB07-007, a novel lipidated NMU analog, potently inhibits food intake and gastric emptying, and improves glycaemic control in mice. The mechanism by which peripheral NMU and NMU analogs inhibit food intake is complicated and warrants further investigation. It is, however, conceivable that part of the effect of the NMU analog is mediated through a profound inhibition of gastric emptying together with central signaling pathways. Unfortunately, the anorectic effect of the NMU analog appeared to be short-lived. Hence, the potential of lipidated NMU analogs in the present form as anti-obesity/diabetes drugs appears to be limited. Finally, we have shown for the first time that NMUR1 is expressed in the lamina propria throughout the entire gastrointestinal tract. Accordingly, these data could help to unravel the physiological implications of NMU in this organ.

Contribution statement Participated in research design: LSD, SLP, NV, JJ, BH. Conducted experiments and performed data analysis: LSD, TS. Wrote or contributed to the writing of the manuscript: LSD, JJ.

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All co-authors performed a critical revision of the manuscript and approved the final manuscript. Conflict of interest The study was funded by Gubra ApS. LSD, SLP and TS are employed by Gubra ApS. JJ and NV are the owners of Gubra ApS and contributed with an unbiased and impartial role in the study design and manuscript revision. Acknowledgements The authors would like to acknowledge the Innovation Fund Denmark for supporting this work, and the Danish Agency for Science, Technology and Innovation for co-financing an industrial Ph.D. stipend to L.S.D. Farida Sahebzadeh and Ena Raahauge Larsen are gratefully acknowledged for their excellent technical assistance, and a special thank to Gitte Hansen for valuable scientific input regarding the in vivo studies. References [1] Domin J, Ghatei MA, Chohan P, Bloom SR. Neuromedin U – a study of its distribution in the rat. Peptides 1987;8:779–84. [2] Fujii R, Hosoya M, Fukusumi S, Kawamata Y, Habata Y, Hinuma S, et al. Identification of neuromedin U as the cognate ligand of the orphan G protein-coupled receptor FM-3. J Biol Chem 2000;275:21068–74. [3] Howard AD, Wang R, Pong SS, Mellin TN, Strack A, Guan XM, et al. Identification of receptors for neuromedin U and its role in feeding. Nature 2000;406:70–4. [4] Raddatz R, Wilson AE, Artymyshyn R, Bonini JA, Borowsky B, Boteju LW, et al. Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system. J Biol Chem 2000;275:32452–9. [5] Szekeres PG, Muir AI, Spinage LD, Miller JE, Butler SI, Smith A, et al. Neuromedin U is a potent agonist at the orphan G protein-coupled receptor FM3. J Biol Chem 2000;275:20247–50. [6] Brighton PJ, Szekeres PG, Willars GB. Neuromedin U and its receptors: structure, function, and physiological roles. Pharmacol Rev 2004;56:231–48. [7] Graham ES, Turnbull Y, Fotheringham P, Nilaweera K, Mercer JG, Morgan PJ, et al. Neuromedin U and Neuromedin U receptor-2 expression in the mouse and rat hypothalamus: effects of nutritional status. J Neurochem 2003;87:1165–73. [8] Hedrick JA, Morse K, Shan L, Qiao X, Pang L, Wang S, et al. Identification of a human gastrointestinal tract and immune system receptor for the peptide neuromedin U. Mol Pharmacol 2000;58:870–5. [9] Hainerova I, Torekov SS, Ek J, Finkova M, Borch-Johnsen K, Jorgensen T, et al. Association between neuromedin U gene variants and overweight and obesity. J Clin Endocrinol Metab 2006;91:5057–63. [10] Hanada R, Teranishi H, Pearson JT, Kurokawa M, Hosoda H, Fukushima N, et al. Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway. Nat Med 2004;10:1067–73. [11] Novak CM, Zhang M, Levine JA. Neuromedin U in the paraventricular and arcuate hypothalamic nuclei increases non-exercise activity thermogenesis. J Neuroendocrinol 2006;18:594–601. [12] Nakazato M, Hanada R, Murakami N, Date Y, Mondal MS, Kojima M, et al. Central effects of neuromedin U in the regulation of energy homeostasis. Biochem Biophys Res Commun 2000;277:191–4. [13] Peier A, Kosinski J, Cox-York K, Qian Y, Desai K, Feng Y, et al. The antiobesity effects of centrally administered neuromedin U and neuromedin S are mediated predominantly by the neuromedin U receptor 2 (NMUR2). Endocrinology 2009;150:3101–9. [14] Kojima M, Haruno R, Nakazato M, Date Y, Murakami N, Hanada R, et al. Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3). Biochem Biophys Res Commun 2000;276:435–8. [15] Peier AM, Desai K, Hubert J, Du X, Yang L, Qian Y, et al. Effects of peripherally administered neuromedin U on energy and glucose homeostasis. Endocrinology 2011;152:2644–54. [16] Hussain SS, Bloom SR. The regulation of food intake by the gut-brain axis: implications for obesity. Int J Obes (Lond) 2013;37:625–33. [17] Suzuki K, Jayasena CN, Bloom SR. Obesity and appetite control. Exp Diab Res 2012;2012:824305. [18] Dalboge LS, Pedersen SL, van Witteloostuijn SB, Rasmussen JE, Rigbolt KT, Jensen KJ, et al. Synthesis and evaluation of novel lipidated neuromedin U analogs with increased stability and effects on food intake. J Pept Sci 2015;21(February (2)):85–94.

65

[19] Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 2012;14:22–9. [20] Bolborea M, Dale N. Hypothalamic tanycytes: potential roles in the control of feeding and energy balance. Trends Neurosci 2013;36:91–100. [21] Ingallinella P, Peier AM, Pocai A, Marco AD, Desai K, Zytko K, et al. PEGylation of neuromedin U yields a promising candidate for the treatment of obesity and diabetes. Bioorg Med Chem 2012;20:4751–9. [22] Guan XM, Yu H, Jiang Q, Van Der Ploeg LH, Liu Q. Distribution of neuromedin U receptor subtype 2 mRNA in the rat brain. Brain Res Gene Expr Patterns 2001;1:1–4. [23] Secher A, Jelsing J, Baquero AF, Hecksher-Sorensen J, Cowley MA, Dalboge LS, et al. The arcuate nucleus mediates GLP-1 receptor agonist liraglutidedependent weight loss. J Clin Invest 2014;124:4473–88. [24] Schaeffer M, Langlet F, Lafont C, Molino F, Hodson DJ, Roux T, et al. Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons. Proc Natl Acad Sci U S A 2013;110:1512–7. [25] Balland E, Dam J, Langlet F, Caron E, Steculorum S, Messina A, et al. Hypothalamic tanycytes are an ERK-gated conduit for leptin into the brain. Cell Metab 2014;19:293–301. [26] Drucker DJ, Shi Q, Crivici A, Sumner-Smith M, Tavares W, Hill M, et al. Regulation of the biological activity of glucagon-like peptide 2 in vivo by dipeptidyl peptidase IV. Nat Biotechnol 1997;15:673–7. [27] Kaczmarek P, Malendowicz LK, Pruszynska-Oszmalek E, Wojciechowicz T, Szczepankiewicz D, Szkudelski T, et al. Neuromedin U receptor 1 expression in the rat endocrine pancreas and evidence suggesting neuromedin U suppressive effect on insulin secretion from isolated rat pancreatic islets. Int J Mol Med 2006;18:951–5. [28] Janssen P, Vanden Berghe P, Verschueren S, Lehmann A, Depoortere I, Tack J. Review article: the role of gastric motility in the control of food intake. Aliment Pharmacol Ther 2011;33:880–94. [29] Nauck MA, Kemmeries G, Holst JJ, Meier JJ. Rapid tachyphylaxis of the glucagonlike peptide 1-induced deceleration of gastric emptying in humans. Diabetes 2011;60:1561–5. [30] Nakagawa A, Satake H, Nakabayashi H, Nishizawa M, Furuya K, Nakano S, et al. Receptor gene expression of glucagon-like peptide-1, but not glucosedependent insulinotropic polypeptide, in rat nodose ganglion cells. Auton Neurosci: Basic Clin 2004;110:36–43. [31] Raybould HE, Tache Y. Cholecystokinin inhibits gastric motility and emptying via a capsaicin-sensitive vagal pathway in rats. Am J Physiol 1988;255:G242–6. [32] Mondal MS, Date Y, Murakami N, Toshinai K, Shimbara T, Kangawa K, et al. Neuromedin U acts in the central nervous system to inhibit gastric acid secretion via CRH system. Am J Physiol Gastrointest Liver Physiol 2003;284:G963–9. [33] Jelsing J, Vrang N, Hansen G, Raun K, Tang-Christensen M, Knudsen LB. Liraglutide: short-lived effect on gastric emptying – long lasting effects on body weight. Diab Obes Metab 2012;14:531–8. [34] Cooke JH, Patterson M, Patel SR, Smith KL, Ghatei MA, Bloom SR, et al. Peripheral and central administration of xenin and neurotensin suppress food intake in rodents. Obesity 2009;17(Silver Spring):1135–43. [35] Crawley JN, Beinfeld MC. Rapid development of tolerance to the behavioural actions of cholecystokinin. Nature 1983;302:703–6. [36] Brown DR, Quito FL. Neuromedin U octapeptide alters ion transport in porcine jejunum. Eur J Pharmacol 1988;155:159–62. [37] Maggi CA, Patacchini R, Giuliani S, Turini D, Barbanti G, Rovero P, et al. Motor response of the human isolated small intestine and urinary bladder to porcine neuromedin U-8. Br J Pharmacol 1990;99:186–8. [38] Neuner P, Peier AM, Talamo F, Ingallinella P, Lahm A, Barbato G, et al. Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide. J Pept Sci 2014;20:7–19. [39] Verbaeys I, Leon-Tamariz F, Buyse J, Decuypere E, Pottel H, Cokelaere M. Lack of tolerance development with long-term administration of PEGylated cholecystokinin. Peptides 2009;30:699–704. [40] Ballesta J, Carlei F, Bishop AE, Steel JH, Gibson SJ, Fahey M, et al. Occurrence and developmental pattern of neuromedin U-immunoreactive nerves in the gastrointestinal tract and brain of the rat. Neuroscience 1988;25:797–816. [41] Nakashima Y, Ida T, Sato T, Nakamura Y, Takahashi T, Mori K, et al. Neuromedin U is necessary for normal gastrointestinal motility and is regulated by serotonin. Ann N Y Acad Sci 2010;1200:104–11. [42] Timmermans JP, Scheuermann DW, Stach W, Adriaensen D, De Groodt-Lasseel MH, Polak JM. Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP. Cell Tissue Res 1989;258:331–7. [43] Austin C, Oka M, Nandha KA, Legon S, Khandan-Nia N, Lo G, et al. Distribution and developmental pattern of neuromedin U expression in the rat gastrointestinal tract. J Mol Endocrinol 1994;12:257–63. [44] Dass NB, Bassil AK, North-Laidler VJ, Morrow R, Aziz E, Tuladhar BR, et al. Neuromedin U can exert colon-specific, enteric nerve-mediated prokinetic activity, via a pathway involving NMU1 receptor activation. Br J Pharmacol 2007;150:502–8.

Neuromedin U inhibits food intake partly by inhibiting gastric emptying.

Neuromedin U (NMU) is a gut-brain peptide, implicated in energy and glucose homeostasis via the peripherally expressed NMU receptor 1 (NMUR1) and the ...
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