Author’s Accepted Manuscript KBP-042 improves bodyweight and glucose homeostasis with indices of increased insulin sensitivity irrespective of route of administration Sara T. Hjuler, Kim V. Andreassen, Sofie Gydesen, Morten A. Karsdal, Kim Henriksen www.elsevier.com/locate/ejphar

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S0014-2999(15)30049-2 http://dx.doi.org/10.1016/j.ejphar.2015.05.051 EJP70015

To appear in: European Journal of Pharmacology Received date: 21 January 2015 Revised date: 7 May 2015 Accepted date: 26 May 2015 Cite this article as: Sara T. Hjuler, Kim V. Andreassen, Sofie Gydesen, Morten A. Karsdal and Kim Henriksen, KBP-042 improves bodyweight and glucose homeostasis with indices of increased insulin sensitivity irrespective of route of a d m i n i s t r a t i o n , European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.05.051 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

KBP-042 improves bodyweight and glucose homeostasis with indices of increased insulin sensitivity irrespective of route of administration Sara T. Hjuler1*, Kim V. Andreassen1, Sofie Gydesen1, Morten A. Karsdal1, Kim Henriksen1

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Nordic Bioscience, Herlev Hovedgade 207, DK-2730 Herlev, Denmark.

*Corresponding Author: Sara Toftegaard Hjuler, Nordic Bioscience, Herlev Hovedgade 207, 2730 Herlev, Denmark, Tel: +45 44 52 52 52, Fax: +45 44 52 52 51, E-mail: [email protected]. Abstract KBP-042 is a synthetic peptide dual amylin- and calcitonin receptor agonist (DACRA) developed to treat type 2 diabetes by inducing a significant weight loss while improving glucose homeostasis. In this study the aim was to compare two different formulations: An oral formulation (1▒mg/kg) to subcutaneous formulations of KBP-042 (2.5▒μg/kg, 5.0▒μg/kg and 7.5▒μg/kg) with comparable pharmacokinetic profiles. Furthermore to examine if differences in mode of action between the two different routes of administration in high-fat fed Sprague-dawley rats were present. It was established that the subcutaneous administrations of KBP-042 were able to dose-dependently cause a significant weight-loss, reduce food intake, and improve glucose homeostasis without increasing insulin secretion, effects comparable to those observed with oral administration. At the same time, s.c. KBP-042 suppressed the inappropriate glucagon response better than the oral formulation. Furthermore, KBP-042 was found to reduce incretins GLP-1 and GIP and considerably, improve gastric emptying, and to alleviate leptin resistance, as well as insulin resistance. In conclusion, the subcutaneous route of administration was found to have the same beneficial effects on blood glucose homeostasis and weight loss as well as resistance towards important insulin and leptin, albeit with a markedly lower variation in both exposure and biological responses. These data support the application of subcutaneously delivered peptide for mechanistic studies, and highlight the potential of developing s.c. KBP-042 as a therapy for T2D.

Keywords Anti-diabetic; Insulin sensitivity; DACRA; Amylin analogue; Weight-loss; Leptin resistance

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1. Introduction Obesity is closely related to insulin resistance and type 2 diabetes (Kahn et al., 2006), and targeting more aspects of the metabolic syndrome is essential during the treatment hereof. Weight-loss together with improvements in glucose control are key targets and can be achieved by many different interventions (diet, exercise, surgery, medication) which will all result in improved metabolic profiles and amelioration of diabetes (Raghow, 2013). The amylin analogue Pramlintide is approved as therapy for both type 1 and type 2 diabetes as an add-on to insulin (Ryan et al., 2005), and amylin agonism in general has been linked with weightloss (Mack et al., 2007) partially driven by an increase in energy expenditure and fat oxidation (Trevaskis et al., 2010). We have previously studied the effects of oral delivery of salmon calcitonin (sCT), a potent agonist of the amylin- and calcitonin receptors, which demonstrated the beneficial effects on glycaemic control, as well as on insulin action. Treatment with oral or injectable sCT can induce weight-loss or prevent weight gain and lower food intake in diet-induced obese rats (Feigh et al., 2011a, 2011b, 2013). Furthermore, orally delivered sCT has been demonstrated to attenuate diabetic hyperglycaemia and preserve pancreatic β-cell function and mass (Feigh et al., 2012) as well as enhance insulin action (Feigh et al., 2014). KBP-042, a novel dual amylin- and calcitonin receptor agonist (DACRA), has recently been found to exert anti-diabetic and anti-obesity effects as seen with sCT; however, the potency was superior on all parameters measured (Andreassen et al., 2014). The effect of KBP-042 is in part mediated through amylin receptors (Lutz et al., 2000) where the effect on eating, gastric emptying, body-weight and suppression of inappropriate glucagon response is well-known (Westermark et al., 2011). KBP-042 does however also potently activate the calcitonin receptor located in various tissues, with a higher affinity than the natural ligand

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(Andreassen, Feigh, Hjuler, Gydesen, Henriksen, Beck-Nielsen, Christiansen, Karsdal, and Henriksen, 2014). KBP-042 improved hyperglycaemia in the fasting, as well as the postprandial state, whereas amylin agonism in general lowers food intake, delays gastric emptying and suppresses glucagon secretion resulting in a lowered blood glucose measured after a meal, but with a modest or no effect in the fasting state (Mack et al., 2007;Edelman et al., 2008;Riddle et al., 2007). While, an oral route of delivery is convenient, the shortcomings of the present oral formulations (low bioavailability and large variability in the pharmacokinetic profiles) (Karsdal et al., 2014) incited us towards examining a subcutaneous administration of KBP-042. In this study we investigate and compare the effects of s.c. and p.o. administration of the dual amylin- calcitonin receptor agonist (DACRA) KBP-042 in high fat diet fed rats with regards to pharmacokinetics, weight loss, adiposity, metabolic hormones, glycaemic control, as well as insulin sensitivity.

2. Materials and Methods 2.1 Peptide therapy Recombinant KBP-042 peptide (Unigene Laboratories, Boonton, NJ, USA) was dissolved in saline for subcutaneous delivery. For oral delivery of KBP-042, the carrier agent N-(5-chlorosalicyloyl)-8aminocaprylic acid (5-CNAC) (vehicle) (Karsdal et al., 2011) was obtained from Biomics Biotechnologies Co. Ltd. (Nantong, China), KBP-042 peptide was mixed with 5-CNAC 150 mg/kg for oral delivery. The doses chosen for KBP-042 administration in the current set of investigations were based on previous comparable studies in animal models of obesity and type 2 diabetes (Feigh et al., 2011a; Andreassen et al., 2014)

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2.2 Animal experiments All animal procedures were performed in accordance with guidelines from the Animal Welfare Division of the Danish Ministry of Justice under the institutional license issued to Nordic Bioscience (2012-15-2934-00094). Male Sprague Dawley rats were obtained at 6 weeks of age and housed under controlled temperature on a normal 12h light-dark cycle with ad libitum access to water and food. Normal diet lean rats (ND) were fed a regular rodent chow and high fat-diet rats (HFD) a 60 % fat kcal diet (#D12495, Research Diets Inc., NJ, USA). The rats were after 10 weeks of high fatfeeding assigned randomly into treatment groups (n=10) by body weight and blood glucose levels. HFD rats received administration of either vehicle (5-CNAC p.o. and saline s.c.), oral KBP-042 1 mg/kg (saline s.c.), KBP-042 (2.5, 5.0 or 7.5 µg/kg s.c. and 5-CNAC p.o.) for 16 days b.i.d. Body weight were recorded daily, and food intake monitored on day 1-8, 13 and 16. Blood glucose was monitored by Accu-Check® Avia monitoring system (Roche Diagnostics, Rotkreuz, Switzerland). After 9 days an oral glucose tolerance test (OGTT, 2 g/kg) (Feigh et al., 2011a) performed after 12h fasting and EDTA-plasma was obtained for hormonal analysis. After 13 days of dosing an insulin tolerance test (0.5 U/kg i.p.; IPITT) was performed after 6 hours fasting as previously described (Andreassen et al., 2014). At the end of the treatment after 3 hours fasting, blood samples were collected for basal plasma hormonal analyses and animals were then euthanized. Excised tissue quick frozen in liquid nitrogen and stored at -80°C and plasma (EDTA) were stored at -20°C samples until further analysis. To assess drug effect on gastric emptying, overnight-fasted rats received 40 mg/kg acetaminophen by oral gavage (4 ml/kg) and the appearance of acetaminophen in plasma was monitored (Hatanaka et al., 1994). Blood was collected 30 min after administration from the tail vein and EDTA-Plasma

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acetaminophen levels were measured (Acetaminophen Direct ELISA Kit, Immuneanalysis, Pomona, CA, USA). Gastric emptying was calculated as % change relative to ND rats. The pharmacokinetic study was performed in ND Sprague Dawley rats. The animals were fasted over-night (18h) and in the morning animals received administration of vehicle (5-CNAC p.o. and saline s.c.), oral KBP-042 1 mg/kg (saline s.c.), or KBP-042 (2.5, 5.0 or 7.5 µg/kg s.c. and 5-CNAC p.o.) and EDTA-plasma was obtained from the tail vein at t = 0, 10, 20, 30, 40, 80 and 120 mins. To detect KBP-042 in rat plasma an in-house sandwich ELISA was developed using two in house antibodies targeting the N-terminal- and C-terminal end of KBP-042 and then optimized for measurement in rat plasma. Bioactive KBP-042 was used as calibrator and prepared covering a range from 0 to 10 ng/mL. 75% horse serum (12449C, Sigma) and 25% 100 mM PBS-BTB was used to imitate rat plasma and serum, and used as buffer (zero calibrator) for calibrator dilution. Streptavidin-coated micro-titer plates (Roche, Germany) were coated with 100 µL biotinylated (704-0010, Innova Biosciences) C-terminal antibody (1 µg/mL) and incubated for 60±5 min followed by 5x wash in washing buffer. 20 µL of the standard or PK serum samples were pipetted into the appropriate micro-titer wells followed by addition of 80 µL incubation buffer (100 mM PBS-BTB) and incubated overnight 20±1 hour at 4 °C followed by x5 wash in washing buffer. 100 µL of peroxidase-conjugated (701-0010, Innova Biosciences) N-terminal antibody (250 ng/mL) was pipetted into appropriate wells and incubated for 60±5 min followed by x5 wash in washing buffer. Finally 100 µL of tetramethylbenzidine solution (TMB) (cat. no. 4380H, Kem-En-Tec, Taastrup, Denmark) was added to each well and incubated for 15 min in darkness. The color reaction was stopped by addition of 100 µL of 0.18 mM H2SO4 and absorbance was measured at 450 nm, with 650 nm as the reference wavelength, using a microtiter plate reader (Molecular Devices, USA). All incubation steps were conducted on a shaker at 300 rpm.

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The effect of leptin on food intake was performed in HFD animals receiving a single dose of vehicle (20 mM Tris-HCl) i.p. + saline s.c.), leptin (500 µg/kg i.p., saline s.c.), KBP-042 (2.5 µg/kg, s.c., Tris-HCl pH = 8, i.p.), or Leptin + KBP-042 (500 µg/kg i.p., 2.5 µg/kg s.c.). Animals received treatment at 6 pm before dark onset and food intake was monitored at 9 am the following day. Homeostasis model assessment of insulin resistance (HOMA-IR) analysis was calculated and was used for the estimation of insulin resistance after high-fat feeding using the formula (Matthews et al., 1985): HOMA-IR = (FI (μU/ml) x FBG (mM)/22.5, where FI and FBG represents fasting insulin and fasting blood glucose, respectively. Plasma levels of insulin (Mercodia Rat Insulin ELISA, Mercodia AB, Uppsala, Sweden), glucagon (Glucagon Quantikine® ELISA, R&D Systems Europe, Abington, UK), and leptin (Rat Leptin ELISA, Millipore Corporation, Billerica, MA, USA), GIP (Rat/Mouse GIP (Total) ELISA, Merck Millipore, Billerica, MA, USA) was analyzed according to manufacturer`s instruction. 2.3 Statistics Data were statistically analyzed by one-way ANOVA multiple comparison with vehicle group followed by Dunnet’s post hoc analysis, except figure 6b which was analyzed by multiple comparison ANOVA followed by Tukey’s test. Values of P