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Micellar Nanomedicine of Novel Fatty Acid Modified Xenopus Glucagon-Like Peptide-1: Improved Physicochemical Characteristics and Therapeutic Utilities for Type 2 Diabetes Jing Han, Yingying Fei, Feng Zhou, Xinyu Chen, Weiwei Zheng, and Junjie Fu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00632 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 27, 2017

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Molecular Pharmaceutics

Title: Micellar Nanomedicine of Novel Fatty Acid Modified Xenopus Glucagon-Like Peptide-1: Improved Physicochemical Characteristics and Therapeutic Utilities for Type 2 Diabetes Authors: Jing Han,a* Yingying Fei,a Feng Zhou,a Xinyu Chen,a Weiwei Zheng,a Junjie Fu.b* Affiliation and Address: a

School of chemistry and materials science, Jiangsu Key Laboratory of Green Synthetic Chemistry

for Functional Materials, Jiangsu Normal University, Xuzhou 221116, PR China b

Department of Medicinal Chemistry, School of Pharmacy, Nanjing Medical University, Nanjing

211166, PR China *Corresponding author: School of chemistry and materials science, Jiangsu Key Laboratory of Green Synthetic Chemistry for Functional Materials, Jiangsu Normal University, Xuzhou 221116, PR China Tel: +86-516-83403166; Fax: +86-516-83403166. E-mail: [email protected] (Jing Han) *Corresponding author: Department of Medicinal Chemistry, School of Pharmacy, Nanjing Medical University, Nanjing 211166, PR China Tel: +86-025-86868480 E-mail: [email protected] (Junjie Fu)

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Table of Contents

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ABSTRACT: To develop novel long-acting antidiabetics with improved therapeutic efficacy, two glucagon-like peptide-1 (GLP-1) analogs were constructed through the hybridization of key sequences of GLP-1, xenGLP-1B, exendin-4 and lixisenatide. Hybrids 1 and 2 demonstrated enhanced in vitro and in

vivo biological activities, and were further site-specifically lipidized at lysine residues to achieve prolonged duration of action and less frequent administration. Compared with their native peptides, compounds 3–6 showed similar in vitro activities but impaired in vivo acute hypoglycemic potencies due to decreased aqueous solubility and retarded absorption in vivo. To circumvent these issues, compound 3 (xenoglutide) was selected to be self-associated with sterically stabilized micelles (SSM). The α-helix and solubility of xenoglutide was significantly improved after self-associated

with

SSM.

Notably,

the

improved

physicochemical

characteristics

of

xenoglutide-SSM led to revival of acute hypoglycemic ability without affecting its long-term glucose-lowering activity. Most importantly, preclinical studies demonstrated improved therapeutic effects and safety of xenoglutide-SSM in diabetic db/db mice. Our work suggests the SSM incorporation as an effective approach to improve the pharmacokinetic and biological properties of hydrophobicity peptide drugs. Furthermore, our data clearly indicate xenoglutide-SSM as a novel nanomedicine for the treatment of type 2 diabetics.

Keywords: Glucagon-like peptide-1; Type 2 diabetes; Lipidation; Sterically stabilized micelles;

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Introduction Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by progressive β-cell dysfunction, decreased insulin production and insulin resistance.1, 2 These features result in consistent hyperglycemia in patients.3 Conventional therapeutic agents currently used for treating T2DM have several side effects, such as hypoglycemia, weight gain and peripheral hyperinsulinemia.4 Moreover, they are not able to maintain or improve pancreatic functions.5 Glucagon-like peptide-1 (GLP-1) is a potent anti-hyperglycemic incretin secreted from intestinal L-cells upon ingestion of glucose.6 GLP-1 normalizes postprandial glucose elevation, suppresses glucagon secretion, decreases body weight through inhibiting gastric emptying and inducing satiety, stimulates β-cell proliferation and/or neogenesis, and inhibits β-cell apoptosis.7 Thus, GLP-1 has garnered increasing interest in T2DM treatment. However, the in vivo circulating half-life (t1/2) of GLP-1 is less than 2 min, due to its rapid degradation by dipeptidyl peptidase IV (DPP-IV) and neutral endopeptidase 24.11 (NEP 24.11), as well as rapid renal clearance by glomerular filtration.8 The short t1/2 makes GLP-1 unsuitable for clinical use. To circumvent this issue, the development of long-acting GLP-1 receptor agonists is an emerging theme.9 The actions of amphibian GLP-1s are similar to that of human GLP-1 (hGLP-1). In a previous report, it was found that the Xenopus proglucagon cDNAs encoded three xenGLP-1s (xenGLP-1A, xenGLP-1B, and xenGLP-1C), which had similar sequences to hGLP-1 and activated GLP-1 receptor.10 Particularly, xenGLP-1B exhibited better GLP-1 receptor activation potency and insulinotropic activity than GLP-1 in vitro. By hybridization of GLP-1, xenGLP-1B and exendin-4, our previous studies developed novel GLP-1 receptor agonists with enhanced glucose-lowering activity. Further PEGylation of the hybrids led to moderately improved stability with prolonged t1/2 values.11 In addition to PEGylation, lipidization is another widely used modification strategy to improve the pharmacokinetic properties of peptides. Through the introduction of fatty acid, lipidization facilitates the noncovalent binding of peptides to human serum albumin (HSA). Thus, the proteolytic degradation and renal clearance of lipidized peptides are minimized.12, 4


13

However,

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conjugates from peptides and fatty acids self-assemble into aggregated peptides, resulting in delayed absorption and increased immunogenic response.14 Moreover, the attachment of fatty acid significantly increases the lipophilicity of peptides, causing decreased aqueous solubility. Fortunately, these issues could be tackled by employing a proper peptide delivery system, such as the sterically stabilized phospholipid micelle (SSM).15 Using PEGylated phospholipid as a peptide delivery vehicle, SSM could enhance the proteolytic stability of loaded peptides, reduce the immunogenicity and toxicity, and improve the bioactivity.16 In one of our previous work, SSM induced a significant conformational change of GLP-1 from random coil to α-helix, resulting in improved antidiabetic activity.17 Based on these findings, in the present study, we designed two novel GLP-1 receptor agonists (peptides 1 and 2) by hybridizing the key sequences of GLP-1, xenGLP-1B, exendin-4 and lixisenatide. In vitro and in vivo biological activities of the two peptides were explored. To further improve their stability, site-specific lipidization was performed, affording compounds 3–6. To overcome the solubility issues and the possible immunogenicity/toxicity concerns, SSM system was used to incorporate compound 3 (named as xenoglutide), generating a nanoformulation xenoglutide-SSM. Our results demonstrated that the α-helix of xenoglutide was significantly enhanced after self-association with SSM, accompanied by improved in vivo stability and biological activities. Most importantly, preclinical studies revealed improved therapeutic effects of xenoglutide-SSM in db/db mice.

Materials and methods

Materials and animals GLP-1(7-36)-NH2, exendin-4, lixisenatide and liraglutide were purchased from GL biochem (Shanghai, China). DSPE-PEG2000 was purchased from Xi'an ruixi Biological Technology Co., Ltd (Xi'an, China). cAMP dynamic kit was obtained from Cisbio (Bedford, MA, USA). Mouse insulin ELISA kit was purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). All other reagents, unless otherwise indicated, were obtained from Sigma-Aldrich Co. (Saint Louis, MO, 5



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USA) and used as received. Sprague-Dawley rats (200–250 g) and normal Kunming (20–25 g) mice were purchased from the Comparative Medical Center of Yangzhou University (Jiangsu, China). Male C57BL/6J-m+/+ Leprdb (db/db) mice (7–9 weeks old) were purchased from Model Animal Research Center of Nanjing University (Jiangsu, China). Animals were housed in a thermostatically controlled room (25 ± 2°C) with a 12:12 h light/dark cycle, and had free access to food (standard laboratory chow) and drinking water. Before the experiment began, all animals were acclimating for one week. All animal experiments protocols were conducted according to the Laboratory Animal Management Regulations in China, the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (revised 2011), and approved by an ethical committee at Jiangsu Normal University.

Peptides 1–2 and fatty acid modified compounds 3–6 synthesis Peptides 1–2 and compounds 3–6 were synthesized on a PSI-200 peptide synthesizer (Peptide Scientific Inc., USA) using the Fmoc solid-phase peptide synthesis (SPPS) methodology.18 For peptides 1–2, 0.1 mmol Rink amide resin (loading: 0.37 mmol·g-1) was placed in synthesizer vessel and swollen with 10 mL CH2Cl2 for 10 min and an additional 10 mL CH2Cl2 for 60 min. Fmoc amino acids (0.4 mmol) were coupled using DIC (0.4 mmol) and HOBT (0.44 mmol) in DMF for 2 h under N2 protected at room temperature. Fmoc group deprotections were performed using 7 mL 20% piperidine/DMF (v/v) for 5 min, followed by an additional 7 mL 20% piperidine/DMF (v/v) for 15 min. After each coupling and deprotecting step, resins were washed with 7 mL DMF for 5 times. For compounds 3–6, the synthesis procedure of peptide backbone was similar, with the exception that the Lys at the acylation site was replaced with Fmoc-Lys(Dde)-OH, and the N-terminal His was replaced with Boc-His(Boc)-OH. Upon completion of the peptide chain, the Dde protecting group was selectively removed by using 7 mL 2% hydrazine hydrate/DMF (v/v). The resin was mixed with 7 mL 2% hydrazine hydrate/DMF for 10 min, washed with 10 mL DMF, and the Dde deprotection procedure was repeated with 5 times. Next, Fmoc-Glu-OtBu (0.4 mmol) was dissolved with DIC (0.4 mmol) and HOBT (0.44 mmol) in DMF, added into the reaction 6


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vessel, and mixed with resin for 2 h. The Fmoc group was removed and palmitic acid (0.4 mmol) was added and coupled using DIC (0.4 mmol) and HOBT (0.44 mmol) in DMF for 2 h. Finally, the peptides 1–2 and compounds 3–6 were cleaved using the same procedure. The resin was washed with

10

mL

DCM

for

four

times,

and

dried

under

N2

flow.

Reagent

K

(EDT/phenol/water/thioanisole/TFA, 2.5:5:5:5:82.5, 5 mL) was added in the vessel and mixed with resin for 2 h, the crude peptides and conjugates were precipitated using ether (30 mL) followed by centrifugation. The crude products were purified by Shimadzu semi preparative RP-HPLC (LC-20AP) and characteristic by Agilent HPLC (1260 infinity), Bruker MicroTOF Q2 or Thermo LC-MS using ESI ionization method.

Preparation, characterization, circular dichroism analysis and solubility test of compound 3 (xenoglutide) in phospholipid nanomicelles DSPE-PEG2000 (56 mg) was dispersed in 500 µL tetrahydrofuran by sonication for 5 min to get solution A, and xenoglutide (0.52 mg) was dissolved in 500 µL tetrahydrofuran and then added to solution A under ultrasonic bath at 25 °C for 5 min. Then, 3 mL saline was slowly added to the above solution under vigorously shaking and the resulting solution was vigorously stirred in the dark for 12 h to evaporate tetrahydrofuran and form xenoglutide-SSM. The micelle size of xenoglutide-SSM was determined by a Zetasizer Nano-ZS from Malvern Instruments. Data were analyzed by intensity and volume weighted distributions.19 The secondary structure of xenoglutide and xenoglutide-SSM was determined using Jasco J-810 Spectropolarimeter (Jasco, Oklahoma City, USA) at room temperature.20 Spectra were scanned in a 1 mm path length fused quartz using the following conditions: 190–250 nm at 1 nm bandwidth and 1 s response time over three runs. The spectra were corrected for blank SSM scans and saline. For solubility test, xenoglutide and xenoglutide-SSM were dissolved in 1 mL of PBS (pH 7.4) until saturation.12 Samples (n = 2) were sonicated for 0.5 h, and the pH values were readjusted to 7.4. After centrifuged at 3500 rpm for 10 min, 20 µL of each samples supernatant were analyzed by Agilent HPLC using a C18 column (Thermo C18 reversed-phase column, 5 µm, 150 mm × 4.6 mm), and the absorbance at 254 nm of 7



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the supernatant was determined. The concentration of xenoglutide was determined according to the absorbance-concentration standard curve.

GLP-1 receptor activation assay The GLP-1 receptor activation assay was performed using a previously described method.21 Briefly, HEK293 cell line that stably expressing GLP-1 receptor was used for functional assay. Cells were kept in DMEM growth medium (Invitrogen, Carlsbad, CA, USA) supplemented with 0.5% FBS, 1% penicillin−streptomycin (Gibco, Grand Island, NY, USA), 20 mmol·L-1 HEPES (Sigma, Saint Louis, MO, USA) and 2 mmol·L-1 L-glutamine (Sangon Biotech, Shanghai, China) at 37 °C in 5% CO2 incubator. On the day of measurement, HEK293 cells were resuspended and transferred to 384-well microplates, and the tested peptides were solubilized in DMSO and further diluted into a range of concentrations, then added into cells followed 20 min incubation. cAMP dynamic 2 kit was used to determine the amount of cAMP in each sample. Fluorescence was determined by Envision 2104 Multilabel Reader using homogenous time resolved fluorescence technology. The fluorescence data in each sample were converted into cAMP concentration according to the standard curve standard curve by plotting delta F% versus cAMP concentration. The potency of the tested peptides (EC50) was determined by GraphPad Prism version 5.0 (GraphPad, San Diego, CA) through sigmoidal curve fitting.

Intraperitoneal glucose tolerance tests (IPGTT) in normal Kunming mice The in vivo hypoglycemic activities of exendin-4, lixisenatide, liraglutide, peptides 1–2 and compounds 3–6 were evaluated by IPGTT on Kunming mice, as previously described.22 In brief, male Kunming mice (20–25 g) were randomly allocated to nine groups (n = 6) and acclimated for one week. Before the experiment began, each group of mice were fasted 12 h followed by i.p. administered with saline (control), exendin-4, lixisenatide, liraglutide, peptides 1–2 and compounds 3–6 (25 nmol·kg-1), respectively, 30 min prior to i.p. glucose challenge (2 g·kg-1). Blood glucose levels were determined by collecting blood samples from the cut tip of the tail vein and analyzing 8


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by one-touch blood glucose monitor (Ascensia, Breeze 2, Bayer, Germany) at -30, 0, 15, 30, 60, 120 and 180 min.

Multiple IPGTT in Kunming mice The long term in vivo hypoglycemic activities of exendin-4, lixisenatide, liraglutide, peptides 1–2 and compounds 3–6 were evaluated by multiple IPGTT on Kunming mice, as previous described.22 In brief, male Kunming mice (20–25 g) were fasted 12 h followed by i.p. injected with saline (control), exendin-4, lixisenatide, liraglutide, peptides 1–2 and compounds 3–6 (25 nmol·kg-1) at t = -0.5 h, respectively. Glucose (2 g·kg-1) was i.p. loaded at t = 0 h. Blood glucose levels were determined at -0.5, 0, 0.25, 0.5, 1, 2 and 3 h by using a one-touch blood glucose monitor. Then mice were subjected to an additional i.p. glucose loads (2 g·kg-1) at 6 h, and the blood collect intervals were identical with the first IPGTT.

Glucose tolerance and insulinotropic activity tests The acute in vivo hypoglycemic and insulinotropic activities of xenoglutide and xenoglutide-SSM were evaluated by IPGTT on db/db mice, using a previously described method.23 Briefly, male db/db mice (9 weeks old) were randomly divided into four groups (n = 6). After fasted 18 h, one group of mice was i.p. injected with xenoglutide (25 nmol·kg-1) 180 min prior to glucose challenge (i.p.), and the other group of mice were i.p. injected with saline (control), liraglutide, and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) 30 min prior to glucose challenge (i.p.). Glucose (1 g·kg-1) was i.p. challenge at 0 min, and blood was collected at -30, 0, 15, 30, 60, and 120 min for blood glucose levels measurement. The plasma insulin levels of each group of mice were determined by collected blood samples (~50 µL) at 0, 5, 10, 15, 30, 60, and 120 min from the cut tip of the tail vein into EDTA-containing microcentrifuge tubes, and plasma was obtained by centrifugation (1250 × g, 5 min). Mouse insulin ELISA kit was used to assay the plasma insulin concentrations.

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Pharmacokinetics Assessment The pharmacokinetic properties of liraglutide, xenoglutide and xenoglutide-SSM were assessed in male SD rats (200–250 g, n = 3).24 Briefly, SD rats were fasted overnight, liraglutide, xenoglutide (50 nmol/rat) and xenoglutide-SSM (50 nmol of xenoglutide/rat) were s.c. injected, and the sterilized saline was used as dosing vehicle. Blood samples (~ 0.1 mL) were obtained at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 h and collected in EDTA-containing tubes. Plasma was prepared by centrifugated at 4 °C and stored at -20 °C until subjected to analysis. Plasma concentrations of liraglutide, xenoglutide and xenoglutide in SSM were determined using the GLP-1 receptors activation assay described above. The cAMP-tested peptide concentrations standard curve was generated by adding different concentrations of tested peptide standards into plasma, and then using the same method described in receptors activation assay to get the according cAMP concentrations. The concentrations of tested peptides in plasma samples were obtained through comparing the concentration of cAMP in each group samples against the cAMP-tested peptide concentrations standard curve. The pharmacokinetic parameters of liraglutide, xenoglutide and xenoglutide in SSM were calculated by Bioavailability Program Package software (BAPP, Version 2.2, Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University).

Food intake tests The acute effects of liraglutide, xenoglutide and xenoglutide-SSM were determined on male

db/db mice (8 weeks old).12 Mice were housed individually in cages and randomly allocated to four groups according to their body weight. During the acclimated period (one week), each group of mice were pricked a couple of times to accustom the i.p. dosing procedure. Mice were fasted 18 h prior to the experiment with free access to water. Each group of mice were i.p. administered with saline (control), liraglutide, xenoglutide (25 nmol·kg-1), and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide), and the pre-weighed chow were immediately placed in the cage. The amount of cumulated food intake was measured at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 18, and 24 h.

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Long-term glucose-lowering duration tests The hypoglycemic duration of liraglutide, xenoglutide and xenoglutide-SSM were measured by multiple oral glucose tolerance tests (OGTT) on db/db mice (8 weeks old), as previously described.25, 11 In order to mimic the multiple-meal-a-day pattern of human diet, glucose was orally administered and repeated every 6 h within 24 h. Briefly, mice were fated 18 h, and i.p. injected with saline (control), liraglutide, xenoglutide (25 nmol·kg-1), and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) at -0.5 h, and then orally administered glucose (1.5 g·kg-1, 0 h). Blood glucose levels of each group mice were measured using the above-mentioned method at 0, 0.25, 0.5, 1, 2, and 3 h after glucose administration. The oral glucose loads were repeated at 6, 12 and 18 h, and the blood collect intervals were identical after each glucose load. Furthermore, the blood glucose areas under the curve (AUC) values of each group were calculated by GraphPad Prism version 5.0 (GraphPad, San Diego, CA).

In vitro cell viability assay and in vivo acute toxicity test Cell viability assay was performed using a previously reported method with some modification.26 Rat pancreatic INS-1 cells were maintained at 37 °C in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 50 U/mL penicillin, 50 µM β-mercaptoethanol and 50 U/mL streptomycin in a humidified atmosphere (5% CO2). Cells were seeded in a 96 well plate and incubate for 24 h (~5000 cells/well), then cells were treated with xenoglutide-SSM (10, 100, 500 nM), or treated with glucotoxicity media (30 mM glucose) followed by the addition of saline, liraglutide (100 nM) or xenoglutide-SSM (10, 100, 500 nM). After incubation for 24 h, MTT solutions (5 mg·ml-1) were added followed by incubation at 37 °C for 5 h. The reaction was stopped and cells were re-suspended in DMSO, then the plates were agitated on plate shaker for 30 min. The viability (color intensity of formazan) was determined at 570 nm by a spectrophotometer (Thermo Labsystems, MA, USA). The acute toxicity assay of xenoglutide-SSM in vivo was performed using a previously reported method with some modification.27 Male db/db mice (9 weeks, 35–45 g, n = 3) were randomly 11



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divided into three groups. After overnight fasting for 18 h, one group of mice were i.p. administered xenoglutide-SSM at a dose of 100 mg·kg-1 (low-dose group), the other group were i.p. administered xenoglutide-SSM at a dose of 1000 mg·kg-1 (high-dose group). The mice in control group were i.p. administered equal volumes of saline. All mice were allowed to free access to food and water. At 48 h after injection, all mice were sacrificed and the blood samples were collected and centrifugation to separate serum samples. The serum creatinine (SCr), blood urea nitrogen (BUN), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were determined by using Hitachi 7060 Automatic Analyzer (Tokyo, Japan).

Chronic in vivo studies and histologic analyses The long-term treatment effects of liraglutide, xenoglutide and xenoglutide-SSM were evaluated on db/db mice.26 Male 8 weeks old db/db mice were randomized divided into four groups (n = 6). Based on the pharmacokinetics profiles of tested peptides and the relative high metabolic rate of rodent, animals were twice daily s.c. injected saline (control), liraglutide, and xenoglutide (25 nmol·kg-1), and xenoglutide-SSM (50 nmol·kg-1 of xenoglutide) was s.c. injected once daily over a 5-week period. The HbA1c values in each group were measured at day 0 and 36 by obtained ~ 10 µL blood samples from the tail vein of mice and determined by a DCA 2000+ chemistry analyzer (Bayer Diagnostics, USA). The amount of food consumption and body weight were recorded every two days. Moreover, in each group of mice, approximately 50 µL of blood was collected at 7 am every two days and the non-fasting blood glucose levels and plasma insulin levels were measured using the method described in glucose tolerance and insulinotropic activity tests section. At the end of the treatment, mice were subjected to an IPGTT to evaluate the glucose tolerance and insulinotropic abilities. Mice were fasted overnight and glucose was i.p. loaded at 0 min. The blood glucose and plasma insulin levels in mice were measured at 0, 15, 30, 60 and 120 min using the same method described above. Finally, the blood in each group of mice was collected in tubes by retroorbital puncture for biochemical analysis. Then mice were immediately sacrificed, and the pancreas was removed. For 12


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biochemical analysis, serum was separated by centrifugation, and several key indexes (triglycerides (TG), serum total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, AST, ALT) were determined by using Hitachi 7060 Automatic Analyzer (Tokyo, Japan). For histochemical analysis, the methodology was described elsewhere.17 Briefly, pancreas samples were embedded in paraffin blocks, then cut in pancreas sections (thickness 3 µm). The pancreas sections were stained with hematoxylin-eosin (HE) for histopathological assessment and the area and number of islets was measured by Olympus DP2-BSW digital camera software (Olympus, Center Valley, PA).

Statistical analysis All data were presented as means ± SD. Statistical analysis was carried out using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA), and one-way ANOVA for multiple comparisons with post hoc Tukey’s tests was used to perform significance test of pharmacology data. P < 0.05 was considered statistically significant.

Results

Synthesis and bioactivities of hybrids 1 and 2 Peptides 1 and 2 were designed by hybridizing the key amino acid sequences of GLP-1, xenGLP-1B, exendin-4 and lixisenatide (Figure 1). Peptide 1 differs from 2 in their C-terminal tails (from position 36), which were adopted from exendin-4 and lixisenatide, respectively. Hybrids 1 and 2 were synthesized by using standard Fmoc SPPS methodology, and Rink amide resin was used to yield peptides with amidated C-terminus. The crude peptides were cleaved from the Rink resin by Reagent K, and purified via semi preparative RP-HPLC. The purified products were further characterized by HPLC and ESI-MS (see Supporting Information). To assess the in vitro receptor activation potency of hybrids 1 and 2, each peptide was subjected to GLP-1 receptor activation potency test by using HEK293 cell line that stably expresses GLP-1 receptor. As shown in Table 1, both 1 (EC50 = 2.1 ± 0.3 nM) and 2 (EC50 = 1.1 ± 0.2 nM) showed 13



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high potency in receptor activation, superior to that of exendin-4 (EC50 = 4.3 ± 0.4 nM) and lixisenatide (EC50 = 3.5 ± 1.0 nM). The in vivo hypoglycemic activities of peptides 1 and 2 were evaluated by IPGTT in Kunming mice. As shown in Figure 2A, the blood glucose level in saline treated mice rapidly increased and peaked at 21.33 ± 1.95 mmol·L-1 after 15 min, while exendin-4, lixisenatide, peptides 1 and 2 dramatically enhanced glucose tolerance and normalized average glucose levels to ~9.4, ~8.4, ~6.9 and ~5.9 mmol·L-1 respectively. Consistent with the receptor activation potency results, calculated glucose AUC0-180

min

values further revealed that the

antidiabetic effects of peptides 1 and 2 were better than exendin-4 and lixisenatide in vivo. Furthermore, a multiple IPGTT was performed to better investigate the in vivo glucose-stabilizing activity of peptides 1–2 (Figure 2C). In the first IPGTT, exendin-4, lixisenatide and peptides 1–2 exhibited similar glucose-lowering activities. Both exendin-4 and lixisenatide showed significantly reduced antidiabetic activities during 6–12 h, while the antidiabetic effects of peptides 1 and 2 were only moderately compromised. Furthermore, the AUC0–12h glucose values showed that the long-term hypoglycemic effects of peptide 2 were slightly better than peptide 1.

Put Figure 1 here Put Figure 2 here Put Table 1 here

Preparation and biological activities of compounds 3–6 Site specific lipidization of 1 and 2 by palmitic acid was performed at Lys20 or Lys28. To this end, Fmoc-Lys(Dde)-OH was introduced into positions 20 or 28 during the peptide backbone construction. The Dde group could be specifically removed by 2% hydrazine/DMF, thereby releasing the free amino group of Lys residue without affecting other protecting groups (Figure 3). To compensate the water solubility decrease resulted from lipidization, γ-L-Glutamic acid (γ-Glu) was incorporated as a hydrophilic spacer between the palmitic acid and the polypeptide. The crude

14


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conjugates were cleaved, purified and characterized using the same method described above (see Supporting Information). The same receptor activation potency test was used to assess the in vitro bioactivities of compounds 3–6. As shown in Table 1, the potency of compounds 3–6 was only slightly reduced as compared with their native peptides 1–2. Moreover, compounds 3 and 5 were more potent than compounds 4 and 6, suggesting that the receptor activation potency of lipidized conjugates could be almost fully maintained when the acylation site was at Lys20 (EC50 values of compounds 3 and 5 were 2.3 ± 0.7 and 1.9 ± 0.4 nM, respectively). The acute and long-term antidiabetic activities of compounds 3–6 were studied in Kunming mice. As shown in Figure 4A, compared with the control group, treatment with liraglutide efficiently reduced the glucose level during 0–180 min. However, compounds 3–6 did not exhibit noticeable glucose-lowering effects. Calculated AUCglucose 0–180 min values revealed that the hypoglycemic effects of compounds 3–6 were weaker than liraglutide. In multiple IPGTT, compounds 3–6 did not show notable antidiabetic activities in the first IPGTT. However, in the second IPGTT, compounds 3–6 treatments significantly enhanced glucose tolerance, better than liraglutide. The average glucose levels in liraglutide and compounds 3–6 treatment groups at 0.25 h after the second glucose challenge were ~10.6, ~8.2, ~9.2, ~6.1 and ~6.9 mmol·L-1, respectively. Taken together, the long-term glucose-lowering activity of compounds 3–6 was guaranteed, but their acute (0.25–3 h post treatment) antidiabetic activity was limited, which might be attributed to their slow absorption after self-aggregation. The acute antidiabetic activity of compounds 5 and 6 was slightly better than compounds 3 and 4 (Figure 4B). This might result from the Lys-rich C-terminal tails (PSSGA PPSKK KKKK) of compounds 5 and 6, which improved their water solubility and absorption rate. Compound 3 (named as xenoglutide), which showed a low acute hypoglycemic activity, but high long-term glucose-controlling ability and receptor activation potency, was selected as an interesting model peptide to study the effects of SSM modification.

Put Figure 3 here Put Figure 4 here 15



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Characterization, secondary structure analysis and solubility test of xenoglutide in phospholipid nanomicelles The mean particle sizes of blank SSM and xenoglutide-SSM in saline were analyzed by dynamic light scattering (DLS). The mean diameter of blank SSM was 19.5 ± 1.9 nm, and xenoglutide-SSM displayed a similar mean diameter of 22.8 ± 0.7 nm (see Supporting Information, Figures S1 and S2). The secondary structures of xenoglutide and xenoglutide-SSM were determined using circular dichroism (CD) spectroscopy. Xenoglutide exhibited a random coil secondary conformation in saline, while the CD spectra of xenoglutide-SSM showed deepened negative bands at approximately 208 and 222 nm, which are representative of a predominantly α-helical secondary conformation (see Supporting Information, Figure S3). The effect of lipidation and self-association with SSM on the aqueous solubility of xenoglutide was tested. Xenoglutide and xenoglutide-SSM were dissolved in PBS until saturation, and the concentrations of xenoglutide and xenoglutide-SSM were calculated by measuring the absorbance at 254 nm. The solubility of xenoglutide was 0.2 mg·mL-1, while the solubility of xenoglutide-SSM reached 3.9 mg·mL-1. These results indicated that the solubility of xenoglutide was significantly improved after SSM encapsulation.

Glucose-lowering and insulin secretion assay in db/db mice To further evaluate the hypoglycemic and insulinotropic activities of xenoglutide and xenoglutide-SSM, an IPGTT was performed in db/db mice, using liraglutide as the positive control. In view of the slow absorption property of xenoglutide, xenoglutide was i.p. administered 180 min prior to glucose challenge, while saline, xenoglutide-SSM, and liraglutide were i.p. administered 30 min prior to glucose challenge. As shown in Figure 5A, blood glucose levels in the saline group rapidly increased to 30.3 ± 0.8 mmol·L-1 0.5 h after glucose load, and then decreased slowly. Liraglutide, xenoglutide and xenoglutide-SSM dramatically reduced average blood glucose levels to ~13.3, ~11.0, and ~8.7 mmol·L-1 0.5 h after the glucose load, respectively. These results suggested that xenoglutide-SSM has a better in vivo hypoglycemic activity than liraglutide and xenoglutide. 16


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The time courses of plasma insulin levels in each group were also recorded. As shown in Figure 5C, the plasma insulin levels in xenoglutide and liraglutide treatments peaked 30 min after the glucose challenge. The plasma insulin levels in mice treated with xenoglutide-SSM increased more rapidly and peaked 15 min post glucose challenge. These results suggested that the absorption rate of xenoglutide-SSM was faster than xenoglutide and liraglutide. Calculated AUC values of glucose and insulin (Figures 5B and D) further confirmed the improved bioactivities of xenoglutide-SSM than xenoglutide.

Put Figure 5 here

In vivo pharmacokinetics The pharmacokinetic profiles of xenoglutide and xenoglutide-SSM were examined in SD rats. As shown in Figure 6A and Table 2, both liraglutide and xenoglutide showed delayed absorption patterns after injection with Tmax values of 3.7 ± 0.6 h and 6.0 ± 0.1 h, respectively. In contrast, xenoglutide-SSM exhibited a different pharmacokinetic profile. The plasma concentrations of xenoglutide-SSM increased rapidly and peaked within 2 h (Tmax = 1.5 ± 0.5 h), followed by a “platform release” pattern during 1−8 h and a slow decrease to baseline within 24 h. The half-life of xenoglutide-SSM (t1/2 = 12.2 ± 0.7 h) was significant longer than liraglutide (t1/2 = 6.6 ± 0.5 h) and xenoglutide (t1/2 = 7.5 ± 0.5 h). Furthermore, dramatic AUC values were observed for xenoglutide-SSM. The AUCinf value of xenoglutide-SMM was approximately 2.0- and 1.9-fold greater than that of liraglutide and xenoglutide, respectively, indicating an improved drug utilization of xenoglutide-SMM.

Put Figure 6 here Put Table 2 here

Anorectic effect of xenoglutide-SSM 17



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A food intake study in db/db mice was conducted to compare the anorectic effects of xenoglutide and xenoglutide-SSM. As shown in Figure 6B, both liraglutide and xenoglutide potently reduced the food intake as compared with saline. The anorectic effect of liraglutide was better than xenoglutide during 0–6 h. However, xenoglutide exhibited a better anorectic effect than liraglutide during 8–24 h. The delayed anorectic effect of xenoglutide might be attributed to the slow absorption rate. Importantly, the acute anorectic effect of xenoglutide was improved after self-association with SSM, and the anorectic effect of xenoglutide-SSM was better than liraglutide and xenoglutide during the whole experiment period. Furthermore, administration of xenoglutide or xenoglutide-SSM decreased cumulative food intake by 48–55%, while liraglutide reduced food intake by 42%.

Long-term glucose-lowering effects of xenoglutide-SSM A multiple OGTT was performed to assess the long-acting hypoglycemic activity of xenoglutide-SSM. As shown in Figure 7, each glucose load led to a hyperglycemic state in db/db mice treated with saline. In line with the results in Figure 4, xenoglutide did not exhibit acute glucose-lowering activity in the first OGTT, but showed comparable hypoglycemic activity to liraglutide in the second and third OGTT. Both liraglutide and xenoglutide showed reduced hypoglycemic activities in the last OGTT. Xenoglutide-SSM excellently maintained the blood glucose levels below 8 mmol·L-1 during the whole experiment period. Calculated AUCglucose values further proved that the long-acting antidiabetic effect of xenoglutide-SSM was much greater than liraglutide and xenoglutide (Figure 7C).

Put Figure 7 here

In vitro and in vivo toxicity tests The in vitro toxicity effect of xenoglutide-SSM was evaluated by MTT assay in INS-1 cells. As shown in Figure 8, 10, 100, and 500 nM concentration of xenoglutide-SSM did not reduce the cell 18


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viability and showed no prominent toxicity effect. We further investigated the protective effect of xenoglutide-SSM on cell viability under glucotoxicity condition (30 mM glucose). The cell viability in saline group was significant decreased by ~23%. In the presence of 100 and 500 nM xenoglutide-SSM, cell viabilities were slightly decreased by < 10%. The protective effect of xenoglutide-SSM was similar to that of liraglutide (100 nM). To test the acute hepatic and renal toxicities of xenoglutide-SSM, SCr and BUN (index for renal toxicity), AST and ALT (index for hepatic toxicity) values in mice treated with low (100 mg·kg-1) or high (1000 mg·kg-1) dose of xenoglutide-SSM were determined. As shown in Table 3, compared with the control group, no significant difference was observed in terms of SCr, BUN, AST and ALT levels after the low or high dose of xenoglutide-SSM administration, suggesting the safety of xenoglutide-SSM.

Put Figure 8 here Put Table 3 here

Chronic in vivo studies To assess the chronic effects of peripherally administered xenoglutide-SSM, a 35-day chronic treatment of liraglutide, xenoglutide and xenoglutide-SSM on db/db mice was performed. Based on their pharmacokinetic properties and the relative high metabolic rate of rodent, liraglutide and xenoglutide were i.p. given twice daily (25 nmol·kg-1), and xenoglutide-SSM was i.p. administrated once a day (50 nmol·kg-1 of xenoglutide). Chronic i.p. treatments with liraglutide, xenoglutide and xenoglutide-SSM decreased HbA1c, food intake and body weight in db/db mice (Figure 9A–C). In addition, non-fasting blood glucose levels were markedly decreased from day 2 after the first injection in liraglutide, xenoglutide and xenoglutide-SSM treatments, accompanied by prominently increased non-fasting plasma insulin concentrations (Figure 9D, E). Interestingly, both the non-fasting plasma insulin concentrations and the non-fasting blood glucose levels in xenoglutide and xenoglutide-SSM treatments were lower than liraglutide group from day 24, suggesting 19



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improved insulin sensitivity. To test the glucose tolerance ability of mice after the chronic treatment, each group of mice were subjected to an IPGTT at day 37. As shown in Figure 9F, mice treated with liraglutide, xenoglutide and xenoglutide-SSM had smaller glucose excursions than the control group. Furthermore, xenoglutide-SSM treatment resulted in the lowest AUCglucose value. Biochemical analysis in follow-up experiments revealed that xenoglutide-SSM treatment significantly decreased serum TG, LDL cholesterol and TC as compared with control (P < 0.001, P < 0.001 and P < 0.001, respectively, Figure 10), but had no prominent effect on HDL cholesterol. Importantly, the AST and ALT levels in xenoglutide-SSM treated mice were slightly lower than saline, liraglutide and xenoglutide groups, indicating the safety of xenoglutide-SSM to be used as a novel antidiabetic formulation. Finally, histological examination further revealed that the islet area and number markedly increased after xenoglutide-SSM treatment (P < 0.001 and P < 0.001, respectively, as compared with control, Figure 10). Representative images of HE staining are shown in Figure S4 (see Supporting Information).

Put Figure 9 here Put Figure 10 here

Discussion The unique antidiabetic effects of GLP-1 receptor agonists make them important therapeutics for treating T2DM.28 Intensive research efforts are focused on developing of novel long-acting GLP-1 receptor agonists. However, all the GLP-1 receptor agonists or their sustained release preparations in clinical use or in clinical development were based on peptide sequences of human GLP-1 or Gila exendin-4.7 The actions of GLP-1 in amphibians are more consistent with those of seen in mammals. Through characterization of the Xenopus proglucagon cDNAs, Michael B. Wheeler et al. successfully encoded three GLP-1-like peptides (xenGLP-1A, xenGLP-1B, and xenGLP-1C), and found that xGLP-1B was a fully GLP-1 receptor agonist with potent insulinotropic activities.10 Inspired by the our previous research on xGLP-1B derivatives, in the present study, with the 20


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specific aims of developing novel long-acting GLP-1 receptor agonists, we explored the hybridization of GLP-1, xenGLP-1B, exendin-4 and lixisenatide into a single molecule. Two hybrid peptides (peptides 1–2) with exendin-4 or lixisenatide C-terminal tail were designed and synthesized. We found that both peptides 1–2 exhibited better GLP-1 receptor activation and glucose lowering potency than exendin-4 and lixisenatide. Importantly, the in vivo long-acting hypoglycemic activities of peptides 1–2 were also superior to those of exendin-4 and lixisenatide. The improved biological activities and stabilities of peptides 1–2 may be attributed to the unique amino acid residues in their sequences, such as Thr11, Glu12, Glu15 and Ile27. Introducing long-chain fatty acids into peptides could facilitate the noncovalent binding of peptides to serum albumin, and hence, prevent renal excretion and proteolytic degradation of peptide drugs.29 Inspired by this, four lipidated hybrid peptides (compounds 3–6) were generated by acylation with a γ-glutamyl-spaced palmitic acid on Lys20 or Lys28. The γ-glutamyl spacer was expected to partly compensate the decrease in aqueous solubility due to lipidization. The GLP-1 receptor activation potency of compounds 3–6 were only slightly reduced as compared with their parent peptides. We observed that the functional potency of compounds 3–6 decreased as the lipidation site was closer to the C-terminus. These findings were in agreement with the previous research concerning lapidated peptide analogs of GLP-1.30 However, compounds 3–6 did not showed prominent acute glucose-lowering activities, reflecting the slow absorption rates of compounds 3–6. Hence, the introduction of a γ-glutamyl spacer was not enough to achieve a desired aqueous solubility, and methods to decrease the hydrophobicity of compounds 3–6 remained to be explored. Previously, we successfully constructed a formulation of GLP-1 in SSM. The α-helical secondary structure of GLP-1 was enhanced after being self-associated with micelles, accompanied by improved in vivo stability and biological activities. Inspired by these results, and considering the high water solubility and the extremely low critical micelle concentration of SSM (~ 1 µM),31 compound 3 (xenoglutide) was selected to be self-associated with SSM. The α-helical secondary structure and water solubility of xenoglutide were significantly improved after being self-associated 21



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with SSM. Importantly, the increased α-helicity of xenoglutide-SSM successfully increased the antidiabetic effect and insulinotropic activity of xenoglutide. The increased water solubility of xenoglutide-SSM markedly altered the pharmacokinetic properties of xenoglutide, as reflected by the significantly improved Tmax and t1/2 of xenoglutide-SSM. The improved Tmax value of xenoglutide-SSM indicated that the self-assembly property of xenoglutide was minimized, and the possible immunogenic response caused by xenoglutide could also be reduced. Furthermore, the MRTs.c. of xenoglutide-SSM was almost twice than that of liraglutide, indicating the potential of xenoglutide-SSM to be used as a once every two days dosing antidiabetic drug. The data on cumulated food intake further proved that the duration of action of xenoglutide-SSM was superior to that of liraglutide. The long-acting hypoglycemic activity of xenoglutide-SSM was further confirmed by multiple OGTT test in db/db mice. The MTT assay revealed that xenoglutide-SSM had no significant effects on the viability of INS-1 cells under normal condition. Importantly, high concentration of xenoglutide-SSM exhibited comparable cell viability restoring ability to liraglutide under high glucose condition. In vivo acute toxicity study showed that xenoglutide-SSM had no adverse effects (hepatic and renal toxicities) on

db/db mice following a single oral dose of 100 or 1000 mg·kg-1. To precisely evaluate the toxicity of xenoglutide-SSM, further studies such as chronic toxicity test may be required. Long-term treatment effects of xenoglutide-SSM on db/db mice were evaluated to assess the potential therapeutic

utility

of

xenoglutide-SSM.

The

improved

acute

biological

activities

of

xenoglutide-SSM was reflected by the long-term treatment data. Once daily administration of xenoglutide-SSM effectively reduced the body weight gain and food intake amount. The non-fasting blood glucose levels in xenoglutide-SSM group were lower than liraglutide and xenoglutide from day 2 to day 24, accompanied by higher non-fasting plasma insulin levels. Moreover, chronic treatment of xenoglutide-SSM achieved beneficial effects on HbA1c lowering, indicating the promising role of xenoglutide-SSM in clinical studies. The histological examination results proved that the β cell neogenesis/proliferation could be induced by xenoglutide-SSM

22


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treatment. Finally, biochemical analysis revealed that there was no significant toxicity in mice treated with xenoglutide-SSM, as xenoglutide-SSM resulted in reduced AST and ALT values. The solubility and immunogenicity of peptide drugs were critical issues for clinical use. In the present study, through hybridization of key structures of GLP-1, xenGLP-1B, exendin-4 and lixisenatide, lipidation and self-association with SSM, we successfully constructed a nanoformulation of xenoglutide. The SSM not only improved the therapeutic utilities of xenoglutide, but also reduced the immunogenic response possibilities. Furthermore, xenoglutide-SSM possessed excellent insulinotropic potency, high acute and long-term glucose-lowering abilities, as well as significant long-term therapeutic effects on db/db mice, making it a promising nanomedicine of novel GLP-1 analogs for T2DM therapeutic application.

Supporting Information Characterization of peptides 1-2 and compounds 3-6, particle size analysis data, CD spectra, representative images of HE staining.

Notes The authors declare no competing financial interest.

ORCID ID Jing Han: 0000-0001-5605-1868 Junjie Fu: 0000-0002-2741-7469

Author contributions Jing Han designed the research study. Jing Han, Yingying Fei and Feng Zhou performed the research and analysed the data. Xinyu Chen and Weiwei Zheng performed some part of the research. Jing Han and Junjie Fu wrote the paper.

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Acknowledgment This work is supported by the National Natural Science Foundation of China (81602964 and 81602960), the Natural Science Foundation of Jiangsu Province (Grants No BK20150243 and BK20161028), PAPD of Jiangsu Higher Education Institutions, the Jiangsu Overseas Visiting Scholar Program for University Prominent Young & Middle-aged Teachers and Presidents, and Startup Funding for Introduced Talents of Nanjing Medical University (KY109RC1602).

Abbreviations: T2DM, type 2 diabetes mellitus; GLP-1, glucagon-like peptide-1; DPP-IV, dipeptidyl peptidase IV; NEP 24.11, neutral endopeptidase 24.11; HSA, human serum albumin; SSM, sterically stabilized phospholipid micelles; DSPE-PEG2000, 1,2-Distearoyl-sn-glycero-3-phosphatidylethanolamine-N[methoxy (polyethyleneglycol)-2000] sodium salt; SPPS, solid-phase peptide synthesis; RP-HPLC, reversed phase high-performance liquid chromatography; HPLC high-performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; FBS, fetal bovine serum; cAMP, cyclic adenosine monophosphate; IPGTT, intraperitoneal glucose tolerance test; AUC, area under the curve; OGTT, oral glucose tolerance tests; CD, circular dichroism; Tmax, time to reach maximum plasma concentration; Cmax, maximum plasma concentration; AUCinf, area under the curve from zero to infinity; t1/2, elimination half-life; MRTs.c., mean residence time by subcutaneous injection; SCr, serum creatinine; BUN, blood urea nitrogen; TC, serum total cholesterol; TG, triglycerides; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HE, hematoxylin-eosin;

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28. Murage, E. N.; Gao, G.; Bisello, A.; Ahn, J.-M., Development of potent glucagon-like peptide-1 agonists with high enzyme stability via introduction of multiple lactam bridges. Journal

of Medicinal Chemistry 2010, 53 (17), 6412-6420. 29. van Witteloostuijn, S. B.; Pedersen, S. L.; Jensen, K. J., Half ‐ Life Extension of Biopharmaceuticals using Chemical Methods: Alternatives to PEGylation. ChemMedChem 2016. 30. Madsen, K.; Knudsen, L. B.; Agersoe, H.; Nielsen, P. F.; Thøgersen, H.; Wilken, M.; Johansen, N. L., Structure−Activity and Protraction Relationship of Long-Acting Glucagon-like Peptide-1 Derivatives: Importance of Fatty Acid Length, Polarity, and Bulkiness. Journal of Medicinal

Chemistry 2007, 50 (24), 6126-6132. 31. Lim, S. B.; Rubinstein, I.; Sadikot, R. T.; Artwohl, J. E.; Önyüksel, H., A Novel Peptide Nanomedicine Against Acute Lung Injury: GLP-1 in Phospholipid Micelles. Pharmaceutical

Research 2011, 28 (3), 662-672.

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Figure legends and Tables

Figure 1. Structures of the peptides 1–2 and compounds 3–6. Figure 2. Acute and long-term hypoglycemic activities of exendin-4, lixisenatide and peptides 1–2 in Kunming mice. Saline, exendin-4, lixisenatide, peptides 1–2 (25 nmol·kg-1) were i.p. administered 30 min prior to i.p. glucose load (2 g·kg-1). (A) The time−glucose response curve in IPGTT (-30 to 180 min). (B) The AUCglucose 0-180 min values of each group. (C) The time−glucose response curve in multiple IPGTT (-0.5 to 12 h). (c) The AUCglucose 0-12 h values of each group.

***

P

< 0.001 vs. Saline, aP < 0.001 vs. exendin-4, bP < 0.01 vs. exendin-4, cP < 0.05 vs. lixisenatide. Means ± SD, n = 6. Figure 3. Synthesis route of compounds 3–6. PG, amino acid protecting group. Figure 4. Acute and long-term hypoglycemic activities of liraglutide and compounds 3–6 in Kunming mice. Saline, liraglutide, compounds 3–6 (25 nmol·kg-1) were i.p. administered 30 min prior to i.p. glucose load (2 g·kg-1). (A) The time−glucose response curve in IPGTT (-30 to 180 min). (B) The AUCglucose 0-180 min values of each group. (C) The time−glucose response curve in multiple IPGTT (-0.5 to 12 h). (D) The AUCglucose 0-12 h values of each group. Means ± SD, n = 6. Figure 5. Glucose-lowering and insulinotropic activities of xenoglutide-SSM in db/db mice by IPGTT. Xenoglutide (25 nmol·kg-1) was i.p. administered 180 min prior to glucose challenge. Saline, liraglutide (25 nmol·kg-1) and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) were i.p. injected 30 min prior to 1 g·kg-1 i.p. glucose load. (A) Hypoglycemic activities of liraglutide, xenoglutide (25 nmol·kg-1), and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) in db/db mice. (B) Calculated blood glucose AUC0−120 min values of each group. (C) The time course of plasma insulin values in each group. (D) Calculated AUCinsulin 0-120 min values of each group. Means ± SD, n = 6. ***

P < 0.001 vs. Saline, aP < 0.001 vs. liraglutide. bP < 0.01 vs. liraglutide.

Figure 6. Pharmacokinetic profiles and anorectic effects of liraglutide, xenoglutide and xenoglutide-SSM in vivo. (A) Pharmacokinetic profiles of liraglutide, xenoglutide and xenoglutide-SSM in SD rats after s.c. administration (50 nmol/rat). Means ± SD, n = 3. (B) 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Anorectic effects of liraglutide, xenoglutide (25 nmol·kg-1) and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) in 18 h fasted db/db mice. Means ± SD, n = 6. Figure 7. Long-term glucose-lowering effects of xenoglutide-SSM was evaluated by using multiple OGTT in db/db mice. Saline, liraglutide, xenoglutide (25 nmol·kg-1) and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) were i.p. administered 30 min prior to first oral glucose challenge (1.5 g·kg-1), and the remaining glucose loads were carried out at 6, 12 and 18 h, respectively. (A) The time course of blood glucose levels in each group during 0-12 h. (B) The time course of blood glucose levels in each group during 12-24 h. (C) The AUCglucose 0-24 h values of each group. Means ± SD, n = 6. ***P < 0.001 vs. Saline, aP < 0.001 vs. liraglutide, a’P < 0.001 vs. xenoglutide. Figure 8. Effects of xenoglutide-SSM on cell viability of INS-1 cells. (A) Effects of xenoglutide-SSM (10, 100, and 500 nM) on INS-1 cells under normal condition. (B) Effects of xenoglutide-SSM (10, 100, and 500 nM) on INS-1 cells under high glucose condition (30 nM). All values are expressed in mean ± SD. *P < 0.05 vs. Saline, ***P < 0.001 vs. Saline. Figure 9. Chronic treatment effects of liraglutide, xenoglutide and xenoglutide-SSM in db/db mice for five weeks. (A) HbA1c measured ay day 0 and day 36. (B) Food intake. (C) Body weight. (D) Non-fasting blood glucose values. (E) Non-fasting plasma insulin values. (F) Excursion of blood glucose in IPGTT test after five weeks of treatment (Calculated AUCglucose 0-120 min values). Means ± SD, n = 6. Figure 10. Biochemical analysis in db/db mice following the five weeks treatment. (A) Total cholesterol (TC). (B) Triglyceride (TG). (C) HDL-cholesterol. (D) LDL-cholesterol. (E) Alanine aminotransferase (ALT). (F) Aspartate aminotransferase (AST). (G) Islet number. (H) Islet area. Means ± SD, n = 6. *P < 0.05 vs. control, **P < 0.01 vs. control, ***P < 0.001 vs. control.

30


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Table 1. The GLP-1 receptor activation activity of peptides 1–2 and compounds 3–6. Peptide

Acylation site

EC50a (nM)

Peptide

Acylation site

EC50a (nM)

Exendin-4

/

4.3 ± 0.4

3

20

2.3 ± 0.7

Lixisenatide

/

3.5 ± 1.0

4

28

5.2 ± 1.4

Liraglutide

/

8.8 ± 2.1

5

20

1.9 ± 0.4

1

/

2.1 ± 0.3

6

28

3.2 ± 0.9

2

/

1.1 ± 0.2

a

The receptor potency data are represented as EC50 (means ± SD). All experiments were performed in triplicate

and repeated three times (n = 3).

31



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Table 2. Pharmacokinetic parameters of liraglutide, xenoglutide and xenoglutide-SSM in SD Ratsa Samples

Cmax

Tmax (h)

t1/2 (h)

MRTs.c. (h)

AUCinf (nM)

Liraglutide

112.9 ± 2.6

3.7 ± 0.6

6.6 ± 0.5

10.9 ± 0.6

1391.3 ± 16.2

Xenoglutide

100.2 ± 2.1

6.0 ± 0.1

7.5 ± 0.5

13.0 ± 0.6

1448.4 ± 25.1

Xenoglutide-SSM

131.5 ± 0.6

1.5 ± 0.5

12.2 ± 0.7

18.4 ± 1.0

2799.4 ± 88.6

a

Data are the Means ± SD (n = 3). Tmax, time to reach maximum plasma concentration; Cmax, maximum plasma

concentration; AUCinf, area under the curve from zero to infinity; t1/2, elimination half-life; MRTs.c., mean residence time by subcutaneous injection.

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Table 3. Effect of the xenoglutide-SSM on SCr, BUN, AST, and ALT levels 48 h after injection. Control

Xenoglutide-SSM Xenoglutide-SSM (100 mg·kg-1)

(1000 mg·kg-1)

SCr (µmol·L-1)

21.3 ± 3.6

23.4 ± 2.1

19.1 ± 1.5

BUN (mmol·L-1)

6.3 ± 0.8

5.7 ± 0.4

7.0 ± 0.6

AST (IU·L-1)

91.3 ± 6.8

84.7 ± 5.1

96.8 ± 4.2

ALT (IU·L-1)

53.9 ± 4.1

56.7 ± 5.9

58.4 ± 6.3

a

Data are the Means ± SD (n = 3). SCr, serum creatinine; BUN, blood urea nitrogen; AST, aspartate

aminotransferase; ALT, alanine aminotransferase.

33



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GLP-1

HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR

X enGLP-1B HAEGT YTNDV TEYLE EKAAK EFIEW LIKGK Exendin-4

HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS

Lixisenatide

HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPSKK KKKK Key sequences hybridization

Peptide 1

HGEGT YTNDV TEYLE EEAAK EFIEW LIKGK PSSGA PPPS

Peptide 2

HGEGT YTNDV TEYLE EEAAK EFIEW LIKGK PSSGA PPSKK KKKK Site-specific lipidation

Compound 3 HGEGT YTNDV TEYLE EEAAX EFIEW LIKGK PSSGA PPPS Compound 4 HGEGT YTNDV TEYLE EEAAK EFIEW LIXGK PSSGA PPPS Compound 5 HGEGT YTNDV TEYLE EEAAX EFIEW LIKGK PSSGA PPSKK KKKK Compound 6 HGEGT YTNDV TEYLE EEAAK EFIEW LIXGK PSSGA PPSKK KKKK H N

O O

X= HN HN

OH O

O

Figure 1. Structures of the peptides 1–2 and compounds 3–6.

34


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Figure 2. Acute and long-term hypoglycemic activities of exendin-4, lixisenatide and peptides 1–2 in Kunming mice. Saline, exendin-4, lixisenatide, peptides 1–2 (25 nmol·kg-1) were i.p. administered 30 min prior to i.p. glucose load (2 g·kg-1). (A) The time−glucose response curve in IPGTT (-30 to 180 min). (B) The AUCglucose 0-180 min values of each group. (C) The time−glucose response curve in multiple IPGTT (-0.5 to 12 h). (c) The AUCglucose 0-12 h values of each group.

***

P

< 0.001 vs. Saline, aP < 0.001 vs. exendin-4, bP < 0.01 vs. exendin-4, cP < 0.05 vs. lixisenatide. Means ± SD, n = 6.

35



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Figure 3. Synthesis route of compounds 3–6. PG, amino acid protecting group.

36


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Figure 4. Acute and long-term hypoglycemic activities of liraglutide and compounds 3–6 in Kunming mice. Saline, liraglutide, compounds 3–6 (25 nmol·kg-1) were i.p. administered 30 min prior to i.p. glucose load (2 g·kg-1). (A) The time−glucose response curve in IPGTT (-30 to 180 min). (B) The AUCglucose 0-180 min values of each group. (C) The time−glucose response curve in multiple IPGTT (-0.5 to 12 h). (D) The AUCglucose 0-12 h values of each group. Means ± SD, n = 6.

37



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Figure 5. Glucose-lowering and insulinotropic activities of xenoglutide-SSM in db/db mice by IPGTT. Xenoglutide (25 nmol·kg-1) was i.p. administered 180 min prior to glucose challenge. Saline, liraglutide (25 nmol·kg-1) and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) were i.p. injected 30 min prior to 1 g·kg-1 i.p. glucose load. (A) Hypoglycemic activities of liraglutide, xenoglutide (25 nmol·kg-1), and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) in db/db mice. (B) Calculated blood glucose AUC0−120 min values of each group. (C) The time course of plasma insulin values in each group. (D) Calculated AUCinsulin 0-120 min values of each group. Means ± SD, n = 6. ***

P < 0.001 vs. Saline, aP < 0.001 vs. liraglutide. bP < 0.01 vs. liraglutide.

38


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Figure 6. Pharmacokinetic profiles and anorectic effects of liraglutide, xenoglutide and xenoglutide-SSM in vivo. (A) Pharmacokinetic profiles of liraglutide, xenoglutide and xenoglutide-SSM in SD rats after s.c. administration (50 nmol/rat). Means ± SD, n = 3. (B) Anorectic effects of liraglutide, xenoglutide (25 nmol·kg-1) and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) in 18 h fasted db/db mice. Means ± SD, n = 6.

39



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Figure 7. Long-term glucose-lowering effects of xenoglutide-SSM was evaluated by using multiple OGTT in db/db mice. Saline, liraglutide, xenoglutide (25 nmol·kg-1) and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) were i.p. administered 30 min prior to first oral glucose challenge (1.5 40


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g·kg-1), and the remaining glucose loads were carried out at 6, 12 and 18 h, respectively. (A) The time course of blood glucose levels in each group during 0-12 h. (B) The time course of blood glucose levels in each group during 12-24 h. (C) The AUCglucose 0-24 h values of each group. Means ± SD, n = 6. ***P < 0.001 vs. Saline, aP < 0.001 vs. liraglutide, a’P < 0.001 vs. xenoglutide.

41



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Figure 8. Effects of xenoglutide-SSM on cell viability of INS-1 cells. (A) Effects of xenoglutide-SSM (10, 100, and 500 nM) on INS-1 cells under normal condition. (B) Effects of xenoglutide-SSM (10, 100, and 500 nM) on INS-1 cells under high glucose condition (30 nM). All values are expressed in mean ± SD. *P < 0.05 vs. Saline, ***P < 0.001 vs. Saline.

42


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Figure 9. Chronic treatment effects of liraglutide, xenoglutide and xenoglutide-SSM in db/db mice for five weeks. (A) HbA1c measured ay day 0 and day 36. (B) Food intake. (C) Body weight. (D) Non-fasting blood glucose values. (E) Non-fasting plasma insulin values. (F) Excursion of blood glucose in IPGTT test after five weeks of treatment (Calculated AUCglucose 0-120 min values). Means ± SD, n = 6. 43



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 10. Biochemical analysis in db/db mice following the five weeks treatment. (A) Total cholesterol (TC). (B) Triglyceride (TG). (C) HDL-cholesterol. (D) LDL-cholesterol. (E) Alanine aminotransferase (ALT). (F) Aspartate aminotransferase (AST). (G) Islet number. (H) Islet area. Means ± SD, n = 6. *P < 0.05 vs. control, **P < 0.01 vs. control, ***P < 0.001 vs. control. 44


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45



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Figure 1. Structures of the peptides 1–2 and compounds 3–6. 137x115mm (300 x 300 DPI)


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Figure 2. Acute and long-term hypoglycemic activities of exendin-4, lixisenatide and peptides 1–2 in Kunming mice. Saline, exendin-4, lixisenatide, peptides 1–2 (25 nmol·kg-1) were i.p. administered 30 min prior to i.p. glucose load (2 g·kg-1). (A) The time−glucose response curve in IPGTT (-30 to 180 min). (B) The AUCglucose 0-180 min values of each group. (C) The time−glucose response curve in multiple IPGTT (0.5 to 12 h). (c) The AUCglucose 0-12 h values of each group. ***P < 0.001 vs. Saline, aP < 0.001 vs. exendin-4, bP < 0.01 vs. exendin-4, cP < 0.05 vs. lixisenatide. Means ± SD, n = 6. 726x528mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Synthesis route of compounds 3–6. PG, amino acid protecting group. 189x122mm (300 x 300 DPI)


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Figure 4. Acute and long-term hypoglycemic activities of liraglutide and compounds 3–6 in Kunming mice. Saline, liraglutide, compounds 3–6 (25 nmol·kg-1) were i.p. administered 30 min prior to i.p. glucose load (2 g·kg-1). (A) The time−glucose response curve in IPGTT (-30 to 180 min). (B) The AUCglucose 0-180 min values of each group. (C) The time−glucose response curve in multiple IPGTT (-0.5 to 12 h). (D) The AUCglucose 0-12 h values of each group. Means ± SD, n = 6. 724x528mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Glucose-lowering and insulinotropic activities of xenoglutide-SSM in db/db mice by IPGTT. Xenoglutide (25 nmol·kg-1) was i.p. administered 180 min prior to glucose challenge. Saline, liraglutide (25 nmol·kg-1) and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) were i.p. injected 30 min prior to 1 g·kg-1 i.p. glucose load. (A) Hypoglycemic activities of liraglutide, xenoglutide (25 nmol·kg-1), and xenoglutideSSM (25 nmol·kg-1 of xenoglutide) in db/db mice. (B) Calculated blood glucose AUC0−120 min values of each group. (C) The time course of plasma insulin values in each group. (D) Calculated AUCinsulin 0-120 min values of each group. Means ± SD, n = 6. ***P < 0.001 vs. Saline, aP < 0.001 vs. liraglutide. bP < 0.01 vs. liraglutide. 1017x793mm (96 x 96 DPI)


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Figure 6. Pharmacokinetic profiles and anorectic effects of liraglutide, xenoglutide and xenoglutide-SSM in vivo. (A) Pharmacokinetic profiles of liraglutide, xenoglutide and xenoglutide-SSM in SD rats after s.c. administration (50 nmol/rat). Means ± SD, n = 3. (B) Anorectic effects of liraglutide, xenoglutide (25 nmol·kg-1) and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) in 18 h fasted db/db mice. Means ± SD, n = 6. 635x893mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7. Long-term glucose-lowering effects of xenoglutide-SSM was evaluated by using multiple OGTT in db/db mice. Saline, liraglutide, xenoglutide (25 nmol·kg-1) and xenoglutide-SSM (25 nmol·kg-1 of xenoglutide) were i.p. administered 30 min prior to first oral glucose challenge (1.5 g·kg-1), and the remaining glucose loads were carried out at 6, 12 and 18 h, respectively. (A) The time course of blood glucose levels in each group during 0-12 h. (B) The time course of blood glucose levels in each group during 12-24 h. (C) The AUCglucose 0-24 h values of each group. Means ± SD, n = 6. ***P < 0.001 vs. Saline, aP < 0.001 vs. liraglutide, a’P < 0.001 vs. xenoglutide. 582x1012mm (96 x 96 DPI)


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Figure 8. Effects of xenoglutide-SSM on cell viability of INS-1 cells. (A) Effects of xenoglutide-SSM (10, 100, and 500 nM) on INS-1 cells under normal condition. (B) Effects of xenoglutide-SSM (10, 100, and 500 nM) on INS-1 cells under high glucose condition (30 nM). All values are expressed in mean ± SD. *P < 0.05 vs. Saline, ***P < 0.001 vs. Saline. 661x978mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 9. Chronic treatment effects of liraglutide, xenoglutide and xenoglutide-SSM in db/db mice for five weeks. (A) HbA1c measured ay day 0 and day 36. (B) Food intake. (C) Body weight. (D) Non-fasting blood glucose values. (E) Non-fasting plasma insulin values. (F) Excursion of blood glucose in IPGTT test after five weeks of treatment (Calculated AUCglucose 0-120 min values). Means ± SD, n = 6. 793x864mm (96 x 96 DPI)


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Figure 10. Biochemical analysis in db/db mice following the five weeks treatment. (A) Total cholesterol (TC). (B) Triglyceride (TG). (C) HDL-cholesterol. (D) LDL-cholesterol. (E) Alanine aminotransferase (ALT). (F) Aspartate aminotransferase (AST). (G) Islet number. (H) Islet area. Means ± SD, n = 6. *P < 0.05 vs. control, **P < 0.01 vs. control, ***P < 0.001 vs. control. 582x901mm (96 x 96 DPI)


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