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Preparation and Pharmaceutical Characterizations of Lipidated Dimeric Xenopus Glucagon-like Peptide-1 Conjugates Jing Han, Feng Zhou, Yingying Fei, Xinyu Chen, Junjie Fu, and Hai Qian Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00712 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017

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Bioconjugate Chemistry

Title: Preparation and Pharmaceutical Characterizations of Lipidated Dimeric Xenopus Glucagon-like Peptide-1 Conjugates

Authors: Jing Han,†* Feng Zhou,† Yingying Fei,† Xinyu Chen,† Junjie Fu,‡ §* Hai Qian,§*

Affiliation and Address: †

School of chemistry and materials science, Jiangsu Normal University, Xuzhou 221116, PR China

‡

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

211166, PR China §

Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China

*Corresponding author: School of chemistry and materials science, 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)

*Corresponding author: Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China Tel: +86-25-83271302; Fax: +86-25-83271480 E-mail: [email protected] (Hai Qian)

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Bioconjugate Chemistry 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

ABSTRACT: Two glucagon-like peptide-1 (GLP-1) analogs (1 and 2) were synthesized by hybridizing the key sequences of GLP-1, exendin-4, lixisenatide and xenGLP-1B (Xenopus GLP-1 analog). To achieve long-acting hypoglycemic effects and to further improve their antidiabetic potencies, lipidization and dimerization strategies were used to afford two lipidated dimeric conjugates (9 and 11). Conjugates 9 and 11 showed stronger receptor activation potency than GLP-1 and exendin-4 in vitro. Moreover, 9 and 11 exhibited superior hypoglycemic and insulinotropic activities to liraglutide in db/db mice. Pharmacokinetic studies revealed that the circulating half-lives (t1/2) of 9 and 11 were 2.3- and 1.7-fold longer than liraglutide. The improved pharmacokinetic profiles led to significantly protracted in vivo antidiabetic effects as confirmed by multiple OGTT test and hypoglycemic duration test. Most importantly, chronic treatment studies found that once daily administration of 9 or 11 in db/db mice achieved more beneficial effects on HbA1c reduction and glucose tolerance normalization than liraglutide. Our research demonstrated lipidization and dimerization as useful tools for the development of novel GLP-1 receptor agonists. The preclinical studies suggested the potential of 9 and 11 to be developed as novel antidiabetic agents.

Keywords: Glucagon-like peptide-1; Dimerization; Lipidation; Type 2 diabetes mellitus;


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INTRODUCTION Glucagon-like peptide 1 (GLP-1) is a potent anti-hyperglycemic and insulinotropic endocrine hormone secreted from both the distal gut and the pancreas into the circulation in response to ingestion.1, 2 GLP-1 normalizes postprandial glucose levels by suppressing glucagon secretion and stimulating insulin secretion in a glucose dependent manner.3, 4 Moreover, GLP-1 decreases body weight by inducing satiety and inhibiting gastric emptying, and plays significant roles in stimulating β cell proliferation and inhibiting β cell apoptosis.5, 6 The above unique pharmacological roles of GLP-1 make it a potent therapeutic agent for type 2 diabetes mellitus (T2DM) treatment. Unfortunately, the major challenge for the clinical use of GLP-1 is the short elimination half-life (t1/2), due principally to rapid enzymatic degradation and/or renal clearance.7, 8 Thus, discovery of novel physiologically stable and potent GLP-1 receptor agonists is actively being pursued. Several GLP-1 receptor agonists, such as exenatide, lixisenatide and liraglutide, have been clinically approved, generating a multi-billion dollar level market globally. Meanwhile, numerous GLP-1 receptor agonists are in clinical development.9 Most recently, semaglutide was approved by FDA as a once-weekly antidiabetic agent. The sequences of GLP-1 are conserved in most mammalian species investigated so far, while those in amphibians appear to be more variable.10 Amphibians also use GLP-1 as an insulinotropic endocrine hormone, and the actions of GLP-1 in amphibians are similar to those seen in mammals. In a previous report, Irwin et al. identified three xenGLP-1s (xenGLP-1A, xenGLP-1B, and xenGLP-1C) from encoded Xenopus proglucagon cDNAs.11 These xenGLP-1s had similar sequences to hGLP-1 (~70% homologous), and showed potent in vitro GLP-1 receptor activation potencies and insulinotropic activities. In our previous research, through hybridizing the key sequences of GLP-1, xenGLP-1B and exendin-4, we successfully developed novel GLP-1 receptor agonists with improved biological activities. Furthermore, the peptide hybrids were conjugated with PEG moiety for t1/2 extension. However, considering high molecular weight PEG will impede the binding of PEGylated peptide to GLP-1 receptor, relatively short PEG chains with a molecular weight of 1–5 kDa were used in our previous research, which only moderately increased the t1/2.12 Lipidization is another widely used strategy to improve the pharmacokinetic profiles of peptide drugs.13 By introducing fatty acid moieties to the peptide chain, lipidization facilitates the noncovalent binding of peptides to human serum albumin (HSA), and protects the peptides from the 3 ACS Paragon Plus Environment


proteolytic degradation and renal clearance.14 Enhanced circulation t1/2 and long-acting characteristics of lipidated GLP-1 receptor agonists have already been demonstrated. However, the fatty acid moiety may hamper the receptor activation potency of lipidated peptides, causing decreased bioactivities.15 Multivalent binding strategies such as dimerization are frequently used to increase the receptor binding affinity of peptides, leading to improved therapeutic potencies compared with the native peptides.16 Previously, several research groups have constructed dimer GLP-1 analogs with enhanced hypoglycemic efficacies. For example, Lee et al. successfully used the dimeric strategy to attenuate the negative effects of PEGylation on receptor binding, and the PEGylated dimeric exendin-4 analogs showed improved in vivo antidiabetic activities.17 On the basis of these findings, in the present study, both the lipidization and dimerization strategies were used to construct novel GLP-1 analogues with improved antidiabetic effects and protracted hypoglycemic efficacies. First, novel GLP-1 receptor agonists 1 and 2 were obtained by hybridizing the key amino acids of GLP-1, xenGLP-1B, exendin-4 and lixisenatide. Next, peptides 1 and 2 were dimerized using a bis-maleimide amine backbone, and further lipidization was carried out on the amine nitrogen. This brings two advantages. First, the bioactivity of 1 and 2 could significantly improve after dimerization, affording 4 and 7. Second, the peptide chains of 4 and 7 were not involved in the lipidization, and the bioactivities of lipidated dimers 9 and 11 were almost fully retained without the need of alanine scanning. The biological characteristics, pharmacokinetic behaviors, and long-term treatment effects of 9 and 11 were explored in vitro and in vivo.

RESULTS Design, Preparation and Characterization of 1 and 2 In our previous research, alanine scanning was conducted to explore the structure-activity relationship (SAR) of xenGLP-1B (unpublished work). It was interesting to find that the in vitro receptor activation potency and in vivo hypoglycemic activities of xenGLP-1B were significantly improved when Lys17 was substituted with either Ala or Cys, indicating that Lys17 might hinder the agonist-receptor interaction and could be replaced with other amino acids. The C-terminus of GLP-1 is known as an important region for receptor interaction and an effective target for sequence modification. For example, the nine-AA sequence (PSSGA PPPS) at the C-terminal of exendin-4 4 ACS Paragon Plus Environment

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plays an important role for its improved bioactivity than GLP-1.18 The C-terminal of lixisenatide (PSSGA PPSKK KKKK), which is similar to that of exendin-4 and is appended with six additional lysine residues, improved both the bioactivity and stability of lixisenatide.19 Based on the above background, the following strategies were comprehensively used for the design of peptides 1 and 2. First, the unfavorable Lys17 in xenGLP-1B was replaced by Glu, which also occupies the position 17 in both exendin-4 and lixisenatide. Next, the C-terminal regions of exendin-4 and lixisenatide were attached after the Lys30 of xenGLP-1B for peptides 1 and 2, respectively. An additional cysteine was further introduced into the C-terminal of both 1 and 2 as a specific site for the following maleimide conjugation. Finally, as a routine modification, the Ala2 was substituted with Gly to avoid DPP-IV mediated degradation. Thus, the rational hybridization of xenGLP-1B, GLP-1, lixisenatide and exendin-4 afforded the sequences of 1 and 2 (Figure 1), which were synthesized according to the standard N-Fmoc/tBu solid phase peptide synthesis (SPPS) methodology (see Supporting Information, Figure S1). The crude products were purified by semi preparative RP-HPLC and characterized by HPLC and LC-MS (see Supporting Information).

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 sequence hybridization C-terminal cysteine modification

1

HGEGT YTNDV TEYLE EEAAK EFIEW LIKGK PSSGA PPPSC

2

HGEGT YTNDV TEYLE EEAAK EFIEW LIKGK PSSGA PPSKK KKKKC

Figure 1. Structures of 1 and 2.

Design, Preparation, Characterization and Biological Activity Tests of 1–7 Multivalent interactions are frequently used to enhance the weak ligand-receptor interactions.20 In order to investigate whether the dimeric strategy could improve the bioactivities of 1 and 2, dimeric hybrids of 1 and 2 were constructed using a bis-maleimide amine backbone, affording 4 and 7 (Figure 2). For comparison, monomeric conjugates of peptides 1 and 2 and bis-maleimide amine were also prepared as 3 and 6. Furthermore, a dimeric peptide 5 consisting of an intact 1 and a 5 ACS Paragon Plus Environment


truncated 1 (deprived of His1 and Gly2) was designed as an analogue of 4 for mechanism of action study. The monomeric and dimeric hybrids (3–7) were synthesized by using chemoselective Michael-addition between the C-terminal cysteine and maleimide (Scheme 1). The reactions were completed within 90 min as monitored by HPLC. The products were purified by preparative RP-HPLC, and their purities and molecular weights were confirmed by HPLC and MicroTOF MS, respectively (see Supporting Information).

Figure 2. Structures of 3–11.


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2: 1

Scheme 1. Synthesis of 3–7.

The receptor activation potency of 1–7 was then measured using HEK293 cells stably expressing GLP-1 receptor. As shown in Figure 3, both 1 (EC50 = 2.14 ± 0.49 nM) and 2 (EC50 = 0.59 ± 0.17 nM) exhibited higher receptor activation potency than GLP-1 (EC50 = 4.18 ± 0.53 nM). Particularly, the receptor activation potency of 2 was 3.9-fold higher than exendin-4 (EC50 = 2.31 ± 0.69 nM). The potency of 3 (EC50 = 3.78 ± 0.85 nM) was slightly decreased as compared with 1 as a consequence of bis-maleimide amine conjugation. The receptor activation potency of 5 (EC50 = 4.58 ± 0.94 nM) was similar to that of 3. Since one of the peptide chains of 5 is truncated without GLP-1 receptor activation potency, the data suggest that the introduction of another peptide chain does not cause steric hinderance to hamper the interaction between the active chain and the receptor. When both of the chains were active peptide 1, the dimer 4 (EC50 = 0.53 ± 0.05 nM) was found to have a 4.0- and 7.1-fold higher activation potency than 1 and 3, respectively. Furthermore, considering that one molar of 4 consists of two molars of peptide 1, the actual dose of 4 at each indicated concentration point was reduced to half for equal comparison, and the dose-response curve of 41/2 was obtained. The result showed that 41/2 (EC50 = 0.91 ± 0.17 nM) still exhibited a 2.4and 4.2-fold higher receptor activation potency than 1 and 3. The above results clearly indicate a synergistic receptor activation potency resulting from the dimerization of 1. Similar to the receptor activation potency results of 1, 3 and 4, compound 7 (EC50 = 0.13 ± 0.03 nM) was found to have a 4.5- and 9.8-fold higher activation potency than 2 and 6 (EC50 = 1.27 ± 0.23 nM).



Figure 3. In vitro bioactivity tests. (A–B) Dose-response relationship of GLP-1, exendin-4, 1–7, 41/2 on GLP-1R. Means ± SD, experiments were performed in triplicate and repeated three times (n = 3).

To further confirm the results of the in vitro receptor activation potency experiments, the in vivo glucose-lowering activities of 1–7 were studied in the intraperitoneal glucose tolerance testing (IPGTT) in Kunming mice. As shown in Figure 4B, the area under the curve (AUC) results clearly indicated that both 4 and 41/2 had significantly increased hypoglycemic activities over the monomer forms (P < 0.001, as compared with 1 and 3) in vivo. The hypoglycemic activity of 5 was very similar to 3 (Figure 4B), further supporting the conclusion that the two peptide chains in the dimeric structure were more likely to individually interact with the receptor. Similarly, 7 exhibited a significantly increased hypoglycemic activity as compared with 2 and 6 (P < 0.05 and P < 0.001,


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respectively, Figure 4D). Importantly, the hypoglycemic activities of 4 and 7 were significantly better than GLP-1 and exendin-4 (P < 0.001, Figure 4B and D).

Figure 4. The glucose lowering effects of GLP-1, exendin-4, 1–7 (25 nmol·kg-1), 41/2 (12.5 nmol·kg-1 of 4) as determined by IPGTT in Kunming mice. (A and C) Blood glucose time-response curve (-30 to 120 min). (B and D) Hypoglycemic effects of GLP-1, exendin-4, 1–7 and 41/2 expressed

as glucose AUC0-120 min. *P < 0.05,

***

P < 0.001, cP < 0.001 vs. GLP-1, c’P < 0.001 vs. exendin-4.

Means ± SD, n = 6.

Design, Preparation, Characterization and Biological Activity Tests of 8–11 The covalent attachment of fatty acids to peptides provides a means of enhancing their in vivo stabilities. As dimeric peptides 4 and 7 exhibited prominent in vitro and in vivo bioactivities, palmitic acid was further introduced into the bis-maleimide amine structure of 4 and 7 to improve their in vivo stabilities, giving lipidated peptides 9 and 11 (Figure 2). To validate the positive effect of dimerization, lipidated monomeric peptides 8 and 10 were also designed for comparison. The 9 ACS Paragon Plus Environment


NHS-activated palmitic acid was first site-specifically conjugated with the amino group of bis-maleimide amine, which was then reacted with one or two equivalents of 1 or 2 to afford 8–11 (Scheme 2). The reactions were completed within 90 min as monitored by HPLC. The representative reaction HPLC chromatograms are shown in Figure S2 (see Supporting Information). The products were purified by preparative RP-HPLC, and their purities and molecular weights were confirmed by HPLC and MicroTOF MS, respectively (see Supporting Information). The receptor activation potency of 8–11 was determined using the same method as described above. As shown in Figures 5A, the introduction of palmitic acid was well tolerated with respect to GLP-1 functional activity, as reflected by the high receptor activation potency of 8 (EC50 = 4.02 ± 1.12 nM), 9 (EC50 = 0.62 ± 0.15 nM), 10 (EC50 = 1.92 ± 0.36 nM) and 11 (EC50 = 0.19 ± 0.03 nM). The receptor activation potency of 9 and 11 were 6.5- and 10.1-fold higher than their lipidated monomeric forms 8 and 11, respectively, indicating again that dimerization significantly improved the in vitro potency. Importantly, the receptor activation potency of 9 and 11 were also much better than GLP-1 (EC50 = 4.18 ± 0.53 nM) and exendin-4 (EC50 = 2.31 ± 0.69 nM). The in vivo hypoglycemic efficacies of 8–11 were tested by IPGTT in Kunming mice. As illustrated in Figure 5B, the administration of GLP-1, exendin-4, 8–11 (25 nmol·kg-1, i.p.) significantly improved the glucose tolerance patterns. The calculated glucose AUC0-120 min values revealed that, in consistence with the in vitro functional potency results, the hypoglycemic efficacies of 9 and 11 were significantly better than their monomer forms (8 and 10), GLP-1 and exendin-4 (P < 0.001, Figure 5C).


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Figure 5. In vitro and in vivo bioactivity tests. (A) Dose-response relationship of GLP-1, exendin-4 and 8–11 on GLP-1R. Means ± SD, experiments were performed in triplicate and repeated three times (n = 3). (B) The glucose lowering effects of GLP-1, exendin-4 and 8–11 (25 nmol·kg-1) as determined by IPGTT in Kunming mice. (C) Hypoglycemic effects of GLP-1, exendin-4 and 8–11



expressed as glucose AUC0-120 min.

***

P < 0.001, cP < 0.001 vs. GLP-1, c’P < 0.001 vs. exendin-4.

Means ± SD, n = 6.

1 1:

Scheme 2. Synthesis of 8–11.

Hypoglycemic and Insulin Secretion Assay in db/db Mice As compounds 9 and 11 exhibited prominent in vitro and in vivo bioactivities, an IPGTT in db/db mice (type 2 diabetic model) was performed to further assess the glucose-lowering and insulinotropic activities of 9 and 11, using exendin-4 and liraglutide as the positive control. As illustrated in Figure 6A, the time course for blood glucose levels in control (saline) mice maintained a hyperglycemic state during the IPGTT. Exendin-4, liraglutide, 9 and 11 (25 nmol·kg-1) injections potently reduced blood glucose levels to ~12.5, ~14.2, ~11.7 and ~10.5 mmol·L-1 15 min after the i.p. glucose load (1 g·kg-1), respectively. Particularly, the average blood glucose levels in 9 and 11 groups were lower than that in exendin-4 and liraglutide group during 15–60 min. These results indicated that the hypoglycemic activities of 9 and 11 were superior to exendin-4 and liraglutide. The time courses of plasma insulin concentrations were also recorded. As illustrated in Figure 6C, the decreases in blood glucose concentrations in exendin-4, liraglutide, 9 and 11 groups were accompanied by increases in plasma insulin levels, indicating a GLP-1-dependent mechanism. AUC calculations of glucose and insulin further revealed that 9 and 11 exhibited similar efficacy profiles and were considerably more potent than exendin-4 and liraglutide in the db/db mice IPGTT model (Figures 6B and D). 12 ACS Paragon Plus Environment

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Figure 6. Antihyperglycemic and insulinotropic activities of 9 and 11 as determine by IPGTT in db/db mice. Saline, exendin-4, liraglutide, and 9 and 11 (25 nmol·kg-1) were i.p. injected 30 min prior to glucose load (1 g·kg-1, i.p.). (A) Blood glucose time-response curve (-30 to 120 min). (B) AUCglucose 0−120 min values of each group. (C) Plasma insulin time-response curve (0 to 120 min). (D) AUCinsulin 0–120 min values of each group.

***

P < 0.001 vs. liraglutide, aP < 0.05 vs. exendin-4, cP
15 mmol·L-1) of blood glucose significantly increased accompanied by the increased doses of 9 and 11 (25 to 100 nmol·kg-1). These results suggest that 9 and 11 have longer in vivo glucose-lowering effects than liraglutide, and the therapeutic effects of 9 and 11 could further extend by properly increasing the administrated doses.


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Figure 8. Long-term glucose-lowering effects of 9 and 11. (A–B) Long-term hypoglycemic effects of liraglutide, 9 and 11 were studied by the multiple OGTT in 18 h fasted db/db mice. Mice were i.p. injected with saline, liraglutide, 9 and 11 (25 nmol·kg-1) 0.5 h prior to oral glucose load (1.5 g·kg-1), and glucose loads were repeated at 6, 12 and 18 h. Blood glucose time-response curves (-0.5 to 24 h) are shown in panels A and B. (C) Hypoglycemic efficacies of liraglutide, 9 and 11 in nonfasted db/db mice. Time response curves for blood glucose lowering effects of liraglutide (25 nmol·kg-1), 9 and 11 (25 or 100 nmol·kg-1) in db/db mice after i.p. dosing. Means ± SD, n = 6.



Chronic Treatment Effects Long-term administration of GLP-1R agonists leads to chronic effects in db/db mice, including HbA1c and body weight lowering, food intake reducing, insulin sensitivity increasing, and β cell neogenesis and/or differentiation promoting.22 As such, the chronic treatment effects of 9 and 11 were studied in diabetic db/db mice. Based on the pharmacokinetic profiles of liraglutide, 9 and 11, liraglutide was administered twice daily at 25 nmol·kg-1, and 9 and 11 were administered once daily at 50 nmol·kg-1 to ensure sufficient and consistent doses over 35 days. Compared with the control group at day 36, treatment with 9 and 11 prohibited the worsening of the HbA1c (sensitive index of glycemic control). The HbA1c in 9 and 11 groups were reduced by ~2.8% and ~2.9%, respectively, better than that in liraglutide group (~0.67%, Figure 9A). The body weight gain and food intake were markedly suppressed by both 9 and 11 treatments (Figures 9B and C). At the end of treatment, the relative body weight loss compared with the control group were found to be ~19%, ~17%, and 12% for 9, 11 and liraglutide, respectively. Furthermore, the non-fasting blood glucose levels in liraglutide, 9 and 11 groups were significantly decreased, accompanied by notably increased non-fasting plasma insulin levels during the treatment period (Figures 9D and E). Moreover, both the non-fasting plasma insulin levels in 9 and 11 groups were lower than liraglutide group from day 20, indicating the improved insulin sensitivity. At the end of the treatment, each group of mice were subjected to an IPGTT to test the glucose tolerance abilities after the chronic treatment. After 5 weeks of therapy, glucose excursions in control group were higher than liraglutide, 9 and 11 groups (Figure 9F). Furthermore, the AUC was lower for 9 and 11 treated mice than for liraglutide treated mice (Figure 9F, inset).


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Figure 9. Chronic treatment effects of liraglutide, 9 and 11 in db/db mice. Saline, liraglutide (25 nmol·kg-1) was administered twice daily, and 9 and 11 (50 nmol·kg-1) were administered once daily over 35 days. (A) HbA1c (%), day 0 vs day 36. (B) Food intake. (C) Body weight. Non-fasting blood glucose levels (D) and non-fasting plasma insulin levels (E) were determined every four days. (F) Blood glucose time-response curves of each group in IPGTT test on day 37. Inset: Calculated AUCglucose 0–120 min values. Means ± SD, n = 6.



DISCUSSION T2DM is a chronic metabolic disease manifested by progressive hyperglycemia and impaired insulin secretion and/or utilization. An ideal T2DM treatment should not only improve the glycemic control but also maintain or restore pancreatic endocrine function.23 GLP-1 receptor agonists are among the most promising therapies for T2DM, due to the unique antidiabetic activities of GLP-1. Although intensive research efforts are focused on developing novel GLP-1 receptor agonists, most of the GLP-1 receptor agonists currently in clinical development are based on the backbone of hGLP-1 or Gila GLP-1 (exendin-4).24 In the present study, based on our previous studies of the structure-activity relationship of xenGLP-1B, two hybrid peptides 1 and 2 were firstly synthesized by hybridizing the key amino acid sequences of GLP-1, xenGLP-1B, exendin-4 and lixisenatide. However, considering the nonmammalian (xenopus) origin of 1 and 2, as well as the introduction of Glu17 and C-terminal sequences from exendin-4 and lixisenatide, the potential immunogenic response of 1 and 2 in long-term treatment requires further investigation.25, 26 Lipidization technology, which introduces fatty acid to peptide drugs, could effectively increase the durations of peptides.27 Considering that the short elimination t1/2 of GLP-1 poses the major obstacle for its clinical use, we employ the lipidization method to modify 1 and 2. Importantly, to avoid any possible negative effects of fatty acid on the bioactivities of 1 and 2, dimerization strategy was also introduced into our design to ensure maintained or even improved therapeutic potencies. Two palmitic acid conjugated dimeric peptides, 9 and 11, were successfully prepared. The nonlipidated dimers, 4 and 7 were also synthesized. To validate the positive effect of dimerization, monomeric or lipidated monomeric peptides 3, 6, 8 and 10 were also designed for comparison. Furthermore, a dimeric peptide 5 consisting of an intact 1 and a truncated 1 was designed as an analogue of 4 for preliminary mechanism of action study. The mechanism study revealed that the two peptide chains on the dimeric structure were more likely to individually interact with the GLP-1 receptor and produced synergistic receptor activation potency. The in vitro receptor activation potency and in vivo hypoglycemic activities of 4, 7, 9 and 11 were significantly improved as compared with their monomeric forms, indicating that dimerization significantly improved the biological activities. We found that the receptor activation potency and glucose-lowering activities of 9 and 11 were much better than GLP-1 and exendin-4. 20 ACS Paragon Plus Environment

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The following hypoglycemic and insulin secretion assays further proved the prominent antidiabetic and insulinotropic activities of 9 and 11. Next, it was found that the pharmacokinetic profiles of 9 and 11 were markedly better than that of liraglutide. The AUCinf of 9 and 11 were 2.0 and 1.9- fold higher than liraglutide, and the t1/2 values 9 and 11 were 2.3 and 1.7- fold greater than liraglutide. The improved pharmacokinetic profiles of 9 and 11 were also manifested in acute food intake, as 9 and 11 exhibited better anorectic effects than liraglutide. Furthermore, the long-acting glucose-lowering activities of 9 and 11 were confirmed by multiple OGTT test and hypoglycemic duration test in db/db mice. Importantly, the hypoglycemic durations of 9 and 11 were longer than that of liraglutide, regardless of the dose administered. The dose-response study showed that the antidiabetic durations of 9 and 11 in db/db mice were nearly 48 h at a dose of 100 nmol·kg-1. As the drug clearance rate in human is much slower than in mice, it is expected that the hypoglycemic durations of 9 and 11 in human could be longer than two days. Finally, the chronic treatment effects of 9 and 11 in db/db mice were tested to evaluate their potential therapeutic utilities. The body weight gain and food intake were effectively suppressed by once daily administration of 9 and 11. In addition, both 9 and 11 improved insulin sensitivity in db/db mice, as reflected by the decreased non-fasting plasma insulin levels from day 20. Moreover, chronic treatment of 9 and 11 improved glucose tolerances and achieved beneficial effects on HbA1c lowering in db/db mice, indicating the promising roles of 9 and 11 in clinical studies. To summarize, we described the design and synthesis of 9 and 11 as novel GLP-1 analogs. The in vitro and in vivo bioactivities of the two lipidated dimeric peptides were extensively explored. Compared with liraglutide, 9 and 11 had higher glucose-lowering and insulinotropic activities, better pharmacokinetics, markedly greater hypoglycemic durations, and more beneficial chronic treatment effects. We believe 9 and 11 as potential antidiabetics for the treatment of T2DM.

MATERIALS AND METHODS Materials Liraglutide, exendin-4 and GLP-1(7-36)-NH2 were purchased from the GL Biochem (Shanghai, China) Ltd. cAMP dynamic kit was purchased from the CIS Bio International (Bedford, MA, USA). Bis-maleimide amine was obtained from the Xi'an Ruixi Biological Technology Co., Ltd (Xi'an, China). Mouse insulin ELISA kit was obtained from Nanjing Jiancheng Bioengineering Institute 21 ACS Paragon Plus Environment


(Jiangsu, China). All other reagents were obtained from Sigma-Aldrich Co. (St. Louis, MO) unless otherwise indicated.

Animals Male Kunming mice and Sprague-Dawley rats, weighing 20–25 g and 200–250 g, respectively, were obtained from the Comparative Medical Center of Yangzhou University (Yangzhou, China). Type 2 diabetic C57BL/6J-m+/+ Leprdb (db/db) mice (male, 8–9 weeks old) were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). Animals were housed in groups (for mice, n = 6; for rats, n = 3) and in a thermostatically controlled room (25 ± 2 °C) under a 12:12 h light/dark cycle. Animals were allowed free access to food and water. All animal experiments protocols were conducted in accordance with 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.

Preparation of Dimeric Hybrids and Lipidated Dimeric Hybrids Hybrids 1–2 were synthesized by standard Fmoc SPPS strategy on a PSI-200 peptide synthesizer (Peptide Scientific Inc., USA).28 Resins used were Rink amide MBHA resin with a loading of 0.37 mmol·g-1. For peptides 3 and 6, bis-maleimide amine (Mw = 546.3) and 1 or 2 (1 eq) were dissolved in 500 µL DMSO, and diluted with 5 mL methanol. Then 200 µL DIPEA was added and the reaction was allowed to react at 25 °C with mixing for 1 h. The reaction was stopped by adding 200 µL 1% TFA/ deionized water. For peptides 4, 5 and 7, bis-maleimide amine and 1 (including truncated 1) or 2 (2 eq) were dissolved in DMSO and the synthesis procedure were the same as describe above. For peptides 8–11, 3.53 mg NHS activated palmitic acid (Mw = 353.3) was reacted with 5.46 mg bis-maleimide amine in DMSO/methanol (1:9, V/V) mixture containing 1% DIPEA at 25 °C for 1 h. Then peptides 1 or 2 (1 eq or 2 eq) were added, and the reaction was monitored by HPLC until completion. The reaction was stopped by adding 500 µL of 1% TFA/ deionized water. The products were purified from reaction mixtures by semi-preparative RP-HPLC (LC-20AP, Shimadzu), and were characterized by Thermo LC-MS or Bruker MicroTOF Q2 using ESI ionization method. 22 ACS Paragon Plus Environment

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GLP-1 Receptor Activation Assay HEK293 cells stably expressing GLP-1 receptor were used for functional assay.29 The receptor assay was performed by measuring cAMP as a response to stimulation, and the methodology was described elsewhere. Briefly, cells were grown at 37 °C in 5% CO2 incubator in Dulbecco’s modified Eagle medium-31053 (Invitrogen, CA) supplemented with 50 µg/mL streptomycin, 50 units/mL penicillin, 20 mM HEPES, 2 mM glutamine and 0.5% fetal bovine serum (FBS). On the experiment day, cells were resuspended and plated in 384-well microplates. The tested compounds were solubilized in DMSO, diluted in medium containing 0.1% bovine serum albumin (BSA) with fraction V to different concentrations. Then HEK293 cells were exposed to different concentrations of tested compounds, followed by 20 min incubation. The cAMP concentrations in cell lysates were assayed using cAMP dynamic 2 kit. Envision 2104 Multilabel Reader was used to determine the fluorescence in each sample by using homogenous time resolved fluorescence technology. cAMP concentration was measured according to the delta F% versus cAMP concentration standard curve. Dose-response curves were plotted and the EC50 values were determined by GraphPad Prism version 5.0.

IPGTT on Kunming Mice The in vivo glucose-lowering activities of GLP-1, exendin-4, 1–11 were evaluated by IPGTT in Kunming mice.30 In brief, 12 h fasted Kunming mice (male, 20–25 g) were randomly divided into 15 groups (n = 6), and each group of mice were i.p. administered with saline (control), GLP-1, exendin-4, 1–11 (25 nmol·kg-1) and 41/2 (12.5 nmol·kg-1 of 4), respectively, at t = -30 min. Glucose was i.p. challenged at t = 0 min (2 g·kg-1). Blood samples were collected from the tail vein and blood glucose levels were monitored at -30, 0, 15, 30, 60, and 120 using a one-touch glucometer (Ascensia, Breeze 2, Bayer, Germany).

Hypoglycemic and Insulinotropic Assay in db/db Mice The effects of 9 and 11 on glucose homeostasis and insulin secretion were evaluated in comparison with liraglutide, exendin-4 and control (saline) by IPGTT on db/db mice.31 Briefly, db/db mice (male, 9 weeks old) were randomly divided into 5 groups (n = 6) and acclimatized for 7 23 ACS Paragon Plus Environment


days before the experiment. After fasted 18 h, each group of mice were i.p. administered with saline (control), liraglutide, exendin-4, 9 and 11 (25 nmol·kg-1) 30 min before i.p. glucose challenged (1 g·kg-1). Blood samples were collected from the tail vein. Blood glucose levels were monitored at -30, 0, 15, 30, 60, and 120 using a one-touch glucometer (Ascensia, Breeze 2, Bayer, Germany). Plasma insulin concentrations were determined at 0, 5, 10, 15, 30, 45, 60, and 120 min using mouse insulin ELISA kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

Pharmacokinetic Tests The pharmacokinetic profiles of s.c. administered liraglutide, 9 and 11 were evaluated as previously described.32 In brief, male Sprague-Dawley rats (200–250 g, 3 rats per group) were fasted 12 h. Then, liraglutide, 9 and 11 (50 nmol/rat) were s.c. administered. Approximately 0.1 mL of blood samples were collected at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 36 h in EDTA-containing tubes from the lateral tail vein. Plasma was obtained through centrifuged at 4°C and plasma proteins were immediately precipitated with two volumes of acetonitrile, and 10 µL supernatants were analyzed by LC−MS/MS (Applied Biosystems Sciex API-4000) to determine plasma peptide levels. The methodology for analysis was described in detail elsewhere.29 The signals of tested peptides were detected by two multiple reaction monitoring (MRM) transitions. Bioavailability Program Package software (BAPP 2.2, China Pharmaceutical University) were used to calculated the pharmacokinetic parameters of liraglutide, 9 and 11.

Acute Food Intake The anorectic effect of 9 and 11 were measured in db/db mice.33 Briefly, 9 weeks old male db/db were randomly divided into 4 groups (n = 6) and mice were housed individually in cages and allowed to acclimate 7 days. To minimize the i.p. dosing effects on food intake, the mice were pricked a couple of times during the acclimated period. After fasted 18 h, mice were i.p. injected with saline (control), liraglutide, 9 and 11 (25 nmol·kg-1), and the pre-weighed food (standard chow) were placed in the cage. The cumulated food intake data were collected at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 18, and 24 h.

Long-Acting Hypoglycemic Efficacy Tests 24 ACS Paragon Plus Environment

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The long-acting hypoglycemic efficacy of liraglutide, 9 and 11 were determined using multiple OGTT on fasted db/db mice and hypoglycemic duration test on non-fasted db/db mice, using previously described method.34 For multiple OGTT, 9 weeks old male db/db mice (n = 6) were fasted 18 h, 0.5 h before glucose load, mice were i.p. administered with saline (control), liraglutide, 9 and 11 (25 nmol·kg-1). The glucose was loaded orally at t = 0 h (1.5 g·kg-1), and blood glucose levels were measured at 0, 0.25, 0.5, 1, 2, and 3 h using a one-touch glucometer (Ascensia, Breeze 2, Bayer, Germany). After the first OGTT, the glucose was loaded at 6, 12, and 18 h, and the blood collect intervals were identical in the remaining OGTT. For hypoglycemic duration test, db/db mice (male, 9 weeks old, n = 6) were under non-fasting conditions and allowed to free access to water and food. Saline (control), liraglutide (25 nmol·kg-1), 9 and 11 (25 or 100 nmol·kg-1) were i.p. administered at t = 0 h, and blood glucose levels were monitored at 0, 2, 4, 6, 12, 24 and 48 h using the same method as mentioned above.

Chronic Treatment Tests Nine weeks old male db/db mice were divided into 4 groups (n = 6) according to their HbA1c (DCA 2000+ chemistry analyzer, Bayer Diagnostics, USA).35, 36 Saline (control) and liraglutide (25 nmol·kg-1) were i.p. administered twice daily (at 7 am and 7 pm), and 9 and 11 (50 nmol·kg-1) were i.p. administered once daily (at 7 am) as according to their pharmacokinetic profiles over 35 days. Food intake and body weight change were measured every two days (at 7 am). Non-fasting blood glucose and insulin was assayed every four days (at 9 am) using the same method mentioned in hypoglycemic and insulinotropic assay section. HbA1c values in each group were measured again at the end of the treatment period. After 35 days of treatment, mice in each group were fasted 18 h, and i.p. challenged with glucose (1 g·kg-1) followed by sampling of blood glucose at 0, 15, 30, 60 and 120 min. The blood glucose levels were measured using a one-touch glucometer (Ascensia, Breeze 2, Bayer, Germany).

Statistical Analysis Data are stated as means ± SD. Pharmacology date analysis were using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Statistical significances of pharmacology data were performed by one-way ANOVA for multiple comparisons with post hoc Tukey’s tests. P 25 ACS Paragon Plus Environment


values < 0.05 were considered statistically significant. Pharmacokinetic parameters were calculated by Bioavailability Program Package software (BAPP 2.2, China Pharmaceutical University).

ASSOCIATED CONTENT Supporting Information Characterization of 1–11. Figure S1-S2. The Supporting Information associated with this article is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Jing Han); Phone: +86-516-83403166; Fax: +86-516-83403166 *E-mail: [email protected] (Junjie Fu); Phone: +86-025-86868480 *E-mail: [email protected] (Hai Qian); Phone: +86-25-83271302; Fax: +86-25-83271480

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

Notes The authors declare no competing financial interest.

Author contributions Jing Han designed the research study. Jing Han, Yingying Fei, Feng Zhou, Xinyu Chen and Hai Qian performed the research and analyzed the data. Jing Han and Junjie Fu wrote the paper.

Acknowledgment This work is supported by the National Natural Science Foundation of China (No. 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, the 26 ACS Paragon Plus Environment

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Open Project of State Key Laboratory of Natural Medicines (SKLNMKF201711), and Startup Funding for Introduced Talents of Nanjing Medical University (KY109RC1602).

Abbreviations GLP-1, glucagon-like peptide-1; T2DM, type 2 diabetes mellitus; HSA, human serum albumin; SPPS, solid-phase peptide synthesis; RP-HPLC, reversed phase high-performance liquid chromatography;

HPLC,

high-performance

liquid

chromatography;

LC−MS/MS,

liquid

chromatography−tandem mass spectrometry; IPGTT, intraperitoneal glucose tolerance test; AUC, area under the curve; 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; OGTT, oral glucose tolerance tests.

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(36) Han, J., Fu, J., Sun, L., Han, Y., Mao, Q., Liao, F., Zheng, X., and Zhu, K. (2017) Synthesis and pharmaceutical characterizations of site-specific mycophenolic acid-modified Xenopus Glucagon-like Peptide-1 analogs. MedChemComm. DOI: 10.1039/c7md00471k.

Graphical Table of Contents



Figure 1. Structures of 1 and 2. 55x21mm (300 x 300 DPI)

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Figure 2. Structures of 3–11. 189x211mm (300 x 300 DPI)



Figure 3. In vitro bioactivity tests. (A–B) Dose-response relationship of GLP-1, exendin-4, 1–7, 41/2 on GLP1R. Means ± SD, experiments were performed in triplicate and repeated three times (n = 3). 267x387mm (300 x 300 DPI)


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Figure 4. The glucose lowering effects of GLP-1, exendin-4, 1–7 (25 nmol·kg-1), 41/2 (12.5 nmol·kg-1 of 4) as determined by IPGTT in Kunming mice. (A and C) Blood glucose time-response curve (-30 to 120 min). (B and D) Hypoglycemic effects of GLP-1, exendin-4, 1–7 and 41/2 expressed as glucose AUC0-120 min. *P < 0.05, ***P < 0.001, cP < 0.001 vs. GLP-1, c’P < 0.001 vs. exendin-4. Means ± SD, n = 6. 187x137mm (300 x 300 DPI)



Figure 5. In vitro and in vivo bioactivity tests. (A) Dose-response relationship of GLP-1, exendin-4 and 8–11 on GLP-1R. Means ± SD, experiments were performed in triplicate and repeated three times (n = 3). (B) The glucose lowering effects of GLP-1, exendin-4 and 8–11 (25 nmol·kg-1) as determined by IPGTT in Kunming mice. (C) Hypoglycemic effects of GLP-1, exendin-4 and 8–11 expressed as glucose AUC0-120 min. ***P < 0.001, cP < 0.001 vs. GLP-1, c’P < 0.001 vs. exendin-4. Means ± SD, n = 6. 244x484mm (300 x 300 DPI)


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Figure 6. Antihyperglycemic and insulinotropic activities of 9 and 11 as determine by IPGTT in db/db mice. Saline, exendin-4, liraglutide, and 9 and 11 (25 nmol·kg-1) were i.p. injected 30 min prior to glucose load (1 g·kg-1, i.p.). (A) Blood glucose time-response curve (-30 to 120 min). (B) AUCglucose 0−120 min values of each group. (C) Plasma insulin time-response curve (0 to 120 min). (D) AUCinsulin 0–120 min values of each group. ***P < 0.001 vs. liraglutide, aP < 0.05 vs. exendin-4, cP < 0.001 vs. exendin-4. Means ± SD, n = 6. 181x139mm (300 x 300 DPI)



Figure 7. In vivo pharmacokinetic properties and anorectic effects of 9 and 11. (A) Pharmacokinetic profiles of liraglutide, 9 and 11 in SD rats. Means ± SD, n = 3. (B) Cumulated food intake following i.p. administered of saline, liraglutide, 9 and 11 (25 nmol·kg-1) in 18 h fasted db/db mice. Means ± SD, n = 6. 190x267mm (300 x 300 DPI)


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Figure 8. Long-term glucose-lowering effects of 9 and 11. (A–B) Long-term hypoglycemic effects of liraglutide, 9 and 11 were studied by the multiple OGTT in 18 h fasted db/db mice. Mice were i.p. injected with saline, liraglutide, 9 and 11 (25 nmol·kg-1) 0.5 h prior to oral glucose load (1.5 g·kg-1), and glucose loads were repeated at 6, 12 and 18 h. Blood glucose time-response curves (-0.5 to 24 h) are shown in panels A and B. (C) Hypoglycemic efficacies of liraglutide, 9 and 11 in nonfasted db/db mice. Time response curves for blood glucose lowering effects of liraglutide (25 nmol·kg-1), 9 and 11 (25 or 100 nmol·kg-1) in db/db mice after i.p. dosing. Means ± SD, n = 6. 242x375mm (300 x 300 DPI)



Figure 9. Chronic treatment effects of liraglutide, 9 and 11 in db/db mice. Saline, liraglutide (25 nmol·kg-1) was administered twice daily, and 9 and 11 (50 nmol·kg-1) were administered once daily over 35 days. (A) HbA1c (%), day 0 vs day 36. (B) Food intake. (C) Body weight. Non-fasting blood glucose levels (D) and non-fasting plasma insulin levels (E) were determined every four days. (F) Blood glucose time-response curves of each group in IPGTT test on day 37. Inset: Calculated AUCglucose 0–120 min values. Means ± SD, n = 6. 194x211mm (300 x 300 DPI)


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Scheme 1. Synthesis of 3–7. 151x70mm (300 x 300 DPI)



Scheme 2. Synthesis of 8–11. 182x86mm (300 x 300 DPI)


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