Development of novel radioiodinated exendin-4 derivatives targeting GLP-1 receptor for detection of ß-cell mass.

In subjects with type 2 diabetes mellitus (T2DM), pancreatic ß-cell mass decreases; however, it is unknown to what extent this decrease contributes to the pathophysiology of T2DM. Therefore, the development of a method for noninvasive detection of ß-cell mass is underway. We previously reported that glucagon-like peptide-1 receptor (GLP-1R) is a promising target molecule for ß-cell imaging. In this study, we attempted to develop a probe targeting GLP-1R for ß-cell imaging using single-photon emission computed tomography (SPECT). For this purpose, we selected exendin-4 as the lead compound and radiolabeled lysine at residue 12 in exendin-4 or additional lysine at the C-terminus using [123I]iodobenzoylation. To evaluate in vitro receptor specificity, binding assay was performed using dispersed mouse islet cells. Biodistribution study was performed in normal ddY mice. Ex vivo autoradiography was performed in transgenic mice expressing green fluorescent protein under control of the mouse insulin I gene promoter. Additionally, SPECT imaging was performed in normal ddY mice. The affinity of novel synthesized derivatives toward pancreatic ß-cells was not affected by iodobenzoylation. The derivatives accumulated in the pancreas after intravenous administration specifically via GLP-1R expressed on the pancreatic ß-cells. Extremely high signal-to-noise ratio was observed during evaluation of biodistribution of [123I]IB12-Ex4. SPECT images using normal mice showed that [123I]IB12-Ex4 accumulated in the pancreas with high contrast between the pancreas and background. These results indicate that [123I]IB12-Ex4 for SPECT is useful for clinical applications because of its preferable kinetics in vivo.

Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4.

Corrination is the conjugation of a corrin ring containing molecule, such as vitamin B12 (B12) or B12 biosynthetic precursor dicyanocobinamide (Cbi), to small molecules, peptides, or proteins with the goal of modifying pharmacology. Recently, a corrinated GLP-1R agonist (GLP-1RA) exendin-4 (Ex4) has been shown in vivo to have reduced penetration into the central nervous system relative to Ex4 alone, producing a glucoregulatory GLP-1RA devoid of anorexia and emesis. The study herein was designed to optimize the lead conjugate for GLP-1R agonism and binding. Two specific conjugation sites were introduced in Ex4, while also utilizing various linkers, so that it was possible to identify Cbi conjugates of Ex4 that exhibit improved binding and agonist activity at the GLP-1R. An optimized conjugate (22), comparable with Ex4, was successfully screened and subsequently assayed for insulin secretion in rat islets and in vivo in shrews for glucoregulatory and emetic behavior, relative to Ex4.

Novel lipid side chain modified exenatide analogs emerged prolonged glucoregulatory activity and potential body weight management properties.

Exenatide is known as the first marketed GLP-1 agonist for antidiabetic treatment, but it need twice injection a day because of its fast clearance. This work aims to prolong the half-life of exenatide by modified with novel lipid chain. Four optimized exenatide analogs named as Cys12-Exenatide (1-39)-NH2, Cys40-Exenatide (1-39)-NH2, Cys12-Tyr22-Gln24-Glu28-Arg35-Exenatide (1-39)-NH2 and Tyr22-Gln24-Glu28-Arg35-Cys40-Exenatide (1-39)-NH2 were selected and applied for conjugation. Then a series of evaluations including GLP-1R activation assay were conducted, conjugation C2 was selected for further investigation. Glucoregulatory and insulin secretion assay and hypoglycemic duration test were accessed and showed that C2 was capable of comparable insulinotropic activities and glucose-lowering abilities with those of liraglutide and exenatide. Cell protective effects in INS-1 cells confirmed that C2 had relatively protection effects. Meanwhile, once daily injection of C2 to STZ-induced diabetic mice achieved long-term beneficial effects on glucose tolerance, body weight and blood chemistry. Acute feeding studies were evaluated in DIO mice. These results suggested that C2 is a promising agent for further investigation of its potential to treat diabetes patients with obese.

Preliminary evaluation of 18F­AlF­NOTA­MAL­Cys40­Exendin­4 in rodent heart after myocardial ischemia and reperfusion.

Glucagon­like peptide­1 (GLP­1) and its receptor (GLP­1R) exert cardioprotective effects after myocardial ischemia and reperfusion (MI/R) in animal models and human clinical trials. Receptor imaging with positron emission tomography (PET) provides a non­invasive method for monitoring GLP­1R expression. In the present study, a fluorine­18­labeled aluminum fluoride exendin­4 analog [18F­AlF conjugated with 1,4,7­triazacyclononanetriacetic acid (NOTA)­maleimide (MAL)­Cys40­exendin­4] was synthesized and evaluated in a rat MI/R model for GLP­1R imaging. NOTA­MAL­Cys40­exendin­4 was synthesized by coupling Cys40­exendin­4 with NOTA­MAL. NOTA­MAL­Cys40­exendin­4 was then conjugated with 18F­AlF to obtain 18F­AlF­NOTA­MAL­Cys40­exendin­4. The yield of 18F­AlF­NOTA­MAL­Cys40­exendin­4 was 18.5±3.4% (not decay corrected). The process was completed within ~30 min. In rat MI/R models, the tracer exhibited specific binding to GLP­1R and an appropriate signal­to­noise ratio. At 8 h post­MI/R, tracer uptake reached its peak [0.35±0.053% of injected dose (%ID)/g; n=6] in ischemic myocardium. Localized tracer uptake decreased 1 day (0.20±0.032 %ID/g; n=6) and 3 days (0.16±0.017 %ID/g; n=6) post­MI/R compared with 8 h post­MI/R, but still remained higher compared with sham­operated groups (0.06±0.012 %ID/g; n=6). Pre­injected unlabeled exendin­4 effectively blocked tracer accumulation (0.09±0.041 %ID/g; n=6). In conclusion, 18F­AlF­NOTA­MAL­Cys40­exendin­4 demonstrated favorable characteristics for GLP­1R imaging following MI/R. PET imaging using 18F­AlF­NOTA­MAL­Cys40­exendin­4 in rodent hearts after MI/R revealed a dynamic pattern of GLP­1R upregulation.

An Albumin Sandwich Enhances in Vivo Circulation and Stability of Metabolically Labile Peptides.

The effectiveness of numerous molecular drugs is hampered by their poor pharmacokinetics. Different from previous approaches with limited effectiveness, most recently, emerging high-affinity albumin binding moieties (ABMs) for in vivo hitchhiking of endogenous albumin opens up an avenue to chaperone small molecules for long-acting therapeutics. Although several FDA-approved fatty acids have shown prolonged residence and therapeutic effect, an easily synthesized, water-soluble, and high-efficiency ABM with versatile drug loading ability is urgently needed to improve the therapeutic efficacy of short-lived constructs. We herein identified an ideal bivalent Evans blue derivative, denoted as N(tEB)2, as a smart ABM-delivery platform to chaperone short-lived molecules, through both computational modeling screening and efficient synthetic schemes. The optimal N(tEB)2 could reversibly link two molecules of albumin through its two binding heads with a preferable spacer, resulting in significantly extended circulation half-life of a preloaded cargo and water-soluble. Notably, this in situ dimerization of albumin was able to sandwich peptide therapeutics to protect them from proteolysis. As an application, we conjugated N(tEB)2 with exendin-4 for long-acting glucose control in a diabetic mouse model, and it was superior to both previously tested NtEB-exendin-4 (Abextide) and the newly FDA-approved semaglutide, which has been arguably the best commercial weekly formula so far. Hence, this novel albumin binder has excellent clinical potential for next-generation biomimetic drug delivery systems.

Formation Mechanism, In vitro and In vivo Evaluation of Dimpled Exenatide Loaded PLGA Microparticles Prepared by Ultra-Fine Particle Processing System.

Spherical poly (D, L-lactic-co-glycolic acid) microparticles (PLGA-MPs) have long been investigated in order to achieve sustained delivery of proteins/peptides. However, the formation mechanism and release characteristics of the specific shape MPs were still unknown. This study aimed to develop a novel-dimpled exenatide-loaded PLGA-MPs (Exe-PLGA-MPs) using an ultra-fine particle processing system (UPPS) and investigate the formation mechanism and release characteristics. Exe-PLGA-MPs were prepared by UPPS and optimized based on their initial burst within the first 24 h and drug release profiles. Physicochemical properties of Exe-PLGA-MPs, including morphology, particle size, and structural integrity of Exe extracted from Exe-PLGA-MPs, were evaluated. Furthermore, pharmacokinetic studies of the optimal formulation were conducted in Sprague-Dawley (SD) rats to establish in vitro-in vivo correlations (IVIVC) of drug release. Exe-PLGA-MPs with dimpled shapes and uniform particle sizes achieved a high encapsulation efficiency (EE%, 91.50 ± 2.65%) and sustained drug release for 2 months in vitro with reduced initial burst (20.42 ± 1.64%). Moreover, the pharmacokinetic studies revealed that effective drug concentration could be maintained for 3 weeks following a single injection of dimpled Exe-PLGA-MPs with high IVIVC. Dimpled PLGA-MPs prepared using the UPPS technique could thus have great potential for sustained delivery of macromolecular proteins/peptides.

C-terminal site-specific PEGylated Exendin-4 analog: A long-acting glucagon like Peptide-1 receptor agonist, on glycemic control and beta cell function in diabetic db/db mice.

PEG modification is a common clinical strategy for prolonging the half-life of therapeutic proteins or polypeptides. In a previous work, we have successfully synthesized PEG-modified Exendin-4 (PE) by conjugating a 20 kDa PEG to the C-terminal of Exendin-4. Then, we introduced an integrative characterization for PE to evaluate its hypoglycemic activity and pharmacokinetic properties. The normoglycemic efficacies and therapeutic activity of PE were investigated in db/db mice. The hypoglycemic time after single administration of PE on db/db mice was prolonged from 8.4 h to 54.9 h. In multiple treatment with PE, the fasting blood glucose in various PE dosages (50, 150, and 250 nmol/kg) were remarkably reduced, and the glycosylated hemoglobin level was decreased to 2.0%. When the in vivo single- and multiple-dose pharmacokinetics of PE were examined in Sprague-Dawley rats, the half-life was prolonged to 31.7 h, and no accumulation effect was observed. Overall, this study provided a novel promising therapeutic approach to improving glucose-controlling ability and extending half-life without accumulation in vivo for long-acting treatment of type-2 diabetes.