ABSTRACT
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.
Subject(s)
Drug Development , Exenatide/pharmacology , Glucagon-Like Peptide-1 Receptor/antagonists & inhibitors , Insulin-Secreting Cells/drug effects , Radiopharmaceuticals/pharmacology , Animals , Dose-Response Relationship, Drug , Exenatide/chemical synthesis , Exenatide/chemistry , Glucagon-Like Peptide-1 Receptor/metabolism , Insulin-Secreting Cells/metabolism , Iodine Radioisotopes , Male , Mice , Mice, Inbred ICR , Mice, Transgenic , Molecular Structure , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/chemistry , Structure-Activity Relationship , Tissue Distribution , Tomography, Emission-Computed, Single-PhotonABSTRACT
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.
Subject(s)
Evans Blue/analogs & derivatives , Evans Blue/metabolism , Exenatide/analogs & derivatives , Exenatide/metabolism , Serum Albumin/metabolism , Animals , Binding Sites , Cell Line, Tumor , Evans Blue/chemical synthesis , Exenatide/blood , Exenatide/chemical synthesis , Humans , Hypoglycemic Agents/blood , Hypoglycemic Agents/chemical synthesis , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/metabolism , Mice , Models, Molecular , Pharmaceutical Preparations/blood , Pharmaceutical Preparations/chemical synthesis , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/metabolism , Protein Binding , Protein Multimerization , Proteolysis , Rats , Serum Albumin/chemistryABSTRACT
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.
Subject(s)
Blood Glucose/drug effects , Body Weight/drug effects , Diabetes Mellitus, Experimental/drug therapy , Exenatide/pharmacology , Hypoglycemic Agents/pharmacology , Lipids/pharmacology , Animals , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/metabolism , Dose-Response Relationship, Drug , Exenatide/chemical synthesis , Exenatide/chemistry , Glucagon-Like Peptide-1 Receptor/agonists , Glucose Tolerance Test , Hypoglycemic Agents/chemical synthesis , Hypoglycemic Agents/chemistry , Lipids/chemistry , Male , Mice , Mice, Inbred Strains , Mice, Obese , Molecular Structure , Rats , Rats, Sprague-Dawley , Streptozocin , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Chemistry, Pharmaceutical/methods , Exenatide/chemical synthesis , Microspheres , Polylactic Acid-Polyglycolic Acid Copolymer/chemical synthesis , Animals , Delayed-Action Preparations/chemical synthesis , Delayed-Action Preparations/pharmacokinetics , Drug Evaluation, Preclinical/methods , Drug Liberation , Exenatide/pharmacokinetics , Hypoglycemic Agents/chemical synthesis , Hypoglycemic Agents/pharmacokinetics , Male , Particle Size , Polylactic Acid-Polyglycolic Acid Copolymer/pharmacokinetics , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Delayed-Action Preparations/administration & dosage , Delayed-Action Preparations/pharmacology , Exenatide/administration & dosage , Exenatide/pharmacology , Hypoglycemic Agents , Animals , Blood Glucose/metabolism , Cells, Cultured , Delayed-Action Preparations/chemical synthesis , Delayed-Action Preparations/pharmacokinetics , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/drug therapy , Dose-Response Relationship, Drug , Exenatide/chemical synthesis , Exenatide/pharmacokinetics , Insulin Resistance , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Rats, Sprague-Dawley , Time FactorsABSTRACT
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.
Subject(s)
Corrinoids/chemistry , Corrinoids/pharmacology , Exenatide/analogs & derivatives , Exenatide/pharmacology , Glucagon-Like Peptide-1 Receptor/agonists , Animals , Cells, Cultured , Corrinoids/chemical synthesis , Exenatide/chemical synthesis , Glucagon-Like Peptide-1 Receptor/metabolism , HEK293 Cells , Humans , Hypoglycemic Agents/chemical synthesis , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacology , Insulin Secretion/drug effects , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Models, Molecular , Rats , Rats, Sprague-DawleyABSTRACT
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.