Design of chimeric GLP-1A using oligomeric bile acids to utilize transporter-mediated endocytosis for oral delivery

Abstract

Background: Despite the effectiveness of glucagon-like peptide-1 agonist (GLP-1A) in the treatment of diabetes, its large molecular weight and high hydrophilicity result in poor cellular permeability, thus limiting its oral bioavailability. To address this, we developed a chimeric GLP-1A that targets transporter-mediated endocytosis to enhance cellular permeability to GLP-1A by utilizing the transporters available in the intestine, particularly the apical sodium-dependent bile acid transporter (ASBT).

Methods: In silico molecular docking and molecular dynamics simulations were used to investigate the binding interactions of mono-, bis-, and tetra-deoxycholic acid (DOCA) (monoDOCA, bisDOCA, and tetraDOCA) with ASBT. After synthesizing the chimeric GLP-1A-conjugated oligomeric DOCAs (mD-G1A, bD-G1A, and tD-G1A) using a maleimide reaction, in vitro cellular permeability and insulinotropic effects were assessed. Furthermore, in vivo oral absorption in rats and hypoglycemic effect on diabetic db/db mice model were evaluated.

Results: In silico results showed that tetraDOCA had the lowest interaction energy, indicating high binding affinity to ASBT. Insulinotropic effects of GLP-1A-conjugated oligomeric DOCAs were not different from those of GLP-1A-Cys or exenatide. Moreover, bD-G1A and tD-G1A exhibited improved in vitro Caco-2 cellular permeability and showed higher in vivo bioavailability (7.58% and 8.63%) after oral administration. Regarding hypoglycemic effects on db/db mice, tD-G1A (50 μg/kg) lowered the glucose level more than bD-G1A (50 μg/kg) compared with the control (35.5% vs. 26.4%).

Conclusion: GLP-1A was conjugated with oligomeric DOCAs, and the resulting chimeric compound showed the potential not only for glucagon-like peptide-1 receptor agonist activity but also for oral delivery. These findings suggest that oligomeric DOCAs can be used as effective carriers for oral delivery of GLP-1A, offering a promising solution for enhancing its oral bioavailability and improving diabetes treatment.

Keywords: ASBT-mediated endocytosis; Chimeric peptide; In silico molecular docking; Oligomeric bile acids; Oral GLP-1 agonist.

Scheme 1

Scheme for developing oral chimeric GLP-1 agonists

Fig. 1

In silico molecular docking analysis and molecular dynamics simulation between oligomer DOCAs and apical sodium bile acid transporter (ASBT). A Substrate-binding cavity colored in back born structure of ASBT and B docking simulation of oligomeric DOCAs (monoDOCA, bisDOCA, and tetraDOCA) to the pore of ASBT. C Binding affinities of oligomeric DOCAs to ASBT. After MD simulation, D the interaction energy over the time range of 0 to 100 ps during MD simulation and average interaction energy. E Stabilized binding poses of oligomeric DOCAs, and F categories and counts of interactions between oligomeric DOCAs and ASBT

Fig. 2

MD simulations of GLP-1A and oligomeric DOCA-G1As. A Conformations of ASBT and GLP-1A were recorded at specific time intervals (baseline to 80 ps) during the MD simulation. B Recorded conformations of ASBT and oligomeric DOCA-G1As. C Continuous change in interaction energy from 0 to 100 ps between ASBT and oligomeric DOCA-G1As. D Interaction energy between ASBT and each DOCA motif in oligomeric DOCA

Fig. 3

Synthesis of oligomeric DOCA-G1As. A Schematic synthesis steps for monoDOCA-G1As (mD-G1A), bisDOCA-G1A (bD-G1A), and tetraDOCA-G1A (tD-G1A); conjugated oligomeric DOCAs to GLP-1A-Cys with 6-maleimidohexanoic NHS (EMCS); and B MALDI-TOF MS identification

Fig. 4

In vitro efficacy of chimeric GLP-1A for ASBT-mediated endocytosis and insulinotropic effect. A Scheme of the cellular uptake mechanism for GLP-1A and oligomeric DOCA-G1As utilizing ASBT-mediated endocytosis, leading to the formation of ASBT vesicles in cytosol. B Cellular permeability in Caco-2 cells after treatment with 1 μM of exenatide, mD-G1A, bD-G1A, and tD-G1A (n = 3; data are presented as means ± standard deviations). ^*p < 0.05 and ^***p < 0.001 compared with exenatide. C Relative P_app of oligomeric DOCA-G1A in the presence of actinomycin D (Act D) alone, clofazimine (CFZ) alone, or both (Act D + CFZ) (n = 4; data are presented as means ± standard deviations). ^***p < 0.001 compared with relative P_app of the non-treated group. D ASBT distribution from the membrane and cytoplasm after administration of 1 μM of tD-G1A. E Relative signal intensity of ASBT in membrane and cytoplasm. ASBT quantification was conducted using ImageJ software (n = 3; data are presented as means ± standard deviations). ^**p < 0.01 and ^***p < 0.001 compared with the control. F Scheme of GLP-1R binding of oligomeric DOCA-G1A. G Insulin secretion (n = 4; data are presented as means ± standard deviations) by islet β cells in low-glucose (2.8 mM) and high-glucose (28 mM) conditions after drug treatment [non-treated; exenatide, 5 nM (control); GLP-1A-Cys, 5 nM; mD-G1A, 5 nM; bD-G1A, 5 nM; tD-G1A, 5 nM] and H the secretion index. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 compared with exenatide

Fig. 5

In vivo oral absorption of GLP-1A-Cys and oligomeric DOCA-G1As. A Time course of GLP-1A concentration after treatment with intravenous GLP-1A-Cys (10 μg/kg, n = 4), oral GLP-1A-Cys (100 μg/kg, n = 4), and B oral oligomeric DOCA-G1As (100 μg/kg, n = 4). Data are presented as mean ± standard deviation. ^**p < 0.01 and ^***p < 0.001 compared with GLP-1A-Cys

Fig. 6

Hypoglycemic effect of oligomeric DOCA-G1As in db/db mice. A IPGTT after treatment with control (saline, oral, n = 4), exenatide (5 µg/kg, subcutaneous, n = 4), bD-G1A (50 μg/kg, oral, n = 4), and tD-G1A (50 μg/kg, oral, n = 4) following glucose (2 g/kg) injection. Data are presented as mean ± standard deviation. ^**p < 0.01 compared with the control. B Time course of changes in blood glucose levels of db/db mice displaying a high fasting glucose level after treatment with exenatide (5 µg/kg, subcutaneous, n = 4), GLP-1A-Cys (20 μg/kg, oral, n = 4), bD-G1A (20 μg/kg, oral, n = 4), and tD-G1A (20 μg/kg, oral, n = 4). Data are presented as means ± standard deviations. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 compared with exenatide. ^###p < 0.001 compared with GLP-1A-Cys