Design And Synthesis Of Long-acting Glucagon-like Peptide-1 Analogs And Their Hypoglycemic Activity Studies

Glucagon-like peptide-1(GLP-1)and its analogs(e.g.Exendin-4,Ex4)are peptide-based incretin agents that have been widely used in the treatment of type 2 diabetes mellitus(T2DM).A major challenge in the clinical use of GLP-1/Exendin-4 based peptides as therapeutic agents is that the inherent properties of peptides,such as susceptibility to proteases and rapid renal clearance,resulting in short half-lives and poor druggability in vivo that requires high doses and increased dosing frequency to achieve therapeutic efficacy.Various strategies have been developed to improve the pharmacokinetic properties of GLP-1based peptides,mainly including PEGylation,long-chain fatty acid or other small molecule modifications to enhance the interaction with albumin,and fusion expression of GLP-1peptides with albumin or Fc,etc.However,while the aforementioned strategies effectively improved the pharmacokinetic properties of GLP-1 and its analogs,they have also generated some new problems and challenges,such as immunogenicity,homogeneity of product and complexity of the preparation process,etc.The development of GLP-1 analogs with novel mechanisms of action for extending half-lives could improve the therapeutic efficacy and window of action of T2DM.Using the clinically approved Ex4 as a model peptide,this thesis explored new strategies via modification of Ex4 with dextran and haptens to improve its pharmacokinetic properties and long-acting hypoglycemic activity,based on orthogonal chemistry and Sortase A(Srt A)site-modification techniques.At the end of this thesis,a semi-synthetic method was developed to synthesize semaglutide by utilizing the Ser/Thr ligation(STL)strategy combined with recombinant expression technology.The main results were as follows:(1)The"chemical mutation"Ex4 analogs(Ex4-12K*,Ex4-27K*,Ex4-12-27K*)were modified by dextran via oxime ligation chemistry to extend the duration of hypoglycemic activity of Ex4.Through solid-phase peptide synthesis(SPPS)technology,the amino acids at positions 12,27 and 12/27 of Ex4 were"chemically mutagenized"to prepare Ex4 analogs containing hydroxylamine lysine,and then dextran with different molecular weights(Mw=6000 Da and 20000 Da)were site-specifically ligated to the hydroxylamine-containing lysine of Ex4 analogs,and six homogeneous dextran-Ex4 conjugates were successfully constructed.In vitro receptor binding assay and in vivo intraperitoneal glucose tolerance test(IPGTT)showed that dextran-modified sites had significant effects on glucagon-like peptide-1 receptor(GLP-1R)activation and acute antidiabetic capacity,and dextran modification at position 12of Ex4 generated the least side-effect on its GLP-1R activation capacity and basically preserved Ex4's ability to promote insulin secretion and thus regulate blood glucose.The results of the antidiabetic duration test showed that the long-acting hypoglycemic ability of all dextran-Ex4 conjugates was improved except dextran-Ex4-12K*-27K*(6000),and the modification sites and the molecular weight of dextrans used for modification had significant effects on the antidiabetic duration ability.The structure-activity relationship study showed that dextran-Ex4-12K*(20000)maintained the lowest blood glucose levels at 4,6,8,12 and24 hour and final AUC glucose 0-24h values,indicating that dextran-Ex4-12K*(20000)has an optimal long-term hypoglycemic effect.(2)A new strategy for utilization of hapten DNP as an endogenous antibody binder to extend the half-life of Ex4 was developed.Three Ex4-DNP conjugates 6-8 and FITC-labelled Ex4-DNP conjugates 9-11 containing different lengths spacer arms were prepared by Sortase A-mediated ligation(SML).In vitro intracellular GLP-1R activation assays showed that DNP site-modification resulted in a slight decrease in Ex4's ability to stimulate GLP-1R,but their EC₅₀ values remained at nanomolar levels(6.1.39 n M;7:1.98 n M;8:4.03 n M).The results of pharmacokinetic experiments showed that Ex4 modification with hapten DNP can significantly extend its half-life in the presence of abundant anti-DNP antibodies in vivo,and the half-life of Ex4-DNP conjugate 10 which contained a medium length PEG linker reached to 3.55 hours,that was 4.9 times longer than that of unmodified Ex4.In addition,the duration of antidiabetic activity of Ex4-DNP conjugates was significantly enhanced in the pre-immunized db/db mice compared to naive db/db mice,with Ex4-DNP conjugate 7containing the PEG₃ linker showing the best long-acting antidiabetic effect,that was consistent with the results of pharmacokinetic experiments.In the final chronic treatment studies,once-daily administration of an optimal Ex4-DNP conjugate 7 demonstrated more beneficial effects on improving HbA1c,glucose tolerance,lipid metabolism,and protecting pancreas islets without prominent toxicity effects and immunogenicity compared to twice daily administration of native Ex4.(3)A new strategy for the utilization of the dual action mechanism of hapten Rha as an endogenous antibody binder and aliphatic chains as an albumin binder to extend the half-life of Ex4 was developed.Ex4-Lipid-Rha conjugates 5-7 were successfully constructed via SML strategy.In vitro intracellular GLP-1R activation assays showed that Rha-Lipid site-modification resulted in a slight decrease in Ex4's ability to stimulate GLP-1R,but their EC₅₀ values remained at sub-nanomolar levels(5:0.14 n M;6:0.19 n M;7:0.21 n M).In vivo IPGTT experiments demonstrated that modification of the C-terminus of Ex4 with Rha-Lipid did not affect its acute antidiabetic activity.The results of antidiabetic duration test showed that the duration of antidiabetic activity of Ex4-Lipid-Rha conjugates 5-7 were all somewhat enhanced in db/db diabetic mice model before pre-immunization with Rha-OVA.On the other hand,the long-acting antidiabetic effect of conjugates 5-7 was further enhanced in db/db diabetic mice model after pre-immunization,and the gain effect at this time was attributed not only to the interaction of aliphatic chains with albumin in vivo,but also to the interaction of Rha with endogenous anti-Rha antibodies.(4)A semi-synthetic method based on STL strategy was developed for preparation of the long-acting hypoglycemic agent semaglutide.The STL reaction conditions between tetrapeptide salicylaldehyde esters 2 and Arg³⁴ GLP-1(11-37)were optimized in terms of solvent,temperature,co-solvent and feeding ratio,and it was finally determined that under the condition of 1:120 ratio of pyridine-acetic acid buffer system(mol/mol),reaction temperature of 40°C,1:3.76 equivalence ratio of Arg³⁴ GLP-1(11-37)to 2 and without the addition of any co-solvent,the peptide backbone of semaglutide was successfully prepared in 85%conversion.On this basis,the acylation reaction conditions between the side chain amino group of Lys²⁶ in the peptide backbone and C18 Diacids and linkers(?Glu-2x OEG)were further optimized in terms of the reaction system,the p H of the reaction solution and the dropping speed of the acylating agent,and it was finally determined that under the condition of 50 m M H₃BO₃(p H=10.5)and dropping speed of the acylation agent within 0.5 h,the conversion of the above acylation reaction was>99%.After removal of the protecting group,the product quality of semaglutide synthesized by this novel method was demonstrated to be consistent with that of the branded drug in terms of mass spectrometry and cellular biological activity level.