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Background and purpose Type 2 diabetes is one of the most severe chronic diseases and is an increasingly important public health problem worldwide. Several agonists of the glucagon-like peptide-1 (GLP-1) receptor have been developed to treat Type 2 diabetes but most of them are administered by injection. This mode of administration seriously reduces patient compliance and increases the risk of infection. Here, we describe the actions of a novel, orally available, GLP-1 receptor agonist - oral hypoglycaemic peptide 2 (OHP2) - derived from exendin-4 by replacing amino acids. We have also investigated its pharmacokinetic profiles, therapeutic effects and absorption mechanism. Experimental approach Healthy Wistar rats were used for pharmacokinetic analyses. In diabetic db/db mice. OHP2 was given for 8 weeks to evaluate its effects on hyperglycaemia, dyslipidaemia, basal metabolism and tissue injury. Possible endocytosis and transcytosis mechanisms of OHP2 uptake were explored in Caco-2 cell monolayers. Key results In rats, the absolute bioavailability of orally administered OHP2 was 20-fold greater than that of orally administered exendin-4. In db/db mice, OHP2 dose-dependently exhibits good potential in glucose-lowering and weight loss after oral administration. OHP2 also alleviated hyperlipidaemia, ameliorated energy metabolism and promoted tissue repair in diabetic mice. Furthermore, uptake of OHP2 by Caco-2 cells was dependent on caveolae-mediated transcytosis rather than endocytosis mediated by GLP-1 receptors. Conclusions and implications OHP2 is a potential, orally bioavailable, candidate drug for the treatment of Type 2 diabetes. Its transcytosis mechanism of uptake could help in the development of absorption enhancers of OHP2.

Given that the biological functions of proteins may decrease or even be lost due to degradation by proteases, it is of great significance to identify potential proteases that degrade protein drugs during systemic circulation. In this work, we describe a method based on high-performance liquid chromatography (HPLC) to identify key proteases that degrade therapeutic proteins in blood, including endopeptidases and exopeptidases. Here, the degradation of proteins was detected by competition with standard substrates of proteases and is shown as the relative residue rate. Four protein drugs were subjected to this method, and the results suggested that growth hormone was degraded by aminopeptidase N and kallikrein-related peptidase 5, pertuzumab was hardly degraded by the proteases, factor VII was degraded by carboxypeptidase B, neprilysin, dipeptidyl peptidase-4 and peptidyl dipeptidase A, and fibrinogen was degraded by carboxypeptidase B and kallikrein-related peptidase 5, findings consistent with the literature. The results were confirmed by microscale thermophoresis; additionally, activity detection in vitro substantiated that the degradation of factor VII decreased its activity. We demonstrate that this method can be used to identify key proteases of proteins with high accuracy, precision and durability.

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. Keywords: GLP-1 mimetic, PEGylation, db/db mice, Pharmacodynamics, Pharmacokinetics

Exendin-4 is a strong therapeutic candidate for the treatment of metabolic syndrome. Related receptor agonist drugs have been on the market since 2005. However, technical limitations and the pain caused by subcutaneous injection have severely limited patient compliance. The goal of the study is to investigate a biologically active exendin-4 analog could be administered orally. Using intraperitoneal glucose tolerance tests, we discovered that exendin4-cysteine administered by oral gavage had a distinct hypoglycemic effect in C57BL/6J mice. Using Rosetta Design and Amber, we designed and screened a series of exendin4-cysteine analogs to identify those that retained biological activity while resisting trypsin digestion. Trypsin Cleavage Site Mutated Exendin4-cysteine 1 (TSME-1), an analog whose bioactivity was similar to exendin-4 and was almost completely resistant to trypsin, was screened out. In addition, TSME-1 significantly normalized the blood glucose levels and the availability of TSME-1 was significantly higher than that of exendin-4 and exendin4-cysteine. Collectively orally administered TSME-1, a trypsin-resistant exendin-4 analog obtained by the system, is a strong candidate for future treatments of type 2 diabetes.