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A brush-polymer/exendin-4 conjugate reduces blood glucose levels for up to five days and eliminates poly(ethylene glycol) antigenicity

Abstract

The delivery of therapeutic peptides and proteins is often challenged by short half-lives and the consequent need for frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. Here, we demonstrate that a single subcutaneous injection of site-specific (C-terminal) conjugates of exendin-4 (exendin)—a therapeutic peptide that is clinically used to treat type 2 diabetes mellitus—and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) with precisely controlled molecular weights lowered blood glucose for up to 120 h in fed mice. Most notably, we show that an exendin-C-POEGMA conjugate with an average of nine side-chain ethylene glycol (EG) repeats exhibits significantly lower reactivity towards patient-derived anti-poly(ethylene glycol) (anti-PEG) antibodies than two US FDA-approved PEGylated drugs, and that reducing the side-chain length to three EG repeats completely eliminates PEG antigenicity without compro­mising in vivo efficacy. Our findings establish the site-specific conjugation of POEGMA as a next-generation PEGylation technology for improving the pharmacological performance of traditional PEGylated drugs, whose safety and efficacy are hindered by pre-existing anti-PEG antibodies in patients.

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Figure 1: Synthesis of exendin-C-POEGMA.
Figure 2: Characterization of the exendin-C-Br macroinitiator and EG9 exendin-C-POEGMA conjugates.
Figure 3: Assessment of the Mw -dependent in vivo efficacy of EG9 exendin-C-POEGMA conjugates.
Figure 4: IPGTT for an EG9 exendin-C-POEGMA conjugate in mice.
Figure 5: Assessment of the reactivity of exendin-C-POEGMA conjugates towards anti-PEG antibodies in patient plasma samples.
Figure 6: Assessment of the in vivo efficacy and pharmacokinetics of exendin-C-POEGMA conjugates.

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Acknowledgements

The authors thank D.M. Gooden at the Duke Small Molecule Synthesis Facility for the synthesis of AEBMP, E.J. Soderblom at the Duke Proteomics Facility for conducting LC/MS-MS, G. Dubay at the Duke Chemistry Mass Spectrometry Facility for LC/ESI-MS support and M.N. Feinglos for discussion of the in vivo results. BHK cells expressing GLP-1R were a gift from the Drucker group (University of Toronto, Canada). This work was supported by the National Institutes of Health (R01-DK092665 to A.C.).

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Y.Q. and A.C. conceived and designed the research. Y.Q., A.S., N.J.G., X.L, I.O. and W.L. performed the experiments. A.S. and K.M. provided technical expertise in polymer chemistry. K.M.L. and W.L. contributed to the design of the in vivo studies. N.J.G. and M.S.H. provided materials and technical expertise for the antigenicity studies. Y.Q., N.J.G., K.M.L., M.S.H., K.M. and A.C. analysed and interpreted the results. Y.Q. and A.C. wrote the manuscript and A.S., N.J.G., K.M.L., M.S.H. and K.M. edited the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Ashutosh Chilkoti.

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Competing interests

A.C. and Y.Q. have a pending patent on the sortase-catalysed C-terminal polymer conjugation technology (WO 2014194244 A1). M.S.H. is a co-inventor of Pegloticase (Krystexxa) and receives royalties from sales of Pegloticase, along with his employer, Duke University. The results reported in this paper form the basis of US provisional patent applications (62/270,401; 62/329,800; 62/310,534; 62/407,403) filled by A.C., Y.Q., M.S.H. and N.J.G. through the Duke University Office of Licensing & Ventures.

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Qi, Y., Simakova, A., Ganson, N. et al. A brush-polymer/exendin-4 conjugate reduces blood glucose levels for up to five days and eliminates poly(ethylene glycol) antigenicity. Nat Biomed Eng 1, 0002 (2017). https://doi.org/10.1038/s41551-016-0002

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