Peptides
`
`The Wave of the Future
`
`MPI EXHIBIT 1013 PAGE 1
`
`MPI EXHIBIT 1013 PAGE 1
`
`

`

`Peptides: The Wave of the Future
`
`MPI EXHIBIT 1013 PAGE 2
`
`MPI EXHIBIT 1013 PAGE 2
`
`

`

`Peptides
`
`The Wave of the Future
`
`Proceedings of the Second International
`and the Seventeenth American
`Peptide Symposium
`
`Edited by
`
`Michal Lebl
`and
`Richard A. Houghten
`
`Springer-Science+Business Media, B.V.
`
`MPI EXHIBIT 1013 PAGE 3
`
`MPI EXHIBIT 1013 PAGE 3
`
`

`

`Peptides: The Wave of the Future
`
`Proceedings of the Second International and the
`Seventeenth American Peptide Symposium
`June 9-14, 2001, San Diego, California, U.S.A.
`
`Edited by
`
`Michal Lebl
`Spyder Instruments, Inc. and Illumina, Inc.
`9885 Towne Centre Drive
`San Diego, CA 92121
`michal@ 5z. com
`
`and
`
`Richard A. Houghten
`Torrey Pines InstituteforMolecu/ar Studies
`Mixture Sciences, Inc.
`3550 General Atomics Court
`San Diego, CA 92121
`rhoughten@tpims.org
`
`Springer-Science+Business Media, B.V.
`
`MPI EXHIBIT 1013 PAGE 4
`
`MPI EXHIBIT 1013 PAGE 4
`
`

`

`A C.I.P. Catalogue record for this book is available from the Library of Congress.
`
`ISBN 978-94-010-0464-0 (eBook)
`ISBN 978-94-010-3905-5
`DOI 10.1007/978-94-010-0464-0
`
`Copyright ©2001 by Springer Science+Business Media Dordrecht
`Originally published by American Peptide Society in 2001.
`Softcover reprint of the bardeover 1st edition 2001
`
`All Rights Reserved. No part of the material protected by this copyright notice may be
`reproduced or utilized in any form or by any means, electronic or mechanical,
`including photocopying, recording or by any information storage and retrieval system,
`without written permission from the copyright owner.
`
`MPI EXHIBIT 1013 PAGE 5
`
`MPI EXHIBIT 1013 PAGE 5
`
`

`

`Peptides: The Wave of the Future
`Michal Lebl and Richard A. Houghten (Editors)
`American Peptide Society, 2001
`
`Glucagon-Like Peptide-1 Analogs with Significantly Improved
`in vivo Activity
`Jesse Z. Dong, Yeelana Shen, John E. Taylor, Michael Culler, Chee-Wai Woon,
`Barry Morgan, Steve Skinner and Jacques-Pierre Moreau
`Biomeasure lncorporated/Beaufour-IPSEN, Milford, MA 01757, USA
`
`Introduction
`Glucagon-like peptide-1 (GLP-1), a potent and strictly glucose-dependent insulino(cid:173)
`tropic agent, has received increasing attention as a possible new treatment for type 2
`diabetes. Although its effectiveness in type 2 diabetes patients has been demonstrated
`in clinical evaluations, the potential use of the native GLP-1 as a therapeutic agent is
`greatly hampered by its short plasma half-life. Physiologically, GLP-1 is rapidly de(cid:173)
`graded by endoproteases. Here we report that a series of novel human GLP-1
`(hGLP-1) analogs have been designed and synthesized, which have greatly improved
`plasma half-life and significantly enhanced in vivo activity.
`Results and Discussion
`One of the enzymes that are responsible for the fast degradation of GLP-1 in vivo is
`DPP-IV, which cleaves the amide bond between Ala8 and Glu9 at theN-terminus of
`hGLP-1 [1]. To prevent this enzymatic cleavage, we replaced Ala8 with some unnatu(cid:173)
`ral amino acids, including N-methyl-D-alanine (N-Me-D-Ala), 1-arninocyclopentane-
`1-carboxylic acid (A5c), and aminoisobutyric acid (Aib). These sterically hindered
`amino acids make the peptide bond between positions 8 and 9 less accessible to the
`enzyme, yielding analogs with greater DPP-IV resistance (compounds 1, 2, and 3,
`Table 1).
`Knowing that the amide bond between Lys34 and Gly35 of hGLP-1(1-36)NH2 may
`also be cleaved in vivo [2], we further substituted the C-terminal Gly35 residue with
`Aib or ~-alanine (~-Ala) with the goal of protecting the peptide bond. The resulting
`analogs bearing modifications at both positions 8 and 35 (compounds 4-8, Table 1)
`have much longer plasma half-life than mono-substituted compounds 1, 2 and 3. These
`
`Table I . hGLP-1 receptor binding affinity and rat plasma half-life.
`
`Peptide
`
`hGLP-1 (7-36)NH2
`I
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`hGLP-l a
`Ki (nM)
`
`Rat plasma
`Tl/2 (h)
`
`Sequence
`
`1.09
`
`1.13
`
`7.23
`
`0.64
`
`0.95
`
`1.26
`
`1.39
`
`1.77
`
`2.12
`
`0.84
`
`4.35
`
`4.86
`
`4.52
`
`9.76
`
`8.34
`
`17.6
`
`7.40
`
`8.91
`
`[N-Me-D-Ala8]hGLP-1(7-36)NH2
`[A5c8]hGLP-1(7-36)NH2
`[Aib8]hGLP-1(7-36)NH2
`[Aib8•35]hGLP-1 (7-36)NH2
`[ Aib8 .~-Aia35]hGLP-l (7-36)NHz
`[Aib8.35,Phe31]hGLP-l (7 -36)NH2
`[Aib8 ,Phe31 .~-Ala35]hGLP-1 (7-36)NH2
`[Aib8·35,Arg26•34,Phe31]hGLP-1 (7-36)NH2
`
`a The assays were done in CHO-Kl cells expressing the human recombinant GLP-1 receptor.
`
`670
`
`MPI EXHIBIT 1013 PAGE 6
`
`MPI EXHIBIT 1013 PAGE 6
`
`

`

`Biologically Active Peptides
`
`novel hGLP-1 analogs with modifications at positions 8 and 35 also retain receptor
`potency of the native hGLP-1 (Table 1). Replacement of Trp31 by chemically more
`stable Phe does not significantly influence receptor affinity (compounds 6 and 7).
`The in vivo studies of this new series of hGLP-1 analogs in normal Sprague(cid:173)
`Dawley rats demonstrated that the efficacy of the analogs, in terms of the glucose(cid:173)
`dependent stimulation of insulin secretion, is highly correlated with their in vitro
`plasma half-life [3]. Among these analogs, compound 4 enhanced the insulin response
`to elevated glucose with a calculated ED50 at 16.0 pmol/kg, compared to that of the
`native hGLP-1(7-36)NH2 at 121 pmol/kg [4]. This 7.6-fold increase in efficacy is
`likely due to its enhanced enzymatic stability, resulting in an increased circulating
`half-life. In studies utilizing the db/db mouse, intraperitoneal administration of com(cid:173)
`pound 4 at 5-50 nmollkg to 5-week old animals produced a dose-dependent reduction
`in blood glucose monitored over a 5-h period [4].
`In conclusion, we have designed and synthesized a novel class of GLP-1 analogs
`that have substantially enhanced plasma half-life, while retaining full receptor potency
`of the native hormone. The representative analog, compound 4, is significantly more
`efficacious than hGLP-1 in vivo, and is effective in lowering blood glucose in the
`db/db mouse model of type 2 diabetes.
`References
`1. Mentlein, R., Gallwitz, B., Schmidt, W.E. Eur. J. Biochem. 214, 829-835 (1993).
`2. Tammem, H., Forssmann, W.-G., Richter, R. J. Chromatogr. A 852, 285-295 (1999).
`3. Culler, M.D., et al. 83rd Annual Meeting of the Endocrine Society, abstract P1-353, 2001.
`4. Culler, M.D., et al. 83rd Annual Meeting of the Endocrine Society, abstract P1-360, 2001.
`
`671
`
`MPI EXHIBIT 1013 PAGE 7
`
`MPI EXHIBIT 1013 PAGE 7
`
`