(12) United States Patent
`Bridon et al.
`
`USOO651450OB1
`(10) Patent No.:
`US 6,514,500 B1
`(45) Date of Patent:
`*Feb. 4, 2003
`
`(54)
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`(75)
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`(73)
`(*)
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`(21)
`(22)
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`(60)
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`(51)
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`(52)
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`(58)
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`(56)
`
`LONG LASTING SYNTHETIC GLUCAGON
`LIKE PEPTIDE {GLP-1}
`Inventors: Dominique P. Bridon, Outremont
`(CA); Benoit L'Archeveque, Laval
`(CA); Alan M. Ezrin, Moraga, CA
`(US); Darren L. Holmes, Montreal
`(CA); Anouk Leblanc, Montreal (CA);
`Serge St. Pierre, Ile Bizard (CA)
`Assignee: Conjuchem, Inc., Montreal (CA)
`Notice:
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is Subject to a terminal dis
`claimer.
`
`Appl. No.: 09/657,332
`Filed:
`Sep. 7, 2000
`Related U.S. Application Data
`Provisional application No. 60/159,783, filed on Oct. 15,
`1999.
`Int. Cl." ...................... A61K 39/385; A61K 38/00;
`AO1N 37/18
`U.S. Cl. ............................... 424/193.1; 424/195.11;
`514/2
`Field of Search ...................... 424/9.1, 488, 193.1;
`514/2, 3, 6, 12, 18, 19; 530/303, 308,399,
`402, 409
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,118,666 A 6/1992 Habener ...................... 514/12
`5,120,712 A 6/1992 Habener ...................... 514/12
`5,493.007 A 2/1996 Burnier et al. .
`... 530/317
`5,545,618 A 8/1996 Buckley et al. ............... 514/12
`5,574,008 A 11/1996 Johnson et al. ............... 514/12
`5,612,034 A 3/1997 Pouletty et al. ...
`424/184.1
`5,614,487 A 3/1997 Battersby et al. .............. 514/2
`5,614,492 A 3/1997 Habener ...................... 514/12
`5,958,909 A 9/1999 Habener ....
`... 514/120
`5,981,488 A 11/1999 Hoffmann .................... 514/12
`6,133,235 A 10/2000 Galloway et al. ............. 514/12
`
`2/2001 DiMarchi et al.
`6,191,102 B1
`FOREIGN PATENT DOCUMENTS
`
`
`
`EP
`EP
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`6/1994 .......... A61 K/47/48
`O 6O2 290
`1/2000
`CO7K/14/605
`O969 O16
`8/1991
`... CO7K/7/34
`9111457
`12/1993
`... CO7K/7/34
`9325579
`4/1995
`A61 K/39/395
`951O3O2
`3/1998 .......... A61 K/38/OO
`98O8531
`3/1998 ......... CO7K/14/605
`98O8871
`3/1998 ......... CO7K/14/605
`98O8873
`10/1998 .......... A61 K/38/OO
`98.43658
`5/1999 .......... A61 K/47/48
`9924O74
`5/1999 .......... A61 K/47/48
`9924O75
`6/1999 .......... A61 K/38/26
`9929.336
`9/1999 .......... A61 K/47/48
`994.8536
`12/2000 .......... A61 K/47/48
`OO/76550
`12/2000 .......... A61 K/47/48
`OO/76551
`OTHER PUBLICATIONS
`Marburg et al., “Intorduction of the Maleimide Function
`onto Resin-Bound Peptides: A simple, High yield Process
`Useful for Discriminating among Several Lysines,” Biocon
`jugate Chemistry, vol. 7, pp. 612-616 (1996).*
`Regulatory Peptides, 1999, 79, 93-102.
`U.S. patent application Ser. No. 09/623,618, Bridon et al.,
`filed Sep. 2000.
`Proc. Natl. Acad. Sci., 1983, 80, 5485-5489.
`Nature, 1983, 302, 716–718.
`Endocrinol., 1984, 115, 2176-2181.
`Anti-Cancer Drugs, 1997, 8, 677-685.
`* cited by examiner
`Primary Examiner James Housel
`ASSistant Examiner Zachariah Lucas
`(74) Attorney, Agent, or Firm Morrison & Foerster LLP
`(57)
`ABSTRACT
`Modified insulinotropic peptides are disclosed. The modi
`fied insulinotropic peptides are capable of forming a pepti
`dase Stabilized insulinotropic peptide. The modified insuli
`notropic peptides are capable of forming covalent bonds
`with one or more blood components to form a conjugate.
`The conjugates may be formed in vivo or ex vivo. The
`modified peptides are administered to treat humans with
`diabetes and other related diseases.
`
`2 Claims, No Drawings
`
`MPI EXHIBIT 1014 PAGE 1
`
`MPI EXHIBIT 1014 PAGE 1
`
`

`

`1
`LONG LASTING SYNTHETIC GLUCAGON
`LIKE PEPTIDE {GLP-1}
`This application claims the benefit under 35 U.S.C.
`$119(e) of United States provisional patent application No.
`60/159,783 filed Oct. 15, 1999, which is hereby incorporated
`by reference in its entirety.
`FIELD OF THE INVENTION
`This invention relates to modified insulinotropic peptides.
`In particular, this invention relates to modified glucagon like
`peptides and eXendin peptides with long duration of action
`for the treatment of diabetes and other insulinotropic peptide
`related diseases, gastrointestinal function and activities asso
`ciated with glucagon levels.
`BACKGROUND OF THE INVENTION
`The insulinotropic peptide hormone glucagon-like pep
`tide (GLP-1) has been implicated gas a possible therapeutic
`agent for the management of type 2 non-insulin-dependent
`diabetes mellitus as well as related metabolic disorders, Such
`as obesity. Other useful insulinotropic peptides include
`exendin 3 and exendin 4. While useful, GLP-1, exendin 3
`and eXendin 4 Suffer from limited duration of action asso
`ciated with short plasma half-lifes in vivo, mainly due to
`rapid Serum clearance and proteolytic degradation. The
`enzyme responsible for the degradation of GLP-1, dipepti
`dyl peptidase IV, has been identified. Extensive work has
`been done in attempts to inhibit the peptidase or to modify
`GLP-1 in such a way that its degradation is slowed down
`while Still maintaining biological activity. Despite these
`extensive efforts, a long lasting, active GLP-1 has not been
`produced. AS Such, the diabetic community has a tremen
`dous need for improved GLP-1, exendin 3 and exendin 4
`peptides.
`There is thus a need to modify GLP-1, exendin 3, exendin
`4 and other insulinotropic peptides to provide longer dura
`tion of action in Vivo, while maintaining their low toxicity
`and therapeutic advantages.
`SUMMARY OF THE INVENTION
`In order to meet those needs, the present invention is
`directed to modified insulinotropic peptides (ITPs). This
`invention relates to novel chemically reactive derivatives of
`insulinotropic peptides that can react with available func
`tionalities on cellular carriers including mobile blood pro
`teins to form covalent linkages. Specifically, the invention
`relates to novel chemically reactive derivatives of insulino
`tropic peptides Such as glucagon like peptide (GLP) and
`exendin 3 and eXendin 4 that can react with available
`functionalities on mobile blood proteins to form covalent
`linkages. The invention also relates to novel chemically
`reactive derivatives or analogs of insulinotropic peptides
`that can react with available functionalities on mobile blood
`proteins to form covalent linkages.
`The present invention relates to modified insulinotropic
`peptides comprising a reactive group which reacts with
`amino groups, hydroxyl groups or thiol groups on blood
`compounds to form stable covalent bonds.
`The present invention relates to an insulinotropic hor
`mone comprising a modified fragment of GLP-1 and deriva
`tives thereof, especially GLP-1 (7-36) amide. The invention
`additionally pertains to the therapeutic uses of Such
`compounds, and especially to the use of modified GLP-1
`(7-36) amide for the treatment of maturity onset diabetes
`mellitus (type II diabetes).
`
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`US 6,514,500 B1
`
`2
`The present invention further relates to modified Exendin
`3 and EXendin 4 fragments and therapeutic uses of Such
`compounds.
`In particular, the present invention is directed to GLP-1
`(1-36)-Lys7 (e-MPA)-NH; GLP-1 (1-36)-Lys7 (e-AAEA
`AEEA-MPA)-NH; GLP-1 (7-36)-Lys7 (e-MPA)-NH;
`GLP-1 (7-36)-Lys7(e-AEEA-AEEA-MPA)-NH; D-Ala
`GLP1 (7-36)-Lys7 (e-MPA)-NH; Exendin-4 (1-39)-Lys'
`(e-MPA)-NH; Exendin-4 (1-39)-Lys" (e-AEEA-AEEA
`MPA)-NH; Exendin-3 (1-39)-Lys" (e-MPA)-NH;
`Exendin-3 (1-39)-Lys' (e-AEEA-AEEA-MPA)-NH; Lys'
`(e-MPA)GLP-1 (7-36)NH; GLP-1 (7-36)-EDA-MPA and
`Exendin-4 (1-39)-EDA-MPA.
`The present invention further relates to compositions
`comprising the derivatives of the insulinotropic peptides and
`the use of the compositions for treating diabetes in humans.
`The invention further pertains to a method for enhancing
`the expression of insulin which comprises providing to a
`mammalian pancreatic Beta-type islet cell an effective
`amount of the modified insulinotropic peptides disclosed
`above.
`The invention further pertains to a method for treating
`maturity-onset diabetes mellitus which comprises adminis
`tration of an effective amount of the insulinotropic peptides
`discussed above to a patient in need of Such treatment.
`The invention further pertains to the treatment of other
`insulinotropic peptide related diseases and conditions with
`the modified insulinotropic peptides of the invention.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`Definitions
`To ensure a complete understanding of the invention the
`following definitions are provided:
`Insulinotropic Peptides: Insulinotropic peptides (ITPs) are
`peptides with insulinotropic activity. Insulinotropic peptides
`Stimulate, or cause the Stimulation of, the Synthesis or
`expression of the hormone insulin. Such peptides include
`precursors, analogues, fragments of peptides Such as
`Glucagon-like peptide, exendin 3 and exendin 4 and other
`peptides with insulinotropic activity.
`Glucagon-Like Peptide: Glucagon-Like Peptide (GLP)
`and GLP derivatives are intestinal hormones which gener
`ally simulate insulin Secretion during hyperglycemia, Sup
`presses glucagon Secretion, Stimulates (pro) insulin biosyn
`thesis and decelerates gastric emptying and acid Secretion.
`Some GLPs and GLP derivatives promote glucose uptake by
`cells but do not Simulate insulin expression as disclosed in
`U.S. Pat. No. 5,574,008 which is hereby incorporated by
`reference.
`Exendin 3 and Exendin 4 Peptides: Exendin 3 and exen
`din 4 peptides and peptide derivatives are 39 amino acid
`peptides which are approximately 53% homologons to
`GLP-1 and have insulinotropic activity.
`Reactive Groups: Reactive groups are chemical groups
`capable of forming a covalent bond. Such reactive agents are
`coupled or bonded to an insulinotropic peptide of interest to
`form a modified insulinotropic peptide. Reactive groups will
`generally be stable in an aqueous environment and will
`usually be carboxy, phosphoryl, or convenient acyl group,
`either as an ester or a mixed anhydride, or an imidate,
`thereby capable of forming a covalent bond with function
`alities Such as an amino group, a hydroxy or a thiol at the
`target Site on mobile blood components. For the most part,
`the esters will involve phenolic compounds, or be thiol
`esters, alkyl esters, phosphate esters, or the like. Reactive
`groups include Succinimidyl and maleimido groups.
`
`MPI EXHIBIT 1014 PAGE 2
`
`MPI EXHIBIT 1014 PAGE 2
`
`

`

`US 6,514,500 B1
`
`4
`
`TABLE 1-continued
`
`3
`Functionalities: Functionalities are groups on blood com
`ponents to which reactive groups on modified insulinotropic
`peptides react to form covalent bonds. Functionalities
`include hydroxyl groups for bonding to ester reactive enti
`ties; thiol groups for bonding to malemides and maleimido
`groups, imidates and thioester groups, amino groups for
`bonding to carboxy, phosphoryl or acyl groups on reactive
`entities and carboxyl groups for bonding to amino groups.
`Such blood components include blood proteins.
`Linking Groups: Linking groups are chemical moieties
`that link or connect reactive groups to ITPS. Linking groups
`may comprise one or more alkyl groupS. Such as methyl,
`ethyl, propyl, butyl, etc. groups, alkoxy groups, alkenyl
`groups, alkynyl groups or amino group Substituted by alkyl
`groups, cycloalkyl groups, polycyclic groups, aryl groups,
`polyaryl groups, Substituted aryl groups, heterocyclic
`groups, and Substituted heterocyclic groups. Linking groups
`may also comprise poly ethoxy aminoacids Such as AEA
`((2-amino)ethoxy acetic acid) or a preferred linking group
`AEEA (2-(2-amino)ethoxy)ethoxyacetic acid).
`Blood Components: Blood components may be either
`fixed or mobile. Fixed blood components are non-mobile
`blood components and include tissues, membrane receptors,
`interstitial proteins, fibrin proteins, collagens, platelets,
`endothelial cells, epithelial cells and their associated mem
`25
`brane and membraneous receptors, Somatic body cells, Skel
`etal and Smooth muscle cells, neuronal components, osteo
`cytes and Osteoclasts and all body tissues especially those
`asSociated with the circulatory and lymphatic Systems.
`Mobile blood components are blood components that do not
`have a fixed situs for any extended period of time, generally
`not exceeding 5, more usually one minute. These blood
`components are not membrane-associated and are present in
`the blood for extended periods of time and are present in a
`minimum concentration of at least 0.1 lug/ml. Mobile blood
`components include Serum albumin, transferrin, ferritin and
`immunoglobulins such as IgM and IgG. The half-life of
`mobile blood components is at least about 12 hours.
`Protective Groups: Protective groups are chemical moi
`eties utilized to protect peptide derivatives from reacting
`with themselves. Various protective groups are disclosed
`herein and in U.S. Pat. No. 5,493,007 which is hereby
`incorporated by reference. Such protective groups include
`acetyl, fluorenylmethyloxycarbonyl (FMOC),
`t-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and
`45
`the like. The Specific protected amino acids are depicted in
`Table 1.
`
`NATURALAMINO ACIDS AND THEIRABBREVIATIONS
`
`Name
`
`Serine
`Threonine
`Tryptophan
`Tyrosine
`Valine
`
`3-Letter
`Abbreviation
`
`1-Letter
`Abbreviation
`
`Protected Amino
`Acids
`
`Ser
`Thr
`Trp
`Tyr
`Val
`
`S
`T
`W
`Y
`V
`
`Fmoc-Ser(thiu)-OH
`Fmoc-Thr(tBu)-OH
`Fmoc-Trp(Boc)-OH
`Boc-Tyr(tBu)-OH
`Fmoc-Val-OH
`
`Sensitive Functional Groups-A sensitive functional
`group is a group of atoms that represents a potential reaction
`Site on an ITP peptide. If present, a Sensitive functional
`group may be chosen as the attachment point for the
`linker-reactive group modification. Sensitive functional
`groups include but are not limited to carboxyl, amino, thiol,
`and hydroxyl groups.
`Modified Peptides-A modified ITP is a peptide that has
`been modified by attaching a reactive group, and is capable
`of forming a peptidase Stabilized peptide through conjuga
`tion to blood components. The reactive group may be
`attached to the therapeutic peptide either via a linking group,
`or optionally without using a linking group. It is also
`contemplated that one or more additional amino acids may
`be added to the therapeutic peptide to facilitage the attach
`ment of the reactive group. Modified peptides may be
`administered in Vivo Such that conjugation with blood
`components occurs in Vivo, or they may be first conjugated
`to blood components in vitro and the resulting peptidase
`stabalized peptide (as defined below) administered in vivo.
`The terms “modified therapeutic peptide’ and “modified
`peptide' may be used interchangeably in this application.
`Peptidase Stabilized ITP-A peptidase stabilized ITP is a
`modified peptide that has been conjugated to a blood com
`ponent via a covalent bond formed between the reactive
`group of the modified peptide and the functionalities of the
`blood component, with or without a linking group. Peptidase
`Stabilized peptides are more stable in the presence of pep
`tidases in Vivo than a non-Stabilized peptide. A peptidase
`Stabilized therapeutic peptide generally has an increased half
`life of at least 10-50% as compared to a non-stabalized
`peptide of identical Sequence. Peptidase Stability is deter
`mined by comparing the half life of the unmodified ITP in
`serum or blood to the half life of a modified counterpart
`therapeutic peptide in serum or blood. Half life is deter
`mined by Sampling the Serum or blood after administration
`of the modified and non-modified peptides and determining
`the activity of the peptide. In addition to determining the
`activity, the length of the ITP may also be measured by
`HPLC and Mass Spectrometry.
`DETAILED DESCRIPTION OF THE
`INVENTION
`Taking into account these definitions the focus of this
`invention is to modify insulinotropic peptides to improve
`bio-availability, extend half-life and distribution through
`Selective conjugation onto a protein carrier but without
`modifying their remarkable therapeutic properties. The car
`rier of choice (but not limited to) for this invention would be
`albumin conjugated through its free thiol by a insulinotropic
`peptide derivatized with a maleimide moiety.
`1. Insulinotropic Peptides
`A. GLP-1 and Its Derivatives The hormone glucagon is
`known to be Synthesized as a high molecular weight pre
`cursor molecule which is Subsequently proteolytically
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`TABLE 1.
`
`NATURALAMINO ACIDS AND THEIRABBREVIATIONS
`
`Name
`
`Alanine
`Arginine
`Asparagine
`Aspartic acid
`Cysteine
`Glutamic acid
`Glutamine
`Glycine
`Histidine
`Isoleucine
`Leucine
`Lysine
`Methionine
`Phenylalanine
`Proline
`
`3-Letter
`Abbreviation
`
`1-Letter
`Abbreviation
`
`Protected Amino
`Acids
`
`Ala
`Arg
`Asn
`Asp
`Cys
`Glu
`Glin
`Gly
`His
`Ile
`Leu
`Lys
`Met
`Phe
`Pro
`
`A.
`R
`N
`D
`C
`E
`O
`G
`H
`I
`L
`K
`M
`F
`P
`
`Fmoc-Ala-OH
`Fmoc-Arg(Pbf)-OH
`Fmoc-Asn(Trt)-OH
`Asp(tBu)-OH
`Fmoc-Cys(Trt)
`Fmoc-Glu(tBu)-OH
`Fmoc-Gln(Trt)-OH
`Fmoc-Gly-OH
`Fmoc-His(Trt)-OH
`Fmoc-Ile-OH
`Fmoc-Leu-OH
`Fmoc-Lys(Mt)-OH
`Fmoc-Met-OH
`Fmoc-Phe-OH
`Fmoc-Pro-OH
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`MPI EXHIBIT 1014 PAGE 3
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`MPI EXHIBIT 1014 PAGE 3
`
`

`

`S
`cleaved into three peptides: glucagon, glucagon-like peptide
`1 (GLP-1), and glucagon-like peptide 2 (GLP-2). GLP-1 has
`37 amino acids in its unprocessed form as shown in SEQ ID
`NO: 1. Unprocessed GLP-1 is essentially unable to mediate
`the induction of insulin biosynthesis. The unprocessed
`GLP-1 peptide is, however, naturally converted to a
`31-amino acid long peptide (7-37 peptide) having amino
`acids 7-37 of GLP-1 (“GLP-1 (7-37)”) SEQ ID NO:2.
`GLP-1 (7-37) can also undergo additional processing by
`proteolytic removal of the C-terminal glycine to produce
`GLP-1 (7-36) which also exists predominantly with the
`C-terminal residue, arginine, in amidated form as
`arginineamide, GLP-1 (7-36) amide. This processing occurs
`in the intestine and to a much lesser extent in the pancreas,
`and results in a polypeptide with the insulinotropic activity
`of GLP-1 (7-37).
`A compound is Said to have an “insulinotropic activity” if
`it is able to Stimulate, or cause the Stimulation of, the
`Synthesis or expression of the hormone insulin. The hor
`monal activity of GLP-1 (7-37) and GLP-1 (7-36) appear to
`be specific for the pancreatic beta cells where it appears to
`induce the biosynthesis of insulin. The glucagon-like
`peptide hormone of the invention is useful in the study of the
`pathogenesis of maturity onset diabetes mellitus, a condition
`characterized by hyperglycemia in which the dynamics of
`insulin Secretion are abnormal. Moreover, the glucagon-like
`peptide is useful in the therapy and treatment of this disease,
`and in the therapy and treatment of hyperglycemia.
`Peptide moieties (fragments) chosen from the determined
`amino acid Sequence of human GLP-1 constitute the Starting
`point in the development comprising the present invention.
`The interchangeable terms "peptide fragment” and "peptide
`moiety' are meant to include both Synthetic and naturally
`occurring amino acid Sequences derivable from a naturally
`occurring amino acid Sequence.
`The amino acid sequence for GLP-1 has been reported by
`Several researchers (Lopez, L. C., et al., Proc. Natl. Acad.
`Sci., USA 80:5485-5489 (1983); Bell, G. I., et al., Nature
`302:716–718 (1983); Heinrich, G., et al., Endocrinol.
`115:2176-2181 (1984)). The structure of the preprogluca
`gon mRNA and its corresponding amino acid Sequence is
`well known. The proteolytic processing of the precursor
`gene product, proglucagon, into glucagon and the two
`insulinotropic peptides has been characterized. AS used
`herein, the notation of GLP-1 (1-37) refers to a GLP-1
`polypeptide having all amino acids from 1 (N-terminus)
`through 37 (C-terminus). Similarly, GLP-1 (7-37) refers to
`a GLP-1 polypeptide having all amino acids from 7
`(N-terminus) through 37 (C-terminus). Similarly, GLP-1
`(7-36) refers to a GLP-1 polypeptide having all amino acids
`from number 7 (N-terminus) through number 36
`(C-terminus).
`In one embodiment, GLP-1 (7-36) and its peptide frag
`ments are Synthesized by conventional means as detailed
`below, Such as by the well-known Solid-phase peptide Syn
`thesis described by Merrifield, J. M. (Chem. Soc. 85:2149
`(1962)), and Stewart and Young (Solid Phase Peptide Syn
`thesis (Freeman, San Francisco, 1969), pages 27-66), which
`are incorporated by reference herein. However, it is also
`possible to obtain fragments of the proglucagon polypeptide,
`or of GLP-1, by fragmenting the naturally occurring amino
`acid Sequence, using, for example, a proteolytic enzyme.
`Further, it is possible to obtain the desired fragments of the
`proglucagon peptide or of GLP-1 through the use of recom
`binant DNA technology, as disclosed by Maniatis, T., et al.,
`Molecular Biology: A Laboratory Manual, Cold Spring
`Harbor, N.Y. (1982), which is hereby incorporated by ref
`CCCC.
`
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`
`6
`The present invention includes peptides which are deriv
`able from GLP-1 such as GLP-1 (1-37) and GLP-1 (7-36).
`A peptide is Said to be "derivable from a naturally occurring
`amino acid Sequence' if it can be obtained by fragmenting
`a naturally occurring Sequence, or if it can be Synthesized
`based upon a knowledge of the Sequence of the naturally
`occurring amino acid Sequence or of the genetic material
`(DNA or RNA) which encodes this sequence.
`Included within the Scope of the present invention are
`those molecules which are said to be "derivatives” of GLP-1
`such as GLP-1 (1-37) and especially GLP-1 (7-36). Such a
`"derivative” has the following characteristics: (1) it shares
`substantial homology with GLP-1 or a similarly sized frag
`ment of GLP-1, (2) it is capable of functioning as an
`insulinotropic hormone and (3) using at least one of the
`assays provided herein, the derivative has either (i) an
`insulinotropic activity which exceeds the insulinotropic
`activity of either GLP-1, or, more preferably, (ii) an insuli
`notropic activity which can be detected even when the
`derivative is present at a concentration of 10' M, or, most
`preferably, (iii) an insulinotropic activity which can be
`detected even when the derivative is present at a concen
`tration of 10' M.
`A derivative of GLP-1 is said to share “Substantial homol
`ogy” with GLP-1 if the amino acid sequences of the deriva
`tive is at least 80%, and more preferably at least 90%, and
`most preferably at least 95%, the same as that of GLP-1
`(1-37).
`The derivatives of the present invention include GLP-1
`fragments which, in addition to containing a Sequence that
`is Substantially homologous to that of a naturally occurring
`GLP-1 peptide may contain one or more additional amino
`acids at their amino and/or their carboxy termini. Thus, the
`invention pertains to polypeptide fragments of GLP-1 that
`may contain one or more amino acids that may not be
`present in a naturally occurring GLP-1 Sequence provided
`that Such polypeptides have an insulinotropic activity which
`exceeds that of GLP-1. The additional amino acids may be
`D-amino acids or L-amino acids or combinations thereof.
`The invention also includes GLP-1 fragments which,
`although containing a Sequence that is Substantially homolo
`gous to that of a naturally occurring GLP-1 peptide may lack
`one or more additional amino acids at their amino and/or
`their carboxy termini that are naturally found on a GLP-1
`peptide. Thus, the invention pertains to polypeptide frag
`ments of GLP-1 that may lack one or more amino acids that
`are normally present in a naturally occurring GLP-1
`Sequence provided that Such polypeptides have an insulino
`tropic activity which exceeds that of GLP-1.
`The invention also encompasses the obvious or trivial
`variants of the above-described fragments which have
`inconsequential amino acid Substitutions (and thus have
`amino acid Sequences which differ from that of the natural
`Sequence) provided that Such variants have an insulinotropic
`activity which is substantially identical to that of the above
`described GLP-1 derivatives. Examples of obvious or trivial
`Substitutions include the Substitution of one basic residue for
`another (i.e. Arg for LyS), the Substitution of one hydropho
`bic residue for another (i.e. Leu for Ile), or the substitution
`of one aromatic residue for another (i.e. Phe for Tyr), etc.
`In addition to those GLP-1 derivatives with insulinotropic
`activity, GLP-1 derivatives which Stimulate glucose uptate
`by cells but do not Stimulate insulin expression or Secretion
`are within the scope of this invention. Such GLP-1 deriva
`tives are described in U.S. Pat. No. 5,574,008.
`GLP-1 derivatives which stimulate glucose uptake by
`cells but do not stimulate insulin expression or Secretion
`which find use in the invention include:
`
`MPI EXHIBIT 1014 PAGE 4
`
`MPI EXHIBIT 1014 PAGE 4
`
`

`

`US 6,514,500 B1
`
`15
`
`25
`
`7
`R-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile
`Ala-Trp-Leu-Val-Xaa-Gly-Arg-R (SEQ ID NO:3)
`wherein R is selected from a) HN; b) HN-Ser; c)
`HN-Val-Ser; d) HN-Asp-Val-Ser; e) HN-Ser-Asp-Val
`Ser (SEQ ID NO:4); f) HN-Thr-Ser-Asp-Val-Ser (SEQ
`ID NO:5); g) HN-Phe-Thr-Ser-Asp-Val-Ser (SEQ ID
`NO:6); h) HN-Thr-Phe-Thr-Ser-Asp-Val-Ser (SEQ ID
`NO:7); i) HN-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser (SEQ
`ID NO:8);j) HN-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser
`(SEQ ID NO:9); or, k) HN-Ala-Glu-Gly-Thr-Phe-Thr
`Ser-Asp-Val-Ser (SEQ ID NO:10). In the peptide, X is
`Selected from LyS or Arg and R2 is Selected from NH2,
`OH, Gly-NH, or Gly-OH. These peptides are C-terminal
`GLP-1 fragments which do not have insulinotropic activ
`ity but which are nonetheless useful for treating diabetes
`and hyperglycemic conditions as described in U.S. Pat.
`No. 5,574,008.
`B. Exendin 3 and Exendin 4 Peptides
`Exendin 3 and Exendin 4 are 39 amino acid peptides
`(differing at residues 2 and 3) which are approximately 53%
`homologous to GLP-1 and find use as insulinotropic agents.
`The Exendin-3 SEQID No:11 sequence is HSDGTFTS
`DLSKOMEEEAVRLFIEWLKNGG PSSGAPPPS and
`The Exendin-4 SEQ ID No:12 sequence is HGEGT
`FTSDLSKOMEEEAVRLFIEWLKNG G PSSGAPPPS.
`The invention also encompasses the insulinotropic frag
`ments of eXendin-4 comprising the amino acid Sequences:
`Exendin-4 (1-31) SEQ ID No:13) HGEGTFTSDL
`SKQMEEAVR LFIEWLKNGGPY and Exendin-4 (1-31)
`SEQ ID No:14 HGEGTFTSDLSKQMEEEAVR
`LFIEWLKNGGY.
`The invention also encompasses the inhibitory fragment
`of exendin-4 comprising the amino acid Sequence:
`Exendin-4(9-39) (SEQ ID No:15) DLSKQMEEEAVR
`LFIEWLKNGGPSSGAPPPS
`35
`Other insulinotropic peptides as presented in the
`Examples are shown as SEQ ID NO:16-22.
`The present invention includes peptides which are deriv
`able from the naturally occurring eXendin 3 and eXendin 4
`peptides. A peptide is said to be "derivable from a naturally
`occurring amino acid Sequence' if it can be obtained by
`fragmenting a naturally occurring Sequence, or if it can be
`Synthesized based upon a knowledge of the Sequence of the
`naturally occurring amino acid Sequence or of the genetic
`material (DNA or RNA) which encodes this sequence.
`Included within the Scope of the present invention are
`those molecules which are said to be "derivatives” of
`exendin 3 and exendin 4. Such a "derivative' has the
`following characteristics: (1) it shares Substantial homology
`with eXendin 3 or eXendin 4 or a similarly sized fragment of
`exendin 3 or exendin 4; (2) it is capable of functioning as an
`insulinotropic hormone and (3) using at least one of the
`assays provided herein, the derivative has either (i) an
`insulinotropic activity which exceeds the insulinotropic
`activity of either eXendin 3 or exendin 4, or, more preferably,
`(ii) an insulinotropic activity which can be detected even
`when the derivative is present at a concentration of 10' M,
`or, most preferably, (iii) an insulinotropic activity which can
`be detected even when the derivative is present at a con
`centration of 10' M.
`A derivative of exendin 3 and exendin 4 is said to share
`“substantial homology” with exendin 3 and exendin 4 if the
`amino acid Sequences of the derivative is at least 80%, and
`more preferably at least 90%, and most preferably at least
`95%, the same as that of either exendin 3 or 4 or a fragment
`of eXendin 3 or 4 having the same number of amino acid
`residues as the derivative.
`
`8
`The derivatives of the present invention include exendin
`3 or exendin 4 fragments which, in addition to containing a
`Sequence that is Substantially homologous to that of a
`naturally occurring eXendin 3 or exendin 4 peptide may
`contain one or more additional amino acids at their amino
`and/or their carboxy termini. Thus, the invention pertains to
`polypeptide fragments of eXendin 3 or exendin 4 that may
`contain one or more amino acids that may not be present in
`a naturally occurring eXendin 3 or exendin 4 Sequences
`provided that Such polypeptides have an insulinotropic
`activity which exceeds that of exendin 3 or exendin 4.
`Similarly, the invention includes exendin 3 or exendin 4
`fragments which, although containing a sequence that is
`Substantially homologous to that of a naturally occurring
`exendin 3 or exendin 4 peptide may lack one or more
`additional amino acids at their amino and/or their carboxy
`termini that are naturally found on a eXendin 3 or exendin 4
`peptide. Thus, the invention pertains to polypeptide frag
`ments of exendin 3 or exendin 4 that may lack one or more
`amino acids that are normally present in a naturally occur
`ring eXendin 3 or exendin 4 Sequence provided that Such
`polypeptides have an insulinotropic activity which exceeds
`that of exendin 3 or exendin 4.
`The invention also encompasses the obvious or trivial
`variants of the above-described fragments which have
`inconsequential amino acid Substitutions (and thus have
`amino acid Sequences which differ from that of the natural
`Sequence) provided that Such variants have an insulinotropic
`activity which is substantially identical to that of the above
`described exendin 3 or exendin 4 derivatives. Examples of
`obvious or trivial Substitutions include the Substitution of
`one basic residue for another (i.e. Arg for LyS), the Substi
`tution of one hydrophobic residue for another (i.e. Leu for
`Ile), or the Substitution of one aromatic residue for another
`(i.e. Phe for Tyr), etc.
`2. Modified Insulinotropic Peptides
`This invention relates to modified insulinotropic peptides
`and their derivatives. The modified insulinotropic peptides
`of the invention include reactive groups which can react
`with available reactive functionalities on blood components
`to form covalent bonds. The invention also relates to Such
`modifications, Such combinations with blood components
`and methods for their use. These methods include extending
`the effective therapeutic in vivo half life of the modified
`insulinotropic peptides.
`To form covalent bonds with the functional group on a
`protein, one may use as a chemically reactive group
`(reactive entity) a wide variety of active carboxyl groups,
`particularly esters, where the hydroxyl moiety is physiologi
`cally acceptable at the levels required to modify the insuli
`notropic peptides. While a number of different hydroxyl
`groups may be employed in these linking agents, the most
`convenient would be N-hydroxysuccinimide (NHS),
`N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimide
`benzoyl-Succinimide (MBS), gamma-maleimido
`butyryloxy succinimide ester (GMBS) and maleimidopro
`pionic acid (MPA).
`Primary amines are the principal targets for NHS esters as
`diagramed in the Schematic below.” Accessible C-amine
`groups present on the N-termini of proteins react with NHS
`esters. However, C.-amino groups on a protein may not be
`desirable or available for the NHS coupling. While five
`amino acids have nitrogen in their side chains, only the
`e-amine of lysine reacts significantly with NHS esters. An
`amide bond is formed when the NHS ester conjugation
`reaction reacts with primary amines releasing
`N-hydroxySuccinimide as demonstrated in the Schematic
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`MPI EXHIBIT 1014 PAGE 5
`
`MPI EXHIBIT 1014 PAGE 5
`
`

`

`US 6,514,500 B1
`
`10
`(the most abundant blood protein) there is only a single thiol
`group. Thus, ITP-maleimide-albumin conjugates will tend
`
`below. These Succinimide containing reactive groups are
`herein referred to as Succinimidyl groups.
`
`NHS-Ester Reaction Scheme
`
`O H
`
`R-C-O-N
`
`+ R-NH. pH 7-9 R-6-N-R
`
`- HO-N
`
`In the preferred embodiments of this invention, the func
`tional group on the protein will be a thiol group and the
`chemically reactive group