PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 00/69911
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 7 :
`C07K 14/605, 14/575, A61K 38/26, A61P Al
`3/08
`
`(11) International Publication Number:
`
`(43) International Publication Date:
`
`23 November 2000 (23.11.00)
`
`(21) International Application Number:
`
`PCT/US00/13563
`
`(22) International Filing Date:
`
`17 May 2000 (17.05.00)
`
`(74) Agents: WARD, Michael, R. et al.; Limbach & Limbach
`L.L.P., 2001 Ferry Building, San Francisco, CA 94111-4207
`(US).
`
`(30) Priority Data:
`60/134,406
`60/159,783
`
`17 May 1999 (17.05.99)
`15 October 1999 (15.10.99)
`
`us
`us
`
`(71) Applicant (for all designated States except US): CONJUCHEM,
`INC. [CNCA]; 225 President Kennedy Avenue West, Third
`floor, Suite 3950, Montreal, Quebec H2X 3Y8 (CA).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): BRIDON, Dominique,
`P. [FR/CA]; 243, chemin cote Sainte-Catherine, Out(cid:173)
`remont, Quebec H2V 2B2 (CA). L' ARCHEVEQUE, Benoit
`[CNCA]; 5875 Peloquin, Laval, Quebec H7H 2Xl (CA).
`EZRIN, Alan, M. [US/US]; 110 Quintas Lane, Moraga, CA
`94556 (US). HOLMES, Darren, L. [US/CA]; 3450 Drum(cid:173)
`mond Street, Montreal, Quebec H3G 1T3 (CA). LEBLANC,
`Anouk [CNCA]; Conjuchem, Inc., 225 President Kennedy
`Avenue West, Third floor, Suite 3950, Montreal, Quebec
`H2X 3Y8 (CA). ST. PIERRE, Serge [CNCA]; 47, place
`Jean-Yves, Ile Bizard, Quebec H9E 1K8 (CA).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE,
`ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP,
`KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA,
`MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU,
`SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG,
`US, UZ, VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE,
`LS, MW, SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM,
`AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT,
`BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU,
`MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM,
`GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: LONG LASTING INSULINOTROPIC PEPTIDES
`
`(57) Abstract
`
`Modified insulinotropic peptides are disclosed. The modified insulinotropic peptides are capable of forming a peptidase stabilized
`insulinotropic peptide. The modified insulinotropic 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.
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
`Page 00001
`
`

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`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cl\te d"Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
`Page 00002
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`LONG LASTING INSULINOTROPIC PEPTIDES
`
`5
`
`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,
`
`10
`
`gastrointestinal function and activities associated with glucagon levels.
`
`BACKGROUND OF THE INVENTION
`The insulinotropic peptide hormone glucagon-like peptide (GLP-1)
`
`has been implicated as a possible therapeutic agent for the
`
`15
`
`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
`
`associated with short plasma half-lifes in vivo, mainly due to rapid serum
`
`20
`
`clearance and proteolytic degradation. The enzyme responsible for the
`
`degradation of GLP-1, dipeptidyl 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
`
`25
`
`long lasting, active GLP-1 has not been produced. As such, the
`
`diabetic community has a tremendous need for improved GLP-1,
`
`exendin 3 and exendin 4 peptides.
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
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`There is thus a need to modify GLP-1, exendin 3, exendin 4 and
`
`other insulinotropic peptides to provide longer duration of action in vivo,
`
`while maintaining their low toxicity and therapeutic advantages.
`
`5
`
`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 functionalities on cellular carriers including mobile blood
`
`10
`
`proteins to form covalent linkages. Specifically, the invention relates to
`
`novel chemically reactive derivatives of insulinotropic 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
`
`15
`
`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
`
`20
`
`groups or thiol groups on blood compounds to form stable covalent
`
`bonds.
`
`The present invention relates to an insulinotropic hormone
`
`comprising a modified fragment of GLP-1 and derivatives thereof,
`
`especially GLP-1 (7-36) amide. The invention additionally pertains to
`
`25
`
`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).
`
`The present invention further relates to modified Exendin 3 and
`
`Exendin 4 fragments and therapeutic uses of such compounds.
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
`Page 00004
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`5
`
`In particular, the present invention is directed to GLP-1(1-36)(cid:173)
`Lys37 (i::-MPA)-NH2; GLP-1 (1-36)-Lys37 (i::-AAEA-AEEA-MPA)-NH2; GLP-
`1 (7-36)-Lys37 (i::-MPA)-NH2; GLP-1 (7-36)-Lys37-(i::-AEEA-AEEA-MPA)(cid:173)
`NH2; D-Ala2 GLP-1 (7-36)-Lys37 (i::-MPA)-NH2; Exendin-4 (1-39)-Lys40 (i::-
`MPA)-NH2; Exendin-4 (1-39)-Lys40 (i::-AEEA-AEEA-MPA)-NH2; Exendin-3
`(1-39)-Lys40 (i::-MPA)-NH2; Exendin-3 (1-39)-Lys40 (i::-AEEA-AEEA-MPA)(cid:173)
`NH2; Lys26(i::-MPA)GLP-1 (7-36)-NH2; GLP-1 (7-36)-EDA-MPA and
`Exendin-4 ( 1-39)-EDA-MP A.
`
`The present invention further relates to compositions comprising
`
`10
`
`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
`
`15
`
`insulinotropic peptides disclosed above.
`
`The invention further pertains to a method for treating maturity(cid:173)
`
`onset diabetes mellitus which comprises administration 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:
`
`20
`
`25
`
`lnsulinotropic Peptides: lnsulinotropic peptides (ITPs) are
`
`30
`
`peptides with insulinotropic activity. lnsulinotropic peptides stimulate, or
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
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`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.
`
`5
`
`Glucagon-Like Peptide: Glucagon-Like Peptide (GLP) and GLP
`
`derivatives are intestinal hormones which generally simulate insulin
`
`secretion during hyperglycemia, suppresses glucagon secretion,
`
`stimulates (pro) insulin biosynthesis and decelerates gastric emptying
`
`1 0
`
`and acid secretion. Some GLPs and GLP derivatives promote glucose
`uptake by cells but do not simulate insulin expression as disclosed in
`
`U.S. Patent No. 5,574,008 which is hereby incorporated by reference.
`
`Exendin 3 and Exendin 4 Peptides: Exendin 3 and exendin 4
`
`15
`
`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
`
`20
`
`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
`
`25
`
`imidate, thereby capable of forming a covalent bond with functionalities
`
`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.
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`30
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`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
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`Functionalities: Functionalities are groups on blood components
`
`to which reactive groups on modified insulinotropic peptides react to
`
`form covalent bonds. Functionalities include hydroxyl groups for
`
`bonding to ester reactive entities; thiol groups for bonding to malemides
`
`5
`
`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.
`
`10
`
`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
`
`15
`
`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)]ethoxy acetic acid).
`
`20
`
`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
`
`membrane and membraneous receptors, somatic body cells, skeletal
`
`25
`
`and smooth muscle cells, neuronal components, osteocytes 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
`
`30
`
`components are not membrane-associated and are present in the blood
`
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`IPR2023-00723
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`for extended periods of time and are present in a minimum concentration
`
`of at least 0.1 µg/ml. Mobile blood components include serum albumin,
`
`transferrin, ferritin and immunoglobulins such as lgM and lgG. The half(cid:173)
`
`life of mobile blood components is at least about 12 hours.
`
`5
`
`Protective Groups: Protective groups are chemical moieties
`
`utilized to protect peptide derivatives from reacting with themselves.
`
`Various protective groups are disclosed herein and in U.S. 5,493,007
`
`which is hereby incorporated by reference. Such protective groups
`
`10
`
`include acetyl, fluorenylmethyloxycarbonyl (FMOC), t-butyloxycarbonyl
`
`(BOC), benzyloxycarbonyl (CBZ), and the like. The specific protected
`
`amino acids are depicted in Table 1.
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`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
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`TABLE 1
`
`NATURAL AMINO ACIDS AND THEIR ABBREVIATIONS
`3-Letter
`1-Letter
`Protected Amino
`Abbreviation Abbreviation
`Acids
`Fmoc-Ala-OH
`Ala
`A
`Arg
`Fmoc-Arg(Pbf)-OH
`R
`N
`Asn
`Fmoc-Asn(Trt)-OH
`Asp
`D
`Asp(tBu)-OH
`Cys
`F moc-Cys(T rt)
`C
`E
`Glu
`Fmoc-Glu(tBu)-OH
`Gin
`Fmoc-Gln(Trt)-OH
`Q
`Gly
`Fmoc-Gly-OH
`G
`His
`Fmoc-His(Trt)-OH
`H
`lie
`Fmoc-lle-OH
`I
`Leu
`Fmoc-Leu-OH
`L
`K
`Lys
`F moc-Lys(Mtt)-OH
`M
`Met
`Fmoc-Met-OH
`Phe
`Fmoc-Phe-OH
`F
`p
`Fmoc-Pro-OH
`Pro
`s
`Ser
`Fmoc-Ser(tBu)-OH
`Thr
`T
`Fmoc-Thr(tBu)-OH
`w
`Trp
`Fmoc-Trp(Boc)-OH
`y
`Tyr
`Boc-Tyr(tBu)-OH
`Val
`Fmoc-Val-OH
`V
`
`Name
`Alanine
`Arginine
`Asparagine
`Aspartic acid
`Cysteine
`Glutamic acid
`Glutamine
`Glycine
`Histidine
`lsoleucine
`Leucine
`Lysine
`Methionine
`Phenylalanine
`Praline
`Serine
`Threonine
`Tryptophan
`Tyrosine
`Valine
`
`Sensitive Functional Groups - A sensitive functional group is a
`
`group of atoms that represents a potential reaction site on an ITP
`
`5
`
`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.
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`10
`
`Modified Peptides - A modified ITP is a peptide that has been
`
`modified by attaching a reactive group, and is capable of forming a
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
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`peptidase stabilized peptide through conjugation 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
`
`5
`
`the therapeutic peptide to facilitage the attachment 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
`
`10
`
`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 component via a
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`15
`
`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 peptidases in vivo than a non-stabilized peptide. A
`
`peptidase stabilized therapeutic peptide generally has an increased half
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`20
`
`life of at least 10-50% as compared to a non-stabalized peptide of
`
`identical sequence. Peptidase stability is determined 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
`
`determined by sampling the serum or blood after administration of the
`
`25
`
`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.
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`30
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`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(cid:173)
`
`life and distribution through selective conjugation onto a protein carrier
`
`5
`
`but without modifying their remarkable therapeutic properties. The
`carrier 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.
`
`10
`
`1.
`
`lnsulinotropic Peptides
`
`15
`
`GLP-1 and Its Derivatives
`A.
`The hormone glucagon is known to be synthesized as a high
`
`molecular weight precursor molecule which is subsequently
`proteolytically 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)
`
`20
`
`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
`
`25
`
`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 hormonal activity of GLP-1(7-37) and GLP-1(7-
`
`30
`
`36) appear to be specific for the pancreatic beta cells where it appears
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
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`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
`
`5
`
`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
`
`10
`
`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
`
`15
`
`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 preproglucagon 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
`
`20
`
`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
`
`25
`
`acids from number 7 (N-terminus) through number 36 (C-terminus).
`
`In one embodiment, GLP-1 (7-36) and its peptide fragments are
`
`synthesized by conventional means as detailed below, such as by the
`
`well-known solid-phase peptide synthesis described by Merrifield, J. M.
`(Chem. Soc. 85:2149 (1962)), and Stewart and Young (Solid Phase
`
`30
`
`Peptide Synthesis (Freeman, San Francisco, 1969), pages 27-66),
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
`Page 00012
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`PCT /US00/13563
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`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
`
`5
`
`desired fragments of the proglucagon peptide or of GLP-1 through the
`
`use of recombinant DNA technology, as disclosed by Maniatis, T., et al.,
`
`Molecular Biology: A Laboratory Manual, Cold Spring Harbor, New York
`
`(1982), which is hereby incorporated by reference.
`
`The present invention includes peptides which are derivable from
`
`10
`
`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)
`
`15
`
`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
`
`20
`
`similarly sized fragment of GLP-1; (2) it is capable of functioning as an
`
`25
`
`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 insulinotropic activity which can be detected even when the
`derivative is present at a concentration of 10-10 M, or, most preferably,
`(iii) an insulinotropic activity which can be detected even when the
`derivative is present at a concentration of 10-11 M.
`A derivative of GLP-1 is said to share "substantial homology" with
`
`GLP-1 if the amino acid sequences of the derivative is at least 80%, and
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
`Page 00013
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`PCT/US00/13563
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`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
`
`5
`
`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
`
`10
`
`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 homologous to that of a
`
`15
`
`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
`
`fragments of GLP-1 that may lack one or more amino acids that are
`
`normally present in a naturally occurring GLP-1 sequence provided that
`
`20
`
`such polypeptides have an insulinotropic 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
`
`25
`
`that of the natural sequence) provided that such variants have an
`
`insulinotropic activity which is substantially identical to that of the above(cid:173)
`
`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 hydrophobic residue for another
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
`Page 00014
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`PCT /US00/13563
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`(i.e. Leu for lie), 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
`
`5
`
`stimulate insulin expression or secretion are within the scope of this
`
`invention. Such GLP-1 derivatives are described in U.S. Patent No.
`
`5,574,008.
`
`10
`
`GLP-1 derivatives which stimulate glucose uptake by cells but do
`
`not stimulate insulin expression or secretion which find use in the
`
`invention include:
`R1-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-lle-Ala-Trp-Leu-Val(cid:173)
`Xaa-Gly-Arg -R2 (SEQ ID NO:3) wherein R1 is selected from a) H2N; b)
`H2N-Ser; c) H2N-Val-Ser; d) H2N-Asp-Val-Ser; e) H2N-Ser-Asp-Val-Ser
`
`(SEQ ID NO:4); f) H2N-Thr-Ser-Asp-Val-Ser (SEQ ID NO:5); g) H2N-
`
`15
`
`Phe-Thr-Ser-Asp-Val-Ser (SEQ ID NO:6); h) H2N-Thr-Phe-Thr-Ser-Asp(cid:173)
`
`Val-Ser (SEQ ID NO:7); i) H2N-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser (SEQ
`ID NO:8); j) H2N-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser (SEQ ID NO:9);
`or, k) H2N-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser (SEQ ID NO:10).
`In the peptide, Xis selected from Lys or Arg and R2 is selected from
`NH2, OH, Gly-NH2, or Gly-OH. These peptides are C-terminal GLP-1
`fragments which do not have insulinotropic activity but which are
`
`nonetheless useful for treating diabetes and hyperglycemic conditions as
`
`described in US Patent No. 5,574,008.
`
`20
`
`25
`
`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 [SEQ ID No:11] sequence is
`
`30
`
`HSDGTFTSDLSKQMEEEAVRLFIEWLKNGG PSSGAPPPS and
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
`Page 00015
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`PCT/US00/13563
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`The Exendin-4 [SEQ ID No:12] sequence is
`
`HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG PSSGAPPPS.
`
`The invention also encompasses the insulinotropic fragments of
`
`exendin-4 comprising the amino acid sequences: Exendin-4(1-31) [SEQ
`
`5
`
`ID No:13] HGEGTFTSDLSKQMEEAVR LFIEWLKNGGPY and Exendin-
`
`4(1-31 ) [SEQ ID No:14] HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGY.
`
`The invention also encompasses the inhibitory fragment of
`
`exendin-4 comprising the amino acid sequence:
`Exendin-4(9-39) [SEQ ID No:15]
`
`10
`
`DLSKQMEEEA VRLFIEWLKNGGPSSGAPPPS
`
`Other insulinotropic peptides as presented in the Examples are
`
`shown as SEQ ID NO:16 - 22.
`
`The present invention includes peptides which are derivable from
`
`the naturally occurring exendin 3 and exendin 4 peptides. A peptide is
`
`15
`
`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.
`
`20
`
`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
`
`25
`
`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 10-10 M, or, most
`
`Novo Nordisk Exhibit 2007
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00723
`Page 00016
`
`

`

`WO 00/69911
`
`PCT /US00/13563
`
`-15-
`
`preferably, (iii) an insulinotropic activity which can be detected even
`when the derivative is present at a concentration of 10-11 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
`
`5
`
`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.
`
`The derivatives of the present invention include exendin 3 or
`
`10
`
`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
`
`15
`
`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
`
`20
`
`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 fragments of exendin 3 or
`
`exendin 4 that may lack one or more amino acids that are normally
`
`25
`
`present in a naturally occurring 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
`
`30
`
`substitutions (and thus have amino acid sequ