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`gear
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`1
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`(81) Designated States: AT (European patent), BE (European
`patent), CA, CH (European patent), DE (European pa-
`tent), DK (European patent), ES (European patent), FR
`(European patent), GB (European patent), GR (Euro-
`pean patent), IT (European patent), JP, LU (European
`patent), NL (European patent), SE (European patent),
`US.
`.
`
`Published
`With international search report.
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`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER TIIE PATENT COOPERATION TREATY (PCT)
`
`. W0 91/11457
`(11') International Publication Number: '
`(51) International Patent Classification 5 :
`C07K 7/34, 7/10, A61K 37/02
`
`
`
`(43) International Publication Date:
`8 August 1991 (08.08.91)
`A61K 37/28
`
`
`(21) International Application Number:
`
`PCT/US91/0050’0
`(74) Agents: MURASHIGE, Kate, H. et al.; Irell & Manella,
`5-45Midd1efield Road, Suite 200, Menlo Park, CA 94025
`
`(US).
`(22) International Filing Date:
`24 January 1991 (24.01.9119 '
`
`
`(30) Priority data:
`
`24 January 1990 (24.01.90)
`US
`468,736
`
`
`
`(60) Parent Application or Grant
`
`(63) Related by Continuation
`
`
`468,736 (CIP)
`US
`Filed on
`24 January 1990 (24.01.90)
`
`
`(71)(72) Applicants and Inventors: BUCKLEY, Douglas, I. [US/
`US]; 215 Brookwood Road, Woodside, CA 94062 (US).
`HABENER, Joel, F. [US/US]; 217 Plymouth Road,
`Newton Highlands, MA 02161 (US). MALLORY,
`, Joanne, B [US/US]; 243 Acalanes, Apt 9, Sunnyvale,
`CA 94086 (US). MOJSOV, Svetlana [YU/YU]; 504 East
`63rd Street, New York, NY 10021 (US).
`.
`
`'
`
`
`
`
`
`(54) Title: GLP-l ANALOGS USEFUL FOR DIABETES TREATMENT
`
`(57) Abstract
`
`The invention provides effective analogs of the active GLP-l peptides, 7-34, 7-35, 7-36, and 7-37, which have improved
`characteristics for treatment of diabetes Type II. These analogs have amino acid substitutions at positions 7-10 and/or are trun-
`cated at the C-terrninus and/or contain various other amino acid substitutions in the basic peptide. The analogs may either have
`an enhanced capacity to stimulate insulin production as compared to gluc‘agon or may exhibit enhanced stability in plasma as
`compared to GLP-l (7-37) or both. Either of these properties will enhance the potency of the analog as a therapeutic. Analogs
`having D-amino acid substitutionsin the 7 and 8 positions and/or N-alkylated or N-acylated amino acidsin the 7 position are
`particularly resistant to degradation in vivo'.
`
`.
`
`x ,ggMYLANINST. EXHIBIT 1041 PAGEVI
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`MYLAN INST. EXHIBIT 1041 PAGE 1
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`MYLAN INST. EXHIBIT 1041 PAGE 1
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`'8!
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`(A,
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCI‘ on the front pages of pamphlets publishing international
`applications under the PCT.
`
`Monaco
`
`AT
`AU
`BB
`BE
`BF
`86
`8.!
`BR
`CA
`CF
`CG ,
`CH
`Cl
`CM
`CS
`DE
`DK
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Canada
`Central African Republic '
`Congo
`.
`Switzerland
`' cane d'lvojre 7
`Cameroon
`Czechoslovakia
`Germany
`Denmark
`
`,
`
`7
`
`'
`
`.
`
`,
`
`‘
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Guinea
`Greece
`Hungary
`ltaly
`7
`,
`Japan
`' Democratic People‘s Republic
`of Korea
`- Republic of Korea
`Liechtenstein
`Sri lanka
`Luxembourg
`
`_
`
`'
`
`Madagascar
`Mali
`Mongolia
`Mauritania
`Malawi
`Netherlands
`Norway
`Poland
`Romania
`Sudan
`Sweden
`Senegal
`Soviet Union
`' Chad
`Togo
`United States of America
`
`.
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`7
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`'
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`MYLAN INST. EXHIBIT 1041 PAGE 2
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`MYLAN INST. EXHIBIT 1041 PAGE 2
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`PCT/US91/00500
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`GLP-l ANALOGS USEE'UL FOR DIABETES TREATMENT
`
`This is-a confiinuation-inbpart of U.S.
`Application Serial No. 468,736, filed 24 January 1990.
`
`10
`
`Technical Field
`
`The invention relates to the field of improved
`
`the invention
`pharmaceutical Compositions. Specifically,
`concerns analogs of the glucagon-like peptide I fragment
`7536 or 7-37 with improvéd pharmacological properties.
`
`,
`_
`‘
`Background Art
`Glucose metabolism is regulated by a number of
`peptide hormones,
`including inSulin, glucagon, and
`gastric inhibitory peptide (GIP). The complex mechanism
`by which these peptidehormOnes regulate this metabolism
`
`(and the manner in which they affecteach other is at
`least partially elutidated. Forexample, glucagon binds
`to receptors on the surface of the pancreatic beta cells
`which produce insulin, andst1mulates insulin secretion.
`
`Glucagon-like peptide I has been suggested to stimulate
`insulin secretion but'this has notfbeen confirmed.
`Several cf trade hormoneSVOriginate from a mam-
`malian glucagon prechrsbr "proglucagon" which is a 180
`amino acid peptide. Proteolysisuand proceSSing of this
`peptide results in a number of these protein hormones;
`’the results of the processing depEnd on the origin of the
`cells in whiCh this odours:
`For example,
`in the pig and
`rat pancreas, proglucagon_ is procésSed to form glucagon
`and glicentin—related pancreaticpeptide, a large peptide
`
`15
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`35
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`MYLAN INST. EXHIBIT 1041 PAGE 3
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`MYLAN INST. EXHIBIT 1041 PAGE 3
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`MYLAN INST. EXHIBIT 1041 PAGE 3
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`PCT/US91/00500 .
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`-2-
`
`which contains both GLP-1 and GLP-2 sequences.
`
`:in:
`
`m
`
`porcine small intestine,
`
`the secreted products are the 69
`
`amino acid glucagon-containing peptide glicentin and the
`
`two glucagon-like sequencesr‘GLP-l and GLP-2 as separate
`
`peptides.
`
`'
`
`In any event, however,
`
`the overall sequence of
`
`proglucagon contains the 29 amino acid sequence of
`
`glucagon,
`
`the 36 or 37 amino acid sequence of GLP—1 and
`
`the 34 amino acid sequence of GLP-2, separated by amino
`
`10
`
`acid spacer sequences.
`
`Early attempts to assign a pattern of activity
`to GLP-l gave ambiguous results, and it was subsequently
`
`concluded that truncated forms of this peptide are bio-
`logically active. Mojsov, S., et al. J Clin Invest
`
`(1987) 13:616-619 disclose that only the 31 amino acid
`peptide GLP-l
`(7-37) strongly stimulates the release of
`
`insulin from pancreas; although both the truncated and
`
`full length 37 amino acid form had earlier been found in
`
`pancreas and intestine.
`
`It has been demonstrated that
`
`GLP-l (7-36), possibly with the carboxy terminus
`
`- amidated, is also a potent mediator of insulin release.
`
`(See, e.g., Holst, J.J., et al. FEBS Letters (1987)
`
`g;;:169-174).
`
`The invention described below concerns analogs
`
`of these truncated forms of GLP-1, which have desirable
`
`combinations of characteristics as they relate to potency
`
`in potentiating glucose-induced insulin secretion and
`
`glucose-induced inhibition of glucagon secretion and to
`
`circulating half-life.
`
`The physiological effects of the
`
`truncated forms in potentiating glucose-induced insulin
`secretion have been shown as described above by Holst,
`J.J., et al. and Mojsov, 5., et al.
`(supra).
`The
`_ activity of the truncated hormones in inhibiting glucagon
`
`release has been shown by Orskov, 0., et al. Endocrinol
`
`15
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`MYLAN INST. EXHIBIT 1041 PAGE 4
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`MYLAN INST. EXHIBIT 1041 PAGE 4
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`MYLAN INST. EXHIBIT 1041 PAGE 4
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`"
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`not?:."'.'Ezj',_;g‘;yt1rlis._q'
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`var,
`’
`>
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`‘ 1
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`wo 91/11457
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`PCP/US91/00500
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`-3-.
`
`The
`
`(1988) 123: 2009—2013; Suzuki, 3., et al. Diabetes
`Research: Clinical Practice (1988) 5(Supp. 1): 830.
`circulating half--life of these truncated forms is
`short--approximately four minutes as shown by Kreymann et
`al. The Lancet
`(December 5,1987) 1300-1303.
`The
`modified forms of these truncated GLP-l peptides provide
`the opportunity to optimite these properties.
`There is some literature relating to the study
`of degradation of peptide harmones in the liver and in
`plasma and the half-lifeof such; ormones Ln va0
`generally.
`An early paper by McDonald JJK. et al., 5
`Biol Chem (1969) 244: 6199-6208 showed that a dipeptidase
`was responsible for the degradation of glucagon in rat
`liver. Studies on the growth hormone releasing factor, a
`member of the general glucagon, GLP-l, GLP-Z family, was
`shown to be rapidly degraded in plasma in vitro and also
`in vao by a dipeptidase, (Frohman, L. A. et al., JClin
`Invest
`(1986) 78: 906-913) Murphy, W. A. et al.,
`in
`Peptide Research (1988)
`1”: 36—41, showed that some but not
`all alkylated growth hormone releasing factor peptides
`had higher potency 15 1:25.
`In particular, for example,
`the triisopropylated GRF429 was found to be 106 times
`
`On the other hand, GRF-'
`more active than GRFeZQ itself.
`29 which.was in methylated at the N-terminus was only 40%
`as potent as the parent.
`It was also shown that
`substitution of D-Ala_position 2 of this hormone enhanced
`its potency.
`It was, of course, ngt certain to what
`effect on properties the enhancement of potency could be
`attributed.
`V
`I
`I
`5
`
`Others have atEempted some modifications of
`GLP-1 (7-37).
`It has been shown that deletion of the
`histidine residue at position 7 greatly diminishes the.
`aCtivity of the hormong (Suzuki, 8., et a1.
`(supra);
`Hendrick, G.K., et a1. Abstract: Endocrine Society
`
`10
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`15
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`20
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`30
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`35
`
`,. ._M){LAN INST. EXHIBIT 1041 PAGE 5
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`MYLAN INST. EXHIBIT 1041 PAGE 5
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`PCT/US91/00500
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`'4-
`
`Meeting. New Orleans, LA (1988)). There have been
`conflicting reports concerning the effect of one or more
`C-terminal deletions (Suzuki, 5., et al.
`(supra);
`Yanaihara, C., et al. Abstract for A Glucagon and Related
`Peptides Satellite Symposium, 8th International Congress
`of Endocrinology, July 15-16, 1988, Osaka, Japan).
`However,
`there is an extensive literature with regard to
`modifications of other members of this peptide hormone
`family, such as GIP, glucagon releasing factor (GRF),
`secretin and vasoactive intestinal peptide (VIP).
`
`Disclosure of the Invention
`
`The invention provides modified forms of the
`GLP-l (7-34);
`(7-35);
`(7-36) or (7-37) human peptide or
`the Citerminal amidated forms thereof.
`The native
`peptides have the amino acid sequence:
`
`25
`20
`15
`1o
`7
`H-A-E-G-T-F-T-S-D-V-S-S-Y—L-E-G-Q-A-A-
`
`37
`3o
`K-E-F-I—A—W-L—V-K-(G)~(R)-(G)
`
`(R), and (G) are present or absent depending
`wherein (G),
`on indicated chain length.
`The modified forms contain
`one or more alterations of the native structure and are
`of improved ability for therapeutic use. Either the
`modified forms have greater potency than glucagon to
`potentiate insulin secretion or enhanced stability in
`plasma or both. This potency and enhanced stability can
`be assessed as described below.
`
`The standard one letter abbreviation code for amino
`_ acids is used.
`The analogs of the invention which show enhanced '
`insulin stimulating properties have the foregoing
`
`5
`
`10
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`15
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`20
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`30
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`35
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`m
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`‘
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`w
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`'*
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`MYLAN INST. EXHIBIT 1041 PAGE 6
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`sequence, or the C-terminal amide thereof, with at least
`one modification selected from the group consisting of:
`
`(a) substitution ofia neutral amino acid, arginine,
`or a D form of lysine for lysine at position 26 and/or 34
`and/or a neutral amino aCid,
`lysine, or a D form of
`
`arginine for arginine at position 36;
`(b) substitution of an oxidation-resistant amino
`acid for tryptophan at poSition 31;
`
`(c) substitution according to at least one of:
`Y for V at position 16;
`
`C)X!mmUin
`
`for S at position 18;
`
`for E at position 21;
`
`for
`
`for WI)Q
`
`for
`
`at position 22;
`
`'at position 23;
`
`at position 24; and
`
`for K at position 26;
`
`(d) a substitution comprising at least one of:
`an alternative small neutral amino acid for A
`
`at position 8;.
`
`an alternative acidic amino acid or neutral
`
`amino acid for E at position 9;
`an alternative neutral amino acid for G at
`
`position 10; and
`‘
`an alternative acidic amino acid for D at
`position 15; and:
`
`(e) substitution of an alternative neutral amino
`
`acid or the D or N-acylated or alkylated form of
`
`histidine for histidine at position 7.‘
`
`With respect to mod1f1cations (a),
`
`(b),
`
`(d) and
`
`the substituted amino acids may be in the D form, as
`(e),
`indicated by a superscript T, e. g. ,CT. The amino acids
`substituted at position 7 can also be in the N-acylated
`or N-alkylated.forms.
`
`,_,MYLAN INST. EXHIBIT 1041 PAGE 7
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`_6_
`
`Thus, one aspect of the invention is directed to
`
`peptides having enhanced insulin stimulating properties
`
`analogous to the above-mentioned truncated forms of GLP-1
`
`(7—34)
`
`to GLP-l (7-37), as described above.
`
`In another aspect,
`
`the invention is directed to
`
`peptides which show enhanced degradation resistance in
`
`plasma as compared to GLP-1'(7-37) wherein this enhanced
`resistance to degradation is defined as set forth below.
`
`In these analogs, any of the above-mentioned truncated
`
`10
`
`forms of GLP-1 (7-34)
`
`to GLP-l
`
`(7-37) or their C-terminal
`
`amidated forms is modified by
`(a) substitution of a D-neutral or D-acidic amino
`
`acid for H at position 7, or
`(b) substitution of a D-amino acid for A at
`
`15
`
`position 8, or
`
`(c) both, or
`
`(d) substitution of an N-acylated or N-alkylated
`
`form of any naturally occurring amino acid for H at
`
`position 7.
`
`Thus, analogs of the invention which are resistant
`to degradation include (N-acyl
`(1-6C) AA)7 GLP-l
`(7-37)
`and (N-alkyl (l-6C) AA)7 GLP-l
`(7-37) wherein when AA is
`
`a lysyl residue, one or both nitrogens may be alkylated
`
`or acylated.
`
`AA symbolizes any amino acid consistent
`
`with retention of insulin stimulating activity.
`
`For substitutions of D-amino acids in the 7 and 8
`
`positions,
`
`the D residue of any acidic or neutral amino
`
`acid can be used at position 7 and of any amino acid at
`
`position 8, again consistent with insulin stimulating
`
`activity. Either or both of position 7 and 8 can be
`
`OJ
`
`substituted by a D-amino acid;.the D-amino acid at
`position 7 can also be acylated or alkylated as set forth
`above. These modified forms are applicable not only to
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`MYLAN INST. EXHIBIT 1041 PAGE 8
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`_7—
`
`(7-37) but also the shorter truncated analogs as-
`GLP—l
`set forth above.
`‘
`‘
`
`In other aspects,
`
`the invention is directed to
`
`pharmaceutical compositions—containing one or more of
`these peptides as active ingredients and to methods to
`treat Type II diabetes using these;peptides or
`
`compositions thereof.
`
`Brief Description of the Drawings%
`
`Figure 1 schematically outlines the classification
`of amino acids as need herein.
`”
`
`Figure 2 gives a list of various compounds of the
`invention.
`1
`_
`Figure 3 shows the resultSiof radiolabel sequencing
`analysis for degradation of two analogs in plasma.
`(7-37)
`Figure 4 shows the results of Various GLP-l
`to
`analogs with changes in the amino terminal region,
`125
`displace
`I-GLP-l
`(7-39)
`from amino terminal specific
`antiserum.
`
`Modes of Carrying Out the Invention
`The analogs of the inVention, which are modified
`forms of the'GLP-l“(7'-3.4),' (73-535),
`(7-36) or (7-37) are
`
`characterized by show1ng greater potency than glucagon in
`
`an in 11339 assay measuring insulin release from isolated
`
`rat islets in culture, or by enhanced stability in plasma
`or both.
`
`Assays for Analogs with Enhanced Insulin Release
`
`“*3
`Stimulating Properties
`One group of analogs.of the invention is more
`potent than glucagon in stimulating insulin release from
`
`islet cells. By being "more_ potent than glucagon in
`stimulating insulin release from iSlet cells" is meant
`that the analog referred to shows greater potency in an
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`in yitgg assay selected from the group consisting of the
`
`w.
`
`following: Rat islets for these assays are isolated by
`
`the method of Sutton, R. et al., Transplantation (1986)
`
`g;:689-691,
`
`incorporated herein by reference. Briefly,
`
`Sprague—Dawley male rats are anesthetized and the lower
`end of the common bile duct is cannulated with a 2 FG
`
`cannula tied in place.
`The left and right hepatic ducts
`are then ligated separately above the region of the entry
`of pancreatic ducts into the biliary tree. The rats are
`
`10
`
`killed by exsanguination and 3 mL Hank's solution
`
`containing 7.5 mM CaClz, 20 mM HEPES buffer and 1-6 mg/mL
`Type I collagenase are run into the cannula to uniformly
`
`distend the pancreas.
`
`The pancreas is then excised and
`
`15
`
`placed in a beaker on ice prior to incubation in Hank's
`solution containing 20 mM HEPES buffer at 37°C.
`
`After 13-25 min of incubation,
`
`the pancreas is
`
`removed and placed in Hank's solution containing 5 g/l
`bovine serum albumin and 20 mM HEPES buffer at 4°C. All
`
`of the pancreatic tissue is then gently syringed through
`
`a 14 FG needle, suspended in further Hank's solution
`
`containing HEPES as above, centrifuged at 50 g for 10 sec
`
`and the supernatant is discarded.
`
`The tissue pellet is
`
`resuspended and again gently syringed, followed by
`
`another wash, after which the dispersed tissue is passed
`
`through a nylon mesh filter of 500 u pore size.
`
`The
`
`filtered tissue is centrifuged at 350 g for 5 sec,
`
`the
`
`supernatant discarded, and the tissue is then suspended
`
`in 25% Ficoll made up in Hank's with HEPES as above, on
`
`which was layered a discontinuous density gradient of
`
`23%, 20%, and 11% Ficoll solutions. This density
`gradient was spun at 750 g for 10 min at 4°C, and the V
`7 tissue obtained from the upper two interfaces was washed
`_three times in Hank's solution.and viewed through a
`dissecting microscope for hand picking of islets.
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`MYLAN INST. EXHIBIT 1041 PAGE 10
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`In one approach the ability of the GLP41 analog to
`potentiate secretion from these Islets is then determined
`
`according to the method of Schatz, H. et al.,
`
`in "Methods
`
`in Diabetes Research" (lfi34r‘Volume l, Part C: pages
`291-307,
`incorporated herein by reference.
`In this
`method, 5-10 islets per test tube are incubated in 1 mL
`Krebs-Ringer-bicarbonate buffer (KRB buffer).
`For
`testing, glucagon or the modified analog of the invention
`is added at 5-10 pg/mL.
`The level of insulin released
`may be measured by the method of Jensen, S.L. et al., M_Q
`
`Physiol (1978).;;§:E381e§386,
`reference.
`‘
`
`incorporated herein by
`
`10
`
`The following protocol is a preferred method to
`
`15
`
`measure stimulation of insulin Secretion. After
`
`collagenase digestion,,the islets are allowed to recover
`
`overnight by incubation in DMEM (Dulbecco's Modified
`Eagle Medium 16 w/o glucose), 2.8 mM glucose,
`10% fetal
`bovine serum (FBSL at 32cc, 5% co}.
`The next day, islets to be used for the experiment
`are transferred to DMEM, no glucose, 012% BSA (Armour,
`clinical grade, made at 5% stock) for a 60 min
`
`Islets
`preincubation in serum—free, glucose-free medium.
`are picked up by Eppendogf pipette and transferred to 60
`mm TC plates containing 8,0 mL medium and returned to the
`
`incubator for 60 min.
`
`Islets are counted during this
`
`transfer.
`
`(Note:
`
`each data paint is 5 islets,
`
`experiments are usually performed in quadruplicate;
`
`therefore, 20 islets arewused per data point.)
`Typically, recoveries are 150-209 islets per pancreas.
`Any suspect islets--too ragged or falling apart—-are not
`used.
`
`During the 60 min preincubation,
`
`the experiment is
`
`set up, so that all that is needed at the end of the
`
`20
`
`25
`
`3O
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`35
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`MYLAN‘INST. EXHIBIT 1041 PAGE 11
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`MYLAN INST. EXHIBIT 1041 PAGE 11
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`MYLAN INST. EXHIBIT 1041 PAGE 11
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`
`preincubation is to tranSfer islets in groups of 5 to‘
`
`m
`
`experimental conditions.
`
`The experiment is set up in 48
`
`well TC plates with 0.5 mL medium per well.
`
`To DMEM-0.2%
`
`BSA is added glucose to desired concentration (usually
`
`2.8 mM for hypoglycemic conditions, 5.6 mM glucose for
`
`euglycemic, or 16.7 mH glucose for hyperglycemic) and
`
`test compound at various dose ranges (typically, 1 pH to
`100 nM). Test compound is diluted from stock stored at
`-80°C and at ~0.3 mM serially into phosphate buffered
`
`saline (PBS) 0.2% BSA to prevent loss on sides of tubes.
`
`After medium plus test compound is mixed, 0.5 mL each is
`
`added to 4 wells for quadruplicate data points.
`After the preincubation period,
`5 islets are added
`per well.
`Islets are picked up by eppendorf pipette in
`
`25 ul volume.
`
`Incubation continues another 60 min, at
`
`which time 0.3 mL is harvested per well with care taken
`
`not to pick up islets. Wells are then rechecked for
`
`islet number. Medium is then assayed for insulin content
`
`10
`
`15
`
`20
`
`If medium is not immediately
`using an insulin RIA.
`assayed, it is stored at -20°C until assay. Dose
`
`response curves for insulin secretion are plotted and
`
`ED50 is calculated from the curves.
`Higher potency as compared to glucagon is defined
`
`as either higher levels of insulin released by the analog
`
`using the same concentrations of glucagon and analog or,
`
`alternatively, the same level of insulin release but
`
`using a lower concentration of analog than glucagon.
`
`While the foregoing assays form specific criteria
`
`for judging enhanced potency, alternative assays can also
`
`be used as substitutes for those set forth above.
`
`An additional test for potency of the compounds of
`
`the invention measures their ability to stimulate CAMP
`production in RIN 1046-38 cells.. This assay can be.
`conducted as follows:
`'
`
`25
`
`3O
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`35
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`MYLAN INST. EXHIBIT 1041 PAGE 12
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`MYLAN INST. EXHIBIT 1041 PAGE 12
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`MYLAN INST. EXHIBIT 1041 PAGE 12
`
`
`
`1
`
`1;
`
`'g,,_;:;2a113m1j1.
`
`aim"
`
`'
`
`.1
`
`‘1“.
`
`.-
`
`y
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`,-
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`W0 91/11457
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`‘1
`
`PCT/US91/00500
`
`-11—
`
`0“ day 1' 5 x 105 RrN 1046-33 cells (Drucker, D. J.,
`et al., Proc Natl Acad Sci USA (1987) 84: 3434-3438) are
`seeded into 1nd1v1dual wells of 6-well dishes with 2.5 mL
`
`M199 culture medium.g on day 4, cells are re-fed with
`fresh medium and on day‘s the assay is performed. At
`this time there are ~2. 0—2. 5 x 196 cells per well.
`Assays are only performed on cell passage <24.
`At time -60 min, monolayers are washed twice with
`2.5 mL PBS, and medium is changed to 1.0 mL of DMEM
`medium plus 4.5 g/l°gluc6se and 011% BSA (assay medium).
`At 0 time, medium is aspirated and fresh assay medium,
`1.0 mL, containing test compound is added. Test compound
`is added in 50 ul volume of PBS plus 0.1% BSA; controls
`are added in vehicle alone.
`Incubation is continued for
`o to 60 min.
`'
`At termination, conditioned medium and monolayer
`are harvested to measure both extra- and intracellular
`
`For extracellular measurement, medium is
`cAMP content.
`removed and centrifuged to remove any cellular debris.
`For intracellular determination, after medium removal,
`1.0 mL of ice cold 95% ethanol is added to monolayer.
`Cells are collected by scrap1ng, lysed by two cycles of
`quick freeze/thawing using liquid N2,and cell debris
`then removed by centrifugation.: Aliquots (1/40th well
`content) of conditioned medium and ethanol cell extract
`are measured in duplicate for cAHB levels using an RIA
`kit by the acetylated protocol.
`1
`‘
`
`As above, higher p6tency as compared to glucagon is
`defined either as higher cAMP stimulation by both the
`analog and glucagon at the same concentration, or the
`same CAMP stimulation by the analog at a lower
`concentration.
`‘
`1
`I
`‘
`Still other assays for measurement of enhanced
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`potency to mediate insulin releaSe can be used.
`
`MYLANJNST. EXHIBIT 1041 PAGE 13
`
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`MYLAN INST. EXHIBIT 1041 PAGE 13
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`MYLAN INST. EXHIBIT 1041 PAGE 13
`
`
`
`WO 91/11457
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`
`-12_
`
`" The ability of the compounds to potentiate the
`
`release of insulin can be tested both in vitro and in
`yiyg.
`Insulin released can be detected using a standard
`antibody assay both in analyzing plasma in in vivo
`studies and in analysing media or perfusion liquid in
`Em.
`
`5
`
`For example, a useful in vitro assay uses the
`pancreatic infusion assay method of Penhos, J.C., et al.
`Diabetes (1969)
`l§:733-738, as employed in the method of
`
`10 Weir, G.C., et al. J Clin Investigat (1974) §A:1403—1412.
`
`Insulin secretion can also be measured by'the method
`
`described by Holst, J.J;, et al. FEBS Letters (1987)
`
`g;;:169-174 (supra). Also useful as an assay for
`
`insulinotropic effect is the measurement of stimulation
`
`15
`
`of adenylate cyclase in the RIN 1046-38 cell line.
`Drucker, D.J. et al., Proc Natl Acad Sci USA (1987)
`785:3434—3438 (supra).
`
`Inhibition of glucagon release can be shown as
`
`described by Orstov, C., et a1. Endocrinol
`
`(1988)
`
`20
`
`;;;:2009—2013; Suzuki, 5., et al. Diabetes Research:
`
`Clinical Practice (1988) §(Supp.
`
`l):S30 (both supra).
`
`Assays for Enhanced Stability to Degradation
`
`The therapeutic efficiency of the GLP—l analogs of
`the invention can also be enhanced by providing analogs
`25 with increased half-lives in yiyg.
`By "enhanced half-
`
`life in yiyg" is meant a demonstrated ability to resist
`
`degradation in the presence of plasma according to an
`
`assay selected from the group consisting of the
`
`following.
`
`In all assays, the plasma is prepared by
`
`30
`
`collecting blood into heparinized tubes, placing the
`
`tubes on ice and centrifuging at about 3,000 rpm for 10
`minutes in.a tabletop centrifuge.
`The Separated plasma
`- is stored at 4‘0.
`
`35
`
`MYLAN INST. EXHIBIT 1041 PAGE 14
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`MYLAN INST. EXHIBIT 1041 PAGE 14
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`MYLAN INST. EXHIBIT 1041 PAGE 14
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`
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`WO 91/11457
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`"—13-~
`
`A. Radiolabel Segggncing:
`The GLP analog is labeledgby radio-iodination in
`position 19 using standard radiplabeling methods. After
`
`exchange into RIA buffer (507mM5NaHP04 pH 7.4. 0.25% BSA
`(Armour insulin and EPA free), 0.5% BME, 0.002%
`polylysine (Sigma 15,000 mw),ao.os% Tween 20, 0.1% NaN3),
`the radioiodinated peptide (about 105 cpm/50 mL) and cold
`uniodinated peptide (20-ul 100 nM) are added into 2 ml of-
`plasma to a final concentration of‘i nM and incubated in
`a circulating water bathgxor preset times. Total RIA
`buffer added to plasma newer exceeds 5% of total volume.
`
`At the end of incubation, 10% hacitracin (w/v)
`in water
`is added to a final concentration of 0.1% to stop the
`reaction.
`1
`,_
`,
`V
`
`The plasma is then extracted using C18 Sep-Pak to
`separate the analog and any fragments from the bulk of
`
`the plasma proteins.
`
`Sep-Pak cartridges (Waters) are
`
`washed with 2.mL of 1-propanol, followed by 2 mL of water
`.and then equilibrated with 2 mL of 20% CH3CN containing
`0.1% trifluoroacetic¢aCid (TFA)
`(Buffer A).
`The bacitracinjtregted plasma is made 20% CH3CN
`with CH3CN containing 0.1% TFA and is expressed slowly
`
`through a 3,mL plastic syringe through the cartridge.
`
`The cartridge is then washed with two 1 mL Buffer A
`
`washes and eluted with a single 2 mL wash of 50% CH3CN
`containing 0.1% TFA (Buffer B)
`intoea siliconized 12 x 75
`glass tube. Recovery of the analog or fragments is more
`than 90%.
`
`The eluates are concentrated to 100 pl in a Speed
`vac and transferred to a 1.5 mL Eppendorf tube to which a
`1 mL RIA buffer rinse of the original tube had been
`
`.added.
`
`To purify any analog or its fragments when the
`analogs of GLP—1
`(7-37) are used, the concentrates are
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
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`MYLAN INST. EXHIBIT 1041 PAGE 15
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`MYLAN INST. EXHIBIT 1041 PAGE 15
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`MYLAN INST. EXHIBIT 1041 PAGE 15
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`'-14-
`
`-_
`
`treated with 5 pl of antiserum prepared to a synthetic
`
`peptide corresponding to residues 24-37 which recognizes
`
`GLP-l, GLP-l
`
`(7-37) but not GLP-l (7-36). When the
`
`shorter forms of analogs are‘used, alternate carboxy
`
`terminal—specific antisera (prepared in the same manner
`
`but using a peptide corresponding to residues 24-34, 24-
`
`35 or 24-36 as immunogen) are used.
`To this is added 100
`pl of a 10% (w/v) solution of protein A¥Sepharose
`(Pharmacia)
`in PBS, and the mixture is incubated
`
`overnight at 4°C with gentle rocking.
`
`The Sepharose is
`
`rthen pelleted with a 5 second spin in an Eppendorf
`
`centrifuge at 4°C after which the pellet is washed two
`
`times with cold RIA buffer_and four times with cold PBS.
`
`Polyclonal antisera were raised in New Zealand
`
`White rabbits against a synthetic peptide fragment
`
`corresponding to residues 24 to 37 of GLP-1 (7-37) using
`
`the method of Mosjoy, S. et al., J Biol Chem (1986)
`
`g§;:ll880-11889.
`
`Initial immunizations were into the
`
`inguinal lymph nodes and used Freund's complete adjuvant.
`
`Two subcutaneous boosts were performed at 1 week
`
`'intervals after the initial immunization and used
`
`Freund's incomplete adjuvant.
`
`For a single immunization
`
`or boost 100 pg peptide and 100 pg methylated BSA
`
`dissolved in 0.3 mL phosphate-buffered saline (PBS) were
`
`emulsified with 0.9 mL adjuvant. Bleeds (50 mL) began at
`
`week 6 after the initial immunization and continued at 1
`
`month intervals thereafter. Repeat boosts were performed
`
`as above when titers dropped noticeably from the level of
`
`the previous bleed.
`
`Serum was prepared by allowing the blood to clot
`
`The clot was pelleted byrr ,
`overnight at 4°C.
`centrifugation at 2000 g for 15 minutes and the serum'
`rremoved.
`Serum is stored in aliquots at 420 or -80°C.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`MYLAN INST. EXHIBIT 1041 PAGE 16
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`MYLAN INST. EXHIBIT 1041 PAGE 16
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`MYLAN INST. EXHIBIT 1041 PAGE 16
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`aa'nfinnwr WWWWWWr5
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`PCT/US91/00500
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`V-15-
`
`The peptides are then eluted from the antibody
`protein-A sepharose complex with three 100 pl washes of
`Buffer B.
`The combined 300 p1 of wash are then applied
`directly to an ABI model 477A sequencer used according to
`the manufacturer's instructions. Fractions from each
`
`cycle are then diverted fior Counting. Counting can be
`effected in 4 mL aqueousscintillant (ACS Amersham).
`The cycle at which label appears indicates the
`extent of degradation from the N-terminus.
`If no
`degradation from the N-terminus has occurred in the GLP—
`1 (7-37) analog, all of the label will appear in the 13th
`cycle, corresponding to the tyrosine at position 19; if
`degradation has occurred, the label will appear in
`earlier cycles.
`a“
`I
`*
`3- Assex_hx_32:flghg:
`‘“33
`While the foregoing method is a clear criterion for
`exhibiting a longer half-life in plasma, alternatiVe
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`forms of the assay for this property can also be used.
`In one convenient assay, the analog Can be assessed for
`degradation into fragments using reverse phase—HPLC,
`since the fragments have different.retention times from
`the analog per se.
`In this assay, the analog is added to
`plasma for various times and reCovered similarly to the
`method described above for rad1oLabel seqUencing
`analysis. Specifically, the analogat a concentration of
`100 nM in RIA buffer is spiked into 1 mL plasma to a
`final concentration of 1 nM and after incubation in 37°C
`circulating water bath for various preset times,
`the
`reaction is stopped by bringing the plasma to 0.1% (w/v)
`
`in bacitracin.
`
`,
`
`'
`
`The peptides are then purifiehfby Sep-Pak
`extraction as described above. urhefieluates are
`concentrated to about 1 mL on a spebd-vac, diluted with 1
`mL distilled water, frozen at 80°C and lyophilized
`
`1.,
`MYLANINST EXHIBIT 1041 PAGE 17
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`MYLAN INST. EXHIBIT 1041 PAGE 17
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`MYLAN INST. EXHIBIT 1041 PAGE 17
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`
`overnight. VThe powder is resuspended in 0.5 mt Buffer C
`
`(0.1% TFA in water) per mL starting plasma and 0.25 mL
`
`are injected on a Hewlett-Packard 1090L liquid
`
`chromatograph using an Alltech C18 column (0.45 x 25 cm;
`
`5
`
`10 um particle size) with a Brownlee 2 cm C18 guard
`
`The extraction is monitored at OD214 throughOut
`column.
`the run and the solvent flow rate was 1 mL/minute.
`A
`gradient between Buffer C and Buffer D (0.1% TFA in
`
`acetonitrile) is set up over a 40 minute run time. The
`
`10
`
`gradient starts at 35% D is held for the first 2 minutes
`
`after injection and then increased to 42% D over 24
`
`minutes.
`
`The gradient is then increased to 60% D over
`
`the next two minutes, held at this level for 2 minutes
`and returned to 35% D over the next 2 minutes.
`The %D
`
`15
`
`remains at 35% for the remaining 8 minutes of the run.
`
`Fractions are collected at 0.5 minute intervals for the
`
`first 30 minutes of each run and dried in a Speed-vac.
`
`The samples can be assayed for the presence of analog or
`
`fragment using RIA (measuring competition with labeled
`
`20
`
`‘
`
`GLP-l (7-37) for binding to C-terminal specific
`antiserum) or by any conventional or convenient
`alternative method.
`
`Radioimmunoassays for the amino or carboxyl
`
`terminus of GLP-1 (7-37) use a single antibody
`displacement format. Binding of 125I-GLP-l
`(7-37)
`
`to
`
`25
`
`antibody is incrementally displaced by increasing
`
`concentrations of unlabeled peptide in.solution.
`
`Antibody bound iodinated peptide is separated from free
`
`iodinated peptide in solution by precipitation of the
`
`30
`
`antibody-peptide complex with Pansorbin‘m (Boheringer
`
`The resulting pellet is then counted on a
`Mannheim).
`gamma counter.
`7'.
`7'
`I
`
`35
`
`MYLAN INST. EXHIBIT 1041 PAGE 18
`
`MYLAN INST. EXHIBIT 1041 PAGE 18
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`MY