PCT
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`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
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`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`
`International
`Bureau
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`
`
`
`
`(51) International Patent Oassification 5 :
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`
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`(11) lntematiqnal Pnblication Number:
`
`WO 91/11457
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`
`
`Al
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`( 43)International Publication Date:
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`
`
`C07K 7 /34, 7 /10, A61K 37 /02
`A61K37/28
`
`8 August 1991 (08.08.91)
`
`(2l)lntemationalApplicationNumber: PCT/US91/00500 (74)Agents: MURASHIGE, Kate, H. et al.; Irell & Manella,
`
`
`
`
`
`
`
`545 Middlefield Road, Suite 200, Menlo Park, CA 94025
`(US).
`1991 (24.01.91) (22)International Filing Date:24 January
`
`
`
`
`
`
`
`(81)Designated States: AT (European
`
`
`
`patent), BE (European
`(30)Priority data:
`
`
`
`
`
`patent), CA, CH (European patent), DE (European pa­
`468,136 24 January 1990 (24.01.90)
`us
`
`
`
`
`
`tent), DK (European patent), ES (European patent), FR
`
`
`
`
`(European patent), GB (European patent), GR (Euro­
`
`
`
`pean patent), IT (European patent), JP, LU (European
`(60)Parent Application or Grant
`
`
`
`
`
`
`patent), NL (European patent), SE (European patent),
`
`(63)Related by Continuation
`468,736 (CIP)
`us.
`usFiled on
`
`24 January 1990 (24.01.90)
`
`Published
`
`
`
`
`(71)(72) Applicants and Inventors: BUCKLEY, Douglas, I. [US/
`
`With intemational search report.
`
`
`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,
`
`
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`CA 94086 (US). MOJSOV, Svetlana [YU/YU]; 504 East
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`63rd Street, New York, NY 10021 (US).
`
`
`
`(54)Title: GLP-1 ANALOGS USEFUL FOR DIABETES TREATMENT
`
`
`
`(57) Abstract
`
`The invention provides effective analogs of the active GLP-1 peptides, 7-34, 7-35, 7-36, and 7-37, which have improved
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`characteristics for treatment of diabetes Type IL These analogs have amino acid substitutions at positions 7-10 and/or are trun­
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`cated at the C-terminus and/ or contain various other amino acid substitutions in the basic peptide. The analogs may either have
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`an enhanced capacity to stimulate insulin production as conipared to glucagon or may exhibit enhanced stability in plasma as
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`compared to GLP-1 (7-37) or both. Either of these properties will enhance the potency of the analog as a therapeutic. Analogs
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`having D-amino acid substitutions in the 7 and 8 positions and/or N-alkylated or N-acylated amino acids in the 7 position are
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`particularly resistant to degradation
`in vivo.
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`FOR THE PURPOSES OF INFORMATION ONLY
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`
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`
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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.
`
`AT Austria
`Madagascar
`ES Spain
`MG
`AU Australia
`ML Mali
`Fl Finland
`MN Mongolia
`Barbados
`FR France
`BB
`Belgium
`Mauritania
`GA Gabon
`BE
`
`Burkina Faso
`Malawi
`United Kingdom
`BF
`GB
`Netherlands
`BG Bulgaria
`Guinea
`GN
`NL
`BJ Benin
`NO Norway
`Greece
`GR
`Brazil
`Poland
`HU Hungary
`BR
`PL
`RO -Romania
`Canada
`IT Italy
`Republic . JP Japan
`CA
`Sudan
`CF Central African
`SD
`CG Congo
`Sweden
`KP Democratic People's Republic
`
`
`SE
`SN Senegal
`
`Switzerland
`of Korlllj
`CH
`SU Soviet Union
`Cote d'Ivoire
`KR Republic of Korea
`
`Cl
`CM Cameroon
`Chad
`Liechtenstein
`LI
`TD
`Sri Lanka
`TC Togo
`cs Czech0&lovakia
`LK
`us United States of America
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`DE Germany
`LU Luxembourg
`DK Denmarll.
`MC Monaco
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`MW
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`MR
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`II!
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`wo 91/11457
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`5
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`I
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`PCT/US91/00500
`
`1'
`
`GLP-l ANALOGS USEFUL FOR DIABETES TREATMENT
`_
`'1,
`5
`
`This is a conflinuation-in—part 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.
`
`mmwml—Art
`
`,
`
`5
`
`Glucose metabolism is regulated by a number of
`
`including insulin, glucagon, and
`peptide hormones,
`gastric inhibitory peptide (GIP).
`'The complex mechanism
`by which these peptide hormbnes regulate this metabolism
`vand the manner in which they affectieach other is at
`least partially elutidated.
`For'5eXamp1e, glucagon binds
`to receptors on the surface of the pancreatic beta cells
`which produce insulin, and stimulates insulin secretion.
`
`Glucagon-like paptide I has been suggested to stimulate
`insulin secretion but this has notdheen confirmed.
`Several Of these hormonesloriginate from a mam-
`malian glucagon precursor "proglficagon"- which is a 180
`amino acid peptide. Proteolysis and process1ng 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 occurs:
`For example,
`in the pig and
`Vrat pancreas, progluCagon_ is proceSSed to form glucagon
`and glicentin—related pancreatic peptide, a large peptide
`
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`WO 91/11457
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`PCT/US91/00500 '
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`-2-
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`which contains both GLP-1 and GLP-2 sequences.
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`:fnl
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`porcine small intestine,
`
`the secreted products are the 69
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`amino acid glucagon—containing peptide glicentin and the
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`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
`
`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) 12:616-619 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
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`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
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`glucose-induced inhibition of glucagon secretion and to
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`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, 8., et al. (supra).' The
`. activity of the truncated hormones in inhibiting glucagon
`release has been shown by Orskov, 0., et al. Endocrinol
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`”~17 1,‘ N .7, gammy?
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`‘ 4x
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`y}.
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`"yin".
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`W0 91/ 11457
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`PCT/US91/00500
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`-3_,
`
`The
`
`(1988) 123:2009—2013; Suzuki, 5., 3? al. Diabetes
`Research: Clinical Practice (1988} 5(Supp. 1):SBO.
`circulating half-life of these truncated forms is
`short--approximately four minutes as shown by Kreymann et
`al. The Lancet
`(December 9, 1987) 1300-1303.
`The
`modified forms of these truncated GLP—1 peptides provide
`the opportunity to optimi2e~these properties.
`There is some literature relating to the study
`of degradation of peptide harmones in the liver and in
`plasma and the half-life of suchhormones Ln vivo
`generally.
`An early paper by McDonald JJK. et al. ,g
`Biol Chem (1969) 244: 61996208 showed that a dipeptidase
`was responsible for the degradatlon of glucagon in rat
`liver. Studies on the grOwth hormone releasing factor, a
`member of the general glucagon, GLPl, GLP-Z family, was
`shown to be rapidly degraded in plasma Ln vitro and also
`in yiyg by a dipeptidase,
`(Frohman, L.A. et al., J Clin
`Invest
`(1986) 1§:906-913).
`.hurphy, 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 in yijg.
`In partiCular, for example,
`the triisopropylated GRF429 was found to be 106 times
`
`‘
`
`0n;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, not certain to what
`effect on properties the enhancement of potency could be
`attributed.
`V
`a
`9
`
`Others have attempted 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 hormone (Suzuki,_s., et al.
`(supra);
`Hendrick, G.K., et al. Abstract: Emgocrine Society
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`W0 91/11457
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`PCI‘/US91/00500
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`, ‘4'
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`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
`10
`7
`H-A-E-G-T-F-T-S-D-V—S-S-Y-L-E-G-Q-A-A-
`
`37
`30
`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
`I_acids is used.
`The analogs of the invention which show enhanced '
`insulin stimulating properties have the foregoing
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`PCT/US91/00500
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`sequence, or the C-terminal amide thereof, with at least
`one modification selected from the group consisting of:
`
`(a) substitution Of‘a 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 adéording to at least one of:
`Y for V at position 16;
`
`ONNUJUN
`
`for S at position 18;
`
`for
`for
`
`for >00!!!
`
`for
`
`at position 21;
`
`at poSition 22;
`
`at position 23;
`
`at position 24; and
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`for K at position 26;
`
`(d) a substitution compriSing at least one of:
`an alternativeysfiall neutral amino acid for A
`
`at position 8;.
`
`an alternative acidic amino acid or neutral
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`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
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`position 15; and
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`(e) substitution of an alternative neutral amino
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`acid or the D or N-acylated or alkylated form of
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`histidine for histidine at position 7.‘
`
`(d) and
`(b),
`With respect to‘modi‘ficatio‘ns (a),
`the substituted amino acidsfmay be in the D form, as
`(e),
`indicated by a superscript T, e.§., CT.
`The amino acids
`substituted at position 7 can also be in the N—acylated
`or N-alkylated.forms;
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`W0 91/ 11457
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`-6-
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`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-l
`(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
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`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
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`15
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`position 8, or
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`(c) both, or
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`20
`
`25
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`(d) substitution of an N-acylated or N-alkylated
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`form of any naturally occurring amino acid for H at
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`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
`(1-6C) 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
`
`30
`
`position 8, again consistent with insulin stimulating
`activity. Either or both of position 7 and 8 can be
`
`,
`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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`lfifl'd“ J
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`»
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`:,‘L:
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`51".“
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`r,-
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`.
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`wo 91/11457
`
`'
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`PCT/US91/00500
`
`_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 activefingredients and to methods to
`treat Type II diabetes using thesegpeptides 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.
`Figure 3 shows the‘resultséof“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.
`
`10
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`15
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`20
`
`Modes of Carrying Out the Invention
`The analogs of the inVention, which are modified
`forms of the“GLP-1“(7'-3.4),' (79:35:),
`(7-36) or (7-37) are
`
`25
`
`characterized by showing? greater potency than glucagon in
`an in vitro 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
`
`‘
`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 is1et cells" is meant
`that the analog referred to shows greater potency in an
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`PCT/US91/00500
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`-8—
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`;n yitgg assay selected from the group consisting of the
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`following: Rat islets for these assays are isolated by
`
`the method of Sutton, R. et al., Transplantation (1986)
`
`32:689-691,
`
`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
`
`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
`
`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
`
`, 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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`W0 91/11457
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`PCT/US91/00500
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`_9_
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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" (lgé4r'Volume 1, 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
`
`(1978) g;§:E381~§386,
`Physiol
`reference.
`‘
`
`incorporated herein by
`
`10
`
`The following protocol is a preferred method to
`measure stimulation of insulin secretion. After
`
`15
`
`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 (PBS), at 31%,, 5% coz.
`The next day, islets to be used for the experiment
`
`are transferred to DMEM, no glucose, 0.2% BSA (Armour,
`clinical grade, made at 5% stock) for a 60 min
`Islets
`preincubation in serum-free, glucose-free medium.
`are picked up by Eppendorf 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 point is 5 islets,
`
`experiments are usually performed in quadruplicate;
`
`therefore, 20 islets are used 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
`
`30
`
`35
`
`
`
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`7
`
`PCT/US91/00500
`
`-10-
`
`preincubation is to transfer islets in groups of 5 to-
`
`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 mM 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 ~O.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
`
`E050 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
`
`35
`
`
`
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`

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`WO 91/11457
`
`‘1
`
`-
`
`PCT/US91/00500
`
`-11...
`
`On day 1' 5 x 105 Rru 1b46——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.
`on day4 , 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 106 cellsper 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 glucose and 0.1% 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 exEra- and intracellular
`cAMP content.
`For extracellular measurement, medium is
`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 scraping, lysed by two cycles of
`quick freeze/thawing using liquid N2,and cell debris
`then removed by centrifugation.r Aliquots (1/40th well
`content) of conditioned medium and ethanol cell extract
`are measured in duplicate for cAMB levels using an RIA
`kit by the acetylated protocol.
`‘
`.
`As above, higher p6tency as compared to glucagon is
`defined either as higher cAMP st1mu1ation by both the
`analog and glucagon at the same concentration, or the
`same CAMP stimulation by the analog at a lower
`concentration.
`‘
`1
`1
`h
`.Still other assays for measurement of enhanced
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`potency to mediate insulin releaSe can be used.
`
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`

`

`W0 91/11457
`
`'
`
`PCI‘/US91/00500 .
`
`-12—
`
`' The ability of the compounds to potentiate the
`
`release of insulin can be tested both in vitro and in
`1119.
`Insulin released can be detected using a standard
`antibody assay both in analyzing plasma in in vivo
`studies and in analyiing media or perfusion liquid in
`lim-
`
`For example, a useful in vitro assay uses the
`pancreatic infusion assay method of Penhos, J.C., et al.
`Diabetes (1969)
`;§:733-738, as employed in the method of
`
`Weir, G.C., et al. J Clin Investigat (1974) fig: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
`
`of adenylate cyclase in the RIN 1046-38 cell line.
`Drucker, D.J. et al., Proc Natl Acad Sci USA (1987)
`fi:3434-3438 (supra).
`
`Inhibition of glucagon release can be shown as
`
`described by Orstov, C., et al. Endocrinol
`
`(1988)
`
`1;;:2009-2013; Suzuki, 8., et al. Diabetes Research:
`
`Clinical Practice (1988) §(Supp. 1):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
`with increased half-lives in yiyg.
`By "enhanced half-
`
`1ife 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
`
`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 49C.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`
`
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`

`

`WO 91/11457
`
`‘
`
`PCT/US9l/00500
`
`1—13-
`
`A. Eadielahel_§ssuensiee:
`The GLP analog is labeledyby radio-iodination in
`position 19 using standard radiolabeling methods. After
`exchange into RIA'buffer'(50—.-mM_NaHP,Q4 pH 7.4,_O.25% BSA
`(Armour insulin and FFA free), 0.5%”BME, 0.002%
`
`polylysine (Sigma 15,000 mw), 0.05% Tween 20, 0.1% NaN3),
`the radioiodinated peptide (about 105 cpm/SO mL) and cold
`uniodinated peptide (ZO-ul 100 in) are added into 2 m1 of-
`plasma to a final concentration of‘i nM and incubated in
`a circulating water bath for preset times; Total RIA
`buffer added to plasma newer exceeds 5%-of total volume.
`
`At the end of incubationJ 10% bacitracin (w/v)
`in water
`is added to a final conCentration of 0.1% to stop the
`reaction.
`H
`,_
`i
`,
`‘
`
`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 bacitracin:treated 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)
`intoaa siliconized 12 x 75
`
`glass tube. Recovery of the analog or fragments is more
`than 90%.
`
`The eluates are concentrated to 100 #1 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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`W0 91/1 1457
`
`‘
`
`'
`
`PCTIU591 I00500
`
`-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
`
`then 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)
`
`&:11880-11889.
`
`Initial immunizations were into the
`
`10
`
`15
`
`20
`
`inguinal lymph nodes and used Freund's complete adjuvant.
`Two subcutaneous boosts were performed at 1 week
`
`7
`
`'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
`
`overnight at 4°C. The clot was pelleted byrr
`centrifugation at 2060 g for 15 minutes and the serum"
`removed.
`Serum is stored in aliquots at -20 or -80°C.
`
`25
`
`30
`
`35
`
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`
`

`

`*
`
`Hr
`;~
`j‘a 1W}?
`
`:"lva,
`2
`
`méfitgtwrvlgm'
`
`‘
`
`'
`
`",9
`1‘
`
`wo 91/11457
`
`'
`
`.
`
`'
`
`PCT/US91/00500
`
`_15-
`
`The peptides are then eluted from the antibody
`protein-A sepharose complex with three 100 pl washes of
`Buffer B.
`The combined300 ul 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 for 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 tyr051ne at position 19; if
`degradation has occurred, the label will appear in
`earlier cycles.
`”
`'
`B. Assay by RP-HPLC:
`“n?
`While the foregoing method iSIa clear criterion for
`exhibiting a longer halfelife in plaSma, alternative
`forms of the assay‘foruthis property can also be used.
`In one convenient assay, the analog‘oan 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 f.cr radioLabel seqUencing
`analysis. Specifically, the analogat a concentration of
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`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‘purifiedfiby Sep-Pak
`extraction as described above. AThemeluates are
`concentrated to about 1 mL on a spEEd-vac, diluted with 1
`mL distilled water, frozen at 80°C and lyophilized
`
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`'
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`’
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`PCT/US91/00500
`
`_-16-'
`
`overnight.
`
`The powder is resuspended in 0.5 mL 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;
`
`10 nm particle size) with a Brownlee 2 cm C18 guard
`
`The extraction is monitored at 0D214 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
`
`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
`
`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
`
`GLP-l (7-37) for binding to C-terminal specific
`
`antiserum) or by any conventional or convenient
`
`alternative method.
`
`10
`
`15
`
`20
`
`Radioimmunoassays for the amino or carboxyl
`
`25
`
`terminus of GLP-1 (7-37) use a single antibody
`f 1251-GLP-1 (7-37)
`
`displacement format. Binding 0
`
`to
`
`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
`
`antibody-peptide complex with Pansorbin” (Boheringer
`
`The resulting pellet is then counted on a
`Mannheim).
`gamma counter.
`7"
`7
`-
`
`30
`
`35
`
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`W0 91/ 11457
`
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`
`PCT/US9l/00500
`
`p -17-
`
`7C. Loss of Binding to N-Terminal Specific'
`Antibodies:
`.
`‘
`4
`’
`A third approachito assessment of half—life in
`
`I
`
`plasma utilizes polyclonal or monoclonal antibodies
`specifically prepared to the N-terminus which will fail
`to bind degraded analogad These antis