PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER Tf{E PATENT COOPERATION TREATY (PCT)
`WO 91/11457
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 5 :
`C07K 7 /34, 7 /10, A61K 37 /02
`A61K37/28
`
`Al
`
`(11) Intematiqnal Publication Number:
`
`(43) International Publication Date:
`
`8 August 1991 (08.08.91)
`
`(21) International Application Number:
`
`PCT/US91/00500
`
`(22) International Filing Date:
`
`24 January 1991 (24.01.91)
`'
`
`(30) Priority data:
`468,136
`
`24 January 1990 (24.01.90)
`
`US
`
`(60) Parent Application or Grant
`(63) Related by Continuation
`us
`Filed on
`
`468,736 (CIP)
`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).
`
`(74)Agents: MURASHIGE, Kate, H. et al.; lrell & Manella,
`545 Middlefield Road, Suite 200, Menlo Park, CA 94025
`(US).
`
`(81) Designated States: AT (European patent), BE (European
`patent), CA, CH (European patent), DE (European pa(cid:173)
`tent), DK (European patent), ES (European patent), FR
`(European patent), GB (European patent), GR (Euro(cid:173)
`pean patent), IT (European patent), JP, LU (European
`patent), NL (European patent), SE (European patent),
`us.
`
`Published
`With international search report.
`
`(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
`characteristics for treatment of diabetes Type II. These analogs have amino acid substitutions at positions 7-10 and/or are trun(cid:173)
`cated at the C-terminus and/ or contain various other amino c1.cid substitutions in the basic peptide. The analogs may either have
`an enhanced capacity to stimulate insulin production as coni:pared to glucagon or may exhibit enhanced stability in plasma as
`compared to GLP-1 (7-37) or both. Either of these properties will enhance the potency of the analog as a therapeutic. Analogs
`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
`particularly resistant to degradation in vivo.
`
`MPI EXHIBIT 1042 PAGE 1
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`MPI EXHIBIT 1042 PAGE 1
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0001
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`CA
`CF
`CG
`CH
`CJ
`CM
`cs
`DE
`DK
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Canada
`Central African Republic
`Congo
`Switzerland
`Cote d'Ivoire
`Cameroon
`C:tech06lovakia
`Germany
`Denmarlt.
`
`F.S
`Fl
`FR
`GA
`GB
`GN
`GR
`HU
`IT
`• JP
`KP
`
`KR
`LI
`LK
`LU
`MC
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Guinea
`Greece
`Hungary
`Italy
`Japan
`Democratic People's Republic
`of Korllij
`Republic of Korea
`Liechtenstein
`Sri Lanka
`Luxembourg
`Monaco
`
`MG
`Madagascar
`ML
`Mali
`MN
`Mongolia
`MR
`Mauritania
`MW Malawi
`NL
`Netherlands
`NO
`Norway
`PL
`Poland
`RO
`•Romania
`SD
`Sudan
`SE
`Sweden
`SN
`Senegal
`Soviet Union
`SU
`TD
`Chad
`TG
`Togo
`us
`United States of America
`
`II!
`
`MPI EXHIBIT 1042 PAGE 2
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`MPI EXHIBIT 1042 PAGE 2
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0002
`
`

`

`W091/11457
`
`PCT /US91/00500
`
`-1-
`
`5
`
`GLP-1 ANALOGS USEFUL FOR DIABETES TREATMENT
`
`ii,
`
`This is· a con'b!i:nuation-tn-part of U.S.
`Application Serial No. 4,68, 736, filed 24 January 1990.
`
`10 Technical Field
`The,inven:tion relates to the field of improved
`pharmaceutical compositions. Specifically, the invention
`concerns analogs of the glucagon-like peptide I fragment
`7-36 or 7-37 with improved pharmacoJ.ogical properties.
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`15
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`Background Art
`Glucose metabolism is reguiated by a number of
`peptide hormones, including insulin, glucagon, and
`gastric inhibitory pe~tide (GIP). The complex mechanism
`by which these peptide·· hormones regulate this metabolism
`and the manner in whic::h they a.f:f:ec1f each other is at
`least partially elu'.cidated. For /&:»:ample, glucagon binds
`: '.
`to receptors on the surface of the pancreatic beta cells
`which produce insuJ:in, and stimulates insulin secretion.
`25 Glucagon-like peptide I has bee.n suggested to stimulate
`insulin secretion but ~his has ndt:'.':b.een confirmed.
`Several of the,~· hormones·:·originate from a mam(cid:173)
`malian glucagon precurs,or "progl'~oagoh"· which is a 180
`amino acid peptid'et.· · P:rot~olysis, anci processing of this
`peptide results :im a number of t:.hesie protein hormones;
`'
`the results of the proce.ssing dep'end on the origin of the
`cells in which tliis~_odcurs·~ For example, in the pig a:nd
`r·at pancreas, prog1u·cagori is ,processed to form glucagon
`.~
`and glicentin-related pancr.eatiC ·peptide, a large peptide
`
`,,.;
`
`.
`
`.
`
`MPI EXHIBIT 1042 PAGE 3
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`MPI EXHIBIT 1042 PAGE 3
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0003
`
`

`

`WO91/11457
`
`PCT/US91/00500
`
`-2-
`
`In
`which contain·s both GLP-1 and GLP-2 sfequences.
`porcine small intestine, the secreted products are the 69
`amino acid glucagon-containing peptide glicentin and the
`two glucagon-like sequences,GLP-1 and GLP-2 as separate
`peptides.
`
`5
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`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-1 gave ambiguous results, and it was subsequently
`concluded that truncated forms of this peptide are bio(cid:173)
`logically active. Mojsov, s., et al. J Clin Invest
`(1987) 79:616-619 disclose that only the 31 amino acid
`peptide GLP-1 (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
`20 GLP-1 (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)
`211:169-174).
`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, s., et al. (supra). The
`activity of the truncated hormones in inhibiting glucagon
`release has been shown by Orskov, c., et al. Endocrinol
`
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`MPI EXHIBIT 1042 PAGE 4
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`MPI EXHIBIT 1042 PAGE 4
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0004
`
`

`

`W091/11457
`
`PCT/US91/00500
`
`-3-
`
`(1°988) 123:2009-2013; Suzuki, s.,. e} ·a1. Diabetes
`Research: Clinical ~ractice (1988) ~(Supp. 1):S30. The
`circulating half-life of these truncated forms is
`short--approximately four m±nutes as shown by Kreymann et
`al. The Lancet (December 9, 1987) 1300-1303. The
`modified forms of these ~runcated GLP-1 peptides provide
`the opportunity to optimize·these properties.
`There.is som~.li1:,eratu7:~ relating to the study
`of degradation of peptid~J'.Aermon~~ Jn the liver and in
`plasma and the half-life .o.f such J~ormones in vivo
`.
`. ~,.,·· <
`generally. An early pap~r by McDbn'ald! J .1<. et al., iI
`Biol Chem ( 1969) 244: 6199::·6208 snowed that a dipeptidase
`was responsible .fo;c th~ q·egradati'oh of glucagon in rat
`.
`.
`liver. Studies on tqe g:r;owth hormone releasing factor, a
`15 member of the general glµ,.cag'?n, GI,,E.-1, GLP-2 family, was
`shown to be rapidly degraded in .Plasma in vitro and. also
`in vivo by a dipeptidase., (Fro:t;unan, L.A. et al., J Clin
`Invest (1986) li:906.;.913).~
`,:Murphy, W.A. et al., in
`Peptide Research (19·88) l,':36·-41, showed that some but not
`all alkylated growth hortn.one re~easing factor peptides
`had higher potency in vi,tti.
`In ~articular, for example,
`the triisopropylated GRF-29 was found to be 106 times
`more active than G~F-29 i~self. On ~he other hand, GRF-·
`29 which. was in methylated at th.e 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, n9t certain to what
`effect on properties th~ enhanceptent of potency could be
`attributed .
`Others have att;empted s.ome modifications of
`GLP-1 (7-37). It ha,s bee·n shown that deletion of the
`histidine residue at position: 7 gre~tly diminishes the
`activity of th$ hormon~. (Suzuki,_S., et al. (supra);
`Hendrick, G.K.; et al. Abstract: Endocrine Society
`
`j
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`•
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`~ -
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`· ,
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`...
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`'
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`•
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`ti;..,
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`..
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`MPI EXHIBIT 1042 PAGE 5
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`MPI EXHIBIT 1042 PAGE 5
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0005
`
`

`

`WO91/11457
`
`PCT/US91/00500
`
`-4-
`
`Meeting. New Orleans. LA (1988)). There have been
`conflicting reports concerning the effect of one or more
`c-terminal deletions (Suzuki, s., et al. (supra);
`Yanaihara, c., et al. Abstra:ct 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 pr~vides modified forms of the
`GLP-1 (7-34); (7-35); (7-36) or (7-37) human peptide or
`the c~terminal amidated forms thereof. The native
`peptides have the amino acid sequence:
`
`7
`15
`10
`20
`25
`H-A-E-G-T-F-T-s-o-v-s-s-Y-L-E-G-Q-A-A-
`30
`37
`K-E-F-I-A-W-L-V-K-(G)-(R)-(G)
`
`wherein (G), (R), and (G) are present or absent depending
`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 lett~r abbreviation code for amino
`acids is used.
`The analogs of the invention which show enhanced
`insulin stimulating properties have the foregoing
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`MPI EXHIBIT 1042 PAGE 6
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`MPI EXHIBIT 1042 PAGE 6
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0006
`
`

`

`W091/11457
`
`PCT/US91/00500
`
`-5-
`
`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 argini:ne at·_position 36;
`(b) substitution of an oxidation-resistant amino
`-
`'
`acid for tryptophan at positfon 31;
`(c) substitution according to at least one of:
`Y for Vat position 16;
`K for s
`at position
`18;
`D for E
`at position
`21;
`s for G
`at posi~ion
`22;
`R for Q at position 23;
`R for A at position 24; and
`Q for Kat position 26;
`(d) a substitution comprising at least one of:
`an alternativet's111all neutral amino acid for A
`at position 8; .
`an alternative acidic amino acid or neutral
`amino acid for~ 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 Dor N-acylated or alkylated form of
`histidine for histidine at position 7.
`With respect tomod.i:fications (a), (b), (d) and
`(e), the substituted amino acids 1may be in the D form, as
`ct
`' t t
`• '·
`' d'
`t d b
`in ica e ya super~crip
`, e.g.,
`T'h
`.
`'
`• e amino aci s
`'d
`substituted at position 7 can als'o be in the N-acylated
`or N-alkylated forms.
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`MPI EXHIBIT 1042 PAGE 7
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`MPI EXHIBIT 1042 PAGE 7
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0007
`
`

`

`WO 91/11457
`
`PCT /US91/00500
`
`-6-
`
`5
`
`10
`
`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-1 (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
`forms of GLP-1 (7-34) to GLP-1 (7-37) or their c-terminal
`amidated forms is modified by
`{a) substitution of a D-neutral or D-acidic amino
`acid for Hat position 7, or
`(b) substitution of a D-amino acid for A at
`positions, or
`(c) both, or
`(d) substitution of an N-acylated or N-alkylated
`form of any naturally occurring amino acid for Hat
`position 7.
`Thus, analogs of the invention which are resistant
`to degradation include (N-acyl (1-6C) AA) 7 GLP-1 (7-37)
`and (N-alkyl (1-6C) AA) 7 GLP-1 (7-37) wherein when AA is
`a lysyl residue, one or both nitrogens may be alkylated
`or acylated. AA symbolizes any amino acid consistent
`25 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
`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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`MPI EXHIBIT 1042 PAGE 8
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`MPI EXHIBIT 1042 PAGE 8
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0008
`
`

`

`W091/11457
`
`PCT /US91/00500
`
`-7-
`
`GLP-1 (7-37) but also the· shorter truncated analogs as·
`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 usi.ng these • peptides or
`compositions thereof.
`
`Brief Description of the Drawings~
`Figure 1 s•chematically outlines the classification
`of amino acids as used herein.
`Figure 2 give.s a list of various compounds of the
`invention.
`Figure 3 show~ the results ,of·radiolabel sequencing
`analysis for degradation .. df two· analogs in plasma.
`Figure 4 shows the results;of various GLP-1 (7-37)
`analogs with changes in the amino terminal region, to
`displace 125I-GLP-1 (7-39) •:. from amino terminal specific
`antiserum.
`
`j" ~,
`
`Modes of carrying Out the.Invention
`The analogs of th.e invention, which are modified
`forms of the GLP-1(7-34), • (7~,35),
`(7-3•6) or (7-37) are
`characterized by showin,g1f<.!freater 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 analogis of the invention is more
`potent than glucagon in stimulating;insulin release from
`islet cells. By: being "more_ po:t:ent than glucagon in
`stimulating insulin rele:a;$~ from -isl~t cells" is meant
`that the analog referred·. to shows greater potency in an
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`ii
`
`MPI EXHIBIT 1042 PAGE 9
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`MPI EXHIBIT 1042 PAGE 9
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0009
`
`

`

`W091/11457
`
`PCT/US91/00500
`
`-8-
`
`in vitro assay selected from the group consisting of the
`following: Rat islets for these assays are isolated by
`the method of Sutton, R. et al., Transplantation {1986)
`42:689-691, 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 cac12, 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 s g/1
`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
`tnree times in Hank's solution .arid vieweq. through a
`dissecting microscope for hand picking of islets.
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`MPI EXHIBIT 1042 PAGE 10
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`MPI EXHIBIT 1042 PAGE 10
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0010
`
`

`

`WO 91/11457
`
`PCT /US91/00500
`
`-9-
`
`s
`
`In one·approach :the ability of the GLP..;.l analog to
`potentiate secretion from these Islets is then determined
`according to the method ort $chatz, H. et al., in "Methods
`in Diabetes Researcht' .. (~~84r Volume ~, Part c: pages
`291-307, incorporate.d h~rfain by reference.
`In this
`method, 5-10 islets per test tube arE;! incubated in 1 mL
`Krebs-Ringer-bicarbpnate }:.)uffer (KRB buffer). For
`testing, glucagon or the modified analog of the invention
`is added at s-10 µ.g/mL. The level of insulin released
`10 may be measured by the .method of Jensen, S.L. et al., M J
`Physiol ( 1978) . 235: E381-:rJJ8.6, incorporated herein by
`reference.
`
`20
`
`The following protocol is a preferred method to
`15 measure stimulation of insulin secretion. After
`collagenase digestio~, ,the islets are allowed to recover
`overnight by incubation in DMEM (Dulbecco•s Modified
`Eagle Medium 16 w/o glucose), 2.8 mM glucose, 10% fetal
`bovine serum (FBSt at 37,0 c, 5% ca2,.
`The next .day, isiets to be used for the experiment
`'nq glucose, o. 2 % BSA (Armour,
`are transferred to DMEM,
`clinical grade, made at 5% stock) for a 60 min
`Islets
`preincubation in serum-free, glucose-free medium.
`are picked up by Eppendotf pipette and transferred to 60
`mm TC plates containing 8,,. o mL medium and returned to the
`incubator for 60 min.
`Islets are counted during this
`transfer.
`(Note:
`each data peimt is 5 islets,
`experiments are usually performed in quadruplicate;
`therefore, 20 islet.s are.ul:;led per d,a.ta point.)
`Typically, recoveries ar~ 150-20 .. 0 .i_$lets per pancreas.
`Any suspect islets--too r.agged or f:alling apart--are not
`used.
`
`2 5
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`30
`
`During the 60 min ;pre.incubation, the experiment is
`set up, so that all that is needed at the end of the
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`MPI EXHIBIT 1042 PAGE 11
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`MPI EXHIBIT 1042 PAGE 11
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0011
`
`

`

`!
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`~
`
`WO 91/11457
`
`PCT/US91/00500
`
`-10-
`
`preincubation is to tran·sfer 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.s 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 pM to
`100 nM). Test compound is diluted from stock stored at
`-so 0 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, -o.s 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
`using an insulin RIA.
`If medium is not immediately
`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 i~vention measures thei~ ability to stimulate cAMP
`production in RIN 1046-38 <?ells. T~is assay can be
`conducted as follows:
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`MPI EXHIBIT 1042 PAGE 12
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`MPI EXHIBIT 1042 PAGE 12
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0012
`
`

`

`I
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`'t"•
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`WO91/11457
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`PCT /US91/00500
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`-11-
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`On day 1 , 5 x 105 RI'N 1046-38 cells (Drucker, D.J.,
`et al., Proc Natl Acad Soi USA (1987) 84:3434-3438) are
`seeded into indivfaual wells of 6-well dishes with 2.5 mL
`., '
`M199 culture medium. 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.s x 106 cells ·per well.
`Assays are only performed :On cell pas·sage S24.
`At time -60 niin, mdrlblayers a~~ washed twice with
`2 . s mL PBS, and medium is.,dhanged to 1. o mL of DMEM
`,~
`'
`medium plus 4.5 g/1 glucose and 0~1%' BSA (assay medium).
`At O 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
`Oto 60 min.
`At termination, conditione·d medium and monolayer
`are harvested to measure both ext~a- and intracellular
`CAMP content. For extradie1lular measurement, medium is
`removed and centrifuged td remove any cellular debris.
`For intracellular determination, after medium removal,
`1. o mL of ice cold 95% ethanol is .. added to l'llonolayer.
`Cells are collected by scraping, flysed by two cycles of
`!\ ': '
`quick freeze/thawing using liquid· N2, a.nd cell debris
`then removed by centrifugation. • 'Aliquots (l/40th well
`content) of conditidned fuedium and ethanol cell extract
`are measured in duplicate for cAMP levels using an RIA
`kit by the acetylated protocol.
`•
`••
`As above, higher p6,tency as compared to glucagon is
`defined either as higher cAMP st~mu1ation by both the
`analog and glucagon at the same concentration, or the
`same cAMP stimulation _by th_e analog at a I lower
`concentration .
`. still other assays ~or measurement of.enhanced
`potency to mediate insulin release can be used.
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`MPI EXHIBIT 1042 PAGE 13
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`MPI EXHIBIT 1042 PAGE 13
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`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0013
`
`

`

`WO91/11457
`
`PCT/US91/00500
`
`-12-
`
`5
`
`The abi°lity of the compounds to potentiate the
`release of insulin can be tested both in vitro and in
`vivo.
`Insulin released can be detected using a standard
`antibody assay both in analyzing plasma in in vivo
`studies and in analyzing media or perfusion liquid in
`vitro.
`For example, a useful in vitro assay uses the
`-
`pancreatic infusion assay method of Penhos, J.C., et al.
`Diabetes (1969) 18:733-738, as employed in the method of
`10 Weir, G.C., et al. J Clin Investigat (1974) M:1403-1412.
`Insulin secretion can also be measured by.the method
`described by Holst, J.J~, et al. FEBS Letters (1987)
`211:169-174 (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., Pree Natl Acad Sci USA (1987)
`84:3434-3438 {supra).
`Inhibition of glucagon release can be shown as
`described by Orstov, c., et al. Endocrinol (1988)
`123:2009-2013; Suzuki, s., et al. Diabetes Research:
`Clinical Practice (1988) ~(Supp. 1):S30 (both supra).
`Assays for Enhanced stability to Degradation
`The therapeutic efficiency of the GLP-1 analogs of
`the invention can also be enhanced by providing analogs
`25 with increased half-lives in vivo. By "enhanced half(cid:173)
`life in vivo" 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 4·o C.
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`MPI EXHIBIT 1042 PAGE 14
`
`MPI EXHIBIT 1042 PAGE 14
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0014
`
`

`

`WO 91/11457
`
`PCT/US91/00500
`
`-13-
`
`,. •
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`•
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`A. Radiolabel sequencing:
`The GLP analog is lrp.bel'ed•i by radio-iodination in
`position 19 using sta.ndard radio labeling methods. After
`exchange into RIA buffer ( 50--: mM .NaHPO 4 pH 7. 4 ,· o. 25% BSA
`(Armour insulin and FFA fret;!), 0.5%. BME, 0.002%
`polylysine (Sigma 1,5,000 mw), 0.05% Tween 20, 0.1% NaN3 ),
`the radioiodinated peptid;e (about 105 cpm/50 mL) and cold
`uniodinated peptide (20 µl 100 nM) are added into 2 ml of
`plasma to a final concent~ation of ,i nM and incubated in
`a circulating water bath .for preset: time.s. Total RIA
`buffer added to plasma n~~er exceeds 5% .of total volume.
`At the end of incubation., 1.0% Qacitracin, (w/v) in water
`is added to a finc\l concentration of 0.1% to stop the
`reaction.
`The plasma is then extz;acted using C18 Sep-Pak to
`separate the analog and any fragment:-s· from the bulk of
`the plasma proteins. Sep-~ak cartridges (Waters) are
`washed with 2 mL .of 1-p:r;qpanol, followed by 2 mL of water
`and then equilibrat~d wi\p. 2 mL of 20% CH3CN containing
`o .1% trifluoroacetiq :a-c.j,.di ··. (TFA) (Buffer A) .
`The bacitraq.i11-:tre't~d. plasma is made 2 0% CH3 CN
`with cH3CN contain•j,:ng 0.1% TFA and is, expressed slowly
`through a 3 mL plastic syringe thrQugh the cartridge.
`The cartridge is then wa:$h~d with two 1 mL Buffer A
`25 washes and eluted with a· single 2 mL wash of 50% CH3CN
`containing o .1%; TFA (Bu.f~er B)
`into.,,;·a siliconized 12 x 75
`glass tube. Recov~ry of the analog or fragments is more
`than 90%.
`The eluates are concentrated to 100 .µl in a Speed
`vac and transferred to a 1.5 mt Ep~endo:r;f tube to which a
`1 mL RIA buffer rinse of the original tube had been
`added.
`To purify any analog or its fragmen.ts when the
`analogs of GLP-1 (7-37) are used, the concentrates are
`
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`MPI EXHIBIT 1042 PAGE 15
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`MPI EXHIBIT 1042 PAGE 15
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0015
`
`

`

`WO91/11457
`
`PCT/US91/00500
`
`-14-
`
`5
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`10
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`treated with 5 µl of antiserum prepared to a synthetic
`peptide corresponding to residues 24-37 which recognizes
`GLP-1, GLP-1 (7-37) but not GLP-1 (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
`µl 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
`15 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)
`261:11880-11889.
`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 µg peptide and 100 µg 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 by
`centrifugation at 2000 g for 15 minutes and the serum
`removed. Serum is stored in aliquots at -20 or -80°C.
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`MPI EXHIBIT 1042 PAGE 16
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`MPI EXHIBIT 1042 PAGE 16
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0016
`
`

`

`WO 91/11457
`
`PCT /US91/00500
`
`-15-
`
`..
`The peptides . are th.en eluted from the antibody
`protein-A sepharose complex with th:n;ee 100 µl washes of
`Buffer B. The combined 300 µl of wash are then applied
`directly to an ABI mod.el 47iA seqµencer used according to
`the manufacturer's instructions. Fractions from each
`cycle are then diverted tor counting. Counting can be
`effected in 4 mL aqueo\ls-scintillant :(ACS, Amersham).
`-
`The cycle at which label appears indicates the
`extent of degradation from the N-term.inus.
`If no
`degradation from the N-terminus has occurred in the GLP-
`1 (7-37) analog, al·l of the label will appear in the 13th
`cycle, corresponding to·the tytosine at position 19; if
`degradation has occurred, 1;'-:h.e label will appear in
`earlier cycles.
`B. Assay by RP~HPLC:
`While the foregoing· metho.d ist 1a cJ.ear criterion for
`exhibiting a lo·nger half~life in· ·plasma, alternative
`forms of the ass~y ,.fol:' . th;is property can also be used.
`In one convenient ass,ay,. the analog' can be assessed for
`2 O degradation into f_ragment.s using reverse· phase-HPLC,
`since the fragments have d:i:,fferent retention times from
`the analog per se.
`In this c:1:ssay, the analog is added to
`plasma for various times and raaov:e:i;-ed similarly to the
`method described above:- for. radi.ol,,abel: sequencing
`analysis. Specifically,, the analog· ·at a concentration of
`100 nM in RIA buff,er is spiked into 1 mL plasma to a
`final concentration of
`1 nM and ·aft.er incubation in 37°C
`circulating water bath
`for varioq,s preset times, the
`reaction is stopped by
`b+inging the plasma to 0.1% (w/v)
`in bacitracin.
`The peptides are then 'purifie:¢1: ·'by Sep-Pak
`extraction as·described.above .. The eluates are
`concentrated to about 1 mL on a Sp~:ed-vac, diluted with 1
`mL distilled water, frozen at 80°C and lyophilized
`
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`MPI EXHIBIT 1042 PAGE 17
`
`MPI EXHIBIT 1042 PAGE 17
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1042-0017
`
`

`

`WO91/11457
`
`PCT /US91/00500
`
`-16-
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`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 109OL liquid
`chromatograph using an Allte-ch ClS column (0.45 x 25 cm;
`10 µm particle size) with a Brownlee 2 cm C18 guard
`column. The extraction is monitored at 00214 throughout
`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