throbber
PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`—
`
`asin
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`(51) International Patent Classification 4 :
`(11) International Publication Number:
`WO 90/11296
`CO7K 7/10, 7/34, 7/40
`A61K 37/02, 37/24
`
`(43) International Publication Date:
`
`4 October 1990 (04.10.90)
`
`
`
`(21) International Application Number:
`
`PCT/US89/01121
`
`(22) International Filing Date:
`
`20 March 1989 (20.03.89)
`
`Published
`With international search report.
`
`(71) Applicant! THE GENERAL HOSPITAL CORPORA-
`TION [US/US]; Fruit Street, Boston, MA 92114 (US).
`
`; 217 Plymouth Road, New-
`(72) Inventor: HABENER,Joel, F.
`ton Highlands, MA 02161 (US).
`
`(74) Agents: FOX, Samuel, L.et al.; Saidman,Sterne, Kessler &
`Goldstein, 1225 Connecticut Avenue, N.W., Suite 300,
`Washington, DC 20036 (US).
`
`(81) Designated States: AT (European patent), BE (European
`+ patent), CH (European patent), DE (European patent),
`FR (European patent), GB (European patent), IT (Euro-
`pean patent), JP, LU (European patent), NL (European
`patent), SE (European patent).
`
`(54) Title: INSULINOTROPIC HORMONE
`
`(57) Abstract
`
`Derivatives of glucagon-like peptide I (GLP-1) have been found to have insulinotropic activity. The invention pertains to
`such derivatives, and to the use of such derivatives as a potential therapy for Diabetes Mellitus.
`
`+ See back of page
`
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`

`

`DESIGNATIONS OF “DE”
`
`Until further notice, any designation of “DE” in any international application
`whose internationalfiling date is prior to October 3, 1990, shall have effect in the
`territory of the Federal Republic of Germany with the exception of the territory of the
`former German Democratic Republic.
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`
`
`RERESSSIRQOZIB
`
`Spain
`Fintand
`France
`Gabon
`United Kingdom
`Hungary
`Italy
`Japan
`Democratic People’s Republic
`of Korea
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`Liechtenstein
`Sri Lanka
`Luemboug
`Monaco
`
`.
`
`RASSUMBRSASEES
`
`Madagascar
`Mali
`Mauritania
`Mabwi
`Netherlands
`Norway
`Romania
`Sudan
`Sweden
`Senegal
`Soviet Union
`Chad
`Togo
`United States of America
`
`Dx
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Canada
`Central African Republic
`Congo
`Swiceriand
`Cameroon
`Genmany, Federal Republic of
`Denmark
`
`BB
`RE
`BF
`BG
`BI
`BR
`CA
`cr
`CG
`aa
`
`™D
`
`E
`
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`

`

`WO 90/11296
`
`PCT/US89/01121
`
`-l-
`
`
`
`ANSULINOTROPTCHORMONE
`
`Cross-Reference to Related Applications
`
`This application is a continuation-in-part of United
`States Patent Application Serial No. 859,928, filed on May
`5, 1987.
`
`BACKGROUND OF THE INVENTION
`
`Field of the Invention
`
`invention is directed to the discovery that
`This
`certain peptide fragments of the prehormone, proglucagon,
`possess hormonal activities and can be used to stimulate
`the
`synthesis
`and
`secretion of
`the hormone,
`insulin.
`These peptide fragments are useful
`in therapy for
`the
`disease Diabetes mellitus.
`
`Description of the Background Art
`
`The endocrine secretions of the pancreatic islets are
`under complex control not only by blood-borne metabolites
`(glucose,
`amino acids, catecholamines, etc.), but also by
`
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`

`

`WO 90/112965
`
`a
`
`- .
`
`PCE/US89/01121
`
`-2-
`
`The major pancreatic islet
`local paracrine influences.
`and somatostatin)
`interact
`hormones
`(glucagon,
`insulin,
`types
`(A, B,
`and D cells,
`among
`their specific cell
`respectively) to modulate secretory responses mediated by
`the metabolites. Although insulin secretion is predomi-
`nantly controlled by blood levels of glucose, glucagon and
`somatostatin
`stimulate
`and
`inhibit
`glucose-mediated
`insulin secretory responses, respectively.
`In addition to
`the proposed interislet paracrine regulation of
`insulin
`secretion,
`there is evidence to support
`the existence of
`insulinotropic factors
`in the intestine.
`This concept
`originates from the observations that glucose taken orally
`is a much more potent stimulant of insulin secretion than
`is a comparable amount of glucose given intravenously.
`acid
`The
`human
`hormone, glucagon,
`is
`a 29-amino
`peptide hormone produced in the A-cells of the pancreas.
`The hormone belongs to a multi-gene family of structurally
`related peptides that include secretin, gastric inhibitory
`peptide, vasoactive
`intestinal peptide,
`and glicentin.
`These peptides variously regulate carbohydrate metabolism,
`gastrointestinal mobility, and secretory processing.
`The
`principal
`recognized
`actions
`of pancreatic
`glucagon,
`however,
`are
`to promote glycogenolysis
`and gluconeo-
`genesis, resulting in an elevation of blood sugar levels.
`In
`this
`regard,
`the actions of glucagon are counter-
`regulatory to those of insulin and may contribute to the
`hyperglycemia that
`accompanies Diabetes mellitus
`(Lund,
`
`P.K., et al., Proc. Natl. Acad. Sci.,
`USA 79:345-349
`(1982)).
`Glucagon has been found to be capable of binding to
`specific receptors which lie on the surface of insulin-
`producing cells. Glucagon, when bound to these receptors,
`Stimulates the rapid synthesis of cAMP,
`by these cells.
`
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`

`WO 90/11296
`
`PCT/US89/01121
`
`-3-
`
`cAMP,
`
`in
`
`turn,
`
`has
`
`been
`
`to stimulate
`
`insulin
`
`found
`
`et al., Diabetes 34:717-722
`(Korman, L.Y.,
`expression
`to inhibit glucagon synthesis
`Insulin acts
`(1985)).
`(Review of Medical Physiology, Ganong, W.F., 1979, Lang
`Publications, Los Altos, California (p. 273)). Thus,
`the
`expression of glucagon is carefully regulated by insulin,
`and ultimately by the serum glucose level.
`The glucagon gene is initially translated from a 630-
`base pair precursor to form the polypeptide, preprogly-
`
`cagon (Lund et al.
`(1982)).
`This polypeptide is subse-
`quently processed to form proglucagon.
`Patzelt, C., et
`
`al.
`(Nature 282:260-266 (1979)) demonstrated that pro-
`glucagon was
`subsequently cleaved into glucagon and
`a
`second polypeptide.
`Subsequent work by Lund, P.K., etal.
`(Proc. Natl. Acad. Sci. USA 79:345-349 (1982)); Lopez,
`
`L.C., et al.
`(Proc. Nati, Acad. Sci. USA 80:5485-5489
`
`(1983)) and Bell, G.I., et.al.
`(Nature 302:716-718 (1983))
`demonstrated that
`the proglucagon molecule: was cleaved
`immediately
`after
`lysine-arginine dipeptide
`residues.
`Studies
`of
`proglucagon
`produced
`by
`channel
`catfish
`(Ictalurus punctata)
`indicated that glucagon from this
`animal was also proteolytically cleaved after adjacent
`lysine-arginine and arginine-arginine dipeptide residues
`
`(Andrews, P.C.,
`et al.,
`J. Biol. Chem.
`260:3910-3914
`
`(1985)).
`Lopez, L.C., et al.
`(Proc. Natl. Acad. Sci. USA
`
`80:5485-5489 (1983)},
`and Bell, G.1., et _al., discovered
`the mammalian proglucagon was cleaved at
`lysine-arginine
`or arginine-arginine dipeptides and demonstrated that the
`proglucagon molecule contained three discrete and highly
`homologous
`peptide molecules which were
`designated
`glucagon, glucagon-like protein 1
`(GLP-1),
`and glucagon-
`
`like protein 2
`(GLP-2).
`Lopez et al.
`concluded that
`glucagon-like protein 1 was 37 amino acid residues long
`
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`WO:90/11296. 22
`
`PCT/US89/01121
`
`-4-
`
`acid
`amino
`34
`2 was
`that glucagon-like peptide
`and
`residues jong. Analogous studies on the structure of rat
`preproglucagon revealed a similar pattern of proteolytic
`cleavage between adjacent
`lysine-arginine or arginine-
`arginine dipeptide residues, resulting in the formation of
`
`glucagon, GLP-1,
`and GLP-2
`(Heinrich, G., et al., Endo-
`
`crinol. 115:2176-2181 (1984)).
`Human rat, bovine,
`and
`hamster sequences of GLP-1 have been found to be identical
`
`(Ghiglione, M., et al., Diabetologia 27:599-600 (1984)).
`
`The conclusion reached by Lopez et al.
`(Proc. Natl.
`Acad. Sci. USA 80:5485-5489 (1983)) regarding the size of
`GLP-1 was confirmed by the work of Uttenthal, L.0., et
`al.,
`(Jd. Clin. Endocrinol. Metabol. 61:472-479 (1985)).
`
`Uttenthal et al.
`examined the molecular
`forms of GLP-1
`
`in the human pancreas. Their research
`which were present
`shows that GLP-1 and GLP-2 are present
`in the pancreas as
`proteins having 37 and 34 amino acid residues,
`respec-
`tively.
`The similarity between GLP-1 and glucagon suggested
`to early investigators that GLP-1 might have biological
`activity.
`Although some
`investigators found that GLP-1
`could induce rat brain cells to synthesize cAMP (Hoosein,
`N.M., et al., FEBS Lett. 178:83-86 (1984)), other inves-
`tigators failed to identify any physiological
`role for
`GLP-1
`(Lopez, L.C., et al., Proc. Natl. Acad. Sci. USA
`80:5485-5489
`(1983)).
`The
`failure
`to identify any
`physiological
`role for GLP-1 caused some investigators to
`question whether GLP-] was
`in fact a hormone and whether
`the
`relatedness
`between
`glucagon
`and GLP-1 might
`be
`artifactual
`(Ghiglione, M., et al., Diabetologia 27:599-
`600 (1984)).
`an
`reveals
`the prior art
`conclusion,
`in
`Thus,
`awareness of
`the processing of a glucagon hormone pre-
`
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`WO 90/11296
`
`PCT/US89/01121
`
`-5-
`
`cursor into a set of peptides sharing extensive homology.
`It has been widely assumed by those of skill
`in the art
`that
`these highly related glucagon-like peptides would
`have
`a biological
`activity.
`Nevertheless,
`extensive
`investigations
`designed
`to
`elucidate
`the
`biological
`effects of these molecules had been unsuccessful.
`
`SUMMARY OF THE INVENTION
`
`invention relates to an insulinotropic
`The present
`hormone
`comprising GLP-1
`and
`its derivatives.
`The
`invention additionally pertains to the therapeutic uses of
`such compounds.
`:
`The invention comprises a compound selected from the
`group consisting of:
`(A)
`a peptide comprising the sequence:
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-
`Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gin-Ala-
`Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-
`Lys
`
`°F His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-
`Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gin-Ala-
`Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-
`Lys-Gly
`
`and
`
`a derivative of the peptide;
`(B)
`wherein the compound is substantially free of
`natural contaminants,
`and has an insulinotropic activity
`which exceeds the insulinotropic activity of GLP-1 (1-36)
`or GLP-1 (1-37).
`the above-
`The
`invention also comprises either of
`described compounds wherein the compound is substantially
`free of natural contaminants,
`and has
`an insulinotropic
`activity at a concentration of at least 19°10 M,
`
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`
`
`
`
`WO 90/11296 .~
`
`PCT/US89/01121
`
`-6-
`
`the above
`invention also comprises either of
`The
`compounds wherein the derivative (B)
`is selected from the
`group consisting of:
`(a)
`pharmaceutically acceptable acid addition salts
`of the peptide;
`(b) pharmaceutically acceptable carboxylate salts of
`the peptide;
`(c) pharmaceutically acceptable lower alkyl] esters
`of the peptide; and
`amides,
`acceptable
`(d) pharmaceutically
`amides, or dialkyl! amides of (a),
`(b), or (c).
`The invention also pertains to a compound having the
`formula:
`
`alkyl
`
`(1) HeN--X--CO-R!
`wherein: R!
`is OH, OM or -NR@R?;
`M is pharmaceutically acceptable cation or
`a lower branched or unbranched alkyl] group;
`R2
`and R? are the same or different
`
`and
`
`selected from the group consisting of hydrogen and a lower
`branched or unbranched alkyl group;
`X is a peptide comprising the sequence:
`
`I.
`
` His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-
`Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
`Ala-Lys-Glu-Phe-I1le-Ala-Trp-Leu-Val -
`Lys
`
`or
`
`II. His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-
`Val -Ser-Ser-Tyr-Leu-Glu-Gly-Gin-Ala-
`Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-
`Lys-Gly
`
`NH2
`terminus of X; and
`
`is
`
`the
`
`amine
`
`group
`
`of
`
`the
`
`amino
`
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`WO 90/11296
`
`PCT/US89/01121
`
`-7-
`
`CO is the carbonyl] group of
`
`the carboxy
`
`terminus of X;
`the acid addition salts thereof; and
`(2)
`the
`protected
`or
`partially
`protected
`(3)
`derivatives thereof;
`insulinotropic
`an
`has
`compound
`wherein
`the
`activity which exceeds the insulintropic activity of GLP-1
`(1-36) or GLP-1 (1-37).
`insulinotropic
`an
`comprises
`The
`invention
`also
`and effective amount of
`the
`medicament which comprises
`above compounds,
`in combination with a suitable physiolog-
`ically acceptable carrier.
`The invention also comprises a method for enhancing
`the expression of insulin which comprises providing to a
`mammalian pancreatic B-type islet cell an effective amount
`of any of the above-described insulinotropic peptides.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`the DNA structure and corresponding
`shows
`Figure J]
`amino acid sequence of human,
`rat,
`and hamster prepro-
`glucagons.
`The preproglucagon precursor
`is proteolyti-
`cally cleaved at sites indicated by circles.
`insulinotropic
`Figure
`2
`shows
`the ability of
`the
`peptides glucagon
`and GLP-1
`(7-37)
`to stimulate cAMP
`formation in the insulinoma line, RIN 1046-38.
`Figure 3
`shows
`a comparison of the insulinotropic
`activity of glucagon with that of GLP-] (7-37).
`Figure 4
`shows
`a comparison of
`the insulinotropic
`activities of GLP-1 (7-34), GLP-] (7-35), and GLP-1 (7-37)
`using the rat pancreas perfusion technique.
`Figure 5
`shows
`the breakdown of GLP-1
`GLP-1 (7-37) under experimental conditions.
`
`(1-37)
`
`into
`
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`“WOSO/LID9EOe of
`
`- peryusso/e1121
`
`DESCRIPTION OF THE PREFERRED EMBODIMENTSee.
`
`A.
`
`GLP-1 and Its Derivatives
`
`The hormone glucagon is known to be synthesized as a
`high molecular weight precursor molecule which is subse-
`quently proteolytically cleaved
`into three peptides:
`glucagon, glucagon-like peptide 1
`(GLP-1),
`and glucagon-
`like peptide 2 (GLP-2).
`GLP-1 has 37 amino acids in its
`unprocessed form.
`The present
`invention discloses that
`the unprocessed GLP-]
`is essentially unable to mediate the
`induction of insulin biosynthesis.
`The unprocessed GLP-1
`peptide is,
`however, naturally converted to a 31-amino
`acid long peptide (7-37 peptide) having amino acids 7-37
`of GLP-1 ("GLP-1 (7-37)"}. This processing occurs in the
`pancreas and the intestine.
`The 7-37 peptide which has
`not
`been previosly described
`is
`a
`hormone
`that
`has
`insulinotropic activity.
`A compound is said to have an
`"insulinotropic activity" if it is able to stimulate, or
`cause the stimulation of,
`the synthesis or expression of
`the hormone insulin.
`The hormonal activity of GLP-1
`(7-
`37) appears to be specific for the pancreatic beta cells
`where it appears to induce the biosynthesis of
`insulin.
`The insulinotropic hormone is useful
`in the study of the
`pathogenesis
`of maturity onset diabetes mellitus,
`a
`condition in which the dynamics of insulin secretion are
`abnormal. Moreover,
`the insulinotropic hormone is useful
`in therapy for this disease.
`Peptide moieties
`(fragments) chosen from the deter-
`mined amino acid sequence of human GLP-1 constitute the
`starting point
`in the development comprising the present
`invention.
`The interchangeable terms "peptide fragment" —
`
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`

`WO 90/11296
`
`PCT/US89/01121
`
`-9-
`
`to include both synthetic
`and "peptide moiety" are meant
`and naturally occurring amino acid sequences derivable
`from a naturally occurring amino acid sequence.
`The amino acid sequence for GLP-1 has been reported
`
`by several
`researchers (Lopez, L.C., et al., Proc. Natl.
`Acad. Sci., USA 80:5485-5489 (1983); Bell, G.I., etal.,
`Nature 302:716-718
`(1983); Heinrich, G., et al., En-
`docrinol. 115:2176-2181
`(1984); Ghiglione, M., et al.,
`Diabetologia 27:599-600 (1984)).
`The structure of
`the
`preprogiucagon gene
`and
`its
`corresponding
`amino
`acid
`sequence
`is
`shown
`in Figure
`1.
`This
`figure further
`displays the proteolytic processing of the precursor gene
`product
`into glucagon and the two glucagon-like peptides.
`As used herein,
`the notation of GLP-1 (1-37) refers to a
`GLP-1 polypeptide
`having all
`amino acids from 1
`(N-
`terminus)
`through 37 (C-terminus).
`Similarly, GLP-1
`(7-
`37)
`refers to a GLP-] polypeptide having all amino acids
`from 7 (N-terminus) through 37 (C-terminus).
`-
`its peptide
`In
`one
`embodiment, GLP-1
`(7-37)
`and
`fragments are synthesized by conventional means,
`such as
`by the well-known solid-phase peptide synthesis described
`by Merrifield,
`J.M.
`(Chem.
`Soc.
`85:2149
`(1962)),
`and
`Stewart and Young (Solid Phase Peptide Synthesis (Freeman,
`San Francisco, 1969), pages 27-66), which are incorporated
`by
`reference herein.
`However,
`it
`is also possible to
`obtain fragments of
`the proglucagon polypeptide, or of
`GLP-1, by fragmenting the naturally occurring amino acid
`sequence,
`using,
`for
`example,
`a proteolytic
`enzyme.
`Further, it is possible to obtain the desired fragments of
`the proglucagon peptide or of GLP-1
`through the use of
`recombinant DNA technology, as disclosed by Maniatis, T.,
`et_al., Molecular Biology:
`A Laboratory Manual, Cold
`
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`

`WO 90/112960";
`
`_
`
`ors
`
`_.
`
` PCT/US89/01121
`
`-10-
`
`Spring Harbor, New York (1982), which is hereby incor-
`porated by reference.
`The present
`invention includes peptides which are
`derivable from GLP-1
`(1-37).
`A peptide is said to be
`"derivable from a naturally occurring amino acid sequence"
`if it can be obtained by fragmenting a naturally occurring
`sequence,
`or
`if
`it
`can
`be
`synthesized based upon
`a
`knowledge of the sequence of the naturally occurring amino
`acid sequence or of
`the genetic material
`(DNA or RNA)
`which encodes this sequence.
`invention
`Included within the scope of the present
`are those molecules which are said to be "derivatives" of
`GLP-1
`(1-37).
`Such
`a "derivative" has
`the following
`characteristics:
`(1)
`it shares substantial homology with
`GLP-1
`(1-37) or
`a similarly sized fragment of GLP-1
`(1-
`37);
`(2) it is capable of functioning as an insulinotropic
`hormone and (3) using at least one of the assays provided
`herein,
`the derivative has either
`(i)
`an insulinotropic
`activity which
`exceeds
`the
`insulinotropic activity of
`either GLP-1 (1-37) or GLP-1 (1-36), or, more preferably,
`(ii) an insulinotropic activity which can be detected even
`when the derivative is present at a concentration of 19°10
`M, or, most preferably, (iii) an insulinotropic activity
`which can
`be detected
`even when
`the derivative is
`present at a concentration of 107
`said to share
`is
`A derivative of GLP-1
`(1-37)
`"substantial homology" with GLP-1 (1-37) if the amino acid
`sequences of
`the derivative is at
`least 80%,
`and more
`preferably at least 90%, and most preferably at least 95%,
`the same as that of either GLP-1
`(1-37) or a fragment of
`GLP-1 (1-37) having the same number of amino acid residues
`as the derivative.
`
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`

`WO 90/11296
`
`PCT/US89/01121
`
`-]I-
`
`The derivatives of the present invention include GLP-
`(1-37)
`fragments which,
`in addition to containing a
`1
`sequence that
`is substantially homologous
`to that of
`a
`naturally occurring GLP-1
`(1-37) peptide may contain one
`or more additional amino acids at their amino and/or their
`carboxy termini. Thus,
`the invention pertains to polypep-
`tide fragments of GLP-1
`(1-37)
`that may contain one or
`more amino acids that may not be present
`in a naturally
`occurring
`GLP-1
`(1-37)
`sequence
`provided
`that
`such
`polypeptides have an insulinotropic activity which exceeds
`that of GLP-1 (1-37) or GLP-1 (1-36).
`(1-37)
`GLP-1
`Similarly,
`the
`invention
`includes
`fragments which,
`although containing a sequence that
`is
`substantially homologous to that of a naturally occurring
`GLP-1 (1-37) peptide may lack one or more additional amino
`acids at their amino and/or their carboxy termini
`that are
`naturally found on
`a GLP-1
`(1-37) peptide.
`. Thus,
`the
`invention pertains to polypeptide fragments of GLP-1
`(1-
`37)
`that may
`lack one or more
`amino
`acids
`that
`are
`normally present
`in a naturally occurring GLP-1
`(1-37)
`sequence provided that such polypeptides have an insulino-
`tropic activity which exceeds that of GLP-1 (1-37) or GLP-
`1 (1-36).
`The invention also encompasses the obvious or trivial
`variants of
`the
`above-described
`fragments which
`have
`inconsequential
`amino acid substitutions (and thus have
`amino acid sequences which differ from that of the natural
`sequence) provided that
`such variants have an insulino-
`tropic activity which is substantially identical
`to that
`of
`the above-described GLP-1 derivatives.
`Examples of
`obvious or trivial substitutions include the substitution
`of one basic residue for another (i.e. Arg for Lys),
`the
`substitution of one hydrophobic residue for another (i.e.
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1040, p. 13 of 53
`
`

`

`WO 90/1129605 Dt
`
`-
`
`PCT/US89/01121 |
`
`-]2-
`
`Leu for Ile), or the substitution of one aromatic residue
`for another (i.e. Phe for Tyr), etc.
`include GLP-1
`Examples of derivatives of GLP-1 (1-37)
`(7-37); GLP-1 (7-36); GLP-1 (7-35); GLP-1 (7-34); and the
`des-Gly amidated forms of these molecules.
`Included as
`well are the use of additional amino acid residues added
`
`to such sequences in order to enhance coupling to carrier
`protein or amino acid residues added to enhance the in-
`sulinotropic effect.
`the amino acid residues may
`As
`is known in the art,
`be in their protected or unprotected form, using appropri-
`ate amino or carboxy] protecting groups. Useful cations
`are alkali or alkaline earth metallic cations (i.e., Na,
`K, Li,
`1/2Ca,
`1/2Ba,
`etc.)
`or
`amine
`cations
`(i.e.,
`tetraalkylammonium,
`trialkylammonium, where alkyl
`can be
`Cy-Cy9).
`The variable length peptides may be in the form of
`the free amines
`(on the N-terminus), or acid-addition
`salts thereof.
`Common acid addition salts are hydrohalic
`acid salts, i.e., HBr, HI, or, more preferably, HCI.
`
`B.
`
`Assays of Insulinotropic Activity
`
`(1-37) deriva-
`invention concerns GLP-1
`The present
`tives which have an insulinotropic activity that exceeds
`the insulinotropic activity of either GLP-1 (1-37) or GLP-
`1
`(1-36).
`The insulinotropic property of a compound may
`be determined by providing that compound to animal cells,
`or injecting that compound into animals and monitoring the
`release of immunoreactive insulin (IRI)
`into the media or
`circulatory system of
`the
`animal,
`respectively.
`The
`presence of
`IRI
`is detected through the use of a radio-
`immunoassay which
`can
`specifically
`detect
`insulin.
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1040, p. 14 of 53
`
`

`

`WO 90/11296
`
`PCT/US89/01121
`
`-13-
`
`Although any radioimmunoassay capable of detecting the
`presence of IRI may be employed, it is preferable to use a
`modification of
`the assay method of Albano,
`J.D.M., et
`al.,
`(Acta Endocrinol.
`70:487-509
`(1972)).
`In
`this
`modification,
`a phosphate/albumin buffer with a pH of 7.4
`was
`employed.
`The
`incubation was prepared with the
`consecutive condition of 500 ul of phosphate buffer, 50 y1
`of perfusate sample or rat insulin standard in perfusate,
`100 wl of anti-insulin antiserum (Wellcome Laboratories;
`1:40,000 dilution), and 100 1 of [2251]
`insulin, giving a
`total volume of 750 ul
`in a 10 x 75-mm disposable glass
`tube. After incubation for 2-3 days at 4°C, free insulin
`was
`separated from antibody-bound
`insulin by charcoal
`Separation.
`The assay sensitivity was 1-2 wU/ml.
`In
`order to measure the release of IRI
`into the cel] culture
`
`medium of cells grown in tissue culture, one preferably
`incorporates radioactive label
`into proinsulin. Although
`any radioactive label capable of
`labeling a polypeptide
`can be used,
`it is preferable to use 3H leucine in order
`to obtain labeling proinsulin.
`Labeling can be done for
`any period of time sufficient to permit the formation of a
`detectably labeled pool of proinsulin molecules; however,
`it
`is preferable to incubate cells in the presence of
`radioactive label
`for a 60-minute time period. Although
`any cell
`line capable of expressing insulin can be used
`for determining whether
`a compound has
`an insulinotropic
`effect,
`it is preferable to use rat insulinoma cells, and
`especially RIN-38 rat insulinoma cells.
`Such cells can be
`
`grown in any suitable medium; however, it is preferable to
`use DME medium containing 0.1% BSA and 25 mM glucose.
`The insulinotropic property of a compound may also be
`determined by pancreatic infusion.
`The in situ isolated
`perfused rat pancreas preparation was
`a modification of
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1040, p. 15 of 53
`
`

`

`WO 90/11296...° °°
`
`a
`
`-14-
`
`PCT/US89/01121
`
`
`(Diabetes 18:733-738
`the method of Penhos, J.C., et al.
`(1969)).
`In accordance with such a method,
`fasted rats
`(preferably male Charles River
`strain albino
`rats),
`weighing
`350-600 g,
`are
`anesthetized with an
`intra-
`peritoneal
`injection of Amytal Sodium (Eli Lilly and Co.,
`160 ng/kg).
`Renal, adrenal, gastric,
`and lower colonic
`blood vessels
`are ligated.
`The entire intestine is
`four
`resected except
`for about
`cm of duodenum and the
`descending colon and rectum. Therefore, only a small part
`of
`the intestine is perfused,
`thus minimizing possible
`interference by enteric substances with glucagon-like
`immunoreactivity.
`The perfusate is preferably a modified
`Krebs-Ringer bicarbonate buffer with 4% dextran 170 and
`0.2% bovine serum albumin (fraction V), and is preferably
`bubbled with 95% O>
`and 5% COp.
`A nonpulsatile flow,
`four-channel
`roller-bearing
`pump
`(Buchler polystatic,
`Buchler
`Instruments Division, Nuclear-Chicago Corp.)
`is
`preferably used, and a switch from one perfusate source to
`another is preferably accomplished by switching a three-
`way stopcock.
`The manner in which perfusion is performed,
`modified,
`and analyzed preferably follows the methods of
`Weir, G.C., et al.,
`(J. Clin.
`Investigat. 54:1403-1412
`(1974)), which are hereby incorporated by reference.
`
`C.
`
`Formulations of Insulinotropic Compounds
`
`The insulinotropic peptides (or peptide derivatives)
`of GLP-1
`(1-37) may be used as therapeutic compositions.
`Such therapeutic compositions may consist solely of the
`insulinotropic peptides (or peptide derivatives) although,
`preferably,
`the compositions will contain the insulino-
`tropic peptides
`(or derivatives
`thereof)
`combined
`in
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1040, p. 16 of 53
`
`

`

`WO 90/11296
`
`PCT/US89/01121
`
`-15-
`
`admixture with
`vehicle,
`
`a
`
`pharmaceutically
`
`acceptable
`
`carrier
`
`inclusive of
`Suitable vehicles and their formulation,
`human proteins, e.g.,
`human
`serum albumin,
`are
`other
`described
`for
`example
`in Remington’s
`Pharmaceutical
`Sciences (16th Ed., A. Oslo Ed. Mack, Easton, PA (1980)}).
`In order to form a pharmaceutically acceptable composition
`Suitable for effective administration,
`such compositions
`will contain an effective amount of GLP-1
`(7-37), or a
`derivative of GLP-1
`(7-37),
`together with a
`suitable
`amount of carrier vehicle.
`The
`GLP-1
`derivatives
`of
`such compounds will preferably have been purified so as to
`be substantially free of natural contaminants.
`A
`material
`is
`said to be “substantially free of natural
`contaminants" if it has been substantially purified from
`materials with which it is normally and naturally found.
`Examples of natural contaminants with which GLP-1
`(7-37)
`might be associated are:
`other peptides, carbohydrates,
`glycosylated peptides,
`lipids, membranes, etc.
`A material
`is also said to be substantially free of natural contami-
`nants if these contaminants are substantially absent
`from
`a sample of the material.
`(7-37) or its deriva-
`Compositions containing GLP-1
`tives may be administered intravenously,
`intramuscularly,
`or subcutaneously at dosages in the range of from about
`1]
`pg/kg to 1,000 pg/kg body weight, or at concentrations
`sufficient to produce serum levels of 102° m to 1072! M,
`although a
`lower or higher dosage may be administered.
`The required dosage will depend upon the severity of the
`condition of the patient and upon such criteria as
`the
`patient’s height, weight, sex, age, and medical history.
`For
`the
`purpose
`of
`parenteral
`administration,
`compositions containing the derivatives of GLP-1
`(1-37)
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1040, p. 17 of 53
`
`

`

`Tea
`Ego,
`a
`
`WO 90/11296
`
`PCT/US89/01121...
`
`-16-
`
`are preferably dissolved in distilled water and the pH-
`value is preferably adjusted to about 6 to 8.
`In order to
`facilitate the
`lyophilization process
`resulting in
`a
`suitable product,
`lactose may be added to the solution.
`Preferably,
`the solution is
`then filtered sterilized,
`introduced into vials, and lyophilized.
`The concentration
`of the GLP-1 (1-37) derivatives in these compositions may
`vary from 107)2H to 107M.
`Additional pharmaceutical methods may be employed to
`control
`the duration of action.
`Controlled release
`
`preparations may be achieved by the use of polymers to
`complex or
`adsorb the GLP-1
`(1-37) derivatives.
`The
`controlled delivery may be exercised by selecting appro-
`priate macromolecules (for example, polyesters, polyamino
`acids,
`polyvinyl
`pyrrolidone,
`ethylenevinylacetate,
`methylcellulose,
`carboxymethyicellulose,
`and
`protamine
`sulfate) and the concentration of macromolecules as well
`as
`the methods of
`incorporation in order.
`to control
`release. Another possible method to control
`the duration
`of
`action
`by
`controlled release preparations
`is
`to
`incorporate the derivatives of GLP-1 (1-37)
`into particles
`of
`a polymeric material
`such as polyesters,
`polyamino
`acids, hydrogels, poly (lactic acid) or ethylene vinyl-
`acetate copolymers.
`Alternatively,
`instead of
`incor-
`porating the GLP-1 (1-37) derivatives into these polymeric
`particles,
`it is possible to entrap these derivatives in
`microcapsules
`prepared,
`for
`example,
`by
`coacervation
`techniques or by interfacial polymerization, for example,
`hydroxymethylcellulose or gelatin-microcapsules and poly
`(methylmethacrylate) microcapsules,
`respectively, or
`in
`colloidal drug delivery systems,
`for example,
`liposomes,
`albumin microspheres, microemulsions, nanoparticles,
`and
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1040, p. 18 of 53
`
`

`

`WO 90/11296
`
`PCT/US89/01121
`
`“17-
`
`Such teachings are
`in macroemulsions.
`nanocapsules or
`disclosed in Remington’s Pharmaceutical Sciences (1980).
`It is possible to enhance the biological half-life of
`the GLP-1
`(1-37) derivatives of
`the present
`invention,
`and,
`thus,
`to increase the retention or Stability of the
`derivatives in a recipient, by bonding such derivatives to
`one or more
`chemical
`"moieties"
`to thereby produce
`a
`compound which can be recognized and processed within a
`recipient
`to yield a GLP-1
`(1-37) derivative.
`The
`‘mojeties" of
`such compounds may
`include one or more
`lipids,
`carbohydrates,
`amino
`acid residues,
`etc.
`A
`preferred “moiety”
`is an amino acid residue.
`The most
`preferred "moiety"
`is
`a peptide.
`The
`amino
`terminal
`(histidine)
`residue of GLP-]
`(7-37)
`is a preferred site
`for the bonding of the "moiety".
`the present
`aspect of
`An
`appreciation
`of
`this
`invention can be obtained through a consideration of the
`natural processing of GLP-!
`(1-37).
`GLP-1 (1-37) has
`a
`biological half-life of 30-50 minutes.
`A natural cleavage
`of the amino terminal hexapeptide, GLP-1 (1-6), occurs to
`yield GLP-1 (7-37) whose biological half-life is only 3-5
`minutes.
`Thus,
`the amino terminal hexapeptide, GLP-1 (1-
`6)
`is a natural "moiety" which when bonded to GLP-1 (7-37)
`increases the biological half-life of GLP-] (7-37).=The
`discovery of
`such
`a natural
`"moiety"
`is disclosed in
`Figure 5,
`and supports
`the concept
`that additional or
`alternative moieties may be employed in the same manner as
`GLP-1
`(1-6)
`to increase the biological half-life of the
`GLP-1
`(1-37)
`derivatives
`of
`the Present
`invention.
`Although the present
`invention does not encompass the use
`of GLP-]
`(1-6) as a "moiety," it does include variants of
`GLP-1
`(1-6)
`as well
`as other peptides of unrelated
`Sequence which are capable of enhancing the half-life of
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1040, p. 19 of 53
`
`

`

`WOSO/MISE™:
`
`~~ °°
`
`- .
`
`os
`
`oe
`
`-
`
`- PCT/US89/01121
`
`-18-
`
`the peptides
`invention.
`
`and peptide derivatives of
`
`the present
`
`insulin secretion from the f-cell of the
`In summary,
`endocrine pancreas is controlled by a complex network of
`metabolic factors.
`This network includes such diverse
`
`and
`amino acids, catecholamines,
`components as glucose,
`peptides.
`The decoding of the glucagon gene has uncovered
`two additional glucagon-like peptides
`encoded in pro-
`glucagon,
`the polypeptide precursor of glucagon.
`One of
`these
`peptides,
`glucagon-like
`peptide-]
`(GLP-1)
`is
`processed from proglucagon in two forms:
`37-amino acids
`GLP-1 (1-37) and 3l-amino acid GLP-1 (7-37).
`The specific
`liberation of GLP-1 peptide’s in the intestine and,
`to
`some degree,
`in the pancreas, suggested to the inventors
`that
`the GLP-1 peptide’s might
`be components of
`the
`entero-insular axis.
`To resolve this-issue,
`the effects
`of the GL

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