PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51}1ntemational Patent Classification 4 =
`(11) International Publication Number:
`W0 90/11296
`21% 3;}3’27ggfi’ZZ/40
`(43) Intematioual Publication Date:
`4 October 1990 (04.10.90)
`
`’
`
`(21) International Application Number:
`
`PCT/USS9/01 121
`
`(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, NW, 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-Iike peptide I (GLP-l) 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.
`
`+ S“ ”a“ “Page
`
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`MYLAN INST. EXHIBIT 1040 PAGE 1
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`

`

`DESIGNATIONS OF “DE”
`
`Until further notice, any designation of “DE” in any international application
`whose international filing 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
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`§55==
`aaaaaaasseggegfl
`
`AT
`AU
`BB
`BE
`BF
`36
`BI
`BR
`CA
`CF
`CG
`CH
`CM
`DE
`DK
`
`Swherhnd
`Cameroon
`Germany. Federal Repubfic of
`Denmark
`
`eaaaeeaaa
`
`Splin
`thnd
`France
`Gabon
`Linked Kingdom
`Hungary
`Italy
`Japan
`Democratic People's Republic
`of Korea
`Republic of Korea
`Liednennein
`Sri Lanka
`Lunmbourg
`Monaco
`
`.
`
`Madagascar
`Mali
`Mauritania
`Mahwi
`Netherlands
`Norway
`Romania
`Sudan
`Sweden
`Senegal
`Soviet Union
`Chad
`Togo
`Un'ted States of America
`
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`W0 90/ 1 1296
`
`PCT/USS9/01121
`
`INSULINOTROPIC HORMONE
`
`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
`
`This
`
`invention is directed to the discovery that
`
`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
`
`MYLAN INST. EXHIBIT 1040 PAGE 3
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`

`w0'9o/1129e-5...
`
`.
`
`'
`
`,
`
`'
`
`,
`
`'
`
`7
`
`PCP/USS9/01121 '
`
`-2-
`
`local paracrine influences.
`
`The major pancreatic islet
`
`hormones
`
`(glucagon,
`
`insulin,
`
`and Somatostatin)
`
`interact
`
`among
`
`their
`
`specific cell
`
`types
`
`(A, B,
`
`and D cells,
`
`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.
`
`The
`
`human
`
`hormone,
`
`glucagon,
`
`is
`
`a
`
`29—amino
`
`acid
`
`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 1g2345-349
`
`(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.
`
`MYLAN INST. EXHIBIT 1040 PAGE 4
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`PCT/USS9/01121
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`-3-
`
`cAMP,
`
`in
`
`turn,
`
`has
`
`been
`
`found
`
`to stimulate
`
`insulin
`
`expression (Korman, L.Y.,
`
`et al., Diabetes gfiz717-722
`
`(1985)).
`
`Insulin acts
`
`to inhibit glucagon synthesis
`
`(Review of Medical Physiology, Ganong, N.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., gt
`
`al‘
`
`(Nature 28;:260-266 (1979)) demonstrated that pro-
`
`glucagon was
`
`subsequently cleaved into glucagon and
`
`a
`
`second polypeptide.
`
`Subsequent work by Lund, P.K., g1_a1;
`
`(Proc. Natl. Acad. Sci. USA 19:345-349 (1982)); Lopez,
`
`L.C., et al.
`
`(Proc. Natl. Acad. Sci. USA 59:5485-5489
`
`(1983)) and Bell, G.I., g;;al; (Ngturg gggz716-718 (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. g§9:3910-3914
`
`(1985)).
`
`Lopez, L.C., gt_al; (Proc. Natl. Acad. Sci. USA
`
`8Q:5485-5489 (1983)),
`
`and Bell, G.I., 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-l),
`
`and glucagon—
`
`like protein 2
`
`(GLP-Z).
`
`Lopez et al.
`
`concluded that
`
`glucagon—like protein 1 was 37 amino acid residues long
`
`MYLAN INST. EXHIBIT 1040 PAGE 5
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`MYLAN INST. EXHIBIT 1040 PAGE 5
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`MYLAN INST. EXHIBIT 1040 PAGE 5
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`

`

`WOW/11296,.
`
`v‘ “'
`
`,
`
`V
`
`'
`
`PCT/USS9/011'21
`
`-4-
`
`and
`
`that glucagon-like peptide
`
`2 was
`
`34
`
`amino
`
`acid
`
`residues long. 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, 6., gt_al;,
`
`fingg;
`
`grjnol. 11§:2176-2181 (1984)).7
`
`Human rat, bovine,
`
`and
`
`hamster sequences of GLP-1 have been found to be identical
`
`(Ghiglione, M., gt_alg, Diabetologia g1:599-600 (1984)).
`
`The conclusion reached by Lopez gt_al; (Erggg_flatlg
`
`Acad. Sci. USA 89:5485-5489 (1983)) regarding the size of
`
`GLP-1 was confirmed by the work of Uttenthal, L.0., gt
`
`al,,
`
`(J. Clin. Endocrinol. Metabol. §;:472-479 (1985)).
`
`Uttenthal et al.
`
`examined the molecular
`
`forms of GLP-1
`
`which were present
`
`in the human pancreas. Their research
`
`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-l might have biological
`
`activity.
`
`Although some
`
`investigators found that GLP-l
`
`could induce rat brain cells to synthesize CAMP (Hoosein,
`
`N.M., et al., FEBS Lett. 118:83-86 (1984)), other inves‘
`
`tigators failed to identify any physiological
`
`role for
`
`GLP-l
`
`(Lopez, L.C., et al., Proc. Natl. Acad. Sci. USA
`
`8Q:5485-5489
`
`(1983)).
`
`The
`
`failure
`
`to
`
`identify any
`
`physiological role for GLP-l caused some investigators to
`
`in fact a hormone and whether
`question whether GLP-l was
`the
`relatedness
`between glucagon
`and GLP-1 might
`be
`
`artifactual
`
`(Ghiglione, M., et al., Diabetologia 21:599-
`
`600 (1984)).
`
`Thus,
`
`in
`
`conclusion,
`
`the .prior art
`
`reveals
`
`an
`
`awareness of
`
`the processing of a glucagon hormone pre-
`
`MYLAN INST. EXHIBIT 1040 PAGE 6
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`W0 90/ 11296
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`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
`
`The present
`
`invention relates to an insulinotropic
`
`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:
`
`7
`
`(A)
`
`a peptide comprising the sequence:
`
`His—Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-
`Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
`Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-
`Lys
`
`or
`
`His-Ala—Glu—Gly-Thr-Phe—Thr-Ser-Asp-
`Val-Ser—Ser-Tyr~Leu-Glu-Gly-Gln-Ala-
`Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-
`Lys-Gly
`
`and
`
`(B)
`
`a derivative of the peptide;
`
`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-l
`
`(1—37).
`
`The
`
`invention also comprises either of
`
`the above-
`
`described compounds wherein the compound is substantially
`
`an insulinotropic
`and has
`free of natural contaminants,
`activity at a concentration of at least 10'10 M.
`
`MYLAN INST. EXHIBIT 1040 PAGE 7
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`MYLAN INST. EXHIBIT 1040 PAGE 7
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`MYLAN INST. EXHIBIT 1040 PAGE 7
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`

`
`
`W0 90/11296 .‘ '
`
`PCP/USS9/0112l
`
`-5-
`
`The
`
`invention also comprises either of
`
`the above
`
`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
`
`(d) pharmaceutically
`
`acceptable
`
`amides,
`
`alkyl
`
`amides, or dialkyl amides of (a),
`
`(b), or (c).
`
`The invention also pertains to a compound having the
`formula:
`I
`
`(1) HzN--X--CO-R1
`wherein: R1 is OH, ON or -NR2R3;
`
`M is pharmaceutically acceptable cation or
`
`a lower branched or unbranched alkyl group;
`R2
`and R3 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:
`
`1.
`
`His-Ala-Glu-Gly—Thr-PheeThr-Ser—Asp-
`Val-Ser-Ser-Tyr—Leu-Glu-Gly-Gln-Ala-
`Ala—Lys-Glu-Phe-Ile-Ala-Trp-Leu—Val~
`Lys
`
`OT
`
`11. His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-
`Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
`Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val—
`Lys-Gly
`
`NH2
`
`is
`
`the
`
`amine
`
`group of
`
`the
`
`amino
`
`terminus of X; and
`
`MYLAN INST. EXHIBIT 1040 PAGE 8
`
`MYLAN INST. EXHIBIT 1040 PAGE 8
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`W0 90/1 1296
`
`PCTIUSS9/01 121
`
`-7-
`
`C0
`
`is the carbonyl group of
`
`the carboxy
`
`terminus of X;
`
`(2)
`
`the acid addition salts thereof; and
`
`the
`(3)
`derivatives thereof;
`
`protected
`
`or partially
`
`protected
`'
`
`insulinotropic
`an
`has
`compound
`the
`wherein
`activity which exceeds the insulintropic activity of GLP-1
`
`(1-36) or GLP-l (1-37).
`
`also
`invention
`The
`medicament which comprises
`
`insulinotropic
`an
`comprises
`and effective amount of
`the
`
`in combination with a suitable physiolog-
`above compounds,
`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 insulinot'ropic peptides.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`the DNA structure and corresponding
`shows
`Figure 1
`amino acid sequence of human,
`rat,
`and hamster prepro—
`glucagons.
`The preproglucagon precursor
`is proteolyti-
`cally cleaved at sites indicated by circles.
`
`the ability of
`shows
`2
`Figure
`peptides glucagon
`and GLP-1
`(7-37)
`
`insulinotropic
`the
`to stimulate CAMP
`
`formation in the insulinoma line, RIN 1046-38.
`
`the insulinotropic
`a comparison of
`shows
`Figure 3
`activity of glucagon with that of GLPel (7-37).
`Figure 4
`shows
`a comparison of
`the insulinotropic
`activities of GLP-1 (7-34), GLP-l
`(7—35), and GLP-1 (7-37)
`using the rat pancreas perfusion technique.
`
`the breakdown of GLP-1
`shows
`Figure 5
`(7-37) under experimental conditions.
`
`GLP-l
`
`(1-37)
`
`into
`
`MYLAN INST. EXHIBIT 1040 PAGE 9
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`MYLAN INST. EXHIBIT 1040 PAGE 9
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`

`

`”7W0 90/11296;2-'T"-'
`
`"
`
`'
`
`‘
`
`‘
`
`'
`
`PCT/USS9/01121
`
`DESCRIPTION OF THE PREFERRED EMBODIMENTS
`
`A.
`
`GLP-1 and Its Derivatives
`
`The hormone gTucagon is known to be synthesized as a
`
`high moTecuTar weight precursor molecuTe which is subse-
`
`quentTy
`
`proteoTyticaTiy cTeaved
`
`into three peptides:
`
`gTucagon, glucagon-Tike peptide 1
`
`(GLP-l),
`
`and gTucagon-
`
`Tike peptide 2
`
`(GLP-Z).
`
`GLP-l has 37 amino acids in its
`
`unprocessed form.
`
`The present
`
`invention discToses that
`
`the unprocessed GLP-1 is essentiaTTy unabTe to mediate the
`
`induction of insuTin biosynthesis.
`
`The unprocessed GLP-l
`
`peptide is,
`
`however, naturaTTy converted to a 31-amino
`
`acid Tong peptide (7-37 peptide) having amino acids 7-37
`
`of GLP—1
`
`("GLP-l
`
`(7-37)"). This processing occurs in the
`
`pancreas and the intestine.
`
`The 7-37 peptide which has
`
`not
`
`been
`
`previosTy described
`
`is
`
`a
`
`hormone
`
`that
`
`has
`
`insuTinotropic activity.
`
`A compound is said to have an
`
`"insuTinotropic activity" if it is abTe to stimulate, or
`
`cause the stimuTation of,
`
`the synthesis or expression of
`
`the hormone insuTin.
`
`The hormonaT activity of GLP-1 (7—
`
`37) appears to be specific for the pancreatic beta ceTTs
`
`where it appears to induce the biosynthesis of insuTin.
`
`The insuTinotropic hormone is usefuT in the study of the
`
`pathogenesis
`
`of maturity onset diabetes meTTitus,
`
`a
`
`condition in which the dynamics of insuTin secretion are
`
`abnormaT. Moreover,
`
`the insuTinotropic hormone is useful
`
`in therapy for this disease.
`Peptide moieties
`(fragments) chosen from the deter-
`
`mined amino acid sequence of human GLP-l constitute the
`
`starting point
`
`in the deveTopment comprising the present
`
`invention.
`
`The interchangeabTe terms "peptide fragment"-
`
`MYLAN INST. EXHIBIT 1040 PAGE 10
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`MYLAN INST. EXHIBIT 1040 PAGE 10
`
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`W0 90/1 1296
`
`PCT/USS9/01121
`
`-9-
`
`and "peptide moiety" are meant
`
`to include both synthetic
`
`and naturally occurring amino acid sequences derivable
`
`from a naturally occurring amino acid sequence.
`
`The amino acid sequence for GLP-1 has been reported
`
`researchers (Lopez, L.C., et_aly, Eyggy_flatly
`by several
`
`Acad. Sci.
`USA §Q=5485-5489 (1983); Bell, 6.1., et al.,
`
`flatyyg §ng716-718 (1983); Heinrich, G., et al.,
`
`fig;
`
`docrinol. 11;:2176-2181
`
`(1984); Ghiglione, M., gfi;_aly,
`
`Diabetologia g1:599-600 (1984)).
`
`The structure of
`
`the
`
`preproglucagon 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-l polypeptide
`having all
`amino ’acids ”from 1
`(N-
`
`(7-
`Similarly, GLP-l
`(C-terminus).
`through 37
`terminus)
`37)
`refers to a GLP-l polypeptide having all amino acids
`
`from 7 (N-terminus) through 37 (C-terminus).
`
`-
`
`In
`
`one
`
`embodiment, GLP-l
`
`(7-37)
`
`and
`
`its peptide
`
`fragments are synthesized by conventional means,
`
`such as
`
`by the well-known solid-phase peptide synthesis described
`
`by Merrifield,
`
`J.M.
`
`(Chem.
`
`Soc.
`
`§§z2149
`
`(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
`
`MYLAN INST. EXHIBIT 1040 PAGE 11
`
`MYLAN INST. EXHIBIT 1040 PAGE 11
`
`MYLAN INST. EXHIBIT 1040 PAGE 11
`
`

`

`W0 90/1129616‘7";
`
`s u
`
`=
`
`,
`
`,
`
`.
`
`PCT/USS9/01121
`
`-10-
`
`Spring Harbor, New York (1982), which is hereby incor-
`
`porated by reference.
`
`The present
`
`invention includes peptides which are
`
`derivable from GLP-l
`
`(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.
`
`Included within the scope of the present
`
`invention
`
`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-l
`
`(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
`
`(1-36), or, more preferably,
`(1-37) or GLP-l
`either GLP-l
`(ii) an insulinotropic activity which can be detected even
`when the derivative is present at a concentration of 10'10
`
`M, or, most preferably, (iii) an insulinotropic activity
`
`even when
`which can be detected
`present at a concentration of 10'11 M.
`
`the derivative is
`
`A derivative of GLP-1
`
`(1-37)
`
`is
`
`said to
`
`share
`
`"substantial homology" with GLP-l
`
`(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-l
`
`(1-37) or a fragment of
`
`GLP-1 (1-37) having the same number of amino acid residues
`
`as the derivative.
`
`MYLAN INST. EXHIBIT 1040 PAGE 12
`
`MYLAN INST. EXHIBIT 1040 PAGE 12
`
`MYLAN INST. EXHIBIT 1040 PAGE 12
`
`

`

`W0 90/1 1296
`
`PCT/USS9/01121
`
`-11-
`
`The derivatives of the present invention incTude GLP-
`
`1
`
`(1—37)
`
`fragments which,
`
`in addition to containing a
`
`sequence that
`
`is substantiaTTy homoTogous
`
`to that of
`
`a
`
`naturaTTy occurring GLP-l
`
`(1-37) peptide may contain one
`
`or more additionaT amino acids at their amino and/or their
`
`carboxy termini. Thus,
`
`the invention pertains to poTypep-
`
`tide fragments of GLP-1
`
`(1-37)
`
`that may contain one or
`
`more amino acids that may not be present
`
`in a naturaTTy
`
`occurring GLP-l
`
`(1-37)
`
`sequence
`
`provided
`
`that
`
`such
`
`poiypeptides have an insuTinotropic activity which exceeds
`
`that of GLP-1 (1-37) or GLP-l
`
`(1-36).
`
`SimiTarTy,
`
`the
`
`invention
`
`incTudes GLP-l
`
`(1-37)
`
`fragments which,
`
`although containing a sequence that
`
`is
`
`substantiaTTy homoiogous to that of a naturaTTy occurring
`
`GLP-l
`
`(1-37) peptide may Tack one or more additionaT amino
`
`acids at their amino and/or their carboxy termini that are
`
`naturaTTy found on
`
`a GLP-l
`
`(1-37) peptide.
`
`Thus,
`
`the
`
`invention pertains to polypeptide fragments-of GLP-l
`
`(1-
`
`37)
`
`that may Tack
`
`one or more
`
`amino acids
`
`that
`
`are
`
`normaTTy present
`
`in a naturaTTy occurring GLP-l
`
`(1-37)
`
`sequence provided that such poTypeptides have an insuTino-
`
`tropic activity which exceeds that of GLP-1 (1-37) or GLP-
`
`1 (1-36).
`
`The invention aTso encompasses the obvious or triviaT
`
`variants
`
`of
`
`the
`
`above-described fragments which
`
`have
`
`inconsequentiaT amino acid substitutions
`
`(and thus have
`
`amino acid sequences which differ from that of the nature]
`
`sequence) provided that
`
`such variants have an insuTino-
`
`tropic activity which is substantiaTTy identicaT to that
`
`of
`
`the above-described GLP-l derivatives.
`
`ExampTes of
`
`obvious or trivia] substitutions incTude the substitution
`
`of one basic residue for another (i.e. Arg for Lys),
`
`the
`
`substitution of one hydrophobic residue for another (i.e.
`
`MYLAN INST. EXHIBIT 1040 PAGE 13
`
`MYLAN INST. EXHIBIT 1040 PAGE 13
`
`MYLAN INST. EXHIBIT 1040 PAGE 13
`
`

`

`'WO 90/112969").- -'
`
`>
`
`'
`
`.
`
`PCT/.USS9/01121’
`
`.
`
`.
`
`-12..
`
`Leu for Ile), or the substitution of one aromatic residue
`
`for another (i.e. Phe for Tyr), etc.
`
`Examples of derivatives of GLP-1 (1-37)
`
`include GLP-l
`
`(7-37); GLP-l
`
`(7-36); GLP-l
`
`(7-35); GLP-l
`
`(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.
`
`As
`
`is known in the art,
`
`the amino acid residues may
`
`be in their protected or unprotected form, using appropri-
`
`ate amino or carboxyl protecting groups. Useful cations
`
`are alkali or alkaline earth metallic cations (i.e., Na,
`
`K, Li,
`
`l/ZCa,
`
`l/ZBa,
`
`etc.)
`
`or
`
`amine
`
`cations
`
`(i.e.,
`
`tetraalkylammonium,
`
`trialkylammonium, where alkyl
`
`can be
`
`Cl'Clzl-
`
`»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, H1, or, more preferably, HCl.
`
`B.
`
`Assays of Insulinotropic Activity
`
`The present
`
`invention concerns GLP-l
`
`(1-37) deriva-
`
`tives which have an insulinotropic activity that exceeds
`
`the insulinotropic activity of either GLP-l
`
`(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.
`
`MYLAN INST. EXHIBIT 1040 PAGE 14
`
`MYLAN INST. EXHIBIT 1040 PAGE 14
`
`MYLAN INST. EXHIBIT 1040 PAGE 14
`
`

`

`WO 90/11296
`
`PCT/U889/01121
`
`-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.
`
`19:487-509
`
`(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 pl of phosphate buffer, 50 ul
`
`of perfusate sample or rat insulin standard in perfusate,
`
`100 pl of anti-insulin antiserum (Wellcome Laboratories;
`1:4o,ooo dilution), and 100 in of [1251]
`insulin, giving a
`
`total volume of 750 pl
`
`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 uU/ml.
`
`In
`
`order to measure the release of IRI
`
`into the cell culture
`
`medium of cells grown in tissue culture, one preferably
`
`incorporates radioactive label
`
`into proinsulin. Although
`
`labeling a polypeptide
`any radioactive label capable of
`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 RIM-38 rat insulinoma cells.
`
`Such cells can be
`
`grown in any suitable medium; however, it is preferable to
`
`use ONE 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
`
`W
`
`MYLAN INST. EXHIBIT 1040 PAGE 15
`
`MYLAN INST. EXHIBIT 1040 PAGE 15
`
`MYLAN INST. EXHIBIT 1040 PAGE 15
`
`

`

`WO 90111296.]...
`
`"3
`
`.~
`
`'.
`
`,
`
`I
`
`'
`
`PCT/USS9/01121
`
`-14-
`
`the method of Penhos, J.C., et al.
`
`(Diabetes 1§:733-738
`
`(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
`
`resected except
`
`for about
`
`four
`
`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 T70 and
`
`0.2% bovine serum albumin (fraction V), and is preferably
`bubbled with 95% oz
`and 5% (:02.
`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
`
`Heir, G.C., et al.,
`
`(J. Clin.
`
`Investigat. 55:1403-1412
`
`(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
`
`MYLAN INST. EXHIBIT 1040 PAGE 16
`
`MYLAN INST. EXHIBIT 1040 PAGE 16
`
`MYLAN INST. EXHIBIT 1040 PAGE 16
`
`

`

`W0 90/ 11296
`
`PCT/USS9/01121
`
`-15-
`
`admixture with
`vehicle.
`
`a
`
`pharmaceutically
`
`acceptable
`
`carrier
`
`Suitable vehicles and their formulation,
`
`inclusive of
`
`other
`
`human proteins, e.g.,
`
`human
`
`serum albumin,
`
`are
`
`described
`
`for
`
`example
`
`in Remington’s
`
`Eharmaceutical
`
`Sciences (16th Ed., A. Oslo Ed. Mack, Easton, PA (1980)).
`In order to form a pharmaceutically acceptable composition
`
`such compositions
`suitable for effective administration,
`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-l
`
`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.
`
`(7-37)
`Examples of natural contaminants with which GLP-l
`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.
`
`Compositions containing GLP-l
`
`(7-37) or its deriva-
`
`tives may be administered intravenously,
`
`intramuscularly,
`
`or subcutaneously at dosages in the range of from about
`
`1
`
`pg/kg to 1,000 ug/kg body weight, or at concentrations
`sufficient to produce serum levels of 10'10 M to 10’11 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)
`
`MYLAN INST. EXHIBIT 1040 PAGE 17
`
`MYLAN INST. EXHIBIT 1040 PAGE 17
`
`MYLAN INST. EXHIBIT 1040 PAGE 17
`
`

`

`W0 90/11296
`
`PCT/US$910! 121 ~
`
`-15-
`
`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-l
`(1-37) derivatives in these compositions may
`vary from IO'IZM to IO'SM.
`
`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-l
`
`(1-37) derivatives.
`
`The
`
`controlled delivery may be exercised by selecting appro-
`
`priate macromolecules
`
`(for example, polyesters, polyamino
`
`acids,
`
`polyvinyl
`
`pyrrolidone,
`
`ethylenevinylacetate,
`
`methylcellulose,
`
`carboxymethylcellulose,
`
`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 (1937)
`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-l
`
`(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
`
`MYLAN INST. EXHIBIT 1040 PAGE 18
`
`MYLAN INST. EXHIBIT 1040 PAGE 18
`
`MYLAN INST. EXHIBIT 1040 PAGE 18
`
`

`

`W0 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-l
`(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
`
`a
`to thereby produce
`"moieties"
`chemical
`one or more
`compound which can be recognized and processed within a
`
`The
`(1-37) derivative.
`to yield a GLP-l
`recipient
`Pmoieties" of
`such compounds may
`include one or more
`lipids,
`carbohydrates,
`amino
`acid residues,
`etc.
`A
`preferred "moiety"
`is an amino acid residue.
`The most
`
`The
`amino
`terminal
`preferred "moiety"
`is
`a peptide.
`is a preferred site
`(histidine)
`residue of GLP-1
`(7-37)
`I
`for the bonding of the "moiety".
`the present
`of
`aspect
`An
`appreciation
`of
`this
`invention can be obtained through a consideration of the
`natural processing of GLP-1
`(1-37).
`GLP-l
`(1-37) has
`a
`biological half-life of 30-50 minutes.
`A natural cleavage
`of the amino terminal hexapeptide, GLP-l
`(1-6), occurs to
`yield GLP-l
`(7-37) whose biological half-life is only 3-5
`minutes.
`Thus,
`the amino terminal hexapeptide, GLP-l
`(1-
`6)
`is a natural "moiety" which when bonded to GLP-l
`(7-37)
`increases the biological half-life of GLP-1 ,(7-37).
`The
`discovery of
`such
`a natural
`"moiety"
`is disclosed in
`
`that additional or
`the concept
`and supports
`Figure 5,
`alternative moieties may be employed in the same manner as
`GLP-l
`(1-6)
`to increase the biological half-life of the
`GLP-l
`(1-37)
`derivatives
`of
`the
`present
`i