PCT
`
`WORLD INTELLECTUAL PROPERlY ORGANIZATION
`International Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC1)
`
`(Sl) International Patent Oassification 4 :
`C07K 7/10, 7/'34, 7/40
`A61K 37 /02, 37 /24
`
`Al
`
`(11) International Publication Number:
`
`WO 90/11296
`
`(43) International Publication Date:
`
`4 October 1990 (04.10.90)
`
`(21) Iatemational Application Number:
`
`(22) International Filing Date:
`
`20 March 1989 (20.03.89)
`
`PCT/ US89/ 0I 121 Published
`With international search report.
`
`(71) Applicant: THE GENERAL HOSPITAL CORPORA(cid:173)
`TION [US/US]; Fruit Street, Boston, MA 92114 (US).
`
`(72) Inventor: HABENER, Joel, F. ; 217 Plymouth Road, New(cid:173)
`ton Highlands, MA 02161 (US).
`
`(74) Agents: FOX, Samuel, L. ct 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(cid:173)
`pean patent), JP, LU (European patent), NL (European
`patent), SE (European patent).
`
`(S4)11tle : INSULIN0TR0PIC 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
`
`FRESENIUS EXHIBIT 1040
`Page 1 of 53
`
`
`
`WORLD INTELLECTUAL PROPERlY ORGANIZATION
`International Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC1)
`
`(Sl) International Patent Oassification 4 :
`C07K 7/10, 7/'34, 7/40
`A61K 37 /02, 37 /24
`
`Al
`
`(11) International Publication Number:
`
`WO 90/11296
`
`(43) International Publication Date:
`
`4 October 1990 (04.10.90)
`
`(21) Iatemational Application Number:
`
`(22) International Filing Date:
`
`20 March 1989 (20.03.89)
`
`PCT/ US89/ 0I 121 Published
`With international search report.
`
`(71) Applicant: THE GENERAL HOSPITAL CORPORA(cid:173)
`TION [US/US]; Fruit Street, Boston, MA 92114 (US).
`
`(72) Inventor: HABENER, Joel, F. ; 217 Plymouth Road, New(cid:173)
`ton Highlands, MA 02161 (US).
`
`(74) Agents: FOX, Samuel, L. ct 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(cid:173)
`pean patent), JP, LU (European patent), NL (European
`patent), SE (European patent).
`
`(S4)11tle : INSULIN0TR0PIC 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
`
`FRESENIUS EXHIBIT 1040
`Page 1 of 53
`
`
`
`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 lo identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`Allllril
`AT
`Allllralia
`AU
`BalW:ls
`BB
`BE
`Beci,rm
`Burkina Fuo
`BF
`Bulpria
`BG
`BJ
`&Din
`Bcazil
`BR
`Canada
`CA
`cr Central A ftao Republic
`COIIIO
`CG
`Of
`Swilzetland
`CM ea-oon
`DE
`Ocrmlny, Fcdctal Republic of
`DK Dcamad
`
`Spain
`Qi
`F1111and
`Fl
`France
`FR
`GA Oabon
`GB Unilcd Kingdom
`W Hunpry
`rr
`Italy
`Japan
`JP
`IP Democralic Pcople·s Republic
`of Korea
`lR Republic of Koru
`u
`LicdJtcmlUI
`LI: Sri l.anu
`w
`lAulcmbowJ
`r.t: Monaco
`
`MG Madagascar
`ML Mali
`MR Maurituia
`MW Malawi
`NL Netherlands
`NO Noiway
`RO Romania
`SD
`Sudan
`SE
`Sweden
`Sencpl
`SIi
`Soviet Un.ion
`!IJ
`1D Chad
`TG
`To,:,
`l5 Unilcd Slates of America
`
`FRESENIUS EXHIBIT 1040
`Page 2 of 53
`
`
`
`WO90/11296
`
`PCT/US89/01121
`
`-1-
`
`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
`
`that
`is directed to the discovery
`invention
`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, catechol amines, etc.), but also by
`
`FRESENIUS EXHIBIT 1040
`Page 3 of 53
`
`
`
`W0'90/11296(,_,
`
`PCT/US89/0t 121
`
`-2-
`
`The major pancreatic is 1 et
`1 oca 1 paracri ne influences.
`hormones (glucagon,
`insulin, and somatostatin) interact
`among
`their specific cell
`types {A, B, and D cells,
`respectively) to modulate secretory responses mediated by
`the rnetabo 1 i tes. A 1 though insulin secretion is predomi -
`nantly controlled by blood levels of glucose, glucagon and
`somatostatin
`stimulate
`and
`inhibit glucose-mediated
`In addition to
`insulin secretory responses, respectively.
`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
`ici d
`peptide hormone produced in the A-cells of the pancreas.
`The hormone belongs to a multi-gene family of st ructurally
`relat ed peptides that include secretin, gast ric inhibitory
`pepti de, vasoactive
`intestinal peptide, and glicentin.
`These peptides variously regulate carbohydrat e metabolism,
`gastrointestinal mobility, and secretory processing. The
`principal
`recognized actions of pancreatic glucagon,
`however, are
`to promote glycogenolysis and gluconeo(cid:173)
`genesis , resulting in an elevat ion of blood sugar levels.
`In
`thi s regard,
`the actions of gl ucagon are counter(cid:173)
`regulatory to those of insulin and may contribute to the
`hyperglycemia that accompanies Diabetes mell itus (Lund,
`P.K., et al . , Proc. Natl. Acad. Sci., USA 79:345-349
`(1982)).
`Gl ucagon has been found to be capab 1 e of binding to
`specific receptors which 1 i e on the surface of insulin(cid:173)
`producing cells. Glucagon , when bound to these receptors,
`stimulates the rapid synthesis of cAMP, by these ce 11 s.
`
`FRESENIUS EXHIBIT 1040
`Page 4 of 53
`
`
`
`W090/11296
`
`PCT/US89/01121
`
`-3-
`
`insulin
`to stimulate
`found
`turn, has been
`in
`cAMP,
`expression
`(Korman, L.Y . , et al., Diabetes 34:717-722
`Insulin acts
`(1985}).
`to
`inhibit glucagon synthesis
`(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(cid:173)
`cagon (Lund et al. (1982)). This polypeptide is subse(cid:173)
`Patzelt, C., tl
`quently processed to form proglucagon.
`AL. (Nature 282:260-266 (1979)) demonstrated that pro(cid:173)
`glucagon was subsequently cleaved
`into glucagon and a
`second polypeptide . Subsequent work by Lund, P.K., et al.
`(Proc. Natl. Acad . Sci. USA 79:345-349 (1982)); Lopez,
`L.C . , et al. (Proc. Natl. Acad. Sci. USA 80 :5485-5489
`(1983)) and Bell, G.l. , et al. (Nature 302:716-718 (1983 ))
`demonstrated that the proglucagon molecule-was cleaved
`lysine-arginine dipeptide
`residues .
`immediately after
`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.I., et al., discovered
`the mammalian proglucagon was cleaved at lysfoe-arginine
`or arginine-arginine dipeptides and demonstrated that the
`progl ucagon molecule contained three discrete and highly
`homologous
`peptide molecules which were designated
`glucagon, glucagon-like protein 1 (GLP-1), and glucagon (cid:173)
`like protein 2 (GLP-2) .
`Lopez et al. concluded that
`glucagon-like protein 1 was 37 amino acid residues long
`
`FRESENIUS EXHIBIT 1040
`Page 5 of 53
`
`
`
`PCT/US89/0lt2l
`
`-4-
`
`that glucagon-like peptide 2 was 34 amino acid
`and
`residues long. Analogous studies on the structure of rat
`preproglucagon revealed a similar pattern of proteolytic
`cleavage between adjacent
`lysine-arginine or arginine(cid:173)
`arginine dipeptide residues, resulting in the formation of
`glucagon, GLP-1, and GLP-2 (Heinrich, G., et al., Endo(cid:173)
`crjnol. lli:2176-2181 (1984)).
`Human rat, bovine, and
`hamster sequences of GLP-1 have been found to be identical
`(Ghiglione, M., et al., Diabetologja Z.I: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 Uttentha 1 , L. O. , et
`al., (J. Clin. Endocrinol. Metabol. fil.: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(cid:173)
`tively.
`The similarity between GLP-1 and gl ucagon 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., FESS Lett. 178:83-86 (1984)), other inves (cid:173)
`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-1 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)).
`the .prior art reveals an
`in conclusion,
`Thus,
`awareness of the processing of a glucagon hormone pre-
`
`FRESENIUS EXHIBIT 1040
`Page 6 of 53
`
`
`
`W090/11296
`
`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
`
`The present invention relates to an insulinotropic
`hormone comprising GLP-1
`and
`its derivatives .
`The
`invention addi tionally pertains t o the therapeuti c uses of
`such compounds.
`The inventi on comprises a compound selected from the
`group consisti ng of :
`(A) a peptide comprising the sequence:
`His-Ala-Gl u-Gly-Thr-Phe-Thr-Ser-Asp(cid:173)
`Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala(cid:173)
`Ala-Lys-Gl u-Phe-I le-Ala-Trp-Leu-Val(cid:173)
`Lys
`
`or
`
`Hi s-Ala-Gl u-Gly-Thr-Phe-Thr-Ser-Asp(cid:173)
`Val -Ser-Ser-Tyr-Leu -Glu-Gly-Gln-Al a(cid:173)
`Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val(cid:173)
`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-1 (1-37).
`The
`invention al so comprises either of the above(cid:173)
`descri bed compounds wherein the compound is substantially
`free of natural contaminants, and has an insul inotropic
`activity at a concentration of at least 10· 10 M.
`
`FRESENIUS EXHIBIT 1040
`Page 7 of 53
`
`
`
`~~~~{! -
`
`·'
`
`WO 90/11296 . ·
`
`PCT/US89/01121
`
`-6-
`
`invention a 1 so comprises either of the above
`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,
`(d) pharmaceutically acceptable
`amides, or dialkyl amides of (a), {b), or {c).
`The invent ion also pertains to a compound having the
`formula:
`
`alkyl
`
`(1) HzN--X--Co-Rl
`wherein : Rl is OH, OM or -NR2R3;
`Mis 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;
`Xis a peptide comprising the sequence:
`
`I. His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp(cid:173)
`Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln•Ala(cid:173)
`Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val(cid:173)
`Lys
`
`or
`
`II. His-Ala-Gl~-Gly-Thr-Phe-Thr-Ser-Asp(cid:173)
`Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala(cid:173)
`Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val(cid:173)
`Lys-Gly
`
`NH2
`terminus of X; and
`
`is
`
`the amine group of
`
`the
`
`amino
`
`FRESENIUS EXHIBIT 1040
`Page 8 of 53
`
`
`
`W090/11296
`
`PCT/US89/0t 121
`
`-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
`wherein
`the compound has an
`activity which exceeds the insulintropic activity of GLP-1
`(1-36) or GLP-1 (1-37).
`insul inotropic
`an
`The
`invention also comprises
`medicament which comprises and effective amount of the
`above compounds , in combination with a suitable physiolog(cid:173)
`ical ly acceptable carrier.
`The invention also compri ses a method for enhancing
`the expression of insul in which comprises providing to a
`mammalian pancreati c 8-type islet cell an effective amount
`of any of the above-described insulinotropic peptides.
`
`BRIEF DESCRIPTION OF THE DRAWINGS·
`
`Figure 1 shows the DNA structure and corresponding
`ami no acid ·sequence of human, rat, and hamster prepro(cid:173)
`gl ucagons.
`The preprog 1 ucagon precursor is proteo 1 yt i -
`cally cleaved at sites indicated by circles.
`the ability of the
`insulinotropic
`Figure 2 shows
`peptides gl ucagon and GLP-1
`(7-37)
`to stimulate cAMP
`formation in the insulinoma l ine , RIN 1046-38.
`Figure 3 shows a comparison of the insul footropic
`activity of glucagon with that of GLP-1 (7-37 }.
`Figure 4 shows a comparison of the insul inotropic
`activi ti es of GLP-1 (7-34), GLP-1 {7-35), and GLP-1 (7 -37)
`using the rat pancreas perfusion technique.
`Figure 5 shows the breakdown of GLP-1 (1-37)
`GLP-1 (7-37) under experimental conditions.
`
`into
`
`FRESENIUS EXHIBIT 1040
`Page 9 of 53
`
`
`
`PCT/US89/0H21
`
`-8-
`
`DESCRIPTION OF THE PREFERRED EMBODIMENTS
`
`A.
`
`GLP-1 and Its Derivatives
`
`The hormone glucagon is known to be synthesized as a
`high molecular weight precursor molecule which is subse(cid:173)
`quently proteolytically cleaved
`into
`three peptides :
`glucagon, glucagon-like peptide 1 (GLP-1), and glucagon-
`1 ike peptide 2 (GLP-2). GLP-1 has 37 amino acids in its
`unprocessed form . The present invention discloses that
`the unprocessed GLP-1 is essentially unable to mediate the
`induction of insulin biosynthesis . The unprocessed GLP- 1
`peptide is, however, natura 11 y converted to a 31-ami no
`acid long pept ide (7-37 peptide ) having amino aci ds 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
`insul inotropic activity. A compound is said to have an
`"insulinotropic activity" if it is able to stimulate , or
`cause the stimulation of, the synthesis or expressi on of
`the hormone insulin. The hormonal activity of GLP-1 (7-
`37) appears to be specific for the pancreatic beta cell s
`where it appears to induce the biosynthesis of insulin .
`The insulinotropic hormone is useful in the study of the
`pathogenesis of maturity onset diabetes melli t us , 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(cid:173)
`mined amino acid sequence of human GLP-1 constitute the
`starting point in the development comprising the present
`invention.
`The interchangeable terms "peptide fragment "
`
`FRESENIUS EXHIBIT 1040
`Page 10 of 53
`
`
`
`WO90/11296
`
`PCT/US89/01121
`
`-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
`by several researchers (Lopez, L.C., et al., Proc. Natl.
`Acad. Sci., USA 80:5485-5489 (1983); Bell, G.I., et al.,
`Nature 302:716-718 (1983); Heinrich, G., et al . , En(cid:173)
`docrinol. 115:2176-2181 (1984); Ghiglione, H., et al.,
`Oiabetoloqia ll: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 ~nd 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(cid:173)
`terminus) through 37 (C-terminus). Similarly, GLP-1 (7-
`37) refers to a GLP-1 polypeptide having all ·amino acids
`from 7 (N-terminus) through 37 (C-terminus). ·
`In one embodiment, GLP-1
`(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. 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 prog l ucagon peptide or of GLP-1 through the use of
`recombinant ONA technology, as disclosed by Maniatis, T.,
`et al., Molecular Biology: A Laboratory Manual, Cold
`
`FRESENIUS EXHIBIT 1040
`Page 11 of 53
`
`
`
`PCT/US89/01121
`
`-10-
`
`Spring Harbor, New York (1982), which is hereby incor(cid:173)
`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.
`Inc 1 uded within the scope of the present invent ion
`are those molecules which are said to be "derivatives" of
`GLP-1 (1-37) .
`Such · a "derivative" has the following
`characteristics: (I) 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
`the
`insulinotropic activity of
`activity which exceeds
`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 10-10
`M, or, most preferably, (iii) an insulinotropic activity
`which can be detected
`even when
`the derivative is
`present at a concentration of 10-11 M.
`to share
`is said
`A derivative of GLP-1
`(1-37)
`"substantial homology" with GLP-1 (1-37) if the amino acid
`sequences of the derivative is at 1 east 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.
`
`FRESENIUS EXHIBIT 1040
`Page 12 of 53
`
`
`
`WO90/11296
`
`PCT/US89/01121
`
`-11-
`
`The derivatives of the present invention include GLP-
`1 (1-37)
`fragments which,
`in addition to containing a
`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(cid:173)
`tide fragments of GLP-1 (1-37) that may contain one or
`more amino acids that may not be present in a naturally
`(1-37)
`sequence provided
`that
`such
`occurring GLP-1
`polypeptides have an insulinotropic activity which exceeds
`that of GLP-1 (1-37) or GLP-1 (1-36).
`(1-37)
`includes GLP-1
`Similarly,
`the
`invention
`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(cid:173)
`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 insul ino(cid:173)
`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.
`
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`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-1
`(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(cid:173)
`sul inotropic effect.
`As is known in the art, the amino acid residues may
`be in their protected or unprotected form, using appropri(cid:173)
`ate amino or carboxyl protecting groups. Useful cations
`are alkali or alkaline earth metallic cations (i.e., Na ,
`l/2Ca, 1/2Ba, etc.) or amine cations (i.e.,
`K, Li,
`tetraalkylammonium, trialkylammonium, where alkyl can be
`C1-C1z).
`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, HCl.
`
`B. Assays of lnsulinotropic Activity
`
`The present invention concerns GLP-1 ( 1-37) deri va(cid:173)
`ti ves 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 irnmunoreactive insulin (IRI) into the media or
`circulatory system of the animal,
`respectively.
`The
`presence of IRI is detected through the use of a radio(cid:173)
`immunoassay which
`can
`specifically detect
`insulin.
`
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`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. ZQ: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 µ1 of phosphate buffer, 50 µ1
`of perfusate sample or rat insulin standard in perfusate,
`100 µ1 of anti-insulin antiserum (Wellcome Laboratories;
`1:40,000 dilution), and 100 µ1 of [1251] insulin, giving a
`in a 10 x 75-mm disposable glass
`total volume of 750 µ1
`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 µU/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
`any radioactive label capable of labeling a polypeptide
`can be used, it is preferable to use 3H leucine in order
`to obtain 1 abe 1 i ng proi nsul in. Labe 1 i ng can be done for
`any period of time sufficient to permit the formation of a
`detectably labeled pool of proinsulin molecules; however,
`it is preferab 1 e to incubate ce 11 s 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
`
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`Page 15 of 53
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`the method of Penhos, J.C. , et a 1 • ( Oj abetes ~: 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(cid:173)
`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% 02 and 5% CO2, 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(cid:173)
`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 (l-37) may be used as therapeutic compositi-ons.
`Such therapeutic compositions may consist solely of the
`insulinotropic peptides (or peptide derivatives) although,
`preferably, the compositions will contain the insulino(cid:173)
`in
`thereof) combined
`tropic peptides
`(or derivatives
`
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`admixture with a pharmaceutically acceptable carrier
`vehicle.
`Suitable vehicles and their formulation, inclusive of
`other human proteins, e.g., human serum albumin, are
`in Remington's Pharmaceutical
`for example
`described
`Sciences (16th Ed., A. Oslo Ed. Hack, Easton, PA (1980}}.
`In order to form a pharmaceutical ly acceptable composition
`suitable for effective administration, such compositions
`will contain an effective amount of GLP-1 (7-37), or a
`(7 -37),
`together with a suitable
`derivative of GLP-1
`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
`is said to be "substantially free of natural
`material
`contaminants" if it has been substantially purified from
`material s with which it is normal ly and natural ly found.
`Examples of natural contaminants with whi ch 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(cid:173)
`nants if these contaminants are substantially absent from
`a sample of the material .
`Compositions containing GLP-1 (7-37} or its deriva(cid:173)
`tives may be administered intravenously, intramuscularly,
`or subcutaneously at dosages in the range of from about 1
`pg/kg to 1,000 µg/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 adn:i nistered.
`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
`t he purpose of parenteral
`administrati on,
`compositions containing the derivatives of GLP-1
`(1-37)
`
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`are preferably dissolved in distilled water and the pH(cid:173)
`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 10·12M to 10·5M.
`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
`contro 11 ed de 1 i very may be exercised by se 1 ect i ng appro(cid:173)
`priate macromolecules (for example, polyesters, polyamino
`acids, polyvinyl pyrrolidone,
`ethylenevinylacetate,
`methylcell ul ose,
`carboxymethylcel 1 ul ose,
`and protami ne
`sulfate) and the concentration of macromo 1 ecul es as we 11
`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(cid:173)
`acetate copolymers. Alternatively,
`instead of incor(cid:173)
`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
`
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`Such teachings are
`nanocapsules or in macroemul sions.
`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
`~moieties" 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" ii a peptide.
`The amino
`terminal
`(histidine) res idue of GLP-1 (7-37) is a preferred site
`for the bonding of the "moiety".
`the present
`An
`appreciation of
`this aspect of
`invention can be obtained through a consideration of the
`natural processing of GLP-1 (1-37). GLP-1 ·(1-37) has a
`biological half-life of 30-50 minutes. A natu~~, 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-1 .(7-37). The
`discovery of such a natural "moiety"
`is disclosed in
`Figure 5, and supports the concept that addi ti ona 1 or
`alternative moieties may be empl oyed in the same manner as
`GLP-1 (1-6) to increase the biological half-1 ife of the
`( 1-37) derivatives of
`the present
`invention .
`GLP-1
`Although the present invention does not encompass the use
`of GLP-1 (1-6) as a "moiety," it does include variants of
`GLP-1
`(1-6) as well as ot her peptides of unrelated
`sequence which are capable of enhancing the half-life of
`
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`the present
`
`the peptides and peptide derivatives of
`invention.
`In summary, insulin secretion from the ~-cell of the
`endocrine pancreas is controlled by a complex network of
`metabolic factors. This network includes such diverse
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