United States Patent 15
`Habener
`
`[54]
`
`[75]
`
`INSULINOTROPIC HORMONE
`
`Inventor:
`
`Joel F. Habener, Newton, Mass.
`
`[73] Assignee:
`
`The General Hospital Corporation,
`Charlestown, Mass.
`
`[21] Appl. No.: 532,111
`
`[22] Filed:
`
`Jun. 1, 1990
`
`[63]
`
`Related U.S, Application Data
`Continuation of Ser. No. 148,517, Jan. 26, 1988, aban-
`doned, which is a continuation-in-part of Ser. No.
`859,928, May 5, 1986, abandoned.
`
`51)
`
`Int. CLS wee A61K 37/02; A61K 37/28;
`CO7K 7/10; CO7TK 7/34
`[52] U.S. Ch. eee essetseeeeeetacsesesanes 514/12; 514/866,
`530/308; 530/324
`(58] Field of Search ........0.. 0000 530/324, 303, 308;
`514/12, 866
`
`[56]
`
`References Cited
`FOREIGN PATENT DOCUMENTS
`
`0044168
`
`.
`1/1982 European Pat. Off.
`OTHER PUBLICATIONS
`
`Uttenthal et al.. J. Clin. End. Metab. 61=472~479
`(1985).
`‘
`Ghiglione et al., Diabetologia, 27 = 599-600 (1984).
`Rudinger, Peptide Hormones, Parsons (Ed.) U. Park
`Press, Baltimore, pp. 1-7 (1976).
`Schmidt et al., Diabetologia 28 = 704-707 (1985).
`
`AAUA
`
`US005118666A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,118,666
`Jun. 2, 1992
`
`Bell et al., Nature, 302=716-718 (1983).
`Andrewset al., J. Biol. Chem. 260= 3910-3914.
`Houghten, R. A. et al., Biotechniques 4:522-524, 526,
`528 (Jul. 1986).
`Meienhofer, J., In: Peptides 1984, Ragnarsson, U. (ed.)
`Almqvist & Wiksell International, Stockholm (1984).
`Sarson, D. L.et al., Diabetologia 22:33 (1982).
`Hauner, H. et al., Ann. Nutr. Metab. 32:282-288 (1989).
`Ganong, W., Review of Medical Physiology. 9th Ed.,
`Lange Medical Publications, Los Altos, CA (1979) pp.
`257-276.
`Drucker, D. J. et al., Proc. Natl Acad. Sci. USA
`84:3434-3438 (1987).
`Mojsov,S.et al., J. Clin. Invest. 79:616-619 (1987).
`Hoist, J. J. et al., FEBS Lett. 211:169-174 (1987).
`Kreymann, B. et al., The Lancet, Dec. 5, 1987, pp.
`1300-1304.
`Weir, G. et al., Diabetes 38(3):338-342 (Mar. 1989).
`Gefel, D. et al, Endocrinology 126 (4):2164-2168
`(1990).
`
`Primary Examiner—John Doll
`Assistant Examiner—Christina Chan
`Attorney, Agent, or Firm—Sterne, Kessler, Goldstein &
`Fox
`
`ABSTRACT
`[57]
`Derivatives of glucagon-like peptide I (GLP-1) have
`been found to have insulinotropic activity. The inven-
`tion pertains to such derivatives, and to the use of such
`derivatives as a potential therapy for Diabetes Mellitus.
`
`21 Claims, 6 Drawing Sheets
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 1
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`SUN EXHIBIT 1056, IPR2024-00107, PAGE 1
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`

`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 1 of 6
`
`5,118,666
`
`
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`SUN EXHIBIT 1056, IPR2024-00107, PAGE 2
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`SUN EXHIBIT 1056, IPR2024-00107, PAGE 2
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`

`

`US. Patent
`
`June 2, 1992
`
`Sheet 2 of 6
`
`5,118,666
`
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`SUN EXHIBIT 1056, IPR2024-00107, PAGE 3
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 3
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`€
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`
`

`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 3 of 6
`
`5,118,666
`
`STIMULATION OF cAMP IN RIN 1046-38 cAMP
`KX
`
`(fmol)
`
`gooO
`
`0 c|s 10 1 12) 9 10.11
`GLPI(7-37)—GLUCOGON
`CONCENTRATION
`(~logM)
`
`FIG. 2
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 4
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 4
`
`

`

`U.S. Patent
`
`June 2, 1992
`
`"Sheet 4 of 6
`
`5,118,666
`
`GLUCOSE 6.6mM
`GLUCOSE 6.6mM
`VLLLLLL VLLLLLA.
`
`@ GLPI (7-37)(n=9)
`@ GLPI (7-37)(n=4)
`o GLUCAGON(n=9)-
`© GLUCAGON (n=9)
`
`5
`
`a4
`&
`>
`= 3
`>
`Be
`1
`
`0
`
`10m
`
`107M
`
`25
`
`00 =
`=
`=
`15 =
`—
`0g
`5
`
`0) 0.65
`TIME (min)
`TIME (min)
`
`0
`
`10
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 5
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 5
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`

`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 5 of 6
`
`5,118,666
`
`SNS
`30 36
`TIME:
`CONDITION:GLPI(7-37)
`10"'M
`
`GLUCOSE (6.6mM)
`DSS
`ISSA
`ON)
`51
`57
`66 72
`81
`87
`GLP1(7-35) GLP1(7-34) GLPI(7-37)
`10°'M
`107M
`10! M
`
`
`
`INSULIN(ng/ml) eS
`
`0
`
`20
`
`30
`
`8640
`
`60
`50
`TIME (min)
`
`70
`
`80
`
`90
`
`FIG.4
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 6
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 6
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`

`

`USS. Patent
`
`June 2, 1992
`
`Sheet 6 of 6
`
`5,118,666
`
`GLPI(1-37)
`
`iiI
`
`
`
`
`
`
`
`IMMUNOREACTIVITY(ng/ml)—--=
`
`I t
`
`l
`
`
`
`4j!|| !t'!I |
`
`GLPI(7-37)
`
`
`
`
`
`
`
`
`
` IMMUNOREACTIVITY——>(ng/ml)
`
`
`
`oO
`
`S
`
`Nn
`
`0
`
`
`
`
`:
`=
`16 20 24 2
`12
`TIME (min)
`
`0
`
`4
`
`8
`
`FIG.5
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 7
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`SUN EXHIBIT 1056, IPR2024-00107, PAGE 7
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`

`

`1
`
`INSULINOTROPIC HORMONE
`
`5,118,666
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`This application is a continuation of application Ser.
`No. 07/148,517, filed Jan. 26, 1988, now abandoned,
`which is a continuation-in-part of U.S. patent applica-
`tion Ser. No. 859,928, filed on May 5, 1986, and now 10
`abandoned.
`
`BACKGROUNDOF THE INVENTION
`1, Field of the Invention
`This invention is directed to the discovery that cer-
`tain peptide fragmentsof the prehormone, proglucagon,
`possess hormonalactivities 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.
`2. Description of the Background Art
`The endocrine secretions of the pancreatic islets are
`under complex control not only by blood-borne metab-
`olites (glucose, amino acids, catecholamines, etc.), but
`also by 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. Althoughinsulin secretion
`is predominantly controlled by blood levels of glucose,
`glucagon and somatostatin stimulate and inhibit glu-
`cose-mediated insulin secretory responses, respectively.
`In addition to the proposedinterislet paracrine regula-
`tion ofinsulin 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.
`is a 29-amino acid
`The human hormone, glucagon,
`peptide hormone produced in the A-cells of the pan-
`creas. The hormone belongs to a multi-gene family of
`structurally related peptides that include secretin, gas-
`tric inhibitory peptide, vasoactive intestinal peptide,
`and glicentin. These peptides variously regulate carbo-
`hydrate metabolism, gastrointestinal mobility, and se-
`cretory processing. The principal recognized actions of
`pancreatic glucagon, however,are to promote glycoge-
`nolysis and gluconeogenesis, resulting in an elevation of
`blood sugar levels. In this regard, the actions of gluca-
`gon are counterregulatory to those of insulin and may
`contribute to the hyperglycemia that accompanies Dia-
`betes 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 boundto these recep-
`tors, stimulates the rapid synthesis of cAMP, by these
`cells. cAMP,in turn, has been found to stimulate insulin
`expression (Korman, L. Y., et al., Diabetes 34:717-722
`(1985)). Insulin acts to inhibit glucagon synthesis (Re-
`view ofMedical Physiology, Ganong, W.F., 1979, Lang
`Publications, Los Altos, California (p. 273)). Thus, the
`expression of glucagonis carefully regulated by insulin,
`and ultimately by the serum glucose level.
`The glucagon geneisinitially translated from a 630-
`base pair precursor to form the polypeptide, prepro-
`glycagon (Lundetal. (1982)). This polypeptideis subse-
`quently processed to form proglucagon.Patzelt, C., et
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`60
`
`65
`
`2
`al. (Nature 282:260-266 (1979) demonstrated that pro-
`glucagon was subsequently cleaved into glucagon anda
`second polypeptide. Subsequent work by Lund, P. K.,
`et al.
`(Proc. Natl. Acad. Sci. USA 79:345-349 (1982)):
`Lopez, L. C., et ah (Proc. Natl Acad. Sci. USA
`80:5485-5489 (1983)) and Bell, G. 1. 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
`channelcatfish (Ictalurus punctata) indicated that gluca-
`gon from this animal was also proteolytically cleaved
`after adjacent lysine-arginine and arginine-arginine di- °
`peptide residues (Andrews, P. C., et al., J. Biol. Chem.
`260:3910-3914 (1985)). Lopez, L. C., et al (Proc. Natl.
`5 Acad. Sci. USA 80:5485-5489 (1983)), and Bell, G. I., et
`al, discovered the mammalian proglucagon was
`cleaved adjacent
`lysine-arginine or arginine-arginine
`dipeptides and demonstrated that the proglucagon mo}-
`ecule 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 gluca-
`gon-like protein 1 was 37 amino acid residues long and
`that glucagon-like peptide 2 was 34 amino acid residues
`long. Analogous studies on the structure of rat prepro-
`glucagonrevealed a similar pattern of proteolytic cleav-
`age between adjacent lysine-arginine or arginine-argi-
`nine dipeptide residues, resulting in the formation of
`glucagon, GLP-1, and GLP-2 (Heinrich,G., er al, En-
`docrinol. 115:2176-2181 (1984)). Humanrat, 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 etal. (Proc. Natl.
`Acad. Sci. USA 80:5485-5489 (1983)) regarding the size
`of GLP-1 was confirmed by the work of Uttenthal, L.
`O., et al,
`(J. Clin. Endocrinol. Metabol. 61:472-479
`(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-2are pres-
`ent in the pancreas as proteins having 37 and 34 amino
`acid residues, respectively.
`The similarity between GLP-1 and glucagon sug-
`gested 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 investigatorsfailed to identify any physio-
`logical 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 wasin fact a
`hormone and whether the relatedness between gluca-
`gon and GLP-1 mightbe artifactual (Ghiglione, M., et
`al, Diabetologia 27:599-600 (1984)).
`Thus, in conclusion, the prior art reveals an aware-
`ness of the processing of a glucagon hormoneprecursor
`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 investiga-
`tions designedto elucidate the biological effects of these
`molecules had been unsuccessful.
`
`SUMMARYOF THE INVENTION
`The present
`invention relates to an insulinotropic
`hormone comprising GLP-1 and its derivatives. The
`
`SUN EXHIBIT 1056, IPR2024-00107, PAGE 8
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`SUN EXHIBIT 1056, IPR2024-00107, PAGE 8
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`

`5,118,666
`
`3
`invention additionally pertains to the therapeutic uses of
`such compounds.
`In detail, the invention pertains to a peptide fragment
`whichis insulinotropic and is derivable from a naturally
`occurring amino acid sequence.
`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-Giu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-
`Ala-Trp-Leu-Val-X
`wherein X is selected from the group consisting of:
`(a) Lys,
`(b) Lys-Gly,
`(c) Lys-Gly-Arg;
`and
`(B) a derivative of the peptide; wherein the com-
`poundis substantially free of natural contaminants, and
`has an insulinotropic activity which exceeds the in-
`sulinotropic activity of GLP-1 (1-36) or GLP-1 (1-37).
`The invention also includes 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-Gln-Ala-Ala-Lys-Glu-Phe-Ile-
`Ala-Trp-Leu-Val-X
`wherein X is selected from the group consisting of:
`(a) Lys,
`(b) Lys-Gly,
`(c) Lys-Gly-Arg;
`and
`(B) a derivative of the peptide: wherein the com-
`poundis substantially free of natural contaminants, and
`has an insulinotropic activity at a concentration of at
`least 10-10M.
`Of particular interest are peptides of the following
`formula:
`
`(1) H2N—X—CO—R!
`
`wherein R! is OH, OM, or —NR2?R:3;
`M is a pharmaceutically acceptable cation or a lower
`branched or unbranched alkyl group;
`R2 and R3 are the same ordifferent and selected from
`the group consisting of hydrogen and a lower branched
`or unbranched alkyl group;
`X is 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-Gly-Arg
`NH?is the amine group of the amino terminusof X;
`and COis the carbonyl group of the carboxy terminus
`of X;
`(2) the acid addition salts thereof; and
`(3) the protected or partially protected derivatives
`thereof;
`wherein said compound has an insulinotropic activity
`which. exceeds the insulinotropic activity of GLP-1!
`(1-36) or GLP-1 (1-37).
`The invention further pertains to a method for en-
`hancing the expression of insulin which comprises pro-
`viding to a mammalian pancreatic B-typeislet cell an
`effective amount of the insulinotropic peptides dis-
`closed above.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 shows the DNAstructure and corresponding
`amino acid sequence of human, rat, and hamster prepro-
`
`4
`glucagons. The preproglucagonprecursoris proteolyti-
`cally cleaved at sites indicated bycircles.
`FIG. 2 showsthe ability of the insulinotropic pep-
`tides glucagon and GLP-1 (7-37) to stimulate cAMP
`formation in the insulinoma line, RIN 1046-38.
`FIG. 3 shows a comparison of the insulinotropic
`activity of glucagon with that of GLP-1 (7-37).
`FIG. 4 shows a comparison of the insulinotropic
`activities of GLP-1 (7-34), GLP-1 (7-35), and GLP-1
`(7-37) using the rat pancreas perfusion technique.
`FIG. 5 shows the breakdown of GLP-1 (1-37) into
`GLP-] (7-37) under experimental conditions.
`
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`A. GLP-1 and Its Derivatives
`
`The hormone glucagon is knownto be synthesized as
`a high molecular weight precursor molecule which is
`subsequently proteolytically cleaved into three pep-
`tides: 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-1 is essentially un-
`able to mediate the induction of insulin biosynthesis.
`The unprocessed GLP-1 peptide is, however, naturally
`converted to a 3l-amino acid long peptide (7-37 pep-
`tide) 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 hormonethat has insulinotropic activity.
`A compoundis 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 spe-
`cific for the pancreatic beta cells where it appears to
`induce the biosynthesis of insulin. The insulinotropic
`hormoneis useful in the study of the pathogenesis of
`maturity onset diabetes mellitus, a condition in which
`the dynamics of insulin secretion are abnormal. More-
`over, the insulinotropic hormoneis 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” and “peptide moiety” are meant to include
`both synthetic and naturally occurring amino acid se-
`quences derivable from a naturally occurring amino
`acid sequence.
`The amino acid sequence for GLP-] has been re-
`ported by several researchers (Lopez, L. C., et al., Proc.
`Natl. Acad. Sci., USA 80:5485- 5489 (1983); Bell, G. I,
`et a., Nature 302:716-718 (1983); Heinrich, G., et al.,
`Endocrinol. 115:2176-2181 (1984); Ghiglione, M., et al,
`Diabetologia 27:599-600 (1984)). The structure of the
`preproglucagon geneand its corresponding aminoacid
`sequence is shown in FIG. 1. This figure further dis-
`plays the proteolytic processing of the precursor gene
`product into glucagon and the two glucagon-like pep-
`tides. As used herein, the notation of GLP-1 (1-37)
`refers to a GLP-1 polypeptide having all amino acids
`from | (N-terminus) through 37 (C-terminus). Similarly,
`GLP-1 (7-37) refers to a GLP-1 polypeptide havingall
`amino acids from 7 (N-terminus) through 37 (C-ter-
`minus).
`_
`In one embodiment, GLP-1 (7-37) and its peptide
`fragments are synthesized by conventional means, such
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`as by the well-knowsolid-phase peptide synthesis de-
`scribed 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 technol-
`ogy, as disclosed by Maniatis, T., et al, Molecular Biol-
`ogy: A Laboratory Manual, Cold Spring Harbor, N.Y.
`(1982), which is hereby incorporated 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 se-
`quence”if it can be obtained byfragmenting 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 mate-
`rial (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-1 (1-37) or a similarly sized fragment of GLP-1
`(1-37); (2) it
`is capable of functioning as an insulino-
`tropic hormoneand (3) using at least one of the assays
`provided herein, the derivative has either(1) an insulino-
`tropic activity which exceedsthe insulinotropic activity
`of either GLP-1 (1-37) or GLP-1 (1-36), or, more pref-
`erably, (ii) an insulinotropic activity which can be de-
`tected even when the derivative is present at a concen-
`tration of 10-10M, or. most preferably, (iii) an insulino-
`tropic activity which can be detected even when the
`derivative is present at a concentration of 10-!1M.
`A derivative of GLP-1 (1-37) is said to share ‘“‘sub-
`stantial 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 frag-
`ment of GLP-1 (1-37) having the same number of amino
`acid residues as the derivative.
`invention include
`The derivatives of the present
`GLP-1 (1-37) fragments which, in addition to contain-
`ing a sequencethat 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 inven-
`tion pertains to polypeptide 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) se-
`quence provided that such polypeptides have an in-
`sulinotropic activity which exceeds that of GLP-1
`(1-37) or GLP-1 (1-36).
`Similarly, the invention includes GLP-1 (1-37) frag-
`ments which, although containing. a sequence that is
`substantially homologous to that of a naturally occur-
`ring GLP-1 (1-37) peptide may lack one or more addi-
`tional amino acids at their amino and/or their carboxy
`termini that are naturally found on a GLP-1 (1-37) pep-
`tide. Thus, the invention pertains to polypeptide frag-
`ments 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 insulinotropic activity which exceeds that of
`GLP-1 (1-37) or GLP-1 (1-36).
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`The invention also encompasses the obviousortrivial
`variants of the above-described fragments which have
`inconsequential aminoacid substitutions (and thus have
`amino acid sequences which differ from that of the
`natural sequence) provided that such variants have an
`insulinotropic activity which is substantially identical to
`that of the above-described GLP-1 derivatives. Exam-
`ples of obviousortrivial substitutions include the substi-
`tution of one basic residue for another(i.e. Arg for Lys),
`the substitution of one hydrophobicresidue for another
`(i.e. Leu for Ile), or the substitution of one aromatic
`residue for another (i.e. Phe for Tyr), etc.
`include
`Examples of derivatives of GLP-1 (1-37)
`GLP-1 (7-37); GLP-1 (7-36); GLP-1 (7-35); GLP-1
`(7-34); and the des-Gly amidated forms of these mole-
`cules. Included as well are the use of additional amino
`acid residues added to such sequences in order to en-
`hance couplingto carrier protein or amino acid residues
`added to enhancetheinsulinotropic effect.
`Asis knownin the art, the amino acid residues may
`be in their protected or unprotected form, using appro-
`priate amino or carboxyl 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 alky!
`can be C}-C}2).
`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 hydro-
`halic acidsalts, i.e., HBr, HI, or, more preferably, HCI.
`
`B. Assays of Insulinotropic Activity
`The present invention concerns GLP-1 (1-37) deriva-
`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 com-
`pound maybe determined by providing that compound
`to animalcells, or injecting that compoundinto 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 radioimmunoassay which can specifically
`detect insulin. 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 pl
`of phosphate buffer, 50 yl of perfusate sample orrat
`insulin standard in perfusate, 100 p! of anti-insulin anti-
`serum (Wellcome Laboratories; 1:40,000 dilution), and
`100 pl of insulin, giving a total volume of 750 pl in a
`10 75-mm disposable glass tube. After incubation for
`2-3 days at 4° C., free insulin was separated from anti-
`body-bound insulin by charcoal separation. The assay
`sensitivity was 1-2 pU/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 radioac-
`tive label capable of labeling a polypeptide can be used,
`it is preferable to use 3H leucine in order to obtain label-
`ing proinsulin. Labeling can be done for any period of
`time sufficient to permit the formation of a detectably
`labeled poo! of proinsulin molecules; however,
`it
`is
`preferable to incubate cells in the presence of radioac-
`tive label for a 60-minute time period. Although anycell
`line capable of expressing insulin can be used for deter-
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`mining whether a compound has an insulinotropic ef-
`fect,
`it
`is preferable to use rat
`insulinoma cells, and
`especially RIN-38 rat insulinomacells. Such cells can be
`grownin any suitable medium; however,it is preferable
`to use DME medium containing 0.1% BSA and 25 mM
`glucose.
`Theinsulinotropic property of a compound mayalso
`be determined by pancreatic infusion. The in situ iso-
`lated perfused rat pancreas preparation was a modifica-
`tion of the method of Penhos, J. C., er al.
`(Diabetes
`18:733-738 (1969)). In accordance with such a method,
`fasted rats (preferably male Charles River strain albino
`rats), weighing 350-600 g, are anesthetized with an
`intraperitoneal 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 possi-
`ble 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% O2 and 5%
`CO:. A nonpulsatile flow, four-channel roller-bearing
`pump (Buchler polystatic, Buchler Instruments Divi-
`sion, Nuclear-Chicago Corp.) is preferably used, and a
`switch from one perfusate source to anotheris prefera-
`bly 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 composi-
`tions. Such therapeutic compositions may consist solely
`of the insulinotropic peptides (or peptide derivatives)
`although, preferably, the compositions will contain the
`insulinotropic peptides (or derivatives thereof) com-
`bined in admixture with a pharmaceutically acceptable
`carrier vehicle.
`Suitable vehicles and their formulation, inclusive of
`other human proteins, e.g., human serum albumin, are
`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 substan-
`tially free of natural contaminantsif these contaminants
`are substantially absent from a sample of the material.
`Compositions containing GLP-1 (7-37)orits deriva-
`tives may be administered intravenously, intramuscu-
`larly, or subcutaneously at dosages in the range of from
`about 1 pg/kg to 1,000 pg/kg body weight, or at con-
`centrations
`sufficient
`to produce serum levels of
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`10-1!0M to 10-1!M. although a loweror higher dosage
`maybe administered. The required dosage will depend
`upon the severity of the condition of the patient and
`upon suchcriteria as the patient’s height. weight, sex,
`age, and medicalhistory.
`Forthe purpose of parenteral administration. compo-
`sitions containing the derivatives of GLP-1 (1-37) are
`preferably dissolved in distilled water and the pH-value
`is preferably adjusted to about 6 to 8. In ordertofacili-
`tate the lyophilization process resulting in a suitable
`product, lactose may be added to the solution. Prefera-
`bly, 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 to 10-12M to 10-5M.
`Additional pharmaceutical methods may be em-
`ployed 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) deriv-
`atives. The controlled delivery may be exercised. by
`selecting appropriate macromolecules (for example,
`polyesters, polyamino acids, polyvinyl pyrrolidone,
`ethylenevinylacetate, methylcellulose, carboxymethy]-
`cellulose, and protamine sulfate) and the concentration
`of macromolecules as well as the methods of incorpora-
`tion in order to control
`release. Another possible
`method to control the duration of action by controlled
`release preparationsis 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 vinylacetate copolymers. Alterna-
`tively, instead of incorporating the GLP-1 (1-37) deriv-
`atives into these polymeric particles, it is possible to
`entrap these derivatives in microcapsules prepared, for
`example, by coacervation techniques or byinterfacial
`polymerization, for example, hydroxymethylcellulose
`or gelatin-microcapsules and poly (methylmethacry-
`late) microcapsules, respectively, or in colloidal drug
`delivery systems, for example, liposomes, albumin mi-
`crospheres, microemulsions, nanoparticles, and nano-
`capsules or in macroemulsions. Such teachings are dis-
`closed in Remington's Pharmaceutical Sciences (1980).
`It is possible to enhancethe biological half-life of the
`GLP-1 (1-37) derivatives of the present invention, and,
`thus. to increase the retention or stability of thederiva-
`tives in a recipient, by bonding such derivatives to one
`or more chemical “moieties” to thereby produce a com-
`pound which can be recognized and processed within a
`recipient to yield a GLP-1 (1-37) derivative. The “moi-
`eties” of such compounds may include one or more
`lipids, carbohydrates, amino acid residues, etc. A pre-
`ferred “moiety” is an amino acid residue. The most
`preferred ‘‘moiety” is a peptide. The amino terminal
`(histidine) residue of GLP-1 (7-37) is a preferred site for
`the bonding of the “moiety”.
`An appreciation of this aspect of the present inven-
`tion 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 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-1 (7-37). The