throbber
PCT
`
`WORLD INTELLECTUALPROPERTY ORGANIZATION
`International Bureau
`
`
`
`Al
`
`
`
`(21) International Application Number: PCT/US87/01005|(81) Designated States: AT (European patent), BE (Euro-
`pean patent), CH (European patent), DE (European
`patent), FR (Europeari patent), GB (European pa-
`tent), IT (European patent), JP, LU (European pa-
`tent), NL (European patent), SE (European patent).
`
`(22) Ynternational Filing Date:
`
`5 May 1987 (05.05.87)
`
`(31) Priority Application Number:
`
`859,928
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 4 :
`(11) International Publication Number:
`WO 87/ 06941
`C07K 7/16, 7/34, A61K 37/02
`.
`son
`(43) International Publication Date:
`A61K 37/28
`19 November 1987 (19.11.87)
`
`nue, N.W., Suite 300, Washington, DC 20036 (US).
`
`
`
`
`(32) Priority Date: 5 May 1986 (05.05.86)|Published
`With international search report.
`
`(33) Priority Country:
`
`US
`
`(71) Applicant: THE GENERAL HOSPITAL CORPORA-
`TION [US/US]; Fruit Street
`(Bar-3), Boston, MA
`02114 (US).
`
`(72) Inventor: HABENER,Joel ; 217 Plymouth Road, New-
`ton Highlands, MA 02161 (US).
`
`(74) Agents: GOLDSTEIN, Jorge, A. et al.; Saidman,
`Sterne, Kessler & Goldstein, 1225 Connecticut Ave-
`
`(54) Title: INSULINOTROPIC HORMONE
`
`(57) Abstract
`
`A fragment of glucagon-like peptide I (GLP-1) has been found to be an insulinotropic hormone. This insulinotropic
`hormone comprises amino acid residues 7-37 of GLP-1. The insulinotropic hormoneis useful as a potential therapy for Di-
`abetes Mellitus.
`
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCTonthe front pages ofpamphlets publishing international appli-
`cations under the PCT.
`
`Madagascar Mali
`
`Austria
`Australia
`Barbados
`Belgium
`Bulgaria
`Benin
`Brazil
`Central African Republic
`Congo
`Switzerland
`Cameroon
`Germany, Federal Republic of
`Denmark
`Finland
`
`AT
`AU
`BB
`BE’
`BG
`BJ
`BR
`CF
`CG
`CH.
`CM
`DE
`DK
`FI
`
`France
`Gabon
`United Kingdom
`Hungary
`Ttaly
`Japan
`Democratic People’s Republic
`of Korea
`Republic of Korea
`Liechtenstein
`Sri Lanka
`Luxembourg
`Monaco
`
`Mauritania
`Malawi
`Netherlands
`Norway
`Romania
`Sudan
`Sweden
`Senegal
`Soviet Union
`Chad
`Togo
`United States of America
`
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`

`WO 87/06941
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`PCT/US87/01005
`
`-l-
`
`INSULINOTROPIC HORMONE
`
`- BACKGROUND OF THE INVENTION
`
`Field of the Invention
`/This invention is directed to the discovery that
`certain peptide fragments of
`the prehormone, proglu-~
`cagon, possess hormonal activities and can be used to
`stimulate the synthesis and secretion of the hormone,
`insulin. These peptide fragments are useful
`in ther-
`apy 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, _catecholemines,
`etc.), but also by local paracrine influences.
`The
`major pancreatic islet hormones (glucagon,
`insulin and
`somatostatin)
`interact
`amongst
`their
`specific cell
`types
`(A, B, and D cells,
`respectively) to modulate
`secretory responses mediated by the metabolites. Al-
`though insulin secretion is predominantly controlled
`by blood levels of glucose, glucagon and somatostatin
`stimulate and inhibit glucose-mediated insulin secre-
`tory responses, respectively.
`In addition to the pro-
`
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`WO 87/06941
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`.
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`insulin se-
`posed interislet paracrine regulation of
`cretion,
`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 se-
`cretion 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,
`gastric inhibitory peptide, vasoactive intestinal pep-
`tide and glicentin.
`These peptides variously regu-
`late carbohydrate metabolism, gastrointestinal mobil-
`ity and secretory processing. The principal recognized
`actions of pancreatic glucagon, however, are to pro-
`mote glycogenolysis and gluconeogenesis, resulting in
`an elevation of blood sugar levels.
`In this regard,
`the actions of glucagon are counterregulatory to those
`of
`insulin and may contribute to the hyperglycemia
`that accompanies Diabetes mellitus
`(Lund, P. K. et
`al., Proc. Natl. Acad. Sci., USA, 79: 345-349 (1982)).
`Glucagon has been found to be capable of binding
`to specific receptors which lie on the surface of in-
`sulin producing cells. Glucagon, when bound to these
`receptors, stimulates the rapid synthesis of CAMP, by
`these cells.
`‘cAMP,
`in turn, has been found to stimul-
`ate insulin expression (Korman, L.¥. etal., Diabetes,
`34:717-722 (1985)).
`Insulin acts to inhibit glucagon
`synthesis (Review of Medical Physiology, Ganong, W.F.,
`1979 Lange Publications, Los Altos, California (p.
`273).
`Thus the expression of glucagon is carefully
`
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`WO87/06941
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`regulated by insulin, and ultimately by the serum glu-
`cose level.
`
`The glucagon gene is initially tranlated from a
`630 base pair precursor to form the polypeptide, pre-
`proglucagon (Lund et al. (1982)). This polypeptide is
`subsequently processed to form proglucagon. Patzelt,
`Cc. et al., Nature, 282: 260-266 (1979), demonstrated
`that proglucagon was subsequently cleaved into gluca-
`gon and a
`second polypeptide.
`Subsequent work by
`Lund, P. K. et al., Lopez L. C. et al., and Bell, G.
`I. et al.,
`(Nature) 302:716~-718
`(1983) demonstrated
`that the proglucagon molecule was cleaved immediately
`after lysine-arginine dipeptide residues.
`Studies of
`proglucagon produced by
`channel catfish (Ictalurus
`punctata) indicated that'glucagon from this animal was
`also proteolytically cleaved after adjacent
`lysine-
`arginine and arginine-arginine dipeptide residues (An-
`drews, 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.etal, dis-
`covered the mammalian proglucagon was cleaved at ly-
`sine-arginine or arginine arginine dipeptides,
`and
`demonstrated that
`the proglucagon molecule contained
`three discreet and highly homologous peptide molecules
`which were designated glucagon, glucagon-like protein
`1 (GLP-1) and glucagon-like protein 2
`(GLP-2). Lopez
`et al. concluded that glucagon-like protein 1 was 37
`amino acid residues long and that glucagon-like pep-
`tide 2 was 34 amino acid residues long.
`Analogous
`studies on the structure of rat preproglucagon reveal~
`ed a similar pattern of proteolytic cleavage between
`adjacent
`lysine-arginine or arginine-arginine dipep-
`
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`WO 87/06941
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`tide residues, resulting in the Formation of glucagon,
`GLP-1l and GLP-2
`(Heinrich, G. et al., Endocrinol.,
`115: 2176-2181 (1984)).
`Human rat, bovine, and ham-
`ster sequences of GLP-l have been found to be identi-
`cal
`(Ghiglione, M. et al., Diabetologia, 27:599-600
`(1984)).
`regarding
`The conclusion reached by Lopez et al.
`the size of GLP-1 was confirmed by the work of Utten-
`thal, L.O. et al. (J. Clin. Endocrinol. Metabol., 61:
`472-479 (1985)). Uttenthal et al. examined the molec~
`ular forms of GLP-l which were present
`in the human
`pancreas. Their research shows that GLP-1 and GLP-2
`are’ present in the pancreas as 37 amino acid and 34
`amino acid peptides, respectively.
`The similarity between GLP-1 and glucagon suggest-
`ed to early investigators that GLP-1 might have bio-
`logical 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
`investigators failed to identify any
`physiological role for GLP=1
`(Lopez, L. C. et al.).
`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 related-
`ness between glucagon and GLP-1 might be artifactual
`(Ghiglione, M. et al.).
`an
`reveals
`the prior art
`Thus,
`in conclusion,
`awareness of the processing of a glucagon hormone pre~
`cursor into a set of peptides sharing extensive homo-
`logy.
`It has been widely assumed by those of skill in
`the art that these highly related glucagon-like pep-
`tides would have a biological activity. Nevertheless,
`extensive investigations designed to elucidate the
`
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`WO 87/06941
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`biological effects of these molecules had been unsuc-
`cessful.
`
`SUMMARY OF THE INVENTION
`The hormone glucagon is known to 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-l has 37 amino
`acids in its unprocessed form.
`This
`invention dis-
`closes that
`the unprocessed GLP-1 is naturally con-
`verted to a 31 amino acid long peptide (7-37 peptide)
`having amino acids 7-37 of GLP-l.
`This processing
`occurs in the pancreas and the intestine.
`The 7-37
`peptide is an insulinotropic hormone which had not
`previously been described.
`The hormone's activity
`appears to be specific for the pancreatic beta cells
`where it appears to induce the biosynthesis of insu-
`lin.
`The unprocessed GLP-l peptide is essentially
`unable to mediate the induction of insulin biosynthe-
`sis,
`The
`insulinotropic hormone
`is useful
`in the
`study of the pathogenesis of maturity onset diabetes
`mellitus, a condition in which the dynamics of insulin
`secretion are abnormal. Moreover,
`the insulinotropic
`hormone is useful in therapy for this disease.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1 shows the DNA structure and corresponding
`amino acid sequence of human, rat and hamster prepro-
`glucagons.
`The preproglucagon precursor
`is proteo-
`lytically cleaved at sites indicated by circles.
`
`os
`
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`WO 87/06941
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`.
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`PCT/US87/01005
`
`the effect of GLP-l peptides on
`shows
`Figure 2
`insulin mRNA levels in rat insulinoma cells.
`Figure 3
`shows
`the effects of GLP-l peptides on
`~ angiotensingen mRNA levels in rat insulinoma cells.
`Figure 4
`shows
`the effects of GLP-l peptides on
`actin mRNA levels in rat insulinoma cells.
`Figure 5 shows the effect of GLP-l1 (1-37) on pro-
`lactin mRNA levels in GH4 cells.
`Figure 6 shows the effects of GLP-1 (1-37) on ACTH
`MRNA levels in AtT-20 cells.
`
`DESCRIPTION OF THE PREFERRED EMBODIMENTS
`_ Peptide moieties (fragments) chosen from the de-
`termined amino acid sequence of human GLP-1 constitute
`the starting point
`in the development comprising the
`present invention.
`The amino acid sequence for GLP-1
`has been reported by several researchers (Lopez, L. C.
`et al. (1983); Bell, G. I. et al.,
`(Nature) 302:716-
`718 (1983); Heinrich, G. et al. (1984); Ghiglione, M.
`et al. (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 GLP-1
`(1-37)
`refers to a
`GLP-1 polypeptide having all
`amino
`acids
`from 1
`(N-terminus)
`-through 37
`(C-terminus).
`Similarly,
`GLP-1 (7-37) refers to a GLP-1 polypeptide having all
`amino acids from 7
`(N-terminus)
`through 37
`(C-term-
`
`inus).
`the peptide fragments are syn-
`In one embodiment,
`thesized by the well-known solid phase peptide syn-
`
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`WO 87/06941
`
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`
`PCT/US87/01005
`
`thesis described by Merrifield, J. M., Chem. Soc., 85:
`2149 (1962), and Stewart and Young, Solid Phase Pep-
`tide 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 pro-
`glucagon peptide or of GLP-1 through the use of recom-
`binant DNA technology, as disclosed by Maniatis, T. et
`al., Molecular Biology:
`A_ Laboratory Manual, Cold
`Spring Harbor, NY 1982, which is hereby incorporated
`by .reference.
`The invention pertains to a peptide fragment which
`is insulinotropie and is derivable from a naturally-
`occurring amino acid sequence.
`The invention comprises a peptide fragment having
`the following amino acid sequences
`
`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-Gly
`
`and functional derivatives thereof,
`
`these fragments
`
`and functional derivatives being substantially free of
`
`natural contaminants and having insulinetropic activ-
`ity.
`:
`Of particular interest are peptides of the follow-
`
`ing formula:
`
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`WO 87/06941
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`CL) H)N--X--CO--R™
`
`wherein i is OH, OM or -nR?R?;
`M is a pharmaceutically acceptable
`cation or a lower (cpg) branched or unbranched
`alkyl group; R? and R”
`are the same or different
`and selected from the group consisting of hydrogen
`and a lower
`(C-C¢) branched or unbranched alkyl
`group; and
`
`X is the amino acid sequence or pep-
`
`&ide fragment described above;
`(2)
`The acid addition salts thereof; and
`(3)
`The protected or partially protected deriva-
`tives thereof.
`The invention further pertains to a method for
`enhancing the expression of insulin which comprises:
`providing to a mammalian pancreatic B-type
`islet cell an effective amount of the insulinotropic
`peptides disclosed above.
`,
`Included within the scope of the present invention”
`are those amino acid sequences in the above peptides
`which are capable of
`functioning as
`insulinotropic
`hormones.
`Included as well are the use of additional
`amino acid residues added to enhance coupling to car-
`rier protein or amino acid residues added to enhance
`the insulinotropic effect.
`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 nat-
`ural contaminants with which GLP-1
`(7-37) might be
`
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`WO87/06941
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`other peptides,’ carbohydrates, gly-
`associated are:
`cosylated peptides, lipids, membrane, etc.
`A material
`is also said to be substantially free of natural con-
`taminants if these contaminants are substantially ab-
`sent from a sample of the material.
`The interchangeable terms "peptide fragment" and
`"peptide moiety" are meant
`to include both synthetic
`and naturally-occurring amino acid sequences derivable
`from a naturally occurring amino acid sequence.
`A peptide is said to be "derivable from a natural-
`ly-occurring amino acid sequence" if it can be obtain-
`ed by fragmenting a naturally-occurring sequence, OF
`if ‘it can be synthesized based upon a knowledge of the
`sequence of the naturally occuring amino acid sequence
`ot of the genetic material
`(DNA or RNA) which encodes
`this sequence.
`The
`invention further pertains to polypeptides
`that,
`in addition to the chosen sequence, may contain
`or lack one or more amino acids that may not be pre-
`sent in the naturally-occurring sequence wherein such
`polypeptides are functionally similar
`to the chosen
`polypeptide.
`Such polypeptides for the present inven-
`tion, are termed "functional derivatives," provided
`that they demonstrate insulinotropic activity which is
`substantially similar to that of GLP-l (7-37).
`An “insulinotropic activity" relates to the abil-
`ity of a substance to stimulate, or cause the stimula-~
`tion of,
`the synthesis or expression of
`the hormone
`insulin.
`the amino acid residues
`As
`is known in the art,
`may be in their protected or unprotected form, using
`appropriate amino or carboxyl protecting groups. Use-
`
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`
`ful cations are alkali or alkaline earth metallic ca-
`‘tions (i.e., Na, K, Li, 1/2Ca, 1/2Ba, etc.) or amine
`cations
`(i.e.,
`tetraalkylammonium,
`trialkylammonium,
`where alkyl can be Cy-Cyo)-
`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 acid salts, i.e., HBr, HI, or more preferably,
`
`HCl.
`
`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, respective~
`ly. 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/al-
`bumin buffer with a pH of 7.4 was employed.
`The in-
`cubation was prepared with the consecutive condition
`of 500 ul of phosphate buffer, 50 ul of perfusate sam-
`ple or rat insulin standard in perfusate, 100 ul of
`anti-insulin
`antiserum
`(Wellcome
`Laboratories;
`1:40,000 dilution), and 100 ul of (17°1] insulin, giv-
`ing a total volume of 750 ul
`in a 10 X 75-mm dispos-
`able 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
`
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`-ll-
`
`into the cell culture medium of cells grown in tissue
`culture, one preferably incorporates radioactive label
`into proinsulin. Although any radioactive label cap-
`able of
`labelling a ‘polypeptide can be used,
`it is
`preferable to use 34 leucine in order to obtain label-
`led proinsulin.
`tabelling can be done for any period
`of time sufficient to permit
`the formation of a de-
`tectably labelled pool of proinsulin molecules; how-
`ever, it is preferable to incubate cells in the pre-
`sence of radioactive label for a 60 minute time per-
`iod.
`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 in-
`sulinoma cells.
`Such cells can be grown in any suit-
`able medium; however, it is preferable to use DME med-
`ium 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 modi-
`fication of the method of Penhos, J. C. etal., (Dia-
`betes, 18:733-738 (1969)).
`Fasted male Charles River
`strain albino rats, weighing 350-600 g, were anesthe-
`tized with an intraperitoneal injection of Amytal Sod-
`ium (Eli Lilly and Co.; 160 ng/kg). Renal, adrenal,
`gastric, and lower colonic blood vessels are ligated.
`The entire intestine was
`resected except
`for about
`four cm of duodenum and the descending colon and rec-
`tum,
`- Therefore, only a small part of
`the intestine
`was perfused,
`thus minimizing possible interference by
`enteric substances with glucagon-like immunoreactiv-
`ity.
`The perfusate was a modified Krebs-Ringer bicar-
`
`a
`
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`-12-
`
`bonate buffer with 4% dextran T70 and 0.2% bovine ser-
`um albumin (fraction V), and was bubbled with 95% 05
`and 5% CO,.
`A nonpulsatile flow, 4-channel roller
`bearing pump (Buchler polystatic, Buchler Instruments
`Division, Nuclear-Chicago Corp.) was used,
`and
`a
`switch from one perfusate source to another was accom-
`plished by switching a 3-way stopcock.
`The manner
`in
`which perfusion was performed, monitored, and analyzed
`followed the method of Weir, G. C. et al.
`(J. Clin,
`Investigat. 54:
`1403-1412
`(1974)), which is hereby
`incorporated by reference.
`The compounds of the present invention can be for-
`mulated according to known methods to prepare pharma~
`ceutically useful compositions, whereby GLP-l
`(7-37)
`or its functional derivatives are combined in admix-
`ture with a pharmaceutically acceptable carrier vehi-
`cle. Suitable vehicles and their formulation,
`inclu-
`sive of other human proteins, e.g. human serum albu-
`min, are described for example in Remington's Pharma-
`ceutical Sciences (16th Ed. A. Oslo Ed. Mack, Easton
`PA (1980)).
`In order to form a pharmaceutically ac- -
`ceptable composition suitable for effective adminis-
`tration,
`such compositions will contain an effective
`amount of the GLP-1 (7-37), or its functional deriva-
`tives,
`together with a suitable amount of carrier ve-
`hicle.
`.
`Compositions containing GLP-1 (7-37) or its func-
`tional derivatives 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, although a lower or higher dosage may be ad-
`ministered.
`The required dosage will depend upon the
`
`MPI EXHIBIT 1057 PAGE 14
`
`MPI EXHIBIT 1057 PAGE 14
`
`MPI EXHIBIT 1057 PAGE 14
`
`

`

`WO87/06941
`
`PCT/US87/01005
`
`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, com-
`positions containing GLP-1
`(7-37) are dissolved in
`distilled water and the pH-value is adjusted to about
`6
`to 8.
`In order
`to facilitate the lyophilization
`process resulting in a suitable product, lactose could
`be added to the solution.
`The solution is then filter
`sterilized, introduced into vials, and lyophilized.
`The concentration of GLP-1 (7-37)
`in these composi-
`tions may vary from 107224 to 107M.
`Additional pharmaceutical methods may be employed
`to control the duration of action. Controlled release
`_ preparations may be achieved by the use of polymers to
`complex or adsorb GLP-1 (7-37) or its functional der-
`ivatives.
`The controlled delivery may be exercised by
`selecting appropriate macromolecules
`(for
`example,
`polyesters, polyamino acids, polyvinyl pyrrolidone,
`ethylenevinylacetate, methylcellulose, carboxymethyl-
`cellulose, and protamine sulfate) and the concentra-
`tion of macromolecules as well as the methods of
`in-
`
`corporation in order to control release. Another pos-
`sible method to control the duration of action by con-
`
`trolled release preparations is to incoporate GLP-1
`(7-37)
`into particles of a polymeric material such as
`polyesters, polyamino acids, hydrogels, poly (lactic
`acid) or ethylene vinylacetate copolymers. Alterna-
`tively,
`instead of
`incorporating GLP-1
`(7-37)
`into
`these polymeric particles,
`it is possible to entrap
`GLP-1 (7-37)
`in microcapsules prepared,
`for example,
`
`MPI EXHIBIT 1057 PAGE15
`
`MPI EXHIBIT 1057 PAGE 15
`
`MPI EXHIBIT 1057 PAGE 15
`
`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-l14-
`
`by coacervation techniques or by interfacial polymer-
`ization, for example, hydroxymethylcellulose or gela-
`tin-microcapsules and poly (methylmethacrylate) micro-
`capsules, respectively, or in colloidal drug delivery
`systems, for example,
`liposomes, albumin microspheres,
`microemulsions, nanoparticles, and nanocapsules or
`in
`macroemulsions.
`Such
`teachings are disclosed in
`Remington's Pharmaceutical Sciences (1980).
`
`SPECIFIC EXAMPLES
`
`EXAMPLE 1
`. Rat
`insulinoma cells of cell line RIN-38 were de-
`rived from a continuous islet cell line, RIN-r, which
`was established from a transplantable rat islet cell
`tumor
`(Gazdar, A. F. et al., Proc. Nat'l Acad. Sci.
`"U.S.A. 77: 3519-3523 (1980)).
`The cells were main-
`tained in DMEM (Gibco) at a glucose concentration of
`4,500 mg/L, and supplemented with 10% heat-inactivated
`fetal bovine serum (Gibco), 100 U/ml of penicillin and
`100 ug/ml of streptomycin.
`Incubations were carried
`out at 37°C in 95% air:
`5% CO). Cells grown in the
`above manner were washed and resuspended in DMEM (Gib-
`co) containing 0.1% bovine serum albumin and 25 mM
`glucose. Cells were incubated with varying concentra-
`tions of GLP=-1
`(1-37), GLP-1
`(7-37) or GLP-1
`(1-36
`des-gly-arg amide) for six hours,
`following which the
`effects of
`these agents on insulin mRNA expression
`were determined. Cellular RNA was analyzed for insu-
`lin specific mRNA as follows:
`cellular RNA was ex-
`
`MPI EXHIBIT 1057 PAGE 16
`
`MPI EXHIBIT 1057 PAGE 16
`
`MPI EXHIBIT 1057 PAGE 16
`
`

`

`WO87/06941
`
`PCT/US87/01005
`
`~-15-
`
`tracted from solid tumors and cells by homogenization
`in guanadine thiocyanate and sedimentation through a
`cesium chloride cushion.
`Poly A’ RNA was isolated by
`oligo dT cellulose chromatography (Aviv, H. et al.,
`Proc, Nat'l Acad. Sci. U.S.A. 69: 1408-1412 (1972)).
`20 ug of total RNA from each sample was fractionated
`by size on a 1.4% agarose gel after denaturation in
`glyoxal,
`followed by electrotransfer to a nylon mem-
`brane (Nytran; Schleicher and Schuell). Blotted mem-
`branes were baked for two hours at 80°C under vacuum,-
`prehybridized in 1M NaCl / 1% SDS/ 10% Dextran sulfate
`at 50°C overnight and hybridized at the same tempera-
`ture for 24 h after addition of
`the labelled probes
`(3-5 x 10° cpm/ml);
`they were then washed at 55°C
`twice in 1 X SSC (0.15 M NaCl / 0.015M Na citrate): /
`1% SDS), and exposed to X-ray film for varying times
`at
`-70°C with an intensifying screen.
`In all cases
`the concentration of peptides was 107’.
`The result of this experiment
`is shown in Figure
`2. Lanes 1-3 (control cells), 4-6 (GLP-1 (1-37)), 7-9
`GLP-1 (7-37), 10-12 (GLP~1(1-36 des- gly arg -amide)
`shows the amount of
`insulin specific mRNA produced.
`Triplicate experimental results are presented for each
`peptide.
`Using a microdensitometer the relative amounts of
`insulin specific mRNA were determined.
`This experi-~
`ment revealed that, at equal peptide concentrations,
`GLP-l
`(7-37)
`stimulated insulin gene expression to
`more than 3 times the level found in control (untreat-
`
`ed) cells.
`
`a
`
`MPI EXHIBIT 1057 PAGE 17
`
`MPI EXHIBIT 1057 PAGE 17
`
`MPI EXHIBIT 1057 PAGE 17
`
`

`

`WO87/06941
`
`PCT/US87/01005
`
`-16-
`
`EXAMPLE 23:
`line RIN-38 were
`Rat
`insulinoma cells of cell
`grown in DME medium as described in Example l. After
`incubation with 107’M GLP=-1
`(1-37, GLP-1
`(7-37) and
`GLP-1
`(1-36),
`the concentrations of
`insulin in the
`cell culture mediums were determined by radioimmun-
`assay (as described above).
`Insulin protein levels
`were determined after incubation for six hours.
`The
`results of this experiment are shown in Table 1.
`
`PEPTIDE ADDED
`
`none
`
`GIP-1 (1-37)
`
`TABLE 1
`
`|
`
`Insulin Produced
`(uUnits/ML)
`
`°
`
`2800
`
`5000
`
`EXAMPLE 3:
`The pancreas of live rat was perfused with vary~_
`ing concentrations of GLP-1 (1-37) and GLP=-1 (7-37) as
`described above. At one minute intervals, rat serum
`insulin levels
`in picograms/ml were determined by
`radioimmunassay (as described above).
`The results of
`this experiment are shown in Table 2. Perfusions were
`1M,
`5 x
`done using peptide concentrations of
`5 x 10°
`“12 yy,
`5 x 104m ana 5 x 10
`107°m, and 5 x 1071,
`Peptides were added after the zero minute serum value
`had been determined.
`
`MPI EXHIBIT 1057 PAGE 18
`
`MPI EXHIBIT 1057 PAGE 18
`
`MPI EXHIBIT 1057 PAGE 18
`
`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-17-
`
`GLP-1 (1-37) was found to mediate a 3.4-fold in-
`crease in serum insulin concentrations when perfused
`into rat pancreas at a concentration of 5 x 107/M; at
`8
`a concentration of 5-x 10 M this peptide was capable
`of mediating only a 2-fold increase in serum insulin
`-10y this peptide
`levels. At a concentration of 5 x 10
`was found to mediate only a 20% increase in serum in-
`
`sulin levels.
`GLP=1 (7-37) was found to be capable of stimulat-
`ing a 132-fold increase in insulin levels when pro-
`vided to rat pancreas at a concentration of 5 x 107M.
`At a 10-fold lower concentration (5 x 107°m) this pep-
`tide was capable of directing a 21l-fold increase in
`the serum concentration of
`insulin.
`At a‘ concen-
`tration of 5 x 1071[m, GLP-1 (7-37) was
`found to be
`capable of mediating an increase in serum insulin
`levels
`(32-fold).
`Even at a concentration of
`5 x
`10724m, GLP-1 (7-37) delivered a 15-fold increase in
`insulin levels whereas GLP-l
`(1-37) was without
`
`effect.
`is more
`(7-37)
`that GLP-1
`shows
`This experiment
`than 1,000-fold more potent than GLP-1 (1-37) in stim-
`ulating insulin expression in vivo.
`In addition,
`the
`GLP-1 peptides had no effects on the release of the
`peptide hormones glucagon and somatostatin in these
`same experiments.
`Thus,
`the stimulatory effects of
`GLP-1 are specific for the beta cells and do not act
`on pancreatic alpha or delta cells.
`
`MPI EXHIBIT 1057 PAGE 19
`
`MPI EXHIBIT 1057 PAGE 19
`
`MPI EXHIBIT 1057 PAGE 19
`
`

`

`WO 87/06941
`
`PCT/US87/01005
`
`~18-
`
`Table 2
`
`Insulin Produced (picograms/ml) at
`’
`Peptide Concentration
`
`Time
`(Minutes) 5x10sxi07’m sx1072°m sxio74m sx10722m
`
`GLP-1
`(7-37)
`
`GLP-1
`(1-37)
`
`+
`
`0
`l
`2
`3
`
`0
`1
`2
`3
`
`50
`6600
`4700
`1700
`
`1400
`4700
`2900
`2200
`
`925
`20,700
`10,500.
`4,000
`
`«3,000
`6,000
`2,000
`2,000
`
`205
`7400
`1800
`760
`
`500
`600
`640
`430
`
`.
`
`160
`«2400
`1700
`1900
`
`340
`180
`230
`340
`
`50
`50
`50
`98
`
`50
`50
`160
`50
`
`7
`
`MPI EXHIBIT 1057 PAGE 20
`
`MPI EXHIBIT 1057 PAGE 20
`
`MPI EXHIBIT 1057 PAGE 20
`
`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-19-
`
`EXAMPLE 4:
`
`to determine whether glucagon-like pro-
`In order
`teins were capable of affecting cellular cAMP levels
`the effects of GLP-l- (7-37) and GLP-1 (1-37) on cAMP
`levels in RINS-38 insulinoma cells was determined.
`
`in 26 well
`Cells were grown as described in Example 1,
`culture dishes. Varying amounts of glucogon-like pep-
`tides were added to culture wells in triplicate.
`A£f-
`ter permitting incubation for’ 10 minutes
`the total
`cell media was examined for cAMP, and the concentra-
`tion of cAMP was determined.
`The results of this ex-
`periment are shown in Table 3.
`20 ul from'each cul-
`ture well was assayed.
`
`Peptide
`
`Concentration
`
`Table 3
`|
`pMOLES OF cAMP PRODUCED
`
`Expt
`
`Expt
`
`
`cM)
`I
`II
`
`0
`
`107°
`107?
`1078
`107?
`10729
`
`yg7tt
`
`,
`
`140
`
`400
`370
`494
`515
`253
`
`|
`
`533
`
`91
`
`170
`120
`160
`100°
`90
`
`90
`
`This experiment reveals that GLP-1 (7-37) was cap-
`able of stimulating cAMP levels even when present at a
`
`MPI EXHIBIT 1057 PAGE21
`
`MPI EXHIBIT 1057 PAGE 21
`
`MPI EXHIBIT 1057 PAGE 21
`
`

`

`WO87/06941
`
`PCT/US87/01005
`
`~20-
`
`The increase in CAMP levels
`concentration of 10724M.
`is an indication that GLP-1 (7-37)
`is capable of
`in-.
`
`teracting with cellular receptors.
`
`EXAMPLE 5
`In order to demonstrate that the effects of GLP-l
`(1-37), GLP-1
`(1-36) and GLP-1
`(7-37) were specific
`for insulin, and were not capable of inducing or pro-
`voking non-specific gene expression,
`the effect of
`these peptides on the levels of actin and angiotens-~
`inogen mRNAs were conducted.
`RIN-38 insulinoma cells
`were grown as described in Example 1 and incubated in
`the presence of GLP-1 (1-37), GLP-1 (7-37), or GLP-1
`(1-36) des-Gly arg (Peninsula Laboratories).
`In all
`cases the concentration of peptides was 107/M.
`Incu-
`bations were for six hours. Messenger RNAs specific
`for insulin, actin, or angiotensinogen were identified
`by Northern hybridization as described in Example 1.
`The results of this experiment are shown in Figure 2
`(insulin mRNA); Figure 3
`(anginotensinogen mRNA); and
`Figure 4 (actin mRNA).
`mRNA levels were determined in_
`arbitrary densitometric units obtained from scanning
`films of
`the RNA gels of Figures 2, 3, and 4.
`The
`mRNA levels are shown in Table 4.
`
`. MPI EXHIBIT 1057 PAGE 22
`
`MPI EXHIBIT 1057 PAGE 22
`
`MPI EXHIBIT 1057 PAGE 22
`
`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-21-
`
`TABLE 4
`
`EFFECTS OF GLUCAGON-LIKE PEPTIDES ON
`CELLULAR LEVELS OF mRNAs ENCODING
`INSULIN, ACTIN AND ANGIOTENSINOGEN
`IN RIN-38 INSULINOMA CELLS
`
`PEPTIDE*
`
`INSULIN
`
`ACTIN
`
`ANGIOTENS INOGEN
`
`
`MESSENGER RNAS
`
`(7-37)
`GLP-I
`(1-37)
`GLP-I
`(1-36) des-Gly
`GLP-I
`Arginineamide
`Control
`(no peptide)
`
`4.2340.74
`1.874+0.56
`2.78+0.80
`
`0.8240.08
`0.9140.02
`0.8840.03
`
`2.78+0.46
`2.25+0.20
`2.5640.22
`
`1.2840.23
`
`0.89+0.05
`
`2.674+0.31
`
`é
`
`EXAMPLE 6
`GLP-1 (1-37) was examined to determine whether it
`could induce the biosynthesis of hormones other than
`insulin.
`Thus, GLP=-1
`(1-37)
`(at a concentration of
`107’™) was added to a rat
`islet glucagon-producing
`cell
`line and two pituitary cell
`lines
`(GH4
`and
`AtT-20) which were capable of producing the hormones
`prolactin and ACTH,
`respectively, and the amount of
`hormone

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