throbber
PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(SI) International Patent Classification 4 :
`C07K 7 /10, 7 /'34, A61K 37 /02
`A61K37/28
`
`(11) lnterD2tional Publication Number:
`
`WO 87/06941
`
`Al
`
`(43) International Publication Date:
`19 November 1987 (19.11.87)
`
`(21) International Application Number:
`
`PCT/US87/01005
`
`(22) International Filing Date:
`
`5 May 1987 (05.05.87)
`
`(31) Priority Application Number:
`
`859,928
`
`(81) Designated States: AT (European patent), BE (Euro•
`pean patent), CH (European patent), DE (European
`patent), FR (European patent), GB (European pa(cid:173)
`tent), IT (European patent), JP, LU (European pa•
`tent), NL (European patent), SE (European patent).
`
`(32) Priority Date:
`
`(33) Priority Country:
`
`5 May 1986 (05.05.86) Published
`With international search report.
`us
`
`(71) Applicant: THE GENERAL HOSPITAL CORPORA·
`TION [US/US]; Fruit Street (Bar-3), Boston, MA
`02ll4 (US).
`
`(72) Inventor: HABENER, Joel ; 217 Plymouth Road, New(cid:173)
`ton Highlands, MA 02161 (US).
`
`(74) Agents: GOLDSTEIN, Jorge, A. et al.; Saidman,
`Sterne, Kessler & Goldstein, 1225 Connecticut Ave(cid:173)
`nue, N.W., Suite 300, Washington, DC 20036 (US).
`
`..
`
`(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 insulinotropi.c hormone is useful as a potential therapy for Di(cid:173)
`abetes Mellitus.
`
`.1
`..
`
`FRESENIUS EXHIBIT 1039
`Page 1 of 36
`
`

`

`•
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international appli(cid:173)
`cations under the PCT.
`
`AT Austria
`AU Australia
`BB Barbados
`BE . Belgium
`BG Bulgaria
`BJ Benin .
`BR Brazil
`CF Central African Republic
`CG Congo
`CH . Switzerland
`CM Cameroon
`DE Gennany, Federal Republic of
`D~ Denmark
`FI Finland
`
`FR France
`GA Gabon
`GB United Kingdom
`HU Hungary
`IT
`Italy
`Japan
`JP
`KP Democratic People's Republic
`of Korea
`KR Republic of Korea
`LI Liechtenstein
`LK Sri Lanka
`LU Luxembourg
`MC Monaco
`MG Madagascar
`
`ML Mali
`MR Mauritania
`MW Malawi
`NL Netherlands
`NO Norway
`RO Romania
`SD Sudan
`SE Sweden
`SN Senegal
`SU Soviet Union
`ID Chad
`TG Togo
`US United States of America
`
`FRESENIUS EXHIBIT 1039
`Page 2 of 36
`
`

`

`WO 87/06941
`
`PCT /US87/01005
`
`-1-
`
`IRSO'LrHOTROPIC HORMONB
`
`BACKGROUND OF THE INVENTION
`
`Field of the Invention
`• This invention is directed to the discovery that
`certain peptide fragments of the prehormone, proglu(cid:173)
`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(cid:173)
`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 CA, B, and D cells, respectively) to modulate
`secretory responses mediated by the metabolites.. Al(cid:173)
`though insulin secretion is predominantly controlled
`by blood levels of glucose, glucagon and somatostatin
`stimulate and inhibit glucose-mediated insulin secre(cid:173)
`tory responses, respectively.
`In addition to the pro-
`
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`WO 87/06941
`
`PCT/US87 /01005
`
`-2-
`
`posed i nterislet paracrine regulqtion of insulin se(cid:173)
`cretion, there is evidence to support the existence of
`i ns ulinot r opic factors in the intestine. This concept
`originates from the -observations that glucose taken
`orally is a much more potent stimulant of insulin se(cid:173)
`cretion than is a comparable amount of glucose given
`intravenously.
`The human hormone, glucagon, is a 29-amino acid
`peptide hormone produced in the A-c·ells of the pan(cid:173)
`creas. The . hormone belongs to a multi-gene family of
`structurally related peptides that include secretin,
`gastric inhibitory peptide, vasoactive intestinal pep(cid:173)
`tide and glicentin.
`These peptides variously regu-
`•
`late carbohydrate metabolism, gastrointestinal mobil-
`ity and secretory processing. The principal recognized
`actions of pancreatic gluca~on, however, are to pro(cid:173)
`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
`(Lund, P. K. !!
`that accompanies Diabetes mellitus
`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(cid:173)
`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(cid:173)
`ate insulin expression (Korman, L.Y. et al., Diabetes,
`l,!:·717-722 (198 5)).
`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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`

`WO 87/06941
`
`PCT/US87/01005
`
`-3-
`
`. - -
`
`I
`
`regulated by insulin, and ultimat~ly by the serum glu(cid:173)
`cose level.
`The glucagon gene is init~ally tranlated from a
`630 base pair precursor to form the polypeptide, pre(cid:173)
`proglucagon (Lund et al. (1982)). This polypeptide is
`subsequently processed to form proglucagon. Patzelt,
`c. et al . , Nature, 282: 260-266 (1979), demonstrated
`that proglucagon was subsequently cleaved into gluca(cid:173)
`gon an~ 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 ·glucl!gon from this animal was
`also . proteolytically cleaved after adjacent lysine(cid:173)
`arginine and arginine-arginine dipeptide residues (An(cid:173)
`drews, P. C. et al., J. Biol. Chem., 260: 3910-3914
`(1985)). Lopez, L. c. et!_!., (Proc. Natl. Acad. Sci.
`~ !Q.: 5485-5489 C 1983)), and Bell, G. I. et al°, dis(cid:173)
`covered the mammalian proglucagon was cleaved at ly(cid:173)
`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
`.
`ami_n~ acid residues long and that glucagon-like_ pep(cid:173)
`tide 2 was 34 amino acid residues long. Analogous
`studies on the structure of rat preproglucagon reveal(cid:173)
`ed a similar pattern of proteolytic cleavage between
`adjacent lysine-arginine or arginine-arginine dipep-
`
`- -
`
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`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-4-
`
`tide residues, resulting in the formation of glucagon,
`GLP-1 and GLP-2
`( Heinrich, G. et al., Endocr inol.,
`-
`115: 2176-2181 (1984)). Human rat, bovine, and ham-
`.
`ster sequences of GLP-1 have been found to be identi-
`cal (Ghiglione, M. et al., Diabetologia, 27:599-600
`( 1984)}.
`The conclusion reached by Lopez il al. regarding
`the size of GLP-1 was confirmed by the work of Utten(cid:173)
`thal, L.O. et al. CJ. Clin. Endocrinol. Metabol., 61:
`472-479 (1985)). Uttenthal i l al. examined the molec(cid:173)
`ular 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 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(cid:173)
`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-l was in fact a hormone and whether the related(cid:173)
`ness between glucagon and GLP-l might be artifactual
`(Ghiglione, M. ~ al.).
`the prior art reveals an
`Thus,
`in conclusion,
`awareness of the processing of a glucagon hormone pre(cid:173)
`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
`
`•
`
`~
`
`FRESENIUS EXHIBIT 1039
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`

`

`WO 87/06941
`
`PCT /US87/01005
`
`-s-
`
`biological effects of these molecules had been unsuc(cid:173)
`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(cid:173)
`tides: glucagon, glucagon-like peptide 1 CGLP-1) and
`glucagon-like peptide 2 (GLP-2). GLP-1 has 37 amino
`acids in its unprocessed form.
`This invention dis(cid:173)
`closes that the unprocessed GLP-1 is naturally con(cid:173)
`verted to a 31 amino acid long peptide (7-37 peptide)
`having amino acids 7-37 of GLP-1.
`This processing
`occurs in the pancreas and the intestine. The 7-37
`peptide i~ an insulinotropic hormone which had not
`previously. been described.
`The hormoneis activity
`appears to be specific for the pancreatid beta cells
`where it appears to induce the biosynthesis of insu(cid:173)
`lin.
`The unprocessed GLP-1 peptide is essentially
`unable to mediate the induction of insulin biosynthe(cid:173)
`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 TBE DRAWINGS
`Figure l shows the DNA structure and corresponding
`amino acid sequence of human, rat and hamster prepro(cid:173)
`glucagons.
`The preproglucagon precursor is proteo(cid:173)
`lytically cleaved at sites indicated by circles.
`
`"
`
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`

`WO 87/06941
`
`PCT/US87/0100S
`
`-6-
`
`Figure 2 shows the effect of GLP-1 peptides on
`i nsul in mRNA levels in rat insulinoma cells.
`Figure 3 shows the effects of GLP-1 peptides on
`angiotensingen mRNA levels in rat insulinoma cells.
`Figure 4 shows the effects of GLP-1 peptides on
`actin mRNA levels in rat insulinoma cells.
`Figure 5 shows the effect of GLP-1 Cl-37) on pro(cid:173)
`lactin mRNA levels in GH4 cells.
`Fig~re 6 shows the effects of GLP-1 Cl-37) on ACTH
`mRNA levels .in AtT-20 cells.
`
`DESCRIPTION OF THE PREFERRED EMBODIMENTS
`Peptide moieties C fragments) chosen from the de-
`•
`termined amino acid sequence of human Gt.P-1 constitute
`the starting point in the dev&lopment comprising the
`presen~ invention. The amino acid seq~ence for GLP-1
`has been reported by several researchers C~opez, L. C.
`tl al. (1983)1 Bell, G. I. et al., (Nature) 302:716-
`718 (1983); Heinrich, G. tl !.h (1984); Ghiglione, M.
`~ al. (1984)). The structure of the preproglucagon
`gene and its corresponding amino acid sequence is
`shown in Figure l. 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
`l
`CN-terminus)
`·throug_h 37 CC-terminus).
`Similarly,
`Gt.P-1 (7-37) refers to a GLP-1 polypeptide having all
`amino acids from 7 (N-terminus) through 37 CC-term(cid:173)
`inus >.
`In one embodiment, the peptide fragments are syn(cid:173)
`thesized by the well-known solid phase peptide syn-
`
`FRESENIUS EXHIBIT 1039
`Page 8 of 36
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`

`

`WO 87/06941
`
`PCT /US87 /0 lOOS
`
`-7-
`
`thesis described by Merrifield, J: M., Chem. Soc.,!§.:
`2149 (1962), and Stewart and Young, Solid Phase Pep(cid:173)
`tide Synthesis (Free~n, 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(cid:173)
`glucagon peptide or of GLP-1 through the use of recom(cid:173)
`binant DNA technology, as disclosed by Maniatis, T. et
`al., Molecular Biology:
`A Laboratory Manual, Cold
`Spring Barbor, NY 1982, which is hereby incorporated
`by . ref er,.ence.
`The invention pertains to a peptide fragment which
`is insulinotropic and is derivable from a naturally(cid:173)
`occurring amino acid sequence.
`The invention comprises a peptide fragment having
`the following amino acid sequence:
`
`Bis-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp(cid:173)
`Val-Ser-Ser-Ty.r-Leu-Glu-Gly-Gln-Ala(cid:173)
`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 ha'ving insulinotropic activ(cid:173)
`
`ity.
`
`Of particular interest are peptides of the follow-
`
`ing formula:
`
`FRESENIUS EXHIBIT 1039
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`

`

`WO87/06941
`
`PCT /US87/01005
`
`-8-
`
`(1)
`
`wherein Rl ls OH, OM or -NR2R3;
`M is a pharmaceutically acceptable
`cation or a lo;er (C~C6) 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 c1 -c6 > branched or unbranched alkyl
`group; and
`
`.
`
`Xis the amino acid sequence or pep(cid:173)
`~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
`w-hich are capable of functioning as insulinotropic
`hormones.
`Included as well are the use of additional
`amino acid residues added to enhance coupling ~o car(cid:173)
`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 C 7-3 7) might be
`
`•
`
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`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-9-
`
`other peptides,· carbohydrates, gly(cid:173)
`associated are:
`cosylated peptides, lipids, membrane, etc. A material
`is also said to be substantially free of natural con(cid:173)
`taminants if these contaminants are substantially ab(cid:173)
`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(cid:173)
`ly-occurring amino acid sequence• if it can be obtain(cid:173)
`ed by fragmenting a naturally-occurring sequence, or
`if 'it can be synthesized based upon a knowledge of the
`sequence of the naturally occuring amino acid sequence
`or of the genetic material (ONA or RNA) waich 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(cid:173)
`sent in the naturally-occurring sequence wherein such
`polypeptides are functionally similar to the chosen
`polypeptide. Such polypeptides for the present inven(cid:173)
`tion, are termed •functional derivatives,• provided
`that they demonstrate insulinotropic activity which is
`substantially similar to that of GLP-1 (7-37).
`An "insulinotropic activity• relates to the abil(cid:173)
`ity of a substance to stimulate, or cause the stimula(cid:173)
`tion- of, the synthesis or expression of the hormone
`insulin.
`As is known in the art, the amino acid residues
`may be in their protected or unprotected form, using
`appropriate amino or carboxyl protecting groups. Use-
`
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`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-lo-
`
`ful cations are alkali or alkaline earth metallic ca(cid:173)
`·tions (i.e., Na, K, Li, l/2Ca, l/2Ba, etc.) or amine
`cations (i.e., tetraalkylammonium,
`trialkylammonium,
`where alkyl can be c1-c12 >.
`The variable length peptides may be in the form of
`the free amines Con the N-terminusl, or acid-addition
`salts thereof. Common acid addition salts are hydro(cid:173)
`halic acid salts, i.e., HBr, HI, or more preferably,
`BCl.
`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 CIRI) into the
`•
`media or circulatory system of the animal, respective-
`ly. · The•presence of IRI is detected through the use
`of a radioimrnunoassay 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 C 1972)).
`In this modification a phosphate/al-
`bumin buffer with a pH of 7.4 was employed. The in(cid:173)
`cubation was prepared with the consecutive condition
`of 500 ul of _phosphate buffer, 50 ul of perfusate sam(cid:173)
`ple or rat insulin standard in perfusate, 100 ul of
`anti-insulin
`antiserum
`(Wellcome
`Laboratories J
`1:40,000 dilution), ~nd 100 ~l of c125I] insulin, giv(cid:173)
`ing a total volume of 750 ul in a 10 X 75-mm dispos(cid:173)
`abie - 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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`

`

`WO 87/06941
`
`. PCT/US87/01005
`
`-11-
`
`into the cell culture medium of c~lls grown in tissue
`culture, one preferably incorporates radioactive label
`into proinsulin. Although any radioactive label cap(cid:173)
`able of labelling a· polypeptide can be used, it is
`preferable to use 3a leucine in order to obtain label(cid:173)
`led proinsulin. Labelling can be done for any period
`of time sufficient to permit the formation of a de(cid:173)
`tectably labelled pool of proinsulin molecules1 how(cid:173)
`ever, it is preferable to incubate cells in the pre(cid:173)
`sence of radioactive label for a 60 minute time per(cid:173)
`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(cid:173)
`able medium1 however, it is preferable to use OM! med(cid:173)
`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(cid:173)
`fication of the method of Penhos, J. c. et al., (Dia(cid:173)
`betes, 18:733-738 (1969)). Fasted male Charles River
`strain albino rats, weighing 350-600 g, were anesthe(cid:173)
`tized with an intraperitoneal injection of Amytal Sod(cid:173)
`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 an~ rec(cid:173)
`tum • . Therefore, only a small part of the intestine
`was perfused, thus minimizing possible interference by
`enteric substances with glucagon-like immunoreactiv(cid:173)
`ity. The perfusate was a modified Krebs-Ringer bicar-
`
`•
`
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`

`WO 87/06941
`
`PCT /US87/01005
`
`-12-
`
`bonate buffer with 4% dextran T70 and 0.2% bovine ser(cid:173)
`um albumin (fraction V), and was bubbled with 95% o2
`and 5% co2• A n~npulsatile flow, 4-channel roller
`bearing pump (Buchler polystatic, Buchler Instruments
`Division, Nuclear-Chicago Corp.) was used, and a
`switch from one perf usate source to another was accom(cid:173)
`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. ll= 1403-1412
`(1974)), which is hereby
`incorporated by reference.
`The compounds of the present invention can be for(cid:173)
`mulated according to known methods to prepare pharma-
`•
`ceutically useful compositions, whereby GLP-1 (7-37)
`or i.ts functional deriyatives are combined in admix(cid:173)
`ture with a pharmaceutically acceptable carrier vehi(cid:173)
`cle. Suitable vehicles and their formulati~n, inclu(cid:173)
`sive of other human proteins, e.g. human serum albu(cid:173)
`min, are described for example in Remington's Pharma(cid:173)
`ceutical Sciences (16th Ed. A. Oslo Ed. Mack, Easton
`PA C 1980)).
`In order to form a pharmaceutically ac- -
`ceptable composition suitable for effective adminis(cid:173)
`tration, such compositions will contain an effective
`amount of the GLP-1 (7-37), or its functional deriva(cid:173)
`tives, together with a suitable amount of carrier ve(cid:173)
`hicle.
`Compositions containing GLP-1 (7-37) or its func(cid:173)
`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(cid:173)
`ministered. The required dosage will depend upon the
`
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`

`WO 87/06941
`
`PCT/US87/0100!
`
`-13-
`
`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(cid:173)
`positions containing GLP-l C 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 C 7-37) in these composi(cid:173)
`tions may vary from 10-12M to 10-sM.
`Additional pharmaceutical methods may be employed
`•
`to control the duration of action. Controlled release
`preparations may be ~chieved by the use of polymers to
`complex or adsorb GLP-1 (7-37) or its functional der(cid:173)
`ivatives. The controlled delivery may be exercised by
`selecting appropriate macromolecules
`(for example,
`polyesters, polyamino acids, polyvinyl pyrrolidone,
`ethylenevinylacetate, methylcellulose, carboxymethyl(cid:173)
`cellulose, and protamine sulfate) and the concentra(cid:173)
`tion of macromolecules as well as the methods of in(cid:173)
`corporation in order to control release. Another pos(cid:173)
`sible method to control the duration of action by con(cid:173)
`trolled release preparations is to incoporate GLP-1
`(7-37) into particles of a polymeric material such as
`polyesters, polyamino acids, hydrogels; poly · c1actic
`aci~)- or ethylene vinylacetate copolymers. Alt_erna(cid:173)
`tively,
`instead of
`incorporating GLP-1
`(7-37)
`into
`these polymeric particles, it is possible to entrap
`GLP-1 ( 7-3 7) in microcapsules prepared, for example,
`
`FRESENIUS EXHIBIT 1039
`Page 15 of 36
`
`

`

`WO87/06941
`
`PCT /US87 /0l00S
`
`..
`
`-14-
`
`by coacervation techniques or by .interfacial polymer(cid:173)
`ization, for example, hydroxymethylcellulose or gela(cid:173)
`tin-microcapsules a~d poly (methylmethacrylate) micro(cid:173)
`capsules, respectiveiy, 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 l
`; Rat insulinoma cells of cell line RIN-38 were de(cid:173)
`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(cid:173)
`tained in DMEM (Gibco) at a glucose concentration of
`4,500 mg/t., and supplemented with 10% heat-inactivated
`fetal bovine serum (Gibco), 100 O/ml of penicillin and
`100 ug/ml of streptomycin. · Incubations were carried
`out at 37°c in 95% air: 5% co2• Cells grown in the
`above manner were washed and resuspended in OMEM (Gib(cid:173)
`co) containing 0.1% bovine serum albumin and 25 mM
`glucose. Cells were incubated with varying concentra(cid:173)
`tions of GLP-1 C 1-3 7), GLP-1
`( 7-3 7) or GLP-1
`( 1-3 6
`des-gly-arg amide) for six hours, following which the
`effec.ts of these agents on insulin mRNA expression
`were determined. Cellular RNA was analyzed for insu(cid:173)
`lin specific mRNA as follows:
`cellular RNA was ex-
`
`FRESENIUS EXHIBIT 1039
`Page 16 of 36
`
`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-15-
`
`tracted from solid tumors and ce~ls 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(cid:173)
`brane (Nytran1 Schleicher and Schuell). Blotted m~(cid:173)
`branes were . baked for two hours at so 0c under vacuum;,(cid:173)
`prehybridized in lM NaCl/ 1% SOS/ 10% Dextran sulfate
`at so0 c overnight and hybridized at the same tempera(cid:173)
`ture for 24 h after addition of the labelled probes
`( 3-•s x 105 cpm/ml);
`they were then washed at ss0 c
`twice in 1 X SSC C0.15 M NaCl/ 0.0lSM Na citrate) · /
`11 SOS), and exposed to X-ray film for varying times
`at -10°c with an intensifying s~reen.
`In all cases
`the concentration of peptides was l0-7M.
`The result of this experiment is shown in Figure
`2. Lanes 1-3 (control cells), 4-6 (GLP-1 Cl-37)), 7-9
`GLP-1 (7-37), 10-12 (GLP-l(·l-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(cid:173)
`ment revealed that, at equal peptide concentrations,
`GLP-1 < 7-3 7 > stimulated insulin gene expressi?n to
`more than 3 times the level found in control (untreat(cid:173)
`ed) cells.
`
`FRESENIUS EXHIBIT 1039
`Page 17 of 36
`
`

`

`WO 87/06941
`
`PCT /US87 /01005
`
`-16-
`
`EXAMPLE 2:
`Rat
`insulinoma cells of cell line RIN-38 were
`grown in DME medium as described in Example 1. After
`·
`·
`-1
`1
`d
`incubation with 10 M GLP-1 (1-37, GLP-
`(7-37) an
`GLP-1 Cl-36),
`the concentrations of insulin in the
`cell culture rnedi urns were determined by radioimmun(cid:173)
`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 Cl-37)
`
`TABLE 1
`
`Insulin Produced
`(uOnits/ ML)
`
`2800
`
`5000
`
`EXAMPLE 3:
`The pancreas o~ live rat was perfused with vary- _
`ing concentrations of GLP-1 Cl-37) and GLP-1 (7-37) as
`described above. At one minute intervals, rat serum
`insulin levels in picograrns/ ml were determined by
`radio_immunassay (as described above). The results of
`this experiment are shown in Table 2. Perfusions were
`done using peptide concentrations of S x l0-7M, 5 x
`10~811, and S x 10-10M, 5 x 10-11M and 5 x 10-12 M.
`Peptides were added after the zero minute serum ·value
`had been determined.
`
`FRESENIUS EXHIBIT 1039
`Page 18 of 36
`
`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-17-
`
`GLP-1 (1-37) was found to me~iate a 3.4-fold in(cid:173)
`crease in serum insulin concentrations when perfused
`- 7
`.
`'
`f 5
`into rat pancreas at a concentration o
`x 10 Mi at
`a concentration of 5 ·x 10-SM this peptide was capable
`of mediating only a 2-fold increase in serum insulin
`levels. At a concentration of 5 x 10-lOM this peptide
`was found to mediate only a 20% increase in serum in(cid:173)
`sulin levels.
`GLP~l (7-37) was found to be capable of stimulat(cid:173)
`ing a 132-fold increase in insulin levels when pro(cid:173)
`vided to rat pancreas at a concentration of 5 x l0-7M.
`At a 10-fold lower concentration (5 x l0-8M) this pep(cid:173)
`tide was capable of directing a 21-fold increase in
`•
`the serum concentration of insulin.
`At a · concen(cid:173)
`tration of 5 x ·10-lOM, GLP-1 (7-37) was found to be
`capable of mediating an
`increase· in serum
`insulin
`levels C 32-fold).
`Even at a concentration of 5 x
`10-11M, GLP-1 (7-37) delivered a 15-fold increase in
`levels whereas GLP-1
`insulin
`(l-37) was without
`effect.
`This experiment shows that GLP-l (7-37) is more
`than 1,000-fold more potent than GLP-1 Cl-37) in stim(cid:173)
`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_p~ncreatic alpha or delta cells.
`
`4
`
`FRESENIUS EXHIBIT 1039
`Page 19 of 36
`
`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-18-
`
`Insulin Produced Cpicograms/rnl) at
`Peptide Concentration
`
`Time
`(Minutes)
`
`Sxl0-7M SxlO-BM sx10-10M SxlO-l~ 5xl0-l2M
`
`GLP-1
`(7-37)
`
`GLP-1
`Cl-37) •
`
`0
`1
`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
`so
`so
`160
`50
`
`FRESENIUS EXHIBIT 1039
`Page 20 of 36
`
`

`

`WO 87/06941
`
`PCT/US87/01005
`
`-19-
`
`EXAMPLE 4:
`In order to determine whether glucagon-like pro-
`I
`teins were capable of affecting cellular cAMP levels ·
`the effects of GLP-1· ~7-37) and GLP-1 (1-37) on cAMP
`levels in RINS-38
`insulinoma cells was determined.
`Cells were grown as described in Example 1, in 26 well
`culture dishes. Varying amounts of glucogon-like pep(cid:173)
`tides were added to culture wells in triplicate. Af(cid:173)
`ter permitting incubation for ! 10 minutes the total
`cell media was examined for cAMP, and the concentra(cid:173)
`tion of cAMP was determined. The results of this ex(cid:173)
`periment are shown in Table 3.
`20 ul from ·each cul-
`ture well was assayed.
`•
`
`Peptide
`Concentration
`
`Table 3
`
`eMOLES OF cAMP PRODUCED
`
`Expt
`
`Exp~
`
`(M)
`
`0
`10-6
`10-1
`10-8
`10-9
`10-10
`
`-10--11
`
`I
`
`140
`
`400
`
`370
`
`494
`
`515
`
`253
`
`533
`
`II
`
`91
`
`170
`
`120
`
`160
`
`100
`
`90
`
`90
`
`This experiment reveals that GLP-1 (7-37) was cap(cid:173)
`able of stimulating cAMP levels even when present at a
`
`FRESENIUS EXHIBIT 1039
`Page 21 of 36
`
`

`

`WO87/06941
`
`PCT/US87/0100S
`
`-20-
`
`concentration of l0-11M. The inc~ease in cAMP levels
`is an indication that GLP-1 {7-37) is capable of in(cid:173)
`teracting with cellular receptors.
`
`EXAMPLE 5
`In order to demonstrate that the effects of GLP-l
`Cl-37), GLP-l Cl-36) and GLP-l (7-37) were specific
`for insulin, and were not capable of inducing or pro(cid:173)
`voking . non-specific gene expression,
`the effect of
`these peptides on the levels of actin and angiotens(cid:173)
`inogen mRNAs were conducted. RIN-38 insulinoma cells
`were grown as described in Example land incubated in
`the presence of GLP-1 Cl-37), GLP-1 (7-37), or GLP-1
`' Cl-36) des-Gly arg (Peninsula Laboratories). 1n all
`~ases the concentration of peptides was l0-7M.
`Incu(cid:173)
`bations were for six hours. Messenger RNAs specific
`for insulin, actin, or angiotensinogen were identified
`by Northern hybridization as de

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