United States Patent [19J
`Habener
`
`INSULI~OTROPIC HORMO:'.'\E
`[54)
`Joel F. Habener, Newton, Mass.
`Inventor:
`[75)
`[73) Assignee: The General Hospital Corporation,
`Charlestown, Mass.
`[21) Appl. No.: 532,111
`Jun. 1, 1990
`[22) Filed:
`
`[51)
`
`[63)
`
`Related U.S. Application Data
`Continuation of Ser. No. 148.517. Jan. 26. 1988. aban(cid:173)
`doned, which is a continuation-in-part of Ser. No.
`859,928, May 5, 1986, abandoned.
`Int. CJ.5 ..................... A61K 37/02; A61K 37/28;
`C07K 7/10; C07K 7/34
`[52) U.S. CI. ...................................... 514/12; 514/866;
`530/308; 530/324
`[58) Field of Search ....................... 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 (I 985).
`
`11111111111111111 IIIII lllll lllll lllll lllll lllll lllll lllll 111111111111111111
`US005 ! ! 8666A
`5,118,666
`[I I] Patent Number:
`Jun. 2, 1992
`[45) Date of Patent:
`
`Bell et al., Nature, 302=716-718 (1983).
`Andrews et 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).
`Holst, 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(cid:173)
`tion pertains to such derivatives, and to the use of such
`derivatives as a potential therapy for Diabetes Mellitus.
`
`21 Claims, 6 Drawing Sheets
`
`MPI EXHIBIT 1056 PAGE 1
`
`MPI EXHIBIT 1056 PAGE 1
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0001
`
`

`

`5 1
`
`-20
`W
`Met Lys Thr Val Tyr
`aaaggagctccacctgtctacacctcctctcagctcagtcccacaaggcagaataaaaaaATG AAG ACC GTT TAC
`------a:E:....:-1;._SIGNAL PEPTIDE------------=i .....--,,---=E ..... -2=----
`-I J +1
`Ile Val Ala Gly Leu Phe Val Met
`Leu Val Gln Gly Ser Trp Gln His Ala Pro Gln Asp
`ATC GTG GCT GGA TTG TTT GTA ATG
`CTG GTA CAA GGC AGC TGG CAG CAT GCC CCT CAG GAC
`NH2 15PEPTIDE
`25
`Thr Glu Glu Asn Ala Arg Ser Phe Pro Ala Ser Gln Thr Glu Pro Leu Glu Asp Pro Asp
`ACG GAG GAG AAC GCC A(iA TCA TTC CCA GCT TCC CAG ACA GAA CCA CTT GAA GAC CCT GAT
`- - - - -35---GLUCAGON--------4
`La\
`-
`5
`His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr
`CAT TCA CAG GGC ACA TTC ACC AGT GAC TAC AGC AAA TAC
`
`Gln Ile Asn Glu Asp
`CAG ATA AAC GAA GAC
`
`55
`65
`Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr
`Asn Arg
`CTA GAC TCC CGC CGT GCT CAA GAT TTT GTG CAG TGG TTG ATG AAC ACC
`AAC C~
`• E-3
`.-IP-I---
`GLP I
`85
`Asn Asn Ile Ala
`His Asp Glu P~: Glut~fHis Ala Glu Gly Thr Phe Thr Ser
`AAC AAC ATT GCC
`CAT GAT GAA TTT GAGeCAT GCT GAA GGG ACC TTT ACC AGT
`
`75
`
`135
`
`195
`
`255
`
`315
`
`375
`
`Asp Val Ser Ser Tyr Leu Glu Gly Gln
`GAT GTG AGT TCT TAC TTG GAG GGC CAG
`
`(NH2)
`ArgtGly
`CGA GGA
`
`Gly
`GGC
`
`Asp Phe
`Gl TTC
`
`95
`105(NH2)
`Ala Ala lys Glu Phe Ile Ala Trp Leu Val Lys ♦
`GCA GCA AAG GAA TTC ATT GCT TGG CTG GTG AAA 435
`IP-ll----=E-_4..,___ __ _____,
`115
`NH2
`Pro Glu Glu Val Ala Ile Ala Glu Glu leufGly
`CCG GAA GAA GTC GCC ATA GCT GAG GAA CTT GGG
`
`495
`
`FIG.1
`
`~ • 00 •
`
`rJ)
`::r
`
`('t)
`
`('t) -t,,,l
`
`0 ...,
`
`O'\
`
`...
`UI
`i (cid:173)
`i -
`...
`00
`0\
`0\
`0\
`
`MPI EXHIBIT 1056 PAGE 2
`
`MPI EXHIBIT 1056 PAGE 2
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0002
`
`

`

`135 GLPJI
`145
`•
`His Ala Asp Gly Ser Phe Ser Asp Glu Met Asn Thr 11 e Leu Asp Asn Leu Ala Thr Arg
`CAT GCT GAT GGA TCC TTC TCT GAT GAG ATG AAC ACG ATT CTC GAT AAC CTT GCC ACC AGA
`E-5
`155
`I
`Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr Asp
`GAC TTC ATC AAC TGG CTG ATT CAA ACC AAG ATC ACT GAC
`
`End
`TAG gaatatttcaccatt 618
`
`555
`
`cacaaccatcttcacaacatctcctgccagtcacttgggatgtacatttgag
`E-6
`atgcggacgaatacatttccctttagcgttgtgtaacccaaaggttgtaaatggaataaagtttttccagggtgttgat 776
`
`697
`
`aaagtaacaactttacagtatgaaaatgctggattctcaaattgtctcctcgttttgaagttaccgccctgagattact 855
`
`tttctgtggtataaattgtaaattatcgcagtcacgacacctggattacaacaacagaagacatggtaacctggtaacc 933
`
`gtagtggtgaacctggaaagagaacttcttccttgaaccctttgtcataaatgcgctcagctttcaatgtatcaagaat 1012
`
`agatttaaataaatatctcat
`3'
`
`1024
`
`FIG. I CONT.
`
`('t)
`
`rJ) =(cid:173)
`('t) ....
`N
`0 ....,
`°'
`
`01
`"' ~
`~
`00
`"' 0\
`0\
`0\
`
`MPI EXHIBIT 1056 PAGE 3
`
`MPI EXHIBIT 1056 PAGE 3
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0003
`
`

`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 3 of 6
`
`5,118',666
`
`STIMULATION OF cAMP IN RIN /046-38
`
`150
`
`-100
`0
`
`.§ -~ <[
`
`<.>
`
`50
`
`oli~~~e
`Cl 9 10 11 ~ 9 10 ·11 j
`GLPI(7-37} GLUCOGON
`CONCENTRATION
`{-JogMJ
`FIG.2
`
`MPI EXHIBIT 1056 PAGE 4
`
`MPI EXHIBIT 1056 PAGE 4
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0004
`
`

`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 4 of 6
`
`5,118,666
`
`I GLUCOSE 6.6mM I
`10/M
`• GLPI (7-37){n=9J
`o GLUCAGON(n=9J-
`
`I GLUCOSE 6.6mM I
`t½o/4
`• GLP I (7-37)(n=4J
`o GLUCAGON (n=9 J
`
`5
`
`1
`
`0
`
`I '
`
`I
`
`'
`
`I
`
`I
`
`'
`I '
`I I
`5
`0
`TIME ( min)
`
`I
`
`I
`
`I
`70
`
`5
`0
`TIME (min)
`
`10
`
`FIG. 3
`
`25
`
`5
`
`0
`
`MPI EXHIBIT 1056 PAGE 5
`
`MPI EXHIBIT 1056 PAGE 5
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0005
`
`

`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 5 of 6
`
`5,118,666
`
`~
`30 36
`TIME:
`CONDITION: GLPI (7·37)
`10·11 M
`
`GLUCOSE (6.6mM J
`5~7 6~2 8~
`GLP1(7·35) GLPI(7-34) GLPIC7·37)
`10·11 M
`10·11 M
`10·11M
`
`6 -
`
`2
`
`0
`
`, -""---'-' ----'-"-' -""---'-' ____._.._, -""---'-' __,
`.... 1 ___ __,_, ____. __ ...
`90
`80
`70
`60
`50
`40
`30
`20
`TIME (min)
`
`FIG.4
`
`MPI EXHIBIT 1056 PAGE 6
`
`MPI EXHIBIT 1056 PAGE 6
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0006
`
`

`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 6 of 6
`
`5,118,666
`
`GLPI(l-37} ' r
`
`,,
`/ I f I
`
`I
`I I
`I
`I
`I
`I
`I
`I I
`I I
`I I
`GLPl(7·37} 1 \
`i
`:
`
`I
`
`I ,s
`--
`~
`en
`.s
`.,_
`:>-
`s
`.::: 10
`0
`-~
`0::
`0
`c!:
`0.6
`::>
`:E
`~ 5 0.4
`Q2
`
`0
`
`0
`
`0 4 8
`
`12
`16 20 24 28
`TIME(minJ
`
`FJG.5
`
`6 T
`I
`l
`5 :::::
`~
`en
`.s
`4~
`s
`i==
`0
`3 i:5
`0::::
`0
`~
`2 :e
`~
`
`1
`
`0
`
`MPI EXHIBIT 1056 PAGE 7
`
`MPI EXHIBIT 1056 PAGE 7
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0007
`
`

`

`1
`
`5,118,666
`
`INSULINOTROPIC HORMONE
`
`2
`al. (Nature 282:260-266 (1979) demonstrated that pro(cid:173)
`glucagon was subsequently cleaved into glucagon and a
`second polypeptide. Subsequent work by Lund, P. K.,
`et al. (Proc. l\·atl. Acad. Sci. USA 79:345-349 (1982)):
`CROSS-REFERENCE TO RELATED
`(Proc. Natl. Acad. Sci. USA
`5 Lopez, L. C., et al.
`APPLICATIONS
`80:5485-5489 (1983)) and Bell. G. I., et al. (.'\'ature
`This application is a continuation of application Ser.
`302:716-718 (1983)) demonstrated that the proglucagon
`No. 07/148,517, filed Jan. 26, 1988, now abandoned,
`molecule was cleaved immediately after lysine-arginine
`which is a continuation-in-part of U.S. patent applica(cid:173)
`dipeptide residues. Studies of proglucagon produced by
`tion Ser. No. 859,928, filed on May 5, 1986, and now
`10 channel catfish (lctalurus punctata) indicated that gluca(cid:173)
`abandoned.
`gon from this animal was also proteolytically cleaved
`BACKGROUND OF THE INVENTION
`after adjacent lysine-arginine and arginine-arginine di- •
`peptide residues (Andrews, P. C., et al., J. Biol. Chem.
`1. Field of the Invention
`260:3910-3914 (1985)). Lopez, L. C., et al. (Proc. Natl.
`This invention is directed to the discovery that cer-
`tain peptide fragments of the prehormone, proglucagon, 15 Acad. Sci. USA 80:5485-5489 (1983)), and Bell, G. I., et
`the mammalian proglucagon was
`al., discovered
`possess hormonal activities and can be used to stimulate
`cleaved adjacent lysine-arginine or arginine-arginine
`the synthesis and secretion of the hormone, insulin.
`dipeptides and demonstrated that the proglucagon mol-
`These peptide fragments are useful in therapy for the
`ecule contained three discrete and highly homologous
`disease Diabetes mellitus.
`20 peptide molecules which were designated glucagon,
`2. Description of the Background Art
`glucagon-like protein 1 (GLP-1), and glucagon-like
`The endocrine secretions of the pancreatic islets are
`protein 2 (GLP-2). Lopez et al. concluded that gluca-
`under complex control not only by blood-borne metab-
`gon-like protein 1 was 37 amino acid residues long and
`olites (glucose, amino acids, catecholamines, etc.), but
`that glucagon-like peptide 2 was 34 amino acid residues
`also by local paracrine influences. The major pancreatic
`islet .hormones (glucagon, insulin, and somatostatin) 25 long. Analogous studies on the structure of rat prepro-
`glucagon revealed a similar pattern of proteolytic cleav-
`interact among their specific cell types (A, B, and D
`age between adjacent lysine-arginine or arginine-argi-
`cells, respectively) to modulate secretory responses
`nine dipeptide residues, resulting in the formation of
`mediated by the metabolites. Although insulin secretion
`glucagon, GLP-l, and GLP-2 (Heinrich, G., et al., En-
`is predominantly controlled by blood levels of glucose,
`glucagon and somatostatin stimulate and inhibit glu- 30 docrinol. 115:2176_2181 (I 984)). Human rat, bovine, and
`cose-mediated insulin secretory responses, respectively.
`hamster sequences of GLP-1 have been found to be
`In addition to the proposed interislet paracrine regula-
`identical (Ghiglione, M., et al., Diabetoloqia 27:599-600
`tion of insulin secretion, there is evidence to support the
`O 984 )).
`existence of insulinotropic factors in the intestine. This
`The conclusion reached by Lopez et al. (Proc. l\'atl.
`concept originates from the observations that glucose 35
`A cad. Sci. USA 80:5485-5489 (1983)) regarding the size
`taken orally is a much more potent stimulant of insulin
`of GLP-1 was confirmed by the work of Uttenthal, L.
`secretion than is a comparable amount of glucose given
`0., et al., (J. Clin. Endocrinol. Metabol. 61 :472-479
`intravenously.
`(1985)). Uttenthal et al. examined the molecular forms
`The human hormone, glucagon, is a 29-amino acid
`peptide hormone produced in the A-cells of the pan- 40 of GLP-1 which were present in the human pancreas.
`Their research shows that GLP-1 and GLP-2 are pres-
`creas. The hormone belongs to a multi-gene family of
`ent in the pancreas as proteins having 37 and 34 amino
`structurally related peptides that include secretin, gas-
`acid residues, respectively.
`tric inhibitory peptide, vasoactive intestinal peptide,
`The similarity between GLP-1 and glucagon sug-
`and glicentin. These peptides variously regulate carbo-
`hydrate metabolism, gastrointestinal mobility, and se- 45 gested to early investigators that GLP-1 might have
`biological activity. Although some investigators found
`cretory processing. The principal recognized actions of
`that GLP-1 could induce rat brain cells to synthesize
`pancreatic glucagon, however, are to promote glycoge-
`cAMP (Hoosein, N. M., et al., FEBS Lett. 178:83-86
`nolysis and gluconeogenesis, resulting in an elevation of
`(1984)), other investigators failed to identify any physio-
`blood sugar levels. In this regard, the actions of gluca-
`gon are counterregulatory to those of insulin and may 50 logical role for GLP-1 (Lopez, L. C., et al., Proc. Natl.
`Acad. Sci. USA 80:5485-5489 (1983)). The failure to
`contribute to the hyperglycemia that accompanies Dia-
`identify any physiological role for GLP-1 caused some
`betes mellitus (Lund, P. K., et al., Proc. Natl. Acad. Sci.,
`investigators to question whether GLP-1 was in fact a
`USA 79:345-349 (1982)).
`hormone and whether the relatedness between gluca-
`Glucagon has been found to be capable of binding to
`specific receptors which lie on the surface of insulin- 55 gon and GLP-1 might be artifactual (Ghiglione, M., et
`al., Diabetologia 27:599-600 (1984)).
`producing cells. Glucagon, when bound to these recep-
`Thus, in conclusion, the prior art reveals an aware-
`tors, stimulates the rapid synthesis of cAMP, by these
`ness of the processing of a glucagon hormone precursor
`cells. cAMP, in turn, has been found to stimulate insulin
`into a set of peptides sharing extensive homology. It has
`expression (Korman, L. Y., et al., Diabetes 34:717-722
`(1985)). Insulin acts to inhibit glucagon synthesis (Re- 60 been widely assumed by those of skill in the art that
`these highly related glucagon-like peptides would have
`view of Medical Physiology, Ganong, W. F., 1979, Lang
`a biological activity. Nevertheless, extensive investiga-
`Publications, Los Altos, California (p. 273)). Thus, the
`tions designed to elucidate the biological effects of these
`expression of glucagon is carefully regulated by insulin,
`molecules had been unsuccessful.
`and ultimately by the serum glucose level.
`The glucagon gene is initially translated from a 630- 65
`SUMMARY OF THE INVENTION
`base pair precursor to form the polypeptide, prepro(cid:173)
`The present invention relates to an insulinotropic
`glycagon (Lund et al. (1982)). This polypeptide is subse(cid:173)
`hormone comprising GLP-1 and its derivatives. The
`quently processed to form proglucagon. Patzelt, C., et
`
`MPI EXHIBIT 1056 PAGE 8
`
`MPI EXHIBIT 1056 PAGE 8
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0008
`
`

`

`3
`invention additionally pertains to the therapeutic uses of
`such compounds.
`In detail, the invention pertains to a peptide fragment
`which is 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(cid:173)
`Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile(cid:173)
`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(cid:173)
`pound is substantially free of natural contaminants, and
`has an insulinotropic activity which exceeds the in(cid:173)
`sulinotropic activity of GLP-1 (1-36) or GLP-1 (1-37). 20
`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(cid:173)
`Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile(cid:173)
`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(cid:173)
`pound is substantially free of natural contaminants, and
`has an insulinotropic activity at a concentration of at
`least JO- !OM.
`Of particular interest are peptides of the following
`formula:
`
`15
`
`wherein R 1 is OH, OM, or -NR2R3:
`M is a pharmaceutically acceptable cation or a lower
`branched or unbranched alkyl group;
`R2 and R3 are the same or different and selected from
`the group consisting of hydrogen and a lower branched
`or unbranched alkyl group;
`X is a peptide comprising the sequence:
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser(cid:173)
`Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile(cid:173)
`Ala-Trp-Leu-Val-Lys-Gly-Arg
`NH2 is the amine group of the amino terminus of X;
`and CO is the carbonyl group of the carboxy terminus
`ofX;
`(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(cid:173)
`hancing the expression of insulin which comprises pro(cid:173)
`viding to a mammalian pancreatic B-type islet cell an
`effective amount of the insulinotropic peptides dis(cid:173)
`closed above.
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 shows the DNA structure and corresponding
`amino acid sequence of human, rat, and hamster prepro-
`
`5,118,666
`
`4
`glucagons. The preproglucagon precursor is proteolyti(cid:173)
`cally cleaved at sites indicated by circles.
`FIG. 2 shows the ability of the insulinotropic pep(cid:173)
`tides glucagon and GLP-1 (7-37) to stimulate cAMP
`5 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
`10 (7-37) using the rat pancreas perfusion technique.
`FIG. 5 shows the breakdown of GLP-1 (1-37) into
`GLP-1 (7-37) under experimental conditions.
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`A. GLP-1 and Its Derivatives
`The hormone glucagon is known to be synthesized as
`a high molecular weight precursor molecule which is
`subsequently proteolytically cleaved into three pep(cid:173)
`tides: glucagon, glucagon-like peptide I (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-
`25 able to mediate the induction of insulin biosynthesis.
`The unprocessed GLP-1 peptide is, however, naturally
`converted to a 31-amino acid long peptide (7-3 7 pep(cid:173)
`tide) having amino acids 7-37 of GLP-1 ("GLP-1
`(7-37)"). This processing occurs in the pancreas and the
`30 intestine. The 7-37 peptide which has not been previosly
`described is a hormone that has insulinotropic activity.
`A compound is 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
`35 hormonal activity of GLP-1 (7-37) appears to be spe(cid:173)
`cific for the pancreatic beta cells where it appears to
`induce the biosynthesis of insulin. The insulinotropic
`hormone is useful in the study of the pathogenesis of
`maturity onset diabetes mellitus, a condition in which
`40 the dynamics of insulin secretion are abnormal. More(cid:173)
`over, the insulinotropic hormone is useful in therapy for
`this disease.
`Peptide moieties (fragments) chosen from the deter(cid:173)
`mined amino acid sequence of human GLP-1 constitute
`45 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(cid:173)
`quences derivable from a naturally occurring amino
`50 acid sequence.
`The amino acid sequence for GLP-1 has been re(cid:173)
`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.,
`55 Endocrinol. 115:2176-2181 (1984); Ghiglione, M., et al.,
`Diabetologia 27:599-600 (1984)). The structure of the
`preproglucagon gene and its corresponding amino acid
`sequence is shown in FIG. 1. This figure further dis(cid:173)
`plays the proteolytic processing of the precursor gene
`60 product into glucagon and the two glucagon-like pep(cid:173)
`tides. As used herein, the notation of 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
`65 amino acids from 7 (N-terminus) through 37 (C-ter(cid:173)
`minus).
`In one embodiment, GLP-1 (7-37) and its peptide
`fragments are synthesized by conventional means, such
`
`MPI EXHIBIT 1056 PAGE 9
`
`MPI EXHIBIT 1056 PAGE 9
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0009
`
`

`

`5,118,666
`
`5
`as by the well-know solid-phase peptide synthesis de(cid:173)
`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 5
`also possible to obtain fragments of the proglucagon
`polypeptide, or ofGLP-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 JO
`GLP-1 through the use of recombinant DNA technol(cid:173)
`ogy, as disclosed by Maniatis, T., et al., Molecular Biol(cid:173)
`ogy: A Laboratory Manual, Cold Spring Harbor, N.Y.
`(1982), which is hereby incorporated by reference.
`The present invention includes peptides which are 15
`derivable from GLP-1 (1-37). A peptide is said to be
`"derivable from a naturally occurring amino acid se(cid:173)
`quence" if it can be obtained by fragmenting a naturally
`occurring sequence, or if it can be synthesized based
`upon a knowledge of the sequence of the naturally 20
`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"
`ofGLP-1 (1-37). Such a "derivative" has the following 25
`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(cid:173)
`tropic hormone and (3) using at least one of the assays
`provided herein, the derivative has either (i) an insulino- 30
`tropic activity which exceeds the insulinotropic activity
`of either GLP-1 (1-37) or GLP-1 (1-36), or, more pref(cid:173)
`erably, (ii) an insulinotropic activity which can be de(cid:173)
`tected even when the derivative is present at a concen(cid:173)
`tration of I0- 10M, or. most preferably, (iii) an insulino- 35
`tropic activity which can be detected even when the
`derivative is present at a concentration of I0- 11 M.
`A derivative of GLP-1 (1-37) is said to share "sub(cid:173)
`stantial homology" with GLP-1 (1-37) if the amino acid
`sequences of the derivative is at least 80o/c, and more 40
`preferably at least 90%, and most preferably at least
`95%, the same as that of either GLP-1 (1-37) or a frag(cid:173)
`ment of GLP-1 (1-37) having the same number of amino
`acid residues as the derivative.
`The derivatives of the present invention include 45
`GLP-1 (1-37) fragments which, in addition to contain-
`ing a sequence that is substantially homologous to that
`of a naturally occurring GLP-1 (1-37) peptide may
`contain one or more additional amino acids at their
`amino and/or their carboxy termini. Thus, the inven- 50
`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(cid:173)
`quence provided that such polypeptides have an in(cid:173)
`sulinotropic activity which exceeds that of GLP-1 55
`(1-37) or GLP-1 (1-36).
`Similarly, the invention includes GLP-1 (l-37) frag(cid:173)
`ments which, although containing a sequence that is
`substantially homologous to that of a naturally occur(cid:173)
`ring GLP-1 (l-37) peptide may lack one or more addi- 60
`tional amino acids at their amino and/or their carboxy
`termini that are naturally found on a GLP-1 (1-37) pep(cid:173)
`tide. Thus, the invention pertains to polypeptide frag(cid:173)
`ments ofGLP-1 (1-37) that may lack one or more amino
`acids that are normally present in a naturally occurring 65
`GLP-1 (1-37) sequence provided that such polypeptides
`have an insulinotropic activity which exceeds that of
`GLP-1 {l-37) or GLP-1 (1-36).
`
`6
`The invention also encompasses the obvious or trivial
`variants of the above-described fragments which have
`inconsequential amino acid substitutions (and thus have
`amino acid sequences which differ from that of the
`natural sequence) provided that such variants have an
`insulinotropic activity which is substantially identical to
`that of the above-described GLP-1 derivatives. Exam(cid:173)
`ples of obvious or trivial substitutions include the substi(cid:173)
`tution of one basic residue for another (i.e. Arg for Lys),
`the substitution of one hydrophobic residue for another
`(i.e. Leu for Ile), or the substitution of one aromatic
`residue for another (i.e. Phe for Tyr), etc.
`Examples of derivatives of GLP-1 (1-37) include
`GLP-1 (7-37); GLP-1 (7-36); GLP-1 (7-35); GLP-1
`(7-34); and the des-Gly amidated forms of these mole(cid:173)
`cules. Included as well are the use of additional amino
`acid residues added to such sequences in order to en(cid:173)
`hance coupling to carrier protein or amino acid residues
`added to enhance the insulinotropic effect.
`As is known in the art, the amino acid residues may
`be in their protected or unprotected form, using appro(cid:173)
`priate amino or carboxyl protecting groups. Useful
`cations are alkali or alkaline earth metallic cations (i.e.,
`Na, K, Li, 1/2Ca, 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 {on the N-terminus), or acid-addition
`salts thereof. Common acid addition salts are hydro(cid:173)
`halic acid salts, i.e., HBr, HI, or, more preferably, HCI.
`B. Assays of Insulinotropic Activity
`The present invention concerns GLP-1 (1-37) deriva(cid:173)
`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(cid:173)
`pound may be determined by providing that compound
`to animal cells, or injecting that compound into animals
`and monitoring the release of immunoreactive insulin
`(IRI) into the media or circulatory system of the animal,
`respectively. The presence of IRI is detected through
`the use of a 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 µI
`of phosphate buffer, 50 µI of perfusate sample or rat
`insulin standard in perfusate, 100 µI of anti-insulin anti(cid:173)
`serum (Wellcome Laboratories; 1:40,000 dilution), and
`100 µI of insulin, giving a total volume of 750 µI in a
`10X75-mm disposable glass tube. After incubation for
`2-3 days at 4° C., free insulin was separated from anti(cid:173)
`body-bound insulin by charcoal separation. The assay
`sensitivity was 1-2 µU/ml. In order to measure the
`release of IRI into the cell culture medium of cells
`grown in tissue culture, one preferably incorporates
`radioactive label into proinsulin. Although any radioac(cid:173)
`tive label capable of labeling a polypeptide can be used,
`it is preferable to use 3H leucine in order to obtain label(cid:173)
`ing proinsulin. Labeling can be done for any period of
`time sufficient to permit the formation of a detectably
`labeled pool of proinsulin molecules; however. it is
`preferable to incubate cells in the presence of radioac(cid:173)
`tive label for a 60-minute time period. Although any cell
`line capable of expressing insulin can be used for deter-
`
`MPI EXHIBIT 1056 PAGE 10
`
`MPI EXHIBIT 1056 PAGE 10
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1056-0010
`
`

`

`5,118,666
`
`7
`mining whether a compound has an insulinotropic ef(cid:173)
`fect, it is preferable to use rat insulinoma cells. and
`especially RIN-38 rat insulinoma cells. Such cells can be
`grown in any suitable medium; however, it is preferable
`to use DME medium containing 0.1 % BSA and 25 mM 5
`glucose.
`The insulinotropic property of a compound may also
`be determined by pancreatic infusion. The in situ iso(cid:173)
`lated perfused rat pancreas preparation was a modifica(cid:173)
`tion of the method of Penhos, J. C., et al. (Diabetes 10
`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 15
`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- 20
`like immunoreactivity. The perfusate is preferably a
`modified Krebs-Ringer bicarbonate buffer with 4%
`dextran T70 and 0.2o/c bovine serum albumin (fraction
`V), and is preferably bubbled with 959c 02 and 5%
`CO2. A nonpulsatile flow, four-channel roller-bearing 25
`pump (Buchler polystatic, Buchler Instruments Divi(cid:173)
`sion, Nuclear-Chicago Corp.) is preferably used, and a
`switch from one perfusate source to another is prefera-
`bly accomplished by switching a three-way stopcock.
`The manner in which perfusion is performed, modified, 30
`and analyzed preferably follows the methods of Weir,
`G. C., et al., (J. Clin. lnvestigat. 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(cid:173)
`tions. Such therapeutic compositions may consist solely
`of the insulinotropic peptides (or peptide derivatives)
`although, preferably, the compositions will contain the 40
`insulinotropic peptides (or derivatives thereof) com(cid:173)
`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 45
`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 50
`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 55
`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, 60
`membranes, etc. A material is also said to be substan(cid:173)
`tially free of natural contaminants if these contaminants
`are substantially absent from a sample of the material.
`Compositions containing GLP-1 (7-37) or its deriva(cid:173)
`tives may be administered intravenously, intramuscu- 65
`larly, or subcutaneously at dosages in the range of from
`about 1 pg/kg to 1,000 µg/kg body weight, or at con(cid:173)
`levels of
`to produce serum
`centrations sufficient
`
`8
`10- 10M to JQ- 11 M. although a lower or higher dosage
`may be administered. The required dosage will depend
`upon the severity of the condition of the patient and
`upon such criteria as the patient's height, weight, sex,
`age, and medical history.
`For the purpose of parenteral administration. compo(cid:173)
`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 order to facili(cid:173)
`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 IQ- 12M to 1Q-5M.
`Additional pharmaceutical methods may be em(cid:173)
`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(cid:173)
`atives. The controlled