United States Patent (19)
`Habener
`
`54 INSULINOTROPIC HORMONE
`75 Inventor: Joel F. Habener, Newton, Mass.
`73) Assignee: The General Hospital Corporation,
`Charlestown, Mass.
`21 Appl. No.: 532,111
`(22
`Filed:
`Jun. 1, 1990
`Related U.S. Application Data
`Continuation of Ser. No. 148.517. Jan. 26, 1988, aban
`doned, which is a continuation-in-part of Ser. No.
`859,928, May 5, 1986, abandoned.
`51) Int. Cl. ..................... A61K 37/02; A61 K 37/28;
`CO7K 7/10; CO7K 7/34
`52 U.S. Cl. ...................................... 514/12; 514/866;
`530/308; 530/324
`58) Field of Search ....................... 530/324, 303, 308;
`514/12, 866
`
`63
`
`(56)
`
`References Cited
`FOREIGN PATENT DOCUMENTS
`0044168 1/1982 European Pat. Off. .
`OTHER PUBLICATIONS
`Uttenthal et al., J. Clin. End. Metab. 61 = 472-479
`(1985).
`Ghiglione et al., Diabetologia, 27=599-600 (1984).
`Rudinger, Peptide Hormones, Parsons (Ed.) U. Park
`Press, Baltimore, pp. 1-7 (1976).
`Schmidt et al., Diabetologia 28-704-707 (1985).
`
`III S005 8666A
`
`11) Patent Number:
`(45) Date of Patent:
`
`5,118,666
`Jun. 2, 1992
`
`Bell et al., Nature, 302 = 716–718 (1983).
`Andrews et al., J. Biol, Chem. 260-390-3914.
`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):2164-21.68
`(1990).
`Primary Examiner-John Doll
`Assistant Examiner-Christina Chan
`Attorney, Agent, or Firm-Sterne, Kessler, Goldstein &
`Fox
`ABSTRACT
`57
`Derivatives of glucagon-like peptide I (GLP-1) have
`been found to have insulinotropic activity. The inven
`tion pertains to such derivatives, and to the use of such
`derivatives as a potential therapy for Diabetes Mellitus,
`
`21 Claims, 6 Drawing Sheets
`
`MPI EXHIBIT 1056 PAGE 1
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`MPI EXHIBIT 1056 PAGE 1
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1056, p. 1 of 24
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`

`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 1 of 6
`
`5,118,666
`
`
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`MPI EXHIBIT 1056 PAGE 2
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`MPI EXHIBIT 1056 PAGE 2
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`MPI EXHIBIT 1056 PAGE 2
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1056, p. 2 of 24
`
`

`

`US. Patent
`
`June 2, 1992
`
`Sheet 2 of 6
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`5,118,666
`
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`MPI EXHIBIT 1056 PAGE 3
`
`MPI EXHIBIT 1056 PAGE 3
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`MPI EXHIBIT 1056 PAGE 3
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1056, p. 3 of 24
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`€
`

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`U.S. Patent
`
`June 2, 1992
`
`Sheet 3 of 6
`
`5,118,666
`
`STIMULATION OF cAMPIN RIN 1046-38
`
`
`
`:
`
`KXX
`
`XXC
`
`Y
`
`Cls O 29 9.
`GLPI(7-37)"GLUCOGON
`CONCENTRATION
`(-log M)
`FIG.2
`
`MPI EXHIBIT 1056 PAGE 4
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`MPI EXHIBIT 1056 PAGE 4
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1056, p. 4 of 24
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`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 4 of 6
`
`5,118,666
`
`5
`S 4
`S
`S.
`S 3
`2
`2
`2
`
`Ot. ,
`
`GLUCOSE66mM
`GLUCOSE 66nM
`2 2
`GLPI (737) (n=9)
`O GLPI (7-37) (n=4)
`o GLUCAGON (n=9).
`O GLUCAGON (n=9)
`
`IOM
`
`lom
`
`25
`2O S
`S
`S.
`75 S.
`N
`IO 5
`2
`5
`
`5
`O
`TIME (min)
`
`O
`
`5
`O
`TIME (min)
`
`O
`
`O
`
`FIG. 3
`
`MPI EXHIBIT 1056 PAGE 5
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`MPI EXHIBIT 1056 PAGE 5
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1056, p. 5 of 24
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`

`U.S. Patent
`
`June 2, 1992
`
`Sheet 5 of 6
`
`5,118,666
`
`TIME:
`
`3O 36
`CONDITION: Ger:7)
`lo"M
`
`GLUCOSE (6.6mM)
`8 87
`5 57
`66 72
`GLPS-35) GP734) GP737)
`lo"M
`lo"M
`O'M
`
`4.6
`
`2
`
`---------------
`2O
`3O
`4O
`5O
`6O
`7O
`8O
`90
`TIME (min)
`
`FIG.4
`
`MPI EXHIBIT 1056 PAGE 6
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`MPI EXHIBIT 1056 PAGE 6
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1056, p. 6 of 24
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`U.S. Patent
`
`June 2, 1992
`
`Sheet 6 of 6
`
`5,118,666
`
`
`
`----> (?ul/6u) ALIMI IOVEJONOWWI
`
`
`
`
`
`
`
`GLPI(-37)
`
`l
`
`6 20 24, 28
`72
`TIME (min)
`
`FIG 5
`
`MPI EXHIBIT 1056 PAGE 7
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`MPI EXHIBIT 1056 PAGE 7
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1056, p. 7 of 24
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`

`

`1.
`
`INSULINOTROPIC HORMONE
`
`30
`
`35
`
`5, 118,666
`2
`al. (Nature 282:260-266 (1979) demonstrated that pro
`glucagon was subsequently cleaved into glucagon and a
`second polypeptide. Subsequent work by Lund, P. K.,
`et al. (Proc. Natl. Acad. Sci. USA 79:345-349 (1982));
`Lopez, L. C., et al. (Proc. Natl. Acad. Sci. USA
`80:5485-5489 (1983)) and Bell, G. 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 gluca
`gon from this animal was also proteolytically cleaved
`after adjacent lysine-arginine and arginine-arginine di
`peptide residues (Andrews, P. C., et al., J. Biol. Chem.
`260:3910-3914 (1985)). Lopez, L. C., et al. (Proc. Natl.
`Acad. Sci. USA 80:5485-5489 (1983)), and Bell, G. I., et
`al, discovered the mammalian proglucagon was
`cleaved adjacent lysine-arginine or arginine-arginine
`dipeptides and demonstrated that the proglucagon mol
`ecule contained three discrete and highly homologous
`peptide molecules which were designated glucagon,
`glucagon-like protein 1 (GLP-1), and glucagon-like
`protein 2 (GLP-2). Lopez et al. concluded that gluca
`gon-like protein 1 was 37 amino acid residues long and
`that glucagon-like peptide 2 was 34 amino acid residues
`long. Analogous studies on the structure of rat prepro
`glucagon revealed a similar pattern of proteolytic cleav
`age between adjacent lysine-arginine or arginine-argi
`nine dipeptide residues, resulting in the formation of
`glucagon, GLP-1, and GLP-2 (Heinrich, G., et al., En
`docrinol. 115:2176-2181 (1984)). Human rat, bovine, and
`hamster sequences of GLP-1 have been found to be
`identical (Ghiglione, M., et al., Diabetologia 27:599–600
`(1984)).
`The conclusion reached by Lopez et al. (Proc. Natl.
`Acad. Sci. USA 80:5485-5489 (1983)) regarding the size
`of GLP-1 was confirmed by the work of Uttenthal, L.
`O., et al., (J. Clin. Endocrinol. Metabol. 6:472-479
`(1985)). Uttenthal et al. examined the molecular forms
`of GLP-1 which were present in the human pancreas.
`Their research shows that GLP-1 and GLP-2 are pres
`ent in the pancreas as proteins having 37 and 34 amino
`acid residues, respectively.
`The similarity between GLP-1 and glucagon sug
`gested to early investigators that GLP-1 might have
`biological activity. Although some investigators found
`that GLP-1 could induce rat brain cells to synthesize
`cAMP (Hoosein, N. M., et al., FEBS Lett. 178:83-86
`(1984)), other investigators failed to identify any physio
`logical role for GLP-1 (Lopez, L. C., et al., Proc. Natl.
`Acad, Sci. USA 80:5485-5489 (1983)). The failure to
`identify any physiological role for GLP-1 caused some
`investigators to question whether GLP-1 was in fact a
`hormone and whether the relatedness between gluca
`gon and GLP-1 might be artifactual (Ghiglione, M., et
`al, Diabetologia 27:599–600 (1984)).
`Thus, in conclusion, the prior art reveals an aware
`ness of the processing of a glucagon hormone precursor
`into a set of peptides sharing extensive homology. It has
`been widely assumed by those of skill in the art that
`these highly related glucagon-like peptides would have
`a biological activity. Nevertheless, extensive investiga
`tions designed to elucidate the biological effects of these
`molecules had been unsuccessful.
`SUMMARY OF THE INVENTION
`The present invention relates to an insulinotropic
`hormone comprising GLP-1 and its derivatives. The
`
`10
`
`15
`
`20
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`This application is a continuation of application Ser.
`No. 07/148,517, filed Jan. 26, 1988, now abandoned,
`which is a continuation-in-part of U.S. patent applica
`tion Ser. No. 859,928, filed on May 5, 1986, and now
`abandoned.
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`This invention is directed to the discovery that cer
`tain peptide fragments of the prehormone, proglucagon,
`possess hormonal activities and can be used to stimulate
`the synthesis and secretion of the hormone, insulin.
`These peptide fragments are useful in therapy for the
`disease Diabetes mellitus.
`2. Description of the Background Art
`The endocrine secretions of the pancreatic islets are
`under complex control not only by blood-borne metab
`olites (glucose, amino acids, catecholamines, etc.), but
`also by local paracrine influences. The major pancreatic
`25
`islet hormones (glucagon, insulin, and somatostatin)
`interact among their specific cell types (A, B, and D
`cells, respectively) to modulate secretory responses
`mediated by the metabolites. Although insulin secretion
`is predominantly controlled by blood levels of glucose,
`glucagon and somatostatin stimulate and inhibit glu
`cose-mediated insulin secretory responses, respectively.
`In addition to the proposed interislet paracrine regula
`tion of insulin secretion, there is evidence to support the
`existence of insulinotropic factors in the intestine. This
`concept originates from the observations that glucose
`taken orally is a much more potent stimulant of insulin
`secretion than is a comparable amount of glucose given
`intravenously.
`The human hormone, glucagon, is a 29-amino acid
`peptide hormone produced in the A-cells of the pan
`40
`creas. The hormone belongs to a multi-gene family of
`structurally related peptides that include secretin, gas
`tric inhibitory peptide, vasoactive intestinal peptide,
`and glicentin. These peptides variously regulate carbo
`hydrate metabolism, gastrointestinal mobility, and se
`45
`cretory processing. The principal recognized actions of
`pancreatic glucagon, however, are to promote glycoge
`nolysis and gluconeogenesis, resulting in an elevation of
`blood sugar levels. In this regard, the actions of gluca
`gon are counterregulatory to those of insulin and may
`50
`contribute to the hyperglycemia that accompanies Dia
`betes mellitus (Lund, P. K., et al., Proc. Natl. Acad, Sci,
`USA 79:345-349 (1982)).
`Glucagon has been found to be capable of binding to
`specific receptors which lie on the surface of insulin
`55
`producing cells. Glucagon, when bound to these recep
`tors, stimulates the rapid synthesis of cAMP, by these
`cells. cAMP, in turn, has been found to stimulate insulin
`expression (Korman, L. Y., et al., Diabetes 34:717-722
`(1985)). Insulin acts to inhibit glucagon synthesis (Re
`view of Medical Physiology, Ganong, W. F., 1979, Lang
`Publications, Los Altos, California (p. 273)). Thus, the
`expression of glucagon is carefully regulated by insulin,
`and ultimately by the serum glucose level.
`The glucagon gene is initially translated from a 630
`65
`base pair precursor to form the polypeptide, prepro
`glycagon (Lund et al. (1982)). This polypeptide is subse
`quently processed to form proglucagon. Patzelt, C., et
`
`MPI EXHIBIT 1056 PAGE 8
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`MPI EXHIBIT 1056 PAGE 8
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`DR. REDDY’S LABORATORIES, INC.
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`5
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`10
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`5,118,666
`4.
`3
`glucagons. The preproglucagon precursor is proteolyti
`invention additionally pertains to the therapeutic uses of
`such compounds.
`cally cleaved at sites indicated by circles.
`FIG. 2 shows the ability of the insulinotropic pep
`In detail, the invention pertains to a peptide fragment
`tides glucagon and GLP-1 (7-37) to stimulate cAMP
`which is insulinotropic and is derivable from a naturally
`formation in the insulinoma line, RIN 1046-38.
`occurring amino acid sequence.
`FIG. 3 shows a comparison of the insulinotropic
`The invention comprises a compound selected from
`activity of glucagon with that of GLP-1 (7-37).
`the group consisting of:
`FIG. 4 shows a comparison of the insulinotropic
`(A) a peptide comprising the sequence:
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser
`activities of GLP-1 (7-34), GLP-1 (7-35), and GLP-1
`(7-37) using the rat pancreas perfusion technique.
`Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-
`Ala-Trp-Leu-Val-X
`FIG. 5 shows the breakdown of GLP-1 (1-37) into
`GLP-1 (7-37) under experimental conditions.
`wherein X is selected from the group consisting of:
`(a) Lys,
`DESCRIPTION OF THE PREFERRED
`(b) Lys-Gly,
`EMBODIMENTS
`(c) Lys-Gly-Arg;
`A. GLP-1 and Its Derivatives
`and
`(B) a derivative of the peptide; wherein the com
`The hormone glucagon is known to be synthesized as
`pound is substantially free of natural contaminants, and
`a high molecular weight precursor molecule which is
`has an insulinotropic activity which exceeds the in
`subsequently proteolytically cleaved into three pep
`sulinotropic activity of GLP-1 (1-36) or GLP-1 (1-37). 20
`tides: glucagon, glucagon-like peptide l (GLP-1), and
`The invention also includes a compound selected
`glucagon-like peptide 2 (GLP-2). GLP-1 has 37 amino
`from the group consisting of:
`acids in its unprocessed form. The present invention
`(A) a peptide comprising the sequence:
`discloses that the unprocessed GLP-1 is essentially un
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser
`able to mediate the induction of insulin biosynthesis.
`Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-
`The unprocessed GLP-1 peptide is, however, naturally
`Ala-Trp-Leu-Val-X
`converted to a 31-amino acid long peptide (7-37 pep
`wherein X is selected from the group consisting of:
`tide) having amino acids 7-37 of GLP-1 ("GLP-1
`(a) Lys,
`(7-37)"). This processing occurs in the pancreas and the
`(b) Lys-Gly,
`intestine. The 7-37 peptide which has not been previosly
`(c) Lys-Gly-Arg:
`described is a hormone that has insulinotropic activity.
`and
`A compound is said to have an "insulinotropic activity"
`(B) a derivative of the peptide: wherein the com
`if it is able to stimulate, or cause the stimulation of, the
`pound is substantially free of natural contaminants, and
`synthesis or expression of the hormone insulin. The
`has an insulinotropic activity at a concentration of at
`hormonal activity of GLP-1 (7-37) appears to be spe
`least 10-10M.
`cific for the pancreatic beta cells where it appears to
`Of particular interest are peptides of the following
`induce the biosynthesis of insulin. The insulinotropic
`formula:
`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. More
`over, the insulinotropic hormone is useful in therapy for
`this disease.
`Peptide moieties (fragments) chosen from the deter
`mined amino acid sequence of human GLP-1 constitute
`the starting point in the development comprising the
`present invention. The interchangeable terms "peptide
`fragment' and "peptide moiety' are meant to include
`both synthetic and naturally occurring amino acid se
`quences derivable from a naturally occurring amino
`acid sequence.
`The amino acid sequence for GLP-1 has been re
`ported by several researchers (Lopez, L. C., et al., Proc.
`Natl. Acad, Sci., USA 80:5485-5489 (1983); Bell, G. I.,
`et a..., Nature 302:716–718 (1983); Heinrich, G., et al.,
`Endocrinol. 115:2176-2181 (1984); Ghiglione, M., et al.,
`Diabetologia 27:599-600 (1984)). The structure of the
`preproglucagon gene and its corresponding amino acid
`sequence is shown in FIG. 1. This figure further dis
`plays the proteolytic processing of the precursor gene
`product into glucagon and the two glucagon-like pep
`tides. As used herein, the notation of GLP-1 (1-37)
`refers to a GLP-1 polypeptide having all amino acids
`from 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-ter
`minus).
`In one embodiment, GLP-1 (7-37) and its peptide
`fragments are synthesized by conventional means, such
`
`(1) HN-X-CO-R
`wherein R 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 45
`or unbranched alkyl group;
`X is a peptide comprising the sequence:
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser
`Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile
`Ala-Trp-Leu-Val-Lys-Gly-Arg
`NH2 is the amine group of the amino terminus of X;
`and CO is the carbonyl group of the carboxy terminus
`of X;
`(2) the acid addition salts thereof; and
`(3) the protected or partially protected derivatives 55
`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- 60
`hancing the expression of insulin which comprises pro
`viding to a mammalian pancreatic B-type islet cell an
`effective amount of the insulinotropic peptides dis
`closed above.
`65
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 shows the DNA structure and corresponding
`amino acid sequence of human, rat, and hamster prepro
`
`25
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`30
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`40
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`50
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`O
`
`15
`
`30
`
`5,118,666
`6
`5
`The invention also encompasses the obvious or trivial
`as by the well-know solid-phase peptide synthesis de
`variants of the above-described fragments which have
`scribed by Merrifield, J. M. (Chem. Soc. 85:2149 (1962)),
`inconsequential amino acid substitutions (and thus have
`and Stewart and Young (Solid Phase Peptide Synthesis
`amino acid sequences which differ from that of the
`(Freeman, San Francisco, 1969), pages 27-66), which
`natural sequence) provided that such variants have an
`are incorporated by reference herein. However, it is
`insulinotropic activity which is substantially identical to
`also possible to obtain fragments of the proglucagon
`polypeptide, or of GLP-1, by fragmenting the naturally
`that of the above-described GLP-1 derivatives. Exam
`ples of obvious or trivial substitutions include the substi
`occurring amino acid sequence, using, for example, a
`tution of one basic residue for another (i.e. Arg for Lys),
`proteolytic enzyme. Further, it is possible to obtain the
`the substitution of one hydrophobic residue for another
`desired fragments of the proglucagon peptide or of
`(i.e. Leu for Ile), or the substitution of one aromatic
`GLP-1 through the use of recombinant DNA technol
`residue for another (i.e. Phe for Tyr), etc.
`ogy, as disclosed by Maniatis, T., et al., Molecular Biol
`Examples of derivatives of GLP-1 (1-37) include
`ogy: A Laboratory Manual, Cold Spring Harbor, N.Y.
`GLP-1 (7-37); GLP-1 (7-36); GLP-1 (7-35); GLP-1
`(1982), which is hereby incorporated by reference.
`(7-34); and the des-Gly amidated forms of these mole
`The present invention includes peptides which are
`cules. Included as well are the use of additional amino
`derivable from GLP-1 (1-37). A peptide is said to be
`acid residues added to such sequences in order to en
`"derivable from a naturally occurring amino acid se
`hance coupling to carrier protein or amino acid residues
`quence' if it can be obtained by fragmenting a naturally
`added to enhance the insulinotropic effect.
`occurring sequence, or if it can be synthesized based
`As is known in the art, the amino acid residues may
`upon a knowledge of the sequence of the naturally
`20
`be in their protected or unprotected form, using appro
`occurring amino acid sequence or of the genetic mate
`priate amino or carboxyl protecting groups. Useful
`rial (DNA or RNA) which encodes this sequence.
`cations are alkali or alkaline earth metallic cations (i.e.,
`Included within the scope of the present invention
`Na, K, Li, 1/2Ca, 1/2Ba, etc.) or amine cations (i.e.,
`are those molecules which are said to be "derivatives'
`tetraalkylammonium, trialkylammonium, where alkyl
`of GLP-1 (1-37). Such a "derivative" has the following
`can be C1-C12).
`characteristics: (1) it shares substantial homology with
`The variable length peptides may be in the form of
`GLP-1 (1-37) or a similarly sized fragment of GLP-1
`the free amines (on the N-terminus), or acid-addition
`(1-37); (2) it is capable of functioning as an insulino
`salts thereof. Common acid addition salts are hydro
`tropic hormone and (3) using at least one of the assays
`halic acid salts, i.e., HBr, HI, or, more preferably, HC.
`provided herein, the derivative has either (i) an insulino
`tropic activity which exceeds the insulinotropic activity
`B. Assays of Insulinotropic Activity
`of either GLP-1 (1-37) or GLP-1 (1-36), or, more pref.
`The present invention concerns GLP-1 (1-37) deriva
`erably, (ii) an insulinotropic activity which can be de
`tives which have an insulinotropic activity that exceeds
`tected even when the derivative is present at a concen
`the insulinotropic activity of either GLP-1 (1-37) or
`tration of 100M, or, most preferably, (iii) an insulino
`35
`GLP-1 (1-36). The insulinotropic property of a com
`tropic activity which can be detected even when the
`pound may be determined by providing that compound
`derivative is present at a concentration of 10-11M.
`to animal cells, or injecting that compound into animals
`A derivative of GLP-1 (1-37) is said to share "sub
`and monitoring the release of immunoreactive insulin
`stantial homology' with GLP-1 (1-37) if the amino acid
`(IRI) into the media or circulatory system of the animal,
`sequences of the derivative is at least 80%, and more
`40
`respectively. The presence of IRI is detected through
`preferably at least 90%, and most preferably at least
`the use of a radioimmunoassay which can specifically
`95%, the same as that of either GLP-1 (1-37) or a frag
`detect insulin. Although any radioimmunoassay capable
`ment of GLP-1 (1-37) having the same number of amino
`of detecting the presence of IRI may be employed, it is
`acid residues as the derivative.
`preferable to use a modification of the assay method of
`The derivatives of the present invention include
`Albano, J. D. M., et al., (Acta Endocrinol. 70:487-509
`GLP-1 (1-37) fragments which, in addition to contain
`(1972)). In this modification, a phosphate/-albumin
`ing a sequence that is substantially homologous to that
`buffer with a pH of 7.4 was employed. The incubation
`of a naturally occurring GLP-1 (1-37) peptide may
`was prepared with the consecutive condition of 500 ul
`contain one or more additional amino acids at their
`of phosphate buffer, 50 ul of perfusate sample or rat
`amino and/or their carboxy termini. Thus, the inven
`insulin standard in perfusate, 100 ul of anti-insulin anti
`tion pertains to polypeptide fragments of GLP-1 (1-37)
`serum (Wellcome Laboratories; 1:40,000 dilution), and
`that may contain one or more amino acids that may not
`100 ul of insulin, giving a total volume of 750 ful in a
`be present in a naturally occurring GLP-1 (1-37) se
`10x75-mm disposable glass tube. After incubation for
`quence provided that such polypeptides have an in
`2-3 days at 4 C., free insulin was separated from anti
`sulinotropic activity which exceeds that of GLP-1
`55
`body-bound insulin by charcoal separation. The assay
`(1-37) or GLP-1 (1-36).
`sensitivity was 1-2 uU/ml. In order to measure the
`Similarly, the invention includes GLP-1 (1-37) frag
`release of IRI into the cell culture medium of cells
`ments which, although containing a sequence that is
`grown in tissue culture, one preferably incorporates
`substantially homologous to that of a naturally occur
`radioactive label into proinsulin. Although any radioac
`ring GLP-1 (1-37) peptide may lack one or more addi
`60
`tive label capable of labeling a polypeptide can be used,
`tional amino acids at their amino and/or their carboxy
`it is preferable to use H leucine in order to obtain label
`termini that are naturally found on a GLP-1 (1-37) pep
`ing proinsulin. Labeling can be done for any period of
`tide. Thus, the invention pertains to polypeptide frag
`time sufficient to permit the formation of a detectably
`ments of GLP-1 (1-37) that may lack one or more amino
`labeled pool of proinsulin molecules; however, it is
`acids that are normally present in a naturally occurring
`preferable to incubate cells in the presence of radioac
`GLP-1 (1-37) sequence provided that such polypeptides
`tive label for a 60-minute time period. Although any cell
`have an insulinotropic activity which exceeds that of
`line capable of expressing insulin can be used for deter
`GLP-1 (1-37) or GLP-1 (1-36).
`
`45
`
`SO
`
`65
`
`MPI EXHIBIT 1056 PAGE 10
`
`MPI EXHIBIT 1056 PAGE 10
`
`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1056, p. 10 of 24
`
`

`

`10
`
`15
`
`30
`
`5,118,666
`8
`7
`10-10M to 10-11M, although a lower or higher dosage
`mining whether a compound has an insulinotropic ef
`may be administered. The required dosage will depend
`fect, it is preferable to use rat insulinoma cells, and
`upon the severity of the condition of the patient and
`especially RIN-38 rat insulinoma cells. Such cells can be
`upon such criteria as the patient's height, weight, sex,
`grown in any suitable medium; however, it is preferable
`age, and medical history.
`to use DME medium containing 0.1% BSA and 25 mM
`For the purpose of parenteral administration, compo
`glucose.
`sitions containing the derivatives of GLP-1 (1-37) are
`The insulinotropic property of a compound may also
`preferably dissolved in distilled water and the pH-value
`be determined by pancreatic infusion. The in situ iso
`is preferably adjusted to about 6 to 8. In order to facili
`lated perfused rat pancreas preparation was a modifica
`tate the lyophilization process resulting in a suitable
`tion of the method of Penhos, J. C., et al. (Diabetes
`product, lactose may be added to the solution. Prefera
`18:733-738 (1969)). In accordance with such a method,
`bly, the solution is then filtered sterilized, introduced
`fasted rats (preferably male Charles River strain albino
`into vials, and lyophilized. The concentration of the
`rats), weighing 350-600 g, are anesthetized with an
`GLP-1 (1-37) derivatives in these compositions may
`intraperitoneal injection of Amytal Sodium (Eli Lilly
`vary from to 10-12M to 10-5M.
`and Co., 160 ng/kg). Renal, adrenal, gastric, and lower
`Additional pharmaceutical methods may be em
`colonic blood vessels are ligated. The entire intestine is
`ployed to control the duration of action. Controlled
`resected except for about four cm of duodenum and the
`release preparations may be achieved by the use of
`descending colon and rectum. Therefore, only a small
`polymers to complex or adsorb the GLP-1 (1-37) deriv
`part of the intestine is perfused, thus minimizing possi
`atives. The controlled delivery may be exercised by
`ble interference by enteric substances with glucagon
`20
`selecting appropriate macromolecules (for example,
`like immunoreactivity. The perfusate is preferably a
`polyesters, polyamino acids, polyvinyl pyrrolidone,
`modified Krebs-Ringer bicarbonate buffer with 4%
`ethylenevinylacetate, methylcellulose, carboxymethyl
`dextran T70 and 0.2% bovine serum albumin (fraction
`cellulose, and protamine sulfate) and the concentration
`V), and is preferably bubbled with 95% O2 and 5%
`of macromolecules as well as the methods of incorpora
`CO2. A nonpulsatile flow, four-channel roller-bearing
`25
`tion in order to control release. Another possible
`pump (Buchler polystatic, Buchler Instruments Divi
`method to control the duration of action by controlled
`sion, Nuclear-Chicago Corp.) is preferably used, and a
`release preparations is to incorporate the derivatives of
`switch from one perfusate source to another is prefera
`GLP-1 (1-37) into particles of a polymeric material such
`bly accomplished by switching a three-way stopcock.
`as polyesters, polyamino acids, hydrogels, poly (lactic
`The manner in which perfusion is performed, modified,
`acid) or ethylene vinylacetate copolymers. Alterna
`and analyzed preferably follows the methods of Weir,
`tively, instead of incorporating the GLP-1 (1-37) deriv
`G. C., et al., (J. Clin. Investigat. 54: 1403-1412 (1974)),
`atives into these polymeric particles, it is possible to
`which are hereby incorporated by reference.
`entrap these derivatives in microcapsules prepared, for
`C. Formulations of Insulinotropic Compounds
`example, by coacervation techniques or by interfacial
`polymerization, for example, hydroxymethylcellulose
`The insulinotropic peptides (or peptide derivatives)
`or gelatin-microcapsules and poly (methylmethacry
`of GLP-1 (1-37) may be used as therapeutic composi
`late) microcapsules, respectively, or in colloidal drug
`tions. Such therapeutic compositions may consist solely
`delivery systems, for example, liposomes, albumin mi
`of the insulinotropic peptides (or peptide derivatives)
`crospheres, microemulsions, nanoparticles, and nano
`although, preferably, the compositions will contain the
`40
`capsules or in macroemulsions. Such teachings are dis
`insulinotropic peptides (or derivatives thereof) com
`closed in Remington's Pharmaceutical Sciences (1980).
`bined in admixture with a pharmaceutically acceptable
`It is possible to enhance the biological half-life of the
`carrier vehicle.
`GLP-1 (1-37) derivatives of the present invention, and,
`Suitable vehicles and their formulation, inclusive of
`thus, to increase the retention or stability of the deriva
`other human proteins, e.g., human serum albumin, are
`45
`tives in a recipient, by bonding such derivatives to one
`described for example in Remington's Pharmaceutical
`or more chemical "moieties' to thereby produce a con
`Sciences (16th Ed., A. Oslo Ed. Mack, Easton, Pa.
`pound which can be recognized and processed within a
`(1980)). In order to form a pharmaceutically acceptable
`recipient to yield a GLP-1 (1-37) derivative. The "moi
`composition suitable for effective administration, such
`eties" of such compounds may include one or more
`compositions will contain an effective amount of
`50
`lipids, carbohydrates, amino acid residues, etc. A pre
`GLP-1 (7-37), or a derivative of GLP-1 (7-37), together
`ferred "moiety' is an amino acid residue. The most
`with a suitable amount of carrier vehicle. The GLP-1
`preferred "moiety' is a peptide. The amino terminal
`derivatives of such compounds will preferably have
`(histidine) residue of GLP-1 (7-37) is a preferred site for
`been purified so as to be substantially free of natural
`the bonding of the "moiety'.
`contaminants. A material is said to be "substantially free
`An appreciation of this aspect of the present inven
`of natural contaminants' if it has been substantially
`tion can be obtained through a consideration of the
`purified from materials with which it is normally and
`natural processing of GLP-1 (1-37). GLP-1 (1-37) h