(19)
`
`(12)
`
`Europäisches Patentamt
`
`European Patent Office
`
`Office européen des brevets
`
`*EP000699686B1*
`EP 0 699 686 B1
`
`(11)
`
`EUROPEAN PATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`08.10.2003 Bulletin 2003/41
`
`(21) Application number: 95305963.1
`
`(22) Date of filing: 25.08.1995
`
`(51) Int Cl.7: C07K 14/605, A61K 38/22
`
`(54) Biologically active fragments of glucagon-like insulinotropic peptide
`
`Biologisch aktive Fragmente des Glucagon ähnlichen, insulinotropen Peptides
`
`Fragments biologiquement actifs de peptide insalinotrope de type glucagon
`
`(84) Designated Contracting States:
`AT BE CH DE DK ES FR GB GR IE IT LI LU NL PT
`SE
`Designated Extension States:
`LT LV SI
`
`(30) Priority: 30.08.1994 US 297731
`
`(43) Date of publication of application:
`06.03.1996 Bulletin 1996/10
`
`(73) Proprietor: ELI LILLY AND COMPANY
`Indianapolis, Indiana 46285 (US)
`
`(72) Inventors:
`• Johnson, William Terry
`Indianapolis, Indiana 46227 (US)
`• Yakubu-Madus, Fatima Emitsela
`Indianapolis, Indiana 46256 (US)
`
`(74) Representative: Denholm, Anna Marie et al
`Eli Lilly and Company Limited,
`Lilli Research Centre,
`Erl Wood Manor
`Windlesham Surrey GU20 6PH (GB)
`
`(56) References cited:
`WO-A-93/25579
`
`• ENDOCRINOLOGY (BALTIMORE) (1989), 125(6),
`3109-14 CODEN: ENDOAO;ISSN: 0013-7227,
`1989, XP000574778 SUZUKI, SEIJI ET AL:
`"Comparison of the effects of various C-terminal
`and N-terminal fragment peptides of
`glucagon-like peptide-1 on insulin and glucagon
`release from the isolated perfused rat pancreas"
`
`• BIOMED. RES. (1988), 9(SUPPL. 3), 213-17
`CODEN: BRESD5;ISSN: 0388-6107, 1988,
`XP000574772 KAWAI, KOICHI ET AL: "The
`biological effects of glucagon-like peptide-1
`(GLP-1) and its structure-activity relationship"
`• INTERNATIONAL JOURNAL OF PEPTIDE AND
`PROTEIN RESEARCH, vol. 40, no. 3 / 04, 1
`September 1992, pages 333-343, XP000311244
`SVETLANA MOJSOV: "STRUCTURAL
`REQUIREMENTS FOR BIOLOGICAL ACTIVITY
`OF GLUCAGON-LIKE PEPTIDE-I"
`• ENDOCRINOLOGY (BALTIMORE) (1990), 126(4),
`2164-8 CODEN: ENDOAO;ISSN: 0013-7227,
`1990, XP000574862 GEFEL, DOV ET AL:
`"Glucagon-like peptide-I analogs: effects on
`insulin secretion and adenosine
`3’,5’-monophosphate formation"
`• J. CLIN. ENDOCRINOL. METAB. (1995), 80(3),
`952-7 CODEN: JCEMAZ;ISSN: 0021-972X, 1995,
`XP000574780 DEACON, CAROLYN F. ET AL:
`"Degradation of glucagon-like peptide-1 by
`human plasma in vitro yields an N-terminally
`truncated peptide that is a major endogenous
`metabolite in vivo"
`• ENDOCRINOLOGY (1995), 136(8), 3585-97
`CODEN: ENDOAO;ISSN: 0013-7227, 1995,
`XP000574863 KIEFFER, TIMOTHY J. ET AL:
`"Degradation of glucose-dependent
`insulinotropic polypeptide and truncated
`glucagon-like peptide 1 in vitro and in vivo by
`dipeptidyl peptidase IV"
`
`Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
`notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
`a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art.
`99(1) European Patent Convention).
`
`Printed by Jouve, 75001 PARIS (FR)
`
`EP0 699 686B1
`
`MYLAN INST. EXHIBIT 1044 PAGE 1
`
`MYLAN INST. EXHIBIT 1044 PAGE 1
`
`

`

`EP 0 699 686 B1
`
`Description
`
`Field of Invention
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`[0001] The present invention relates to medicinal chemistry and provides novel peptides and compositions thereof
`that are useful for treating diabetes.
`
`Background of the Invention
`
`[0002] Endocrine secretions of pancreatic islets are regulated by complex control mechanisms driven not only by
`blood-borne metabolites such as glucose, amino acids, and catecholamines, but also by local paracrine influences.
`The major pancreatic islet hormones, glucagon, insulin and somatostatin, interact with specific pancreatic cell types
`(A, B, and D cells, respectively) to modulate the secretory response. Although insulin secretion is predominantly con-
`trolled by blood glucose levels, somatostatin inhibits glucose-mediated insulin secretion.
`[0003] The human hormone glucagon is a 29-amino acid hormone produced in pancreatic A-cells. The hormone
`belongs to a multi-gene family of structurally related peptides that include secretin, gastric inhibitory peptide, vasoactive
`intestinal peptide and glicentin. These peptides variously regulate carbohydrate metabolism, gastrointestinal motility
`and secretory processing. However, the principal recognized actions of pancreatic glucagon are to promote hepatic
`glycogenolysis and glyconeogenesis, resulting in an elevation of blood sugar levels. In this regard, the actions of glu-
`cagon are counter regulatory to those of insulin and may contribute to the hyperglycemia that accompanies Diabetes
`mellitus (Lund, P.K., et al., Proc. Natl. Acad. Sci. U.S.A., 79:345-349 (1982)).
`[0004] When glucagon binds to its receptor on insulin producing cells, cAMP production increases which in turn
`stimulates insulin expression (Korman, L.Y., et al., Diabetes, 34:717-722 (1985)). Moreover, high levels of insulin down-
`regulate glucagon synthesis by a feedback inhibition mechanism (Ganong, W.F., Review of Medical Physiology, Lange
`Publications, Los Altos, California, p. 273 (1979)). Thus, the expression of glucagon is carefully regulated by insulin,
`and ultimately by serum glucose levels.
`[0005] Preproglucagon, the zymogen form of glucagon, is translated from a 360 base pair gene and is processed to
`form proglucagon (Lund, et al., Proc. Natl. Acad. Sci. U.S.A. 79:345-349 (1982)). Patzelt, et al. (Nature, 282:260-266
`(1979)) demonstrated that proglucagon is further processed into glucagon and a second peptide. Later experiments
`demonstrated that proglucagon is cleaved carboxyl to Lys-Arg or Arg-Arg residues (Lund, P.K., et al., Lopez L.C., et
`al., Proc. Natl. Acad. Sci. U.S.A., 80:5485-5489 (1983), and Bell, G.I., et al., Nature 302:716-718 (1983)). Bell, G.I., et
`al., also discovered that proglucagon contained three discrete and highly homologous peptide regions which were
`designated glucagon, glucagon-like peptide 1 (GLP-1), and glucagon-like peptide 2 (GLP-2). Lopez, et al., demon-
`strated that GLP-1 was a 37 amino acid peptide and that GLP-2 was a 34 amino acid peptide. Analogous studies on
`the structure of rat preproglucagon revealed a similar pattern of proteolytic cleavage at Lys-Arg or Arg-Arg residues,
`resulting in the formation of glucagon, GLP-1, and GLP-2 (Heinrich, G., et al., Endocrinol., 115:2176-2181 (1984)).
`Finally, human, rat, bovine, and hamster sequences of GLP-1 have been found to be identical (Ghiglione, M., et al.,
`Diabetologia, 27:599-600 (1984)).
`[0006] The conclusion reached by Lopez, et al., regarding the size of GLP-1 was confirmed by studying the molecular
`forms of GLP-1 found in the human pancreas (Uttenthal, L.O., et al. J. Clin. Endocrinol. Metabol., 61:472-479 (1985)).
`Their research showed that GLP-1 and GLP-2 are present in the pancreas as 37 and 34 amino acid peptides respec-
`tively.
`[0007] The similarity between GLP-1 and glucagon suggested 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 physiological role for GLP-1 (Lopez,
`L.C., et al. supra). However, GLP-1 is now known to stimulate insulin secretion (insulinotropic action) causing glucose
`uptake by cells which decreases serum glucose levels (see, e g., Mojsov, S., Int. J. Peptide Protein Research, 40:
`333-343 (1992)).
`[0008] Numerous GLP-1 analogs demonstrating insulinotropic action are known in the art. These variants and ana-
`logs include, for example, GLP-1(7-36), Gln9-GLP-1(7-37), D-Gln9-GLP-1(7-37), acetyl-Lys9-GLP-1(7-37), Thr16-
`Lys18-GLP-1(7-37), and Lys18-GLP-1(7-37). Derivatives of GLP-1 include, for example, acid addition salts, carboxylate
`salts, lower alkyl esters, and amides (see, e.g., WO91/11457 (1991)). More importantly, it was demonstrated using
`GLP-1(8-37)OH that the histidine residue at position 7 is very important to insulinotropic activity of GLP-1 (Suzuki, S.,
`et al. Diabetes Res.; Clinical Practice 5 (Supp. 1):S30 (1988).
`[0009] Endocrinology, Volume 125, Number 6, Issued 1989, Suzuki et al., "Comparison of the Effects of Various C
`Terminal and N-Terminal Fragment Peptides of Glucagen-Like Peptide-1 on Insulin and Glucagon Release from the
`Isolated Perfused Rat Pancreas", pages 3109-3114 teaches that truncated glucagon-like peptide-1 (GLP-1) posseses
`a potent stimulatory activity for insulin production. The activities of N and C-terminal fragments were examined.
`
`2
`
`MYLAN INST. EXHIBIT 1044 PAGE 2
`
`MYLAN INST. EXHIBIT 1044 PAGE 2
`
`

`

`EP 0 699 686 B1
`
`[0010]
`In view of the above, it was most surprising when the present inventors discovered that administering N-
`terminal deletion mutants of GLP-1 to experimental animals caused an increase in serum glucose uptake in the absence
`of any insulinotropic activity. This discovery suggests that an entirely new mechanism for lowering elevated blood
`glucose levels may exist and directly lead to the present invention.
`[0011] Accordingly, the primary object of this invention is to provide novel, C-terminal GLP-1 fragments having no
`insulinotropic action but which are nonetheless useful for treating diabetes and hyperglycemic conditions. Further ob-
`jects of the present invention are pharmaceutical compositions that contain biologically-active GLP-1 fragments, as
`well as methods for using such compounds to treat diabetes.
`
`5
`
`10
`
`Summary of the Invention
`
`[0012] According to a first aspect of the present invention there is provided a protected, unprotected, or partially
`protected GLP-1 fragment selected from the following formulae:
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`wherein R1 is selected from NH2, OH, Gly-NH2, and Gly-OH;1 R2 is selected from OH and NH2.
`[0013] Preferably, the formula of the GLP-1 fragment is:
`
`3
`
`MYLAN INST. EXHIBIT 1044 PAGE 3
`
`MYLAN INST. EXHIBIT 1044 PAGE 3
`
`

`

`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`EP 0 699 686 B1
`
`wherein R1 is selected from NH2, OH, Gly-NH2, and Gly-OH;
`
`wherein R2 is selected from NH2 and OH.
`[0014] The GLP-1 fragment is preferably protected or partially protected. In particular, the side chain of the amino
`acid Lys may be protected. The side chain of the amino acid Lys may be protected by acetyl or C1-Z.
`[0015] According to a second aspect of the present invention, there is provided a GLP-1 fragment of the present
`invention, for use in treating diabetes or hyperglycemia in a mammal.
`[0016] According to a third aspect of the present invention, there is provided a pharmaceutical composition compris-
`ing a GLP-1 fragment of the present invention, in combination with a pharmaceutical carrier, diluent or excipient.
`[0017] The composition may be used for use in treating diabetes or hyperglycemia in a mammal.
`[0018] According to a further aspect of the present invention, there is provided the use of a GLP-1 fragment according
`to the present invention in the preparation of a medicament for the treatment of diabetes.
`
`Detailed Description of the Invention
`
`[0019]
`In one embodiment, the present invention provides novel, biologically-active, C-terminal fragments of GLP-
`1. For purposes of this specification, the term "biologically-active" refers to the ability of a substance to lower elevated
`levels of blood glucose in a mammal without stimulating insulin secretion.
`[0020] Given the sequence information herein disclosed and the state of the art in solid phase protein synthesis,
`biologically-active GLP-1 fragments can be obtained via chemical synthesis. However, it also is possible to obtain a
`biologically-active GLP-1 fragment by fragmenting proglucagon using, for example, proteolytic enzymes. Moreover,
`recombinant DNA techniques may be used to express biologically-active GLP-1 fragments.
`[0021] The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found
`in general texts in the area such as Dugas, H. and Penney, C., Bioorganic Chemistry (1981) Springer-Verlag, New
`York, pgs. 54-92, Merrifield, J.M., Chem. Soc., 85:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis,
`pp. 24-66, Freeman (San Francisco, 1969).
`[0022] For example, a biologically-active GLP-1 fragment may be synthesized by solid-phase methodology utilizing
`an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA
`94404) and synthesis cycles supplied by Applied Biosystems. Boc amino acids and other reagents are commercially
`available from Applied Biosystems and other chemical supply houses. Sequential Boc chemistry using double couple
`protocols are applied to the starting p-methyl benzhydryl amine resins for the production of C-terminal carboxamides.
`For the production of C-terminal acids, the corresponding PAM resin is used. Asp, Gln, and Arg are coupled using
`preformed hydroxy benzotriazole esters. The following side chain protecting groups may be used:
`
`Arg, Tosyl
`Asp, cyclohexyl
`Glu, cyclohexyl
`Ser, Benzyl
`Thr, Benzyl
`Tyr, 4-bromo carbobenzoxy
`
`[0023] Boc deprotection may be accomplished with trifluoroacetic acid in methylene chloride. Following completion
`of the synthesis the peptides may be deprotected and cleaved from the resin with anhydrous hydrogen fluoride (HF)
`containing 10% meta-cresol. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried
`
`4
`
`MYLAN INST. EXHIBIT 1044 PAGE 4
`
`MYLAN INST. EXHIBIT 1044 PAGE 4
`
`

`

`EP 0 699 686 B1
`
`out at zero degrees centigrade or below, preferably -20°C for thirty minutes followed by thirty minutes at 0°C. After
`removal of the HF, the peptide/resin is washed with ether, and the peptide extracted with glacial acetic acid and lyophi-
`lized.
`[0024] The preparation of protected, unprotected, and partially protected GLP-1 has been described in the art. See
`U.S. Pat. No. 5,120,712 and 5,118,666, herein incorporated by reference, and Orskov, C., et al., J. Biol. Chem., 264
`(22):12826-12829 (1989) and WO 91/11457 (Buckley, D.I., et al., published August 8, 1991).
`[0025]
`Likewise, the state of the art in molecular biology provides the ordinarily skilled artisan another means by
`which biologically-active GLP-1 fragments can be obtained. Although GLP-1 fragments may be produced by solid
`phase peptide synthesis, recombinant methods, or by fragmenting glucagon, recombinant methods may be preferable
`because higher yields are possible. The basic steps in the recombinant production of a biologically-active GLP-1 frag-
`ment are:
`
`a) isolating a natural DNA sequence encoding GLP-1 or constructing a synthetic or semi-synthetic DNA coding
`sequence for GLP-1,
`
`b) placing the coding sequence into an expression vector in a manner suitable for expressing proteins either alone
`or as a fusion proteins,
`
`c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector,
`
`d) culturing the transformed host cell under conditions that will permit expression of a GLP-1 intermediate, and
`
`e) recovering and purifying the recombinantly produced protein.
`
`[0026] As previously stated, the coding sequences for GLP-1 fragments may be wholly synthetic or the result of
`modifications to the larger, native glucagon-encoding DNA. A DNA sequence that encodes preproglucagon is presented
`in Lund, et al., Proc. Natl. Acad. Sci. U.S.A. 79:345-349 (1982) and may be used as starting material in the recombinant
`production of a biologically-active GLP-1 fragment by altering the native sequence to achieve the desired results.
`[0027] Synthetic genes, the in vitro or in vivo transcription and translation of which results in the production of a
`biologically-active GLP-1 fragment, may be constructed by techniques well known in the art. Owing to the natural
`degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences
`may be constructed which encode GLP-1 intermediates.
`[0028] The methodology of synthetic gene construction is well known in the art. See Brown, et al. (1979) Methods
`in Enzymology, Academic Press, N.Y., Vol. 68, pgs. 109-151. DNA sequences that encode GLP-1 intermediates can
`be designed based on the amino acid sequences herein disclosed. Once designed, the sequence itself may be gen-
`erated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA
`synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404).
`[0029] To effect the expression of a biologically-active GPL-1 fragment, one inserts the engineered synthetic DNA
`sequence in any one of many appropriate recombinant DNA expression vectors through the use of appropriate restric-
`tion endonucleases. See generally Maniatis et al. (1989) Molecular Cloning; A Laboratory Manual, Cold Springs Harbor
`Laboratory Press, N.Y., Vol. 1-3. Restriction endonuclease cleavage sites are engineered into either end of the DNA
`encoding the GLP-1 fragment to facilitate isolation from, and integration into, known amplification and expression vec-
`tors. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the
`parent expression vector to be employed. The choice of restriction sites are chosen so as to properly orient the coding
`sequence with control sequences to achieve proper in-frame reading and expression of the protein of interest. The
`coding sequence must be positioned so as to be in proper reading frame with the promoter and ribosome binding site
`of the expression vector, both of which are functional in the host cell in which the protein is to be expressed.
`[0030] To achieve efficient transcription of the coding region, it must be operably associated with a promoter-operator
`region. Therefore, the promoter-operator region of the gene is placed in the same sequential orientation with respect
`to the ATG start codon of the coding region.
`[0031] A variety of expression vectors useful for transforming prokaryotic and eukaryotic cells are well known in the
`art. See The Promega Biological Research Products Catalogue (1992) (Promega Corp., 2800 Woods Hollow Road,
`Madison, WI, 53711-5399); and The Stratagene Cloning Systems Catalogue (1992) (Stratagene Corp., 11011 North
`Torrey Pines Road, La Jolla, CA, 92037). Also, U.S. Patent No. 4,710,473 describes circular DNA plasmid transfor-
`mation vectors useful for expression of exogenous genes in E. coli at high levels. These plasmids are useful as trans-
`formation vectors in recombinant DNA procedures and:
`
`(a) confer on the plasmid the capacity for autonomous replication in a host cell;
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`5
`
`MYLAN INST. EXHIBIT 1044 PAGE 5
`
`MYLAN INST. EXHIBIT 1044 PAGE 5
`
`

`

`(b) control autonomous plasmid replication in relation to the temperature at which host cell cultures are maintained;
`
`EP 0 699 686 B1
`
`(c) stabilize maintenance of the plasmid in host cell populations;
`
`5
`
`(d) direct synthesis of a protein prod. indicative of plasmid maintenance in a host cell population;
`
`(e) provide in series restriction endonuclease recognition sites unique to the plasmid; and
`
`(f) terminate mRNA transcription.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`These circular DNA plasmids are useful as vectors in recombinant DNA procedures for securing high levels of expres-
`sion of exogenous genes.
`[0032] Having constructed an expression vector for a biologically-active GLP-1 fragment, the next step is to place
`the vector into a suitable cell and thereby construct a recombinant host cell useful for expressing a biologically-active
`GLP-1 fragment. Techniques for transforming cells with recombinant DNA vectors are well known in the art and may
`be found in such general references as Maniatis, et al. supra. Host cells made be constructed from either eukaryotic
`or prokaryotic cells. Eukaryotic host cells are capable of carrying out post-translational glycosylations on expressed
`proteins and some are capable of secreting the desired protein into the culture medium.
`[0033] Prokaryotic host cells generally produce the protein at higher rates, are easier to culture but are not capable
`of glycosylating the final protein. Proteins which are expressed in high-level bacterial expression systems may aggre-
`gate in granules or inclusion bodies which contain high levels of the overexpressed protein. Such protein aggregates
`must be solubilized, denatured and refolded using techniques well known in the art. See Kreuger, et al. (1990) in Protein
`Folding, Gierasch and King, eds., pgs 136-142, American Association for the Advancement of Science Publication No.
`89-18S, Washington, D.C.; and U.S. Patent No. 4,923,967.
`[0034] Regardless of the methods used to produce a biologically-active GLP-1 fragment, purification of the protein
`generally will be required. Methods for purifying proteins are well known in the art and include conventional chroma-
`tography, including ion and cation exchange, hydrophobic interaction, and immuno-affinity chromatographic media.
`The amino acid sequences herein disclosed in conjunction with well known protein purification methods will enable
`the ordinarily skilled artisan to purify biologically-active GLP-1 fragments claimed herein.
`[0035] The present invention also includes salt forms of biologically-active GLP-1 fragments. A biologically-active
`GLP-1 fragment of the invention may be sufficiently acidic or sufficiently basic to react with any of a number of inorganic
`bases, and inorganic and organic acids, to form a salt. Acids commonly employed to form acid addition salts are
`inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the
`like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid,
`carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include the
`sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, meta-
`phosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,
`isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate,
`butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate,
`methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lac-
`tate, gamma-hydroxybutyrate, glycolate,
`tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,
`naphthalene-2-sulfonate, mandelate, and the like. Preferred acid addition salts are those formed with mineral acids
`such as hydrochloric acid and hydrobromic acid, and, especially, hydrochloric acid.
`[0036] Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth
`metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention
`thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like. Salt
`forms of GLP-1 analogs are particularly preferred. Of course, when the compounds of this invention are used for
`therapeutic purposes, those compounds may also be in the form of a salt, but the salt must be pharmaceutically ac-
`ceptable.
`[0037] The inability of a GLP-1 fragment to stimulate insulin secretion may be determined by providing a GLP-1
`fragment to cultured animal cells, such as the RIN-38 rat insulinoma cell line, and monitoring the release of immuno-
`reactive insulin (IRI) into the media. Alternatively one can inject a GLP-1 fragment into an animal and monitor plasma
`levels of immunoreactive insulin (IRI).
`[0038] The presence of IRI is detected through the use of a radioimmunoassay which can specifically detect insulin.
`Any radioimmunoassay capable of detecting the presence of IRI may be employed; one such assay is a modification
`of the 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 is employed. The incubation is prepared with the consecutive addition of 500 µl of phosphate
`buffer, 50 µl of perfusate sample or rat insulin standard in perfusate, 100 µl of anti-insulin antiserum (Wellcome Labo-
`
`6
`
`MYLAN INST. EXHIBIT 1044 PAGE 6
`
`MYLAN INST. EXHIBIT 1044 PAGE 6
`
`

`

`EP 0 699 686 B1
`
`ratories; 1:40,000 dilution), and 100 µl of [125I) insulin, giving a total volume of 750 µl in a 10x75 mm disposable glass
`tube. After incubation for 2-3 days at 4° C, free insulin is separated from antibody-bound insulin by charcoal separation.
`The assay sensitivity is 1-2 uU/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 radioactive label capable
`of labeling a polypeptide can be used, it is preferable to use 3H leucine in order to obtain labeled proinsulin.
`[0039] To determine whether a GLP-1 fragment has insulinotropic properties may also be determined by pancreatic
`infusion. The in situ isolated perfused rat pancreas assay is a modification of the method of Penhos, J.C., et al., Dia-
`betes, 18:733-738 (1969). Fasted male Charles River strain albino rats, weighing 350-600 g, are anesthetized with an
`intraperitoneal injection of Amytal Sodium (Eli Lilly and Co.: 160 ng/kg). Renal, adrenal, gastric, and lower colonic
`blood vessels are ligated. The entire intestine is resected except for about four cm of duodenum and the descending
`colon and rectum. Therefore, only a small part of the intestine is perfused, minimizing possible interference by enteric
`substances with glucagon-like immunoreactivity. The perfusate is a modified Krebs-Ringer bicarbonate buffer with 4%
`dextran T70 and 0.2% bovine serum albumin (fraction V), and is bubbled with 95% O2 and 5% CO2. A nonpulsatile
`flow, 4-channel roller bearing pump (Buchler polystatic, Buchler Instruments Division, Nuclear-Chicago Corp.) is used,
`and a switch from one perfusate source to another is accomplished by switching a 3-way stopcock. The manner in
`which perfusion is performed, monitored, and analyzed follow the method of Weir, G.C., et al., J. Clin. Inestigat. 54:
`1403-1412 (1974), which is hereby incorporated by reference.
`[0040] The present invention also provides pharmaceutical compositions comprising a GLP-1 fragment of the present
`invention in combination with a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical com-
`positions are prepared in a manner well known in the pharmaceutical art, and are administered individually or in com-
`bination with other therapeutic agents, preferably via parenteral routes. Especially preferred routes include intramus-
`cular and subcutaneous administration.
`[0041] Parenteral daily dosages, preferably a single, daily dose, are in the range from about 1 pg/kg to about 1,000
`µg/kg of body weight, although lower or higher dosages 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.
`[0042]
`In making the compositions of the present invention, the active ingredient, which comprises at least one protein
`of the present invention, is usually mixed with an excipient or diluted by an excipient. When an excipient is used as a
`diluent, it may be a solid, semi-solid, or liquid material which acts as a vehicle, carrier, or medium for the active ingre-
`dient.
`[0043]
`In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle
`size prior to combining with the other ingredients. If the active protein is substantially insoluble, it ordinarily is milled to
`particle size of less than about 200 mesh. If the active compound is substantially water soluble, the particle size is
`normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.
`[0044] Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, mannitol, starch-
`es, gum acacia, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl
`cellulose. The formulations can additionally include lubricating agents such as talc, magnesium stearate and mineral
`oil, wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxyben-
`zoates, sweetening agents or flavoring agents. The compositions of the invention can be formulated so as to provide
`quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures
`well known in the art.
`[0045] The compositions are preferably formulated in a unit dosage form with each dosage normally containing from
`about 50 µg to about 100 mg, more usually from about 1 mg to about 10 mg of the active ingredient. The term "unit
`dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals,
`each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect
`in association with a suitable pharmaceutical excipient.
`[0046] For the purpose of parenteral administration, compositions containing a protein of the present invention pref-
`erably are combined with distilled water and the pH is adjusted to about 6.0 to about 9.0.
`[0047] Additional pharmaceutical methods may be employed to control the duration of action. Controlled release
`preparations may be achieved by the use of polymers to complex or absorb a compound of the present invention. The
`controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino
`acids, polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose, carboxymethylcellulose, and protamine sulfate) and
`the concentration of macromolecules as well as the methods of incorporation in order to control release.
`[0048] Another possible method to control the duration of action by controlled release preparations is to incorporate
`a protein of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels,
`poly (lactic acid) or ethylene vinylacetate copolymers.
`[0049] Alternatively, instead of incorporating a compound into these polymeric particles, it is possible to entrap a
`compound of the present invention in microcapsules prepared, for example, by coacervation techniques or by interfacial
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`7
`
`MYLAN INST. EXHIBIT 1044 PAGE 7
`
`MYLAN INST. EXHIBIT 1044 PAGE 7
`
`

`

`EP 0 699 686 B1
`
`polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules, respectively, or in colloidal drug delivery
`systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or in mac-
`roemulsions. Such teachings are disclosed in Remington's Pharmaceutical Sciences (1980).
`[0050] Similarly, the present invention provides a method for treating diabetes or hyperglycemia in a mammal, pref-
`erably a human, in need of such treatment comprising administering an effective amount of a GLP-1 fragment or
`composition of the present invention, to such a mammal.
`[0051] By way of illustration, the following examples are provided to help describe how to make and practice the
`various embodiments of the invention. These example are in no way meant to limit the scope of the invention.
`
`5
`
`10
`
`Example 1
`
`Synthesis of GLP-1(9-36)NH2
`
`[0052] GLP-1(9-36)NH2 (SEQ ID NO:9) was produced by solid phase peptide chemistry on an Applied Biosystems
`(ABI) 460A peptide synthesizer using a MBHA resin (Applied Biosystems Inc., lot # AlA023, 0.77 mmol/g). All amino
`acids had their α-amino groups protected by the tert-butyloxycarbonyl (t-Boc) group. Those with reactive side chains
`had them protected as follows: Arg(Tos); Lys(Cl-Z); Trp(CHO); Glu(CHex); Tyr(Br-Z); Ser(Bzl); Asp(OBzl); Thr(Bzl).
`[0053] The protected amino acids were activated in dichloromethane (DCM) with one half an equivalent of dicy-
`clohexylcarbodiimide (DCC) per equivalent of amino acid to give the symmetric anhydride of the amino acid. However,
`arginine, glutamine, and glycine residues were activated by forming the 1-hydroxybenzotriazole (HOBt) esters of these
`amino acids (1:1:1 equivalents of amino acid, HOBt, and DCC in dimtethylformamid