(19)
`
`(12)
`
`Europäisches Patentamt
`
`European Patent Office
`
`Office européen des brevets
`
`*EP000708179B1*
`EP 0 708 179 B1
`
`(11)
`
`EUROPEAN PATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`22.12.2004 Bulletin 2004/52
`
`(21) Application number: 95307299.8
`
`(22) Date of filing: 13.10.1995
`
`(51) Int Cl.7: C12N 15/16, C07K 14/605,
`A61K 38/26
`
`(54) Glucagon-like insulinotropic peptide analogs, compositions, and methods of use
`
`Glucagon-ähnliche insulinotrope Peptid-Analoge, Zusammensetzungen und Verwendungsverfahren
`
`Analogues de peptides insulinotropes de type glucagon, compositions et méthodes d’utilisation
`
`(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: 18.10.1994 US 324960
`
`(43) Date of publication of application:
`24.04.1996 Bulletin 1996/17
`
`(60) Divisional application:
`02000257.2 / 1 227 151
`
`(73) Proprietor: ELI LILLY AND COMPANY
`Indianapolis, Indiana 46285 (US)
`
`(72) Inventors:
`• Chen, Victor John
`Indianapolis, Indiana 46140 (US)
`
`• Dimarchi, Richard D.
`Carmel, Indiana 46033 (US)
`• Kriauciunas, Aidas V.
`Indianapolis, Indiana 46254 (US)
`• Smiley, David L.
`Greenfield, Indiana 46140 (US)
`• Stucky, Russell D.
`Indianapolis, Indiana 46227 (US)
`
`(74) Representative: Kent, Lindsey Ruth et al
`Eli Lilly and Company Limited
`Lilly Research Centre
`Erl Wood Manor
`Sunninghill Road
`Windlesham, Surrey GU20 6PH (GB)
`
`(56) References cited:
`EP-A- 0 658 568
`WO-A-92/18531
`
`WO-A-91/11457
`
`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 708 179B1
`
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`Description
`
`EP 0 708 179 B1
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`[0001] The present invention relates to organic and peptide chemistry as applied to pharmaceutical research and
`development. The invention provides novel peptide derivatives and compositions that are useful for up-regulating in-
`sulin expression in mammals and for treating diabetes.
`[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. In addition to inter-islet para-
`crine regulation of insulin secretion, there is evidence to support the existence of insulinotropic factors in the intestine.
`This concept originates from observations that glucose taken orally is a much more potent stimulant of insulin secretion
`than is a comparable amount of glucose given intravenously.
`[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 mobility
`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 precursor form of glucagon, is encoded by 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). 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 glucagon and GLP-1 might be artifactual.
`[0008]
`It has now been shown that biologically processed forms of GLP-1 have insulinotropic properties and may
`delay gastric emptying. GLP-1(7-34) and GLP-1(7-35) are disclosed in U.S. Patent No: 5,118,666. GLP-1(7-37) is
`disclosed in U.S. Patent No: 5,120,712.
`[0009] Variants and analogs of GLP-1 are known in the art. These variants and analogs 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). Generally, the various disclosed forms of GLP-1 are known to stimulate insulin secretion
`(insulinotropic action) and cAMP formation (see, e g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)).
`[0010] EP - A - 0658568 published after the priority date of the present application relates to glucagon- like peptide-
`1 molecules having a modified histidine functionality at the 7-position.
`[0011] More importantly, numerous investigators have demonstrated a predictable relationship between various in
`vitro laboratory experiments and mammalian, especially human, insulinotropic responses to exogenous administration
`
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`EP 0 708 179 B1
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`of GLP-1, GLP-1(7-36) amide, and GLP-1(7-37) acid (see, e.g., Nauck, M.A., et al., Diabetologia, 36:741-744 (1993);
`Gutniak, M., et al., New England J. of Medicine, 326(20):1316-1322 (1992); Nauck, M.A., et al., J. Clin. Invest., 91:
`301-307 (1993); and Thorens, B., et al., Diabetes, 42:1219-1225 (1993)).
`[0012] The fundamental defects responsible for causing hyperglycemia in mature onset diabetes include impaired
`secretion of endogenous insulin and resistance to the effects of insulin by muscle and liver tissue (Galloway, J.S.,
`Diabetes Care, 13:1209-1239, (1990)). The latter defect results in excess glucose production in the liver. Thus, whereas
`a normal individual releases glucose at the rate of approximately 2 mg/kg/minute, a patient with mature onset diabetes
`releases glucose at a rate exceeding 2.5 mg/kg/minute, resulting in a net excess of at least 70 grams of glucose per
`24 hours.
`[0013] Because there exists exceedingly high correlations between hepatic glucose production, fasting blood glucose
`levels, and overall metabolic control as indicated by glycohemoglobin measurements (Galloway, J.A., supra; and Gal-
`loway, J.A., et al., Clin. Therap., 12:460-472 (1990)), it is readily apparent that control of fasting blood glucose is
`essential for achieving overall normalization of metabolism sufficient to prevent hyperglycemic complications. Since
`existing insulin therapies rarely normalize hepatic glucose production without producing significant hyperinsulinemia
`and hypoglycemia (Galloway, J.A., and Galloway, J.A., et al., supra) alternative approaches are needed. Therapy based
`on administration of GLP-1 analogs is one such approach and is an object of the present invention.
`[0014] Presently, therapy involving the use of GLP-1 type molecules has presented a significant problem because
`the serum half-life of such peptides is quite short. For example, GLP-1(7-37) has a serum half-life of only 3 to 5 minutes.
`Presently, the activity of dipeptidyl-peptidase IV (DPP IV) is believed to readily inactivate GLP-1(7-37) in addition to
`rapid absorption and clearance following parenteral administration. Thus, there exists a critical need for biologically
`active GLP-1(7-37) analogs that possess extended pharmacodynamic profiles following parenteral administration.
`[0015] Accordingly, the primary object of this invention is to provide novel, chemically modified peptides that not only
`stimulate insulin secretion in type II diabetics but also produce other beneficial insulinotropic responses. The com-
`pounds of the present invention persist in the serum for longer periods than native GLP-1(7-37) either by showing
`resistance to DPP IV or by being absorbed and cleared slower than native GLP-1(7-37) following parenteral adminis-
`tration. Most surprisingly, some compounds of the present invention demonstrated a synergistic effect as individual
`alterations to GLP-1(7-37) failed to add-up to the biological performance of compounds that contained all of the alter-
`ations.
`[0016] The present invention provides compounds of the general formula:
`
`wherein
`
`R1 is selected from 4-imidazopropionyl (des-amino-histidyl), 4-imidazoacetyl, or 4-imidazo-α,α dimethyl-acetyl;
`R2 is selected from C6-C10 unbranched acyl, or is absent;
`R3 is selected from Gly-OH or NH2; and,
`Xaa is Lys or Arg,
`
`with the proviso when R1 is 4-imidazopropionyl and Xaa is Lys, R2 is selected from C6-C10 unbranched acyl.
`[0017] The present invention also provide pharmaceutical compositions comprising a compound of the present in-
`vention in combination with a pharmaceutically acceptable carrier, diluent, or excipient. The present invention further
`provides for the use of a compound of the present invention for treating non-insulin dependant diabetes mellitus in a
`mammal in need of such treatment comprising administering an effective amount of a compound of the present invention
`to said mammal.
`[0018]
`In one embodiment, the present invention provides analogs of naturally-occuring GLP-1(7-37) that arise from
`adding various R groups via a peptide bond to the amino terminus of the peptide portion of Formula 1. Optionally,
`further compounds of the invention are made by acylating the epsilon amino group of the Lys34 residue and by making
`limited amino acid substitutions at position 26 or by altering the carboxy terminus. Therefore, preparing the polypeptide
`backbone of Formula 1 is a logical first step when preparing compounds of the present invention.
`[0019]
`It should be noted that this specification uses the nomenclature scheme that has developed around processed
`
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`EP 0 708 179 B1
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`forms of GLP-1. In this scheme, the amino terminus of the known GLP-1(7-37)OH has been assigned number 7 and
`the carboxy terminus number 37. Therefore, the first Ala residue of Formula 1 corresponds to residue 8 of GLP-1(7-37)
`OH. Likewise Xaa in Formula 1 corresponds to residue 26 of GLP-1(7-37)OH and so forth.
`[0020] Given the sequence information herein disclosed and the state of the art in solid phase protein synthesis, the
`protein portion of Formula 1 can be prepared via chemical synthesis. Also, recombinant DNA techniques may be used
`to express the protein backbone of Formula 1.
`[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, the protein portion of Formula 1 may be synthesized by solid-phase methodology utilizing a
`430A peptide synthesizer (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) and synthesis
`cycles supplied by PE-Applied Biosystems. Boc amino acids and other reagents are commercially available from PE-Ap-
`plied 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 pro-
`duction of C-terminal acids, the corresponding PAM resin is used. Asn, 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
`out at -5°C to 5°C, preferably on ice for 60 minutes. After removal of the HF, the peptide/resin is washed with ether,
`and the peptide extracted with glacial acetic acid and lyophilized.
`[0024] The preparation of protected, unprotected, and partially protected GLP-1 molecules has been described in
`the art. See U.S. Pat. No. 5,120,712 and 5,118,666 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 the protein portion of Formula 1 can be obtained. Although it may be produced by solid phase peptide synthesis
`or recombinant methods, recombinant methods may be preferable because higher yields are possible. The basic steps
`in recombinant production are:
`
`a) isolating a natural DNA sequence encoding GLP-1 or constructing a synthetic or semisynthetic 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,
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`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.
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`[0026] As previously stated, the coding sequences 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 semisynthetic production of the
`compounds of the present invention 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 the
`protein portion of Formula 1, 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, all of which encode the polypeptide of Formula 1.
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`[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 the protein backbone of
`Formula 1 can be designed based on the amino acid sequences herein disclosed. Once designed, the sequence itself
`may be generated using conventional DNA synthesizing apparatus such as the Model 380A or 380B DNA synthesizers
`(PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404).
`[0029] To effect expression of the polypeptide of Formula 1, one inserts the engineered synthetic DNA sequence in
`any one of many appropriate recombinant DNA expression vectors through the use of appropriate restriction endonu-
`cleases. 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 GLP-1 intermediate-
`encoding DNA to facilitate isolation from, and integration into, known amplification and expression vectors. The par-
`ticular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent ex-
`pression 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 se-
`quence 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 synthetic gene, it must be operably associated with a promoter-operator
`region. Therefore, the promoter-operator region of the synthetic gene is placed in the same sequential orientation with
`respect to the ATG start codon of the synthetic gene.
`[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;
`
`(b) control autonomous plasmid replication in relation to the temperature at which host cell cultures are maintained;
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`(c) stabilize maintenance of the plasmid in host cell populations;
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`(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.
`
`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 the protein of Formula 1, the next step is to place the vector into
`a suitable cell and thereby construct a recombinant host cell useful for expressing the polypeptide. 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.
`[0033] Prokaryotic host cells generally produce the protein at higher rates and are easier to culture. Proteins which
`are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies
`which contain high levels of the overexpressed protein. Such protein aggregates typically must be solubilized, dena-
`tured 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] Having preparing the polypeptide backbone of Formula 1, an imidazole, as defined above in the "Summary
`of the Invention," is added to the amino terminus to produce various embodiments of the present invention. Coupling
`the imidazolic group to the polypeptide of Formula 1 is accomplished by synthetic chemical means. Because all of the
`various organic groups contemplated in this invention contain a carboxylic acid, the imidazolic group can be added by
`solid phase protein synthesis analogous to adding an amino acid to the N-terminus of a polypeptide. Alternatively, an
`activated ester of the imidazolic group can be added by standard chemical reaction methods.
`[0035] Preferred imidazolic groups of the present invention are:
`
`4-imidazopropionyl (des-amino-histidyl)
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`4-imidazoacetyl
`
`and
`
`4-imidazo-α, α dimethyl-acetyl
`
`The most preferred group is 4-imidazopropionyl.
`[0036] Further embodiments of the present invention are made by acylating the epsilon amino group of the Lys34
`residue. Straight chain acyl additions containing between 6 to 10 carbon atoms are preferred and unbranched C8 is
`most preferred.
`[0037] Other embodiment of the present invention include amino acid substitutions at position 26 (Xaa) of Formula
`1. Lys, and Arg are acceptable at this position, though Arg is preferred.
`[0038] Modifications at the carboxy terminus are also included in the present invention. As such R3 may be Gly-OH
`or NH2; Gly-OH is preferred over the carboxy terminal amide embodiments.
`[0039] Addition of an acyl group to the epsilon amino group of Lys34 may be accomplished using any one of a variety
`of methods known in the art. See Bioconjugate Chem. "Chemical Modifications of Proteins: History and Applications"
`pages 1, 2-12 (1990); Hashimoto et al., Pharmacuetical Res. "Synthesis of Palmitoyl Derivatives of Insulin and their
`Biological Activity" Vol. 6, No:2 pp.171-176 (1989).
`[0040] For example, the N-hydroxy-succinimide ester of octanoic acid can be added to the lysyl-epsilon amine using
`50% acetonitrile in borate buffer. The peptide can be acylated either before or after the imidazolic group is added.
`Moreover, if the peptide is prepared recombinantly, acylation prior to enzymatic cleavage is possible.
`[0041] The present invention also includes salt forms of GLP-1(7-37) analogs. Compounds 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-toluenesul-
`
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`fonic 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, metaphosphate, pyrophosphate, chloride, bromide, io-
`dide, 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, xyle-
`nesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tar-
`trate, 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.
`[0042] 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(7-37) analogs are particularly preferred. When the compounds of the invention are used for therapeutic
`purposes, those compounds may also be in the form of a salt, but the salt must be pharmaceutically acceptable.
`[0043] GLP-1(7-37) analogs of the present invention demonstrate insulinotropic activity. The term "insulinotropic
`activity" relates to the ability of a substance to stimulate, or cause the stimulation of, the synthesis or expression of the
`hormone insulin.
`[0044] The insulinotropic property of a compound may be determined by providing that compound to animal cells,
`or injecting that compound into animals and monitoring the release of immunoreactive insulin (IRI) into the media or
`circulatory system of the animal, respectively. The presence of IRI is detected through the use of a radioimmunoassay
`which can specifically detect insulin.
`[0045] Although any radioimmunoassay capable of detecting the presence of IRI may be employed, a modification
`of the assay may also be used. See J.D.M., et al., Acta Endocrinol., 70:487-509 (1972). The insulinotropic property of
`a compound may also be determined by pancreatic infusion. See Penhos, J.C., et al., Diabetes, 18:733-738 (1969).
`[0046] The present invention also provides pharmaceutical compositions comprising a compound 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. An especially preferred route is by subcutane-
`ous administration.
`[0047] Parenteral dosages may 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.
`[0048]
`In making the compositions of the present invention, the active ingredient, which comprises at least one com-
`pound 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 semi-solid or liquid material which acts as a vehicle, carrier, or medium for the active ingredient.
`Liquid excipients are preferred.
`[0049]
`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 compound 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.
`[0050] 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. 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.
`[0051] For the purpose of parenteral administration, compositions containing a compound of the present invention
`preferably are combined with distilled water at an approriate pH. 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, methylcellu-
`lose, carboxymethylcellulose, and protamine sulfate) and the concentration of macromolecules as well as the methods
`of incorporation in order to control release.
`[0052] Another possible method to control the duration of action by controlled release preparations is to incorporate
`a compound of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydro-
`gels, poly (lactic acid) or ethylene vinylacetate copolymers.
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` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1043, p. 7 of 22
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`EP 0 708 179 B1
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`[0053] 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
`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).
`[0054] The compounds of the present invention have insulinotropic activity. Thus, another aspect of the present
`invention provides a method for enhancing the expression of insulin comprising providing to a mammalian pancreatic
`B-type islet cell an effective amount of a compound of the present invention.
`[0055] Similarly, the present invention provides a method for treating diabetes mellitus in a mammal, preferably a
`human, in need of such treatment comprising administering an effective amount of a compound or composition of the
`present invention, to such a mammal.
`[0056] 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.
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`Example 1
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`Synthesis of (Arg26)GLP-1(8-37)OH
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`[0057] The polypeptide portion of Formula 1 wherein Xaa is Arg and R3 is Gly-OH was prepared by solid phase
`synthesis on a Model 430A peptide synthesizer (PE-Applied Biosystems, Foster City, CA) using the Boc protecting
`strategy. The side chain protecting groups were: Asp (C