United States Patent | 119
`Chenetal.
`[45] Date of Patent:
`
`[11] Patent Number:
`
`5,512,549
`Apr. 30, 1996
`
`NCOATTA
`
`US005512549A
`
`[54] GLUCAGON-LIKE INSULINOTROPIC
`PEPTIDE ANALOGS, COMPOSITIONS, AND
`METHODSOF USE
`
`(75]
`
`Inventors: Victor J. Chen, Indianapolis; Richard
`D. DiMarchi, Carmel; David L.
`Smiley, Greenfield; Russell D. Stucky;
`Aidas V. Kriauciunas, Indianapolis, all
`of Ind.
`
`[73] Assignee: Eli Lilly and Company,Indianapolis,
`Ind.
`
`[21] Appl. No.: 324,960
`
`[22] Filed:
`
`Oct. 18, 1994
`
`[51]
`
`Int. Cho A61K 38/00; A61K 38/26;
`CO7K 14/605
`
`[52] U.S. Ch. cccccsesssesseceeeees 514/12; 530/308; 530/324
`[58] Field of Search .uu.......c.cceccccsecssseeeeee 530/308, 324;
`514/12
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,070,188
`5,118,666
`5,120,712
`
`12/1991 Nijieha et al. oeesssceesceees 530/324
`
`we 514/12
`6/1992 Habener............
`6/1992 Habener ..........csccsesescescseoeserse 514/12
`
`FOREIGN PATENT DOCUMENTS
`
`12/1982
`0082731
`2/1994
`0619322A2
`11/1987
`WO087/06941
`10/1990
`WO090/11296
`8/1991
`WO91/11457
`9/1993
`WO093/18786
`W093/25579 12/1993
`
`.
`.
`
`European Pat. Off.
`European Pat. Off.
`WIPO.
`WIPO .
`WIPO.
`WIPO.
`WIPO .
`
`OTHER PUBLICATIONS
`
`Ser. No. 08/164,277, Galloway, et al, filing date Dec. 9,
`1993.
`Kreymann,et. al., “Glucagon—Like Peptide 7-36 A Physi-
`ological Incretin In Man”, The Lancet, vol. 2, pp. 1300-1303
`(Dec. 5, 1987).
`Holst,et. al., “Truncated glucagon-like peptide J, an insulin-
`—releasing hormonefrom the distal gut”, FEBS Letters, vol.
`211, No. 2, pp. 169-174 (Jan. 1987).
`Mojsov, et. al., “Insulinotropic: Glucagon—like Peptide I
`(7-37) Co.-encoded in the Glucagon Gene Is a Potent
`Stimulator of Insulin Release in the Perfused Rat Pancreas”,
`The American Society for Clinical Investigation, Inc., vol.
`79, pp. 616-619 (Feb. 1987).
`.
`Goke,et. al., “Glucagon like peptide-1 (7-36) amide is a
`new incretin/enterogastrone candidate”, European Journal
`of Clinical Investigation, vol. 21, pp. 135-144 (1991).
`Suzuki, et. al., “Effects Of GLP-1 And Its Fragment Pep-
`tides On Pancreatic Hormone Release”, Diabetes Research
`and Clinical Practice, Supp. 1, vol. 5, ORA-007-007,p.
`S30 (1988).
`Weir, et. al., “Glucagonlike Peptide I (7-37) Actions on
`Endocrine Pancreas”, Diabetes, vol. 38, pp. 338-342 (Mar.
`1989).
`
`Komatsu,et. al., “Glucagonostatic and Insulinotropic Action
`of Glucagonlike Peptide I-(7-36)~Amide”, Diabetes, vol.
`38, pp. 903-905, (Jul. 1989).
`Orskov, et. al., “Complete Sequences of Glucagon-like
`Peptide—1 from Human and Pig Small Intestine”, The Jour-
`nal of Biological Chemistry, vol. 264, No. 22, pp.
`12826-12929, (Aug. 5, 1989).
`Takahashi, et. al., “Radioimmunoassay For Glucagon—Like
`Peptide—1 In Human Plasma Using N-Terminal And C—Ter-
`minal Directed Antibodies: A Physiologic Insulinotropic
`Role of GLP-1 (7-36 Amide)”, Biomedical Research vol. 11
`(2), pp. 99-108, (1990).
`Mojsov, “Structural requirements for biological activity of
`glucagon-like peptide-I’”, Int J Peptide Protein Res, vol. 40,
`pp. 333-343 (1992).
`Orskov, “Glucagon-like peptide-1, a new hormone of the
`entero—insular axis”, Diabetologia, vol. 35, pp. 701-711 -
`(1992).
`Thorens, et. al., “Glucagon—Like Peptide-I and the Control
`of Insulin Secretion in the Normal State and in NIDDM”,
`Diabetes, vol. 42, pp. 1219-1225 (Sep. 1992).
`Nauck,et. al., “Normalization of fasting hyperglycaemia by
`exogenous glucagon-like peptide 1 (7-36 amide in Type 2
`(non-insulin-dependent) diabetic patients”, Diabetologia,
`vol. 36, pp. 741-744 (1993).
`Nauck,et. al., “Preserved Incretin Activity of Glucagon—like
`Peptide 1 (7-36 Amide) but Not of Synthetic Human Gastric
`Inhibitory Polypeptide in Patients with Type-2 Diabetes
`Mellitus”, The American Society for Clinical Investigation,
`Inc., vol. 91, pp. 301-307, (Jan. 1993).
`Hvidberg, et al., “Effect of Glucagon-like Peptide-1 (pro-
`glucagon 78-107 amide) on Hepatic Glucose Production in
`Healthy Man”, Metabolism, vol. 43, No. 1, pp. 104-108,
`(Jan. 1994).
`Fehmann, et al, “Insulinotropic Glucagonlike Peptide-I
`(7-37) (7-36) Amide A New Incretin Hormone”, TEM, vol.
`3, No. 5, 158-163, (1992).
`Hashimoto, et al, “Synthesis of Palmitoyl Derivatives of
`Insulin and Their Biological Activities”, Pharmaceutical
`Research, vol. 6, No. 2, 171-176 (1989).
`Suzuki, S., et al. “Comparison of the Effects of Various
`C-Terminal and N-Terminal Fragment Peptides of Glu-
`cagon—Like Peptide—-1 on Insulin and Glucagon Release
`from the Isolated Perfused Rat Pancreas” Endocrinology,
`vol. 125, No. 6, 3109-3114 (1989).
`
`Primary Examiner—Jill Warden
`Assistant Examiner—Benet Prickril
`Attorney, Agent, or Firm—Ronald S. Maciak; David E.
`Boone
`
`[57]
`
`ABSTRACT,
`
`Glucagon-like insulinotropic peptide (GLP-1(7-37)) analogs
`and derivatives are disclosed. The analogs include amino
`acid substitutions, amino and carboxyl terminal modifica-
`tions, and C,-C,, acylations. The claimed compounds
`stimulate the secretion or biosynthesis of insulin in poorly
`functioning beta cells and are therefore useful in treating
`Type If diabetics
`
`28 Claims, No Drawings
`
`MPI EXHIBIT 1017 PAGE 1
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`MPI EXHIBIT 1017 PAGE 1
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`MPI EXHIBIT 1017 PAGE 1
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`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1017, p. 1 of 15
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`

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`5,512,549
`
`1
`GLUCAGON-LIKE INSULINOTROPIC
`PEPTIDE ANALOGS, COMPOSITIONS, AND
`METHODS OF USE
`
`FIELD OF INVENTION
`
`invention relates to organic and peptide
`The present
`chemistry as applied to pharmaceutical research and devel-
`opment. The invention provides novel peptide derivatives
`and compositions that are useful for up-regulating insulin
`expression in mammals and for treating diabetes.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`15
`
`2
`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 foundto be identical (Ghiglione, M., et al.,
`Diabetologia, 27:599-600 (1984)).
`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 presentin
`the pancreas as 37 and 34 aminoacid peptides respectively.
`The similarity between GLP-1 and glucagon suggested to
`early investigators that GLP-1 might have biological activ-
`ity. Although some investigators found that GLP-1 could
`inducerat 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 beartifac-
`tual.
`
`Endocrine secretions of pancreatic islets are regulated by
`complex control mechanisms driven not only by blood-
`bome metabolites such as glucose, amino acids, and cat-
`echolamines, 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 glu-
`cose-mediated insulin secretion. In addition to inter-islet
`paracrine regulationofinsulin secretion, there is evidence to
`support the existence of insulinotropic factors in the intes-
`tine. This concept originates from observations that glucose
`It has now been shownthat biologically processed forms
`taken orally is a much more potent stimulant of insulin
`of GLP-1 have insulinotropic properties and may delay
`secretion than is a comparable amount of glucose given
`gastric emptying. GLP-1(7-34) and GLP-1(7-35) are dis-
`intravenously.
`closed in U.S. Pat. No. 5,118,666, herein incorporated by
`The human hormone glucagon is a 29-amino acid hor-
`reference. GLP-1(7—37) is disclosed in U.S. Pat. No: 5,120,
`mone produced in pancreatic A-cells. The hormonebelongs
`712, herein incorporated by reference.
`to a multigene family of structurally related peptides that
`Variants and analogs of GLP-1 are knownin the art. These
`include secretin, gastric inhibitory peptide, vasoactive intes-
`variants and analogs include, for example, GLP-1(7—36),
`tinal peptide and glicentin. These peptides variously regulate
`Gin?-GLP-1(7-37),|D-Gln?-GLP-1(7-37),_acetyl-Lys?-
`carbohydrate metabolism, gastrointestinal mobility and
`GLP-1(7-37), Thr'®-Lys?8-GLP-1(7-37), and Lys'®-GLP-
`secretory processing. However,
`the principal recognized
`1(7-37). Derivatives of GLP-1 include, for example, acid
`actions of pancreatic glucagon are to promote hepatic gly-
`addition salts, carboxylate salts,
`lower alkyl esters, and
`cogenolysis and glyconeogenesis, resulting in an elevation
`amides (see, e.g., WO91/11457). Generally,
`the various
`of blood sugar levels. In this regard, the actions of glucagon
`disclosed forms of GLP-1 are known to stimulate insulin
`are counter regulatory to those of insulin and may contribute
`secretion (insulinotropic action) and cAMP formation (see,
`to the hyperglycemia that accompanies Diabetes mellitus
`e.g., Mojsov, S.,
`Int.
`J. Peptide Protein Research,
`(Lund, P. K., et al, Proc. Natl Acad. Sci. U.S.A.,
`40:333-343 (1992)).
`79:345-349 (1982)).
`More importantly, numerous investigators have demon-
`Whenglucagonbindsto its receptor on insulin producing
`strated a predictable relationship between various in vitro
`cells, cAMP production increases which in turn stimulates
`laboratory experiments and mammalian, especially human,
`insulin expression (Korman, L. Y., et al., Diabetes,
`insulinotropic responses to exogenous administration of
`34:717—722 (1985)). Moreover, high levels of insulin down-
`GLP-1, GLP-1(7-36) amide, and GLP-1(7-37) acid (see,
`regulate glucagon synthesis by a feedback inhibition mecha-
`e.g., Nauck, M.A., et al., Diabetologia, 36:741—744 (1993);
`nism (Ganong, W. F.,, Review ofMedical Physiology, Lange
`Gutniak, M., et al, New England J. of Medicine,
`Publications, Los Altos, Calif., p. 273 (1979)). Thus, the
`326(20):1316—-1322 (1992); Nauck, M. A., et al., J. Clin.
`expression of glucagonis carefully regulated by insulin, and
`Invest., 91:301-307 (1993); and Thorens, B., et al., Diabe-
`ultimately by serum glucoselevels.
`tes, 42:1219-1225 (1993)).
`is
`form of glucagon,
`Preproglucagon,
`the precursor
`The fundamental defects responsible for causing hyper-
`encoded by a 360 base pair gene and is processed to form
`glycemia in mature onset diabetes include impaired secre-
`proglucagon (Lund, et al., Proc. Natl. Acad. Sci. U.S.A.
`tion of endogenous insulin and resistance to the effects of
`79:345—349 (1982)). Patzelt, et al. (Nature, 282:260-266
`insulin by muscle andliver tissue (Galloway, J. S., Diabetes
`(1979)) demonstrated that proglucagonis further processed
`Care, 13:1209-1239, (1990)). The latter defect results in
`into glucagon and a second peptide. Later experiments
`excess glucose production in the liver. Thus, whereas a
`demonstrated that proglucagon is cleaved carboxyl to Lys-
`normal individual releases glucose at the rate of approxi-
`Arg or Arg-Arg residues (Lund,P. K., et al., Lopez L. C., et
`mately 2 mg/kg/minute, a patient with mature onset diabetes
`al., Proc. Natl. Acad. Sci. U.S.A., 80:5485-5489 (1983), and
`releases glucose at a rate exceeding 2.5 mg/kg/minute,
`Bell, G. 1, et al., Nature 302:716—-718 (1983)). Bell, G.L, et
`resulting in a net excess of at least 70 grams of glucose per
`al., also discovered that proglucagon contained three dis-
`24 hours.
`crete 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-
`
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`Because there exists exceedingly high correlations
`between hepatic glucose production, fasting blood glucose
`
`MPI EXHIBIT 1017 PAGE 2
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`DR. REDDY’S LABORATORIES, INC.
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`5,912,549
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`3
`4
`adding various R groups via a peptide bond to the amino
`levels, and overall metabolic control as indicated by glyco-
`terminus of the peptide portion of Formula 1. Optionally,
`hemoglobin measurements (Galloway, J. A., supra; and
`Galloway,J. A., et al., Clin. Therap., 12:460-472 (1990)),it
`further compoundsof the invention are made by acylating
`the epsilon amino group ofthe Lys** residue and by making
`is readily apparent that control of fasting blood glucose is
`limited aminoacid substitutionsat position 26 or byaltering
`essential for achieving overall normalization of metabolism
`sufficient to prevent hyperglycemic complications. Since
`the carboxy terminus. Therefore, preparing the polypeptide
`backboneof Formula1is a logical first step when preparing
`existing insulin therapies rarely normalize hepatic glucose
`production without producing significant hyperinsulinemia
`compoundsof the present invention.
`and hypoglycemia (Galloway, J. A., and Galloway,J. A., et
`It should be noted that this specification uses the nomen-
`al., supra) alternative approaches are needed. Thereapy
`clature scheme that has developed around processed forms-
`based on administration of GLP-1 analogs is one such
`of GLP-1. In this scheme, the amino terminusof the known
`approach and is an object of the present invention.
`GLP-1(7-37) OH has been assigned number 7 and the
`Presently, therapy involving the use of GLP-1 type. mol-
`carboxy terminus number37. Therefore,the first Ala residue
`of Formula 1 correspondsto residue 8 of GLP-1(7-37)OH.
`ecules has presented a significant problem because the
`serum half-life of such peptides is quite short. For example,
`Likewise Xaa in Formula 1 corresponds to residue 26 of
`GLP-1 (7-37) has a serum half-life of only 3 to 5 minutes.
`GLP-1(7-37)OH and so forth.
`Presently, the activity of dipeptidyl-peptidase IV (DPP IV)
`Given the sequence information herein disclosed and the
`is believed to readily inactivate GLP-1(7-37) in addition to
`state of the art in solid phase protein synthesis, the protein
`rapid absorption and clearance following parenteral admin-
`portion of Formula 1 can be prepared via chemical synthesis.
`istration. Thus, there exists a critical need for biologically
`Also, recombinant DNAtechniques may be used to express
`active GLP-1 (7-37) analogs that possess extended pharma-
`the protein backbone of Formula 1.
`codynamic profiles following parenteral administration.
`The principles of solid phase chemical synthesis of
`Accordingly, the primary object of this invention is to
`polypeptides are well knownin the art and may be found in
`provide novel, chemically modified peptides that not only
`general texts in the area such as Dugas, H. and Penney, C.,
`stimulate insulin secretion in type I diabetics but also
`Biooraanic Chemistry (1981) Springer-Verlag, New York,
`produceother beneficial insulinotropic responses. The com-
`pgs. 54-92, Merrifield, J. M., Chem. Soc., 85:2149 (1962),
`pounds of the present invention persist in the serum for
`and Stewart and Young, Solid Phase Peptide Synthesis, pp.
`longer periods than native GLP-1(7-37) either by showing
`24-66, Freeman (San Francisco, 1969).
`resistance to DPP IV or by being absorbed and cleared
`For example, the protein portion of Formula 1 may be
`slower
`than native GLP-1(7-37)
`following parenteral
`synthesized by solid-phase methodology utilizing a 430A
`administration. Most surprisingly, some compoundsof the
`peptide synthesizer (PE-Applied Biosystems, Inc., 850 Lin-
`present invention demonstrated a synergistic effect as indi-
`coln Center Drive, Foster City, Calif. 94404) and synthesis
`vidual alterations to GLP-1(7-37) failed to add-up to the
`cycles supplied by PE-Applied Biosystems. Boc amino
`biological performance of compoundsthat contained all of
`acids and other reagents are commercially available from
`the alterations.
`PE-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 produc-
`tion 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
`Boc deprotection may be accomplished with trifluoroace-
`tic acid in methylene chloride. Following completion of the
`synthesis the peptides may be deprotected and cleaved from
`the resin with anhydrous hydrogenfluoride (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 lyo-
`philized.
`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, herein incorpo-
`rated 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 Aug. 8, 1991).
`Likewise, the state of the art in molecular biology pro-
`vides the ordinarily skilled artisan another means by which
`
`wherein R’ is selected from the group consisting of
`4-imidazopropionyl (des-amino-histidyl), 4-imidazoacety]l,
`or 4-imidazo-a, adimethyl-acety];
`R? is selected from the group consisting of C.-C,
`unbranched acyl, or is absent;
`R? is selected from the group consisting of Gly-OH or
`NH,; and,
`Xaa is Lys or Arg.
`The present invention also provides pharmaceutical com-
`positions comprising a compoundofthe presentinventionin
`combination with a pharmaceutically acceptable carrier,
`diluent, or excipient. The present invention further provides
`a method for treating non-insulin dependent diabetes mel-
`litus in a mammal in need of such treatment comprising
`administering an effective amount of a compound of the
`present invention to said mammal.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`In one embodiment, the present invention provides ana-
`logs of naturally-occurring GLP-1(7-37) that arise from
`
`MPI EXHIBIT 1017 PAGE 3
`
`SUMMARY OF THE INVENTION
`
`The present invention provides compoundsof the general
`formula:
`
`(Formula 1)
`R!—Ala—Glu—Gly—Thr—Phe~Thr— Ser—Asp— Val—
`Ser — Ser — Tyr — Leu — Glu — Gly — Gln — Ala Ala — Xaa—
`
`GluPhe—He—Ala~Tsp—Leu—Val—Tys—Gly—Arg—R?
`R2
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`DR. REDDY’S LABORATORIES, INC.
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`5,512,549
`
`5
`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 bepref-
`erable 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 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.
`Aspreviously 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
`compoundsof the present invention by altering the native
`sequence to achieve the desired results.
`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 Formula1.
`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.
`DNAsequences that encode the protein backbone of For-
`mula 1 can be designed based on the amino acid sequences
`herein disclosed. Once designed, the sequenceitself may be
`generated using conventional DNA synthesizing apparatus
`such as the Model 380A or 380B DNAsynthesizers (PE-
`Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster
`City, Calif. 94404).
`To effect expression of the polypeptide of Formula 1, one
`inserts the engineered synthetic DNA sequencein any one of
`many appropriate recombinant DNA expression vectors
`through the use of appropriate restriction 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 GLP-1 intermediate-en-
`coding DNAto facilitate isolation from, and integration into,
`known amplification and expression vectors. The particular
`endonucleases employed will be dictated by the restriction
`endonuclease cleavage pattern of the parent expression
`vector to be employed. The choice ofrestriction sites are
`chosen so as to properly orient the coding sequence with
`contro] sequences to achieve proper in-frame reading and
`expression of the protein of interest. The coding sequence
`mustbe 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.
`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 syn-
`
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`6
`thetic gene is placed in the same sequential orientation with
`respect to the ATG start codon of the synthetic gene.
`A variety of expression vectors useful for transforming
`prokaryotic and eukaryotic cells are well knownin theart.
`See The Promega Biological Research Products Catalogue
`(1992) (Promega Corp., 2800 Woods Hollow Road, Madi-
`son, Wis., 53711-5399); and The Stratagene Cloning Sys-
`tems Catalogue (1992) (Stratagene Corp., 11011 North
`Torrey Pines Road, La Jolla, Calif., 92037). Also, U.S. Pat.
`No. 4,710,473 describes circular DNA plasmid transforma-
`tion vectors useful for expression of exogenousgenesin E.
`coli at high levels. These plasmids are useful as transfor-
`mation vectors in recombinant DNA procedures and
`(a) confer on the plasmid the capacity for autonomous
`replication in a host cell;
`(b) control autonomousplasmid replication in relation to
`the temperature at which host cell cultures are main-
`tained;
`
`(c) stabilize maintenance of the plasmid in host cell
`populations;
`(d) direct synthesis of a protein prod. indicative of plas-
`mid maintenance in a hostcell population;
`(e) provide in series restriction endonuclease recognition
`sites unique to the plasmid; and
`(f) terminate mRNAtranscription.
`These circular DNA plasmids are useful as vectors in
`recombinant DNA procedures for securing. high levels of
`expression of exogenous genes.
`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 recombinanthost 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.
`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 char-
`acteristically aggregate in granules or inclusion bodies
`whichcontain highlevels of the overexpressed protein. Such
`protein aggregates typically 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. 89-188, Washing-
`ton, D.C.; and U.S. Pat. No. 4,923,967.
`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 accom-
`plished by synthetic chemical means. Because all of the
`various organic groups contemplated in this invention con-
`tain 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.
`
`MPI EXHIBIT 1017 PAGE 4
`
`MPI EXHIBIT 1017 PAGE 4
`
`MPI EXHIBIT 1017 PAGE 4
`
`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1017, p. 4 of 15
`
`

`

`5,512,549
`
`8
`oxalate, malonate, succinate, suberate, sebacate, fumarate,
`maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate,
`chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxy-
`benzoate, methoxybenzoate, phthalate, sulfonate, xylene-
`sulfonate, phenylacetate, phenylpropionate, phenylbutyrate,
`citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate,
`methanesutfonate,
`propanesulfonate,
`naphthalene-1-sul-
`fonate, 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.
`Base addition salts include those derived from inorganic
`bases, such as ammonium oralkali 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, ammo-
`nium hydroxide, potassium carbonate, and the like. Salt
`forms of GLP-1(7-37) analogs are particularly preferred.
`When the compoundsof the invention are used for thera-
`peutic purposes, those compounds mayalso be in the form
`of a salt, but the salt must be pharmaceutically acceptable.
`GLP-1(7—37) analogs of the present invention demon-
`strate insulinotropic activity. The term “‘insulinotropic activ-
`ity” relates to the ability of a substance to stimulate, or cause
`the stimulation of, the synthesis or expression of the hor-
`mone insulin.
`The insulinotropic property of a compound maybe deter-
`mined by providing that compound to animal cells, or
`injecting that compound into animals and monitoring the
`release of immunoreactive insulin (IRI) into the media or
`circulatory system of the animal, respectively. The presence
`of IRI is detected through the use of a radioimmunoassay
`which can specifically detect insulin.
`Although any radioimmunoassay capable of detecting the
`presence of IRI may be employed, a modification of the
`assay may also be used. See J. D. M., et al., Acta Endo-
`’ crinol., 70:487-509 (1972). The insulinotropic property of a
`compound mayalso be determined by pancreatic infusion.
`See Penhos, J. C., et al., Diabetes, 18:733-738 (1969).
`The present invention also provides pharmaceutical com-
`positions comprising a compoundofthe present invention in
`combination with a pharmaceutically acceptable carrier,
`diluent, or excipient. Such pharmaceutical compositions are
`prepared in a manner well knownin the pharmaceutical art,
`and are administered individually or in combination with
`other therapeutic agents, preferably via parenteral routes. An
`especially preferred route is by subcutaneous administration.
`Parenteral dosages may range from about1 pg/kg to about
`1,000 g/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 suchcriteria as the patient’s height, weight, sex, age,
`and medical history.
`In making the compositions of the present invention, the
`active ingredient, which comprisesat least one compound 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.
`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
`compoundis substantially insoluble, it ordinarily is milled to
`particle size of less than about 200 mesh. If the active
`compoundis substantially water soluble, the particle size is
`normally adjusted by milling to provide a substantially
`uniform distributionin the formulation, e.g., about 40 mesh.
`
`10
`
`15
`
`30
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`7
`Preferred imidazolic groups of the present invention are:
`
`co
`
`N
`
`( T~N
`
`4-imidazopropionyl (des-amino-histidy])
`4-imidazoacetyl
`
`N
`
`(tTN
`
`Co,
`
`and
`4-imidazo-a,adimethyl-acetyl
`
`H3C
`
`8=—CH3
`
`co
`
`N (
`
`N
`
`|
`
`The most preferred group is 4-imidazopropionyl.
`Further embodiments of the present invention are made
`by acylating the epsilon amino groupof the Lys** residue.
`Straight chain acyl additions containing between 6 to 10
`carbon atoms are preferred and unbranched C,
`is most
`preferred.
`Other embodimentof 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.
`Modificationsat the carboxy terminusare also included in
`the present invention. As such R® may be Gly-OH or NH,;
`Gly-OH is preferred over the carboxy terminal amide
`embodiments.
`Addition of an acyl group to the epsilon amino group of
`Lys** may be accomplished using any one of a variety of
`methods knowninthe art. See Bioconjugate Chem. “Chemi-
`cal Modifications of Proteins: History and Applications”
`pages 1, 2-12 (1990); Hashimotoet al., PharmacueticalRes.
`“Synthesis of Palmitoyl Derivatives of Insulin and their
`Biological Activity” Vol. 6, No: 2 pp.171-176 (1989).
`For
`example,
`the N-hydroxy-succinimide
`ester of
`octanoic acid can be addedto the lysyl-epsilon amine using
`50% acetonitrile in borate buffer. The peptide can be acy-
`lated either before or after the imidazolic group is added.
`Moreover, if the peptide is prepared recombinantly, acyla-
`tion prior to enzymatic cleavageis. possible.
`The present invention also includes salt forms of GLP-
`1(7-37) analogs. Compounds of the invention may be suf-
`ficiently acidic or sufficiently basic to react with any of a
`numberof inorganic bases, and inorganic and organicacids,
`to form a salt. Acids commonly employed to form acid
`addition salts are inorganic acids such as hydrochloricacid,
`hydrobromic acid, hydroiodic acid,sulfuric acid, phosphoric
`acid, and the like, and organic acids such as p-toluene-
`sulfonic acid, methanesulfonic acid, oxalic acid, p-bro-
`mophenyl-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, dihydrogen-
`phosphate, metaphosphate, pyrophosphate, chloride, bro-
`mide, iodide, acetate, propionate, decanoate, caprylate, acry-
`late, formate, isobutyrate, caproate, heptanoate, propiolate,
`
`MPI EXHIBIT 1017 PAGE 5
`
`MPI EXHIBIT 1017 PAGE 5
`
`MPI EXHIBIT 1017 PAGE 5
`
`DR. REDDY’S LABORATORIES, INC.
`IPR2024-00009
`Ex. 1017, p. 5 of 15
`
`

`

`5,512,549
`
`9
`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 yg to about 100
`mg, more usually from about 1 mg to about 10 mgofthe
`active ingredient. The term “unit dosage form” refers to
`physically discrete units suitable as unitary dosages fo