United States Patent 19
`Chen et al.
`
`(54) GLUCAGON-LIKE INSULINOTROPIC
`PEPTIDE ANALOGS, COMPOSITIONS, AND
`METHODS OF 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,
`End.
`
`21) Appl. No.: 324,960
`22 Filed:
`Oct. 18, 1994
`(51) Int. Cl.' ........................ A61K38/00; A61K 38/26;
`CO7K 141605
`52 U.S. Cl. ............................. 514/12; 530/308; 530/324
`58) Field of Search ..................................... 530/308, 324;
`514/12
`
`(56)
`
`
`
`References Cited
`U.S. PATENT DOCUMENTS
`5,070,188 12/1991 Njieha et al. ........................... 530/324
`5,118,666
`6/1992 Habener .................................... 54/2
`5,120,712 6/1992 Habener .................................... 514f12
`FOREIGN PATENT DOCUMENTS
`European Pat. Off..
`0082731, 12/1982
`European Pat. Off..
`0619322A2 2/1994
`WO87106941 11/1987
`WIPO.
`WO90/1296 10/1990
`WIPO.
`WO91/11457 8/1991
`WIPO.
`WO93/18786 9/1993 WIPO
`WO93/25579 12/1993 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 I, an insulin
`-releasing hormone from 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).
`
`USOO5512549A
`11) Patent Number:
`(45) Date of Patent:
`
`5,512,549
`Apr. 30, 1996
`
`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
`
`ABSTRACT
`57)
`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 Co-Co acylations. The claimed compounds
`stimulate the secretion or biosynthesis of insulin in poorly
`functioning beta cells and are therefore useful in treating
`Type II diabetics
`
`28 Claims, No Drawings
`
`MPI EXHIBIT 1046 PAGE 1
`
`MPI EXHIBIT 1046 PAGE 1
`
`

`

`5,512,549
`
`1.
`GLUCAGON-LKE NSULNOTROPIC
`PEPTIDE ANALOGS, COMPOSITIONS, AND
`METHODS OF USE
`
`FIELD OF INVENTION
`The present invention relates to organic and peptide
`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
`
`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 found to 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 present in
`the pancreas as 37 and 34 amino acid 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
`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 artifac
`tual.
`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 dis
`closed in U.S. Pat. No. 5,118,666, herein incorporated by
`reference. GLP-1 (7-37) is disclosed in U.S. Pat. No. 5,120,
`712, herein incorporated by reference.
`Variants and analogs of GLP-1 are known in the art. These
`variants and analogs include, for example, GLP-1 (7-36),
`Gln-GLP-1(7–37), D-Gln-GLP-1 (7-37), acetyl-Lys
`GLP-1 (7-37), Thr-Lys-GLP-1(7–37), and Lys-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)).
`More importantly, numerous investigators have demon
`strated a predictable relationship between various in vitro
`laboratory experiments and mammalian, especially human,
`insulinotropic responses to exogenous administration 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., Diabe
`tes, 42:1219-1225 (1993)).
`The fundamental defects responsible for causing hyper
`glycemia in mature onset diabetes include impaired secre
`tion 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 approxi
`mately 2 mg/kg/minute, apatient 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.
`Because there exists exceedingly high correlations
`between hepatic glucose production, fasting blood glucose
`
`15
`
`BACKGROUND OF THE INVENTION
`Endocrine secretions of pancreatic islets are regulated by
`complex control mechanisms driven not only by blood
`borne 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,
`20
`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 regulation of insulin secretion, there is evidence to
`support the existence of insulinotropic factors in the intes
`tine. 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.
`The human hormone glucagon is a 29-amino acid hor
`mone produced in pancreatic A-cells. The hormone belongs
`to a multigene family of structurally related peptides that
`include secretin, gastric inhibitory peptide, vasoactive intes
`tinal 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 gly
`cogenolysis and glyconeogenesis, resulting in an elevation
`of blood sugar levels. In this regard, the actions of glucagon
`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)).
`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 mecha
`nism (Ganong, W. F., Review of Medical Physiology, Lange
`Publications, Los Altos, Calif., p. 273 (1979)). Thus, the
`expression of glucagon is carefully regulated by insulin, and
`ultimately by serum glucose levels.
`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 dis
`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
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`MPI EXHIBIT 1046 PAGE 2
`
`MPI EXHIBIT 1046 PAGE 2
`
`

`

`5,512,549
`
`3
`levels, and overall metabolic control as indicated by glyco
`hemoglobin measurements (Galloway, J. A., supra; and
`Galloway, 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. Thereapy
`based on administration of GLP-1 analogs is one such
`approach and is an object of the present invention.
`Presently, therapy involving the use of GLP-1 type mol
`ecules 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 admin
`istration. Thus, there exists a critical need for biologically
`active GLP-1 (7-37) analogs that possess extended pharma
`codynamic profiles following parenteral administration.
`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
`25
`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
`administration. Most surprisingly, some compounds of the
`present invention demonstrated a synergistic effect as indi
`vidual alterations to GLP-1 (7-37) failed to add-up to the
`biological performance of compounds that contained all of
`the alterations.
`SUMMARY OF THE INVENTION
`The present invention provides compounds of the general
`formula:
`
`10
`
`15
`
`20
`
`4.
`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 Lys' 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.
`It should be noted that this specification uses the nomen
`clature scheme that has developed around processed 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.
`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.
`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.,
`Biooraanic 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).
`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 Lin
`coln Center Drive, Foster City, Calif. 94404) and synthesis
`cycles supplied by PE-Applied Biosystems. Boc amino
`acids and other reagents are commercially available from
`PE-Applied Biosystems and other chemical supply houses.
`Sequential Boc chemistry using double couple protocols are
`applied to the starting p-methylbenzhydryl 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 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 peptidelresin 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. Chen.,
`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
`
`30
`
`35
`
`(Formula 1)
`R-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val
`Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa
`
`Glu-Phe-Ile-Ala-Tip-la-va-y-Gly-As-R
`
`R2
`
`40
`
`45
`
`50
`
`wherein R' is selected from the group consisting of
`4-imidazopropionyl (des-amino-histidyl), 4-imidazoacetyl,
`or 4-imidazo-O, Odimethyl-acetyl;
`R’ is selected from the group consisting of Co-Co
`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
`55
`positions comprising a compound of the present invention in
`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.
`DETALED DESCRIPTION OF THE
`INVENTION
`In one embodiment, the present invention provides ana
`logs of naturally-occurring GLP-1 (7-37) that arise from
`
`60
`
`65
`
`MPI EXHIBIT 1046 PAGE 3
`
`MPI EXHIBIT 1046 PAGE 3
`
`

`

`5,512,549
`
`10
`
`15
`
`20
`
`25
`
`30
`
`S
`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 pref
`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.
`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.
`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.
`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 For
`mula 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, Calif. 94.404).
`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 endonucleases. See
`generally Maniatis et al. (1989) Molecular Cloning. A
`Laboratory Manual, Cold Springs Harbor Laboratory Press,
`50
`N.Y., Vol. 1-3. Restriction endonuclease cleavage sites are
`engineered into either end of the GLP-1 intermediate-en
`coding DNA to 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 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.
`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
`
`60
`
`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 known in the art.
`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 exogenous genes in 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 autonomous plasmid 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 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
`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 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.
`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
`which contain high levels 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. 89-18S, 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.
`
`35
`
`40
`
`45
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`55
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`65
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`MPI EXHIBIT 1046 PAGE 4
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`MPI EXHIBIT 1046 PAGE 4
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`

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`5,512,549
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`7
`Preferred imidazolic groups of the present invention are:
`
`N
`
`CO
`
`( r N
`
`4-imidazopropionyl (des-amino-histidyl)
`4-imidazoacetyl
`
`N
`
`CO, er N
`
`and
`4-imidazo-O,Odimethyl-acetyl
`
`HC CH3
`
`CO
`
`N
`
`(
`
`N
`
`10
`
`15
`
`20
`
`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 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, ammo
`nium 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 thera
`peutic purposes, those compounds may also 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 may be 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 may also 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 compound of the present invention in
`combination with a pharmaceutically acceptable carrier,
`diluent, or excipient. Such pharmaceutical compositions are
`prepared in a manner well known in 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 about 1 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 such criteria as the patient's height, weight, sex, age,
`and medical history.
`In making the compositions of the present invention, the
`active ingredient, which comprises at 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
`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.
`
`30
`
`40
`
`45
`
`The most preferred group is 4-imidazopropionyl.
`25
`Further embodiments of the present invention are made
`by acylating the epsilon amino group of the Lys' residue.
`Straight chain acyl additions containing between 6 to 10
`carbon atoms are preferred and unbranched C is most
`preferred.
`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.
`Modifications at the carboxy terminus are also included in
`35
`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 known in the art. See Bioconjugate Chem. "Chemi
`cal 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).
`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 acy
`lated either before or after the imidazolic group is added.
`Moreover, if the peptide is prepared recombinantly, acyla
`tion prior to enzymatic cleavage is 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
`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-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,
`
`50
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`60
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`
`MPI EXHIBIT 1046 PAGE 5
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`MPI EXHIBIT 1046 PAGE 5
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`

`5,512,549
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`10
`Tyr (Br-Z), and Arg (Tos). All except for Asp (Chxl)
`(Peptides International) were obtained from PE-Applied
`Biosystems. Each residue was double coupled using either
`DCC initiated symmetric anhydride or HOBT activation.
`The 30 residue intermediate was left attached to the