`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 98/43658
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 6 :
`A61K 38/00
`
`Al
`
`(11) International Publication Number:
`
`(43) International Publication Date:
`
`8 October 1998 (08.10.98)
`
`(21) International Application Number:
`
`PCT/US98/05945
`
`(22) International Filing Date:
`
`25 March 1998 (25.03.98)
`
`(30) Priority Data:
`60/041,167
`
`31 March 1997 (3 1.03.97)
`
`us
`
`(71) Applicant (for all designated States except US): ELI LILLY
`AND COMPANY [US/US]; Lilly Corporate Center, Indi(cid:173)
`anapolis, IN 46285 (US).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): HOFFMANN, James, A.
`[US/US]; 4272 Woodland Streams Drive, Greenwood, IN
`46 143 (US).
`
`(74) Agents: MACIAK, Ronald, S. et al.; Eli Lilly and Company,
`Lilly Corporate Center, Indianapolis, IN 46285 (US).
`
`(81) Designated States: AL, AM. AU, AZ, BA, BB, BG, BR, BY,
`CA, CN, CU, CZ, EB, GE, GH, GM, GW, HU, ID, IL, IS,
`JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LV, MD,
`MG, MK, MN, MW, MX, NO, NZ, PL, RU, SD, SG, SJ,
`SK, SL, TJ, TM, TR, TI, UA, UG, US, UZ, VN, YU, ZW,
`ARIPO patent (GH, GM, KE, LS, MW, SD, SZ, UG, ZW),
`Eurasian patent (AM, Az, BY, KG, KZ, MD, RU, TJ, TM),
`OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR,
`NE, SN, TD, TG).
`
`Published
`With international search report.
`
`(54) Title: GLUCAGON- LIKE PEPTIDE-I ANALOGS
`
`(57) Abstract
`
`The invention provides extended- action GLP-1 based peptides and compositions that are useful for treating diabetes and minimize
`the risk of hypoglycemia.
`
`FRESENIUS EXHIBIT 1042
`Page 1 of 26
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Anncnia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`C6te d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The fonner Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
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`FRESENIUS EXHIBIT 1042
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`PCT /US98/05945
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`GLUCAGON-LIKE PEPTIDE-1 ANALOGS
`
`Field of Invention
`
`The present invention relates to protein chemistry
`as applied to pharmaceutical research and development. The
`invention provides novel peptides and compositions that are
`useful for treating diabetes.
`
`5
`
`Background of the Invention
`
`20
`
`The World Health Organization estimates that there
`10 may be as many of 100 million patients with type II diabetes
`worldwide, although only 23 million have been diagnosed and
`are receiving therapy. Type II diabetes (n on-insulin
`dependent diabetes mellitus - NIDDM) is characterized by a
`resist ance to insulin action in peripheral tissues such as
`15 muscle, adipose and liver and by a progressive failure in
`the ability of the islet ~-cell to secrete insulin. Because
`current therapeutics do not halt the progression of ~-cell
`failure, virtually all NIDDM patients eventually require
`insulin to control blood glucose level s. The most commonly
`described therapeutics for such patients are the
`sulfonylureas, so called oral agents, that stimulate insulin
`secretion. Each year, 10-20% of the patients on
`sulfonylureas fail to maintain acceptable blood glucose
`levels and switch to insulin therapy.
`Insulin however, is a difficult drug for patients
`to self-administer for several reasons. First, i nsulin has
`a narrow therapeutic index. This l eads to poor control of
`blood glucose levels since most patients and physicians
`prefer elevated glucose levels to the risk of hypoglycemia
`and coma. Second, proper insulin dosing is complicated.
`The insulin dose that a diabeti c patient should administer
`is dependent on the amount of food consumed, the time
`between meals, the amount o f physical exercise, and the
`prevailing blood glucose level which requires blood glucose
`35 monitoring to determine. The general diabetic population is
`
`25
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`30
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`i l l-equipped to correlate these factors to the proper
`insulin dose. Third, as a parenteral product, insulin is
`inconvenient to administer. Alternative delivery methods
`are made even more difficult by the narrow therapeutic index
`for insulin . Thus, many diabetic patients lack good control
`of blood glucose.
`The Diabetes Control and Complication Trial
`definitively established for Type I diabetics that disease -
`complications (retinopathy, neuropathy and nephropathy) are
`10 directly correlated to blood glucose control. A clinical
`trial is currently underway in the UK to determine if this
`link also holds true to Type II diabetes. A positive result
`is reasonable to anticipate, and with it will come a desire
`for agents that improve the ability for the patient with
`15 diabetes to control blood glucose levels tightly.
`In
`addi tion, a standard of care for diabetes is being developed
`by the US government in coordination with care-givers and
`the pharmaceutical industry. Thus, there is clearly a need
`for agents that truly are able to tightly control blood
`glucose levels in the normal range .
`Glucagon-like peptide-1 (GLP -1 ) was first
`identified in 1987 as a incretin hormone, a peptide secreted
`by the gut upon ingestion of food. GLP - 1 is secreted by the
`L-cells of the inte stine after being proteolytically
`25 processed from the 160 amino acid precursor protein,
`preproglucagon. Cleavage of preprogl ucagon first yields
`GLP-1, a 37 amino acid peptide, GLP -1(1-37)OH, that is
`poorly active. A subsequent cleavage at the 7-position
`yields biologically active GLP-1(7-37)OH. Approximately 80%
`of the GLP-1(7-37)OH that is synthesized is amidated at the
`C-terminal after removal of the termi n al glycine residue in
`the L-cells. The biological effects and metabolic turnover
`of the free aci d GLP-1(7-37)OH, and t he amide, GLP-1
`(7-36)NH2, are indistinguishable.
`GLP -1 is known to stimulate insulin secretion
`(insulinotropic action) causing glucose uptake by cells
`which decreases serum glucose levels (see, e g., Mojsov, S.,
`
`30
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`15
`
`Int. J. Peptide Protein Research, 40:333-343 (1992)).
`Numerous GLP-1 analogs demonstrating insulinotropic action
`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-Lys 9 -GLP-1(7-37), Thr16 - Lys 18 - GLP-1(7-37), and
`LyslB-GLP-1(7-37). Derivatives of GLP-1 include, for
`example, acid addition salts, carboxylate salts, lower alkyl
`esters, and amides (see, e.g., WO91/11457 (1991); EP o
`733,644 (1996); and US Patent No: 5,512,549 (1996)). It has
`also been demonstrated that the N-terminal histidine residue
`(His 7) is very important to insulinotropic activity of GLP-
`1 (Suzuki, S., et al. Diabetes Res.; Clinical Practice 5
`( 19 8 8 ) .
`( Supp . 1 ) : S 3 0
`Multiple authors have demonstrated the nexus
`between laboratory experimentation and mammalian,
`particularly human, insulinotropic responses to exogenous
`administration of GLP-1, particularly GLP-1 (7-36)NH2 and
`GLP-1 (7-37) [~ , e.g., Nauck, M.A., et al., Diabetologia,
`36:741-744 (1993); Gutniak, M., et al., New England J. of
`20 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)].
`GLP-1 based peptides hold great promise as
`alternatives to insulin therapy for patients with diabetes
`25 who have failed on sul fonylureas. GLP-1 has been studied
`intensively by academic investigators, and this research has
`established the following for patients with type II diabetes
`who have failed on sulfonylureas:
`1) GLP - 1 stimulates insulin secretion, but only
`during periods of hyperglycemia. The safety of GLP-1
`compared to insulin is enhanced by this property of GLP-1
`and by the observation that the amount of insulin secreted
`is proportional to the magnitude of the hyperglycemia.
`In
`addition, GLP - 1 therapy will result in pancreatic release of
`insulin and first-pass insulin action at the liver. This
`results in lower circulating levels of insulin in the
`periphery compared to subcutaneous insulin injections. 2)
`
`30
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`35
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`GLP-1 suppresses glucagon secretion, and this, in addition
`to the delivery of insulin via the portal vein helps
`suppress the excessive hepatic glucose output in diabetic
`patients. 3) GLP-1 slows gastric emptying which is
`desirable in that it spreads nutrient absorption over a
`longer time period, decreasing the postprandial glucose
`peak.
`4) Several reports have suggested that GLP-1 may
`enhance insulin sensitivity in peripheral tissues such as
`muscle and fat.
`5) Finally, GLP-1 has been s hown to be a
`10 potential regulator of appetite.
`Meal-time use of GLP-1 based peptides offers
`several advantages over insulin therapy.
`Insulin therapy
`requires blood glucose monitoring, which is both expensive
`and painful. The glucose-dependency of GLP-1 provides an
`enhanced therapeutic window in comparison to insulin, and
`should minimize the need to monitor blood glucose . Weight
`gain also can be a problem with intensive insulin therapy,
`particularl y in the obese type II diabetic patients.
`The therapeutic potential for native GLP-1 is
`further increased if one considers its use in patients with
`type I diabetes. A number of studies have demonstrated the
`effectiveness of native GLP-1 in the treatment of insulin
`dependent diabetes mellitus (IDDM). Similar to NIDDM
`patients, GLP - 1 is effective in reducing fasting
`hyperglycemia through its glucagonostatic properties.
`Additional studies have indicated that GLP-1 also reduces
`postprandial glycemic excursions in IDDM, most likely
`through a delay in gastr i c emptying. These observations
`indicate that GLP-1 is may be useful as a treatment in IDDM
`as well as in NIDDM.
`However, the biologi cal half - life of native GLP-1
`molecules which are affected by the activity of dipeptidyl(cid:173)
`peptidase IV (DPP IV) is quite short. For example, the
`biological half - life of GLP-1(7-37)OH is a mere 3 to 5
`35 minutes (U.S. Pat. No. 5,118,666). Therefore extended(cid:173)
`action GLP - 1 based peptides are needed to enhance glycemic
`
`20
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`25
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`30
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`control during the night while minimizing the risk of
`hypoglycemia.
`In one embodiment, the present invention provides
`GLP-1 analogs that have extended time action relative to
`5 native GLP-1, show resistance to the action of DPP-IV, and
`retain affinity for the GLP-1 receptor.
`
`Summary of the Invention
`
`The invention includes a GLP-1 compound of the
`
`formula:
`
`10
`
`R1-X-Glu-Gly- Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-
`Gly-Gln -Ala- Ala - Lys-Z-Phe - Ile - Ala - Trp - Leu- Val - Lys - Gly(cid:173)
`Arg-R2
`
`( SEQ ID NO: 1)
`
`or a pharmacuetically accetable salt thereof, wherein:
`
`15 R1 is selected from the group consisting of His,
`D-histidine, desarnino-his t idine, 2-amino-histidine,
`g-hydroxy-histidine, homohistidine, alpha-fluoromethyl(cid:173)
`h istidine, and alpha-methyl-histidine;
`
`Xis selected from the group consisting of Met, Asp, Lys,
`20 Thr , Leu, Asn, Gln, Phe, Val, and Tyr
`
`Y and Z are independently selected from the group consisting
`of Glu, Gln, Ala , Thr, Ser, and Gly, and;
`
`R is selected from the group consisting of NH , and Gly-OH;
`2
`provided that, if R1 is His, Xis Val, Y is Glu, and Z is
`25 Glu, then R2 is NH2 ;
`
`2
`
`Also provided by the present invention are
`pharmaceutical compositions comprising a GLP-1 compound of
`the present invention in combination wi th one or more
`pharmaceutical l y acceptable carriers, diluents, or
`excipients.
`
`3 0
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`The present invention further provides a method of
`treating diabetes which comprises admini stering to a mammal
`in need of such treatment an effective amount of a GLP-1
`compound of the present invention.
`
`Detailed Description of the Invention
`
`In one embodiment, the present invention provides
`novel, biologically-act ive GLP-1 based peptides. It should
`be noted that this specification uses the nomenclature
`scheme that has developed around p rocessed forms of GLP-1.
`In this scheme, the amino terminus of native GLP - 1(7-37)OH
`has been assigned number 7 and the carboxy terminus number
`37. Therefore, R1 of SEQ ID NO: 1 corresponds to residue 7
`of GLP-1(7-37)OH. Likewise X in SEQ I D NO:l corresponds to
`residue 8 of GLP -1(7-37)OH and Y corresponds to res idue 2 1
`and so forth . Moreover, all amino acids referred to in this
`specificat ion are in the L form , unless otherwise specified.
`In a preferred embodiment , R1 is His, and Y and Z
`are Glu. Another preferred group i s when R1 is His, R2 is
`
`5
`
`10
`
`15
`
`20
`
`Gly-OH , and any one or more of X, Y, and Z dif fer from the
`residues present in native GLP - 1( 7-3 7)OH. Another preferred
`group is when R1 is His, Xis Met , Asp, Lys, Thr, Leu, Asn,
`Gln, Phe or Typ, Y and Z are Glu, and R2 is Gly-OH.
`Given the sequence information herein discl osed
`and the state of the art in solid phase protein synthesis,
`25 GLP-1 analogs can be obtained via chemical synthesis.
`However, it also is possible to obtain a GLP-1 analog by
`fragment i ng proglucagon using, f or example, proteolytic
`enzymes. Moreover, recombinant DNA techniques may be used
`to express GLP-1 analogs of the invention.
`The princ iples of solid phase chemical synthesis
`of polypeptides are well known in the art and may be found
`in general texts in the area s uch as Dugas , H. and Penney,
`C., Bioorganic Chemistry (1981) Spri nger - Verlag, New York,
`pgs. 54 - 92, Merrifield, J.M., Chem . Soc., 85:2149 (1962),
`
`30
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`and Stewart and Young, Solid Phase Peptide Synthesis, pp.
`24-66, Freeman (San Francisco, 1969).
`For example, a GLP-1 analog of the invention may
`be synthesized by solid-phase methodology utilizing an
`5 Applied Biosystems 430A peptide synthesizer (Applied
`Biosystems, Inc. , 85 0 Lincoln Center Drive, Foster City, CA
`94404) and synthesis cycles supplied by Applied Biosystems.
`Boe amino acids and other reagents are commercially
`available from Applied Biosystems and other chemical supply
`houses. Sequential Boe chemistry using double couple
`protocols are applied to the starting p - methyl benzhydryl
`amine resins for the production of C-terminal carboxamides.
`For the production of C- terminal acids, the corresponding
`PAM resin is used. Asp, Gln, and Arg are coupled using
`preformed hydroxy benzotri azole esters. The following side
`chain protecting groups may be used:
`
`10
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`15
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`20
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`25
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`30
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`35
`
`Arg, Tosyl
`Asp, cyclohexyl
`Glu, cyclohexyl
`Ser, Benzyl
`Thr, Benzyl
`Tyr, 4 -bromo carbobenzoxy
`
`Boe deprotection may be accomplished with
`trifluoroacetic acid i n methylene chloride. Following
`completion of the synthesis the peptides may be depro tected
`and c leaved from the resin with anhydrous hydrogen fluoride
`(HF) containing 10% meta-cresol . Cleavage of the side chain
`protecting group(s) a nd of the peptide from the resin is
`carried out at zero degrees centigrade or below, preferably
`-20°c fo r thirty minutes foll owed by thirty minutes at o0 c.
`After removal of the HF, the peptide/resin is washed wit h
`ether, and the peptide extracted with glacial acetic acid
`and lyophilized.
`The preparation of protected, unprotected, and
`partially protected GLP - 1 has been described in the art.
`See U.S. Pat. No. 5,120,712 and 5, 118,666, herein
`incorporated by reference, and Orskov, c., et al., J. Biol.
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`Chem., 264 (22) :12826-12829 (1989) and WO 91/11457 (Buckley,
`D.I., et al., published August 8, 1991).
`Likewise, the state of the art in molecular
`biology provides the ordinarily skilled artisan another
`5 means by which GLP-1 analogs can be obtained. Although GLP-
`1 analogs may be produced by solid phase peptide synthesis,
`recombinant methods, or by fragmenting glucagon, recombinant
`methods may be preferable because higher yields are
`possible. The basic steps in the recombinant production of
`a GLP-1 analog are:
`
`10
`
`a)
`
`b)
`
`c)
`
`d)
`
`e)
`
`isolating a natural DNA sequence encoding
`GLP-1 or constructing a synthetic or semi(cid:173)
`synthetic DNA coding sequence for GLP-1,
`
`placing the coding sequence into an
`expression vector in a manner suitable for
`expressing proteins either alone or as a
`fus ion proteins,
`
`transforming an appropriate eukaryotic or
`prokaryotic host cell with the expression
`vector,
`
`culturing the transformed host cell under
`conditions that will permit expression of a
`GLP-1 intermediate, and
`
`recovering and purifying the recombinantly
`produced protein.
`
`. 15
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`20
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`25
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`30
`
`As previously stated, the coding sequences for
`GLP- 1 analogs 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
`35 Lund, et al., Proc. Natl. Acad. Sci. U.S.A. 79:345-349
`(1982) and may be used as starting material in the
`recombinant production of a GLP-1 analog 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 a GLP- 1 analog, may be constructed by
`techniques well known in the art. Owing to the natural
`
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`degeneracy of the genetic code, the skilled artisan will
`recognize that a sizable yet definite number of DNA
`sequences may be constructed which encode GLP-1
`intermediates.
`The methodology of synthetic gene construction is
`well known in the art. See Brown, et al. (1979) Methods in
`Enzymology, Academic Press, N.Y., Vol. 68, pgs. 109-151.
`DNA sequences that encode GLP-1 intermediates can be
`designed based on the amino acid sequences herein disclosed.
`10 Once designed, the sequence itself may be generated us i ng
`conventional DNA synthesizing apparatus such as the Applied
`Biosystems Model 380A or 380B DNA synthesizers (Applied
`Biosystems, Inc., 850 Lincol n Center Drive, Foster City, CA
`94404).
`
`15
`
`20
`
`To effect the expression of a biologically-active
`GPL- 1 analog, 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, N.Y., Vol. 1 - 3. Restriction
`endonuclease cleavage sites are engineered into either end
`of the DNA encoding the GLP- 1 analog 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 codi ng sequence with control sequences to achieve
`30 proper in-frame reading and expression of the protein of
`interest. The coding sequence must be positioned so as t o
`be in proper reading frame with the promoter and ribosome
`binding sit e of the expression vector, both of which are
`functional in the host cell in which the protein is to be
`expressed.
`
`25
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`3 5
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`To achieve efficient transcription of the coding
`region, it must be operably associated with a promoter-
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`operator region. Therefore, the promoter-operator region of
`the gene is placed in the same sequential orientation with
`respect to the ATG start codon of the coding region.
`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
`10 Road, La Jolla, CA, 92037). Also, U.S. Patent No. 4,710,473
`describes circular DNA plasmid transformation vectors useful
`for expression of exogenous genes in E . coli at high levels.
`These plasmids are useful as transformation vectors in
`recombinant DNA procedures and:
`
`15
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`20
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`25
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`30
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`35
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`40
`
`(a)
`
`(b)
`
`confer on the plasmid the capacity for autonomous
`replication in a host cell;
`
`control autonomous plasmid replication in relation
`to the temperature at which host cell cultures are
`maintained;
`
`(c)
`
`stabil ize maintenance of the plasmid in host cell
`populations;
`
`(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 p l asmid; 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 a
`GLP-1 analog, the next step is to place the vector into a
`suitable cell and thereby construct a recombinant host cell
`useful for expressing a GLP- 1 analog. 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
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`from either eukaryotic or prokaryotic cells. Eukaryotic
`host cells are capable of carrying out post -translational
`glycosylations on expressed proteins and some are capable of
`secreting the desired protein into the culture medium.
`Prokaryotic host cells generally produce the
`protein at higher rates, are easier to culture but are not
`capable of glycosylating the final protein. Proteins which
`are expressed in high-level bacterial expression systems may
`aggregate in granules or inclusion bodies which contain high
`levels of the overexpressed protein. Such protein
`aggregates must be solubilized, denatured and refolded using
`techniques well known in the art. See Kreuger, et al.
`(1990) in Protein Folding, Gierasch and King, eds. , pgs 136-
`142, American Association for the Advancement of Science
`15 Publication No. 89-lBS, Washington, D.C.; and U.S . Patent
`No . 4,923,967.
`Regardless of the methods used to produce a GLP-1
`analog, purification of the protein generally will be
`required. Methods for purifying proteins are well known in
`the art and include conventional chromatography, including
`ion and cation exchange, hydrophobic interaction, and
`immuno-affinity chromatographic media. The amino acid
`sequences herein disclosed in conjunction with well known
`protein purification methods will enable the ordinarily
`skilled artisan to purify GLP-1 analogs claimed he rein.
`The present invention also includes salt forms
`of GLP-1 analogs. A GLP-1 analog of the invention may be
`sufficiently acidic or sufficiently basic to react with
`any of a number of inorganic bases, and inorganic and
`30 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 :e-toluenesulfonic acid, methanesulfonic
`acid, oxalic acid, e-bromophenyl-sulfonic acid, carbonic
`acid, succinic acid, citric acid, benzoic acid, acetic
`acid, and the like. Examples of such salts include the
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`sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,
`phosphate, monohydrogenphosphate, dihydrogenphosphate,
`metaphosphate, pyrophosphate, chloride, bromide, iodide,
`acetate, propionate, decanoate, caprylate, acrylate,
`fo r mate, 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,
`10 phthalate, sulfonate, xylenesulfonate, phenylacetate,
`phenylpropionate, phenylbutyrate, citrate, lactate,
`gamma-hydroxybutyrate, glycolate, tartrate,
`methanesulfonate, propanesulfonate, naphthalene- 1 -
`sulfonate, naphthalene-2-sulfonate, mandelate, and the
`like. Preferred acid addition salts are those formed
`with mineral acids such as hydrochloric acid and
`hydrobromic acid, and, especially, hydrochloric acid .
`Base addition salts include those derived from
`inorganic bases, such as ammonium or a lkali 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
`l ike. Salt forms of GLP- 1 analogs are particularly
`25 preferred. Of course, when the compounds of this invention
`are used for therapeutic purposes, those compounds may also
`be in the form of a salt, but the salt must be
`pharmaceutically acceptable.
`The ability of a GLP- 1 analog to stimulate i n sulin
`secretion may be determined by providing a GLP-1 analog to
`cultured anima l cells, such as the RIN- 38 rat insulinoma
`cell line , and monitoring the release of immunoreactive
`insulin (IRI) into the media. Alternativel y one can inject
`a GLP-1 anal og into an animal and monitor plasma levels of
`immunoreacti ve insulin ( IRI ) .
`The presence of IRI is detected through the use of
`a radioimmunoassay which can specifically detect insulin.
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`Any radioimmunoassay capable of detecting the presence of
`IRI may be employed; one such assay is a modification of
`the method of Albano, J.D.M., et al., Acta Endocrinol.,
`70:487-509 (1972) .
`In this modification, a
`phosphate/albumin buffer with a pH of 7.4 is employed. The
`incubation is prepared with the consecutive addition of 500
`µl of phosphate buffer, 50 µl of perfusate sample or rat
`insulin standard in perfusate, 100 µl of anti - insulin
`antiserum (Wellcome Laboratories; 1:40,000 dilution), and
`100 µl of [1251) insulin, giving a total volume of 750 µl in
`a 10x75 mm disposable glass tube. After incubation for 2 - 3
`days at 4° C, free insulin is separated from antibody-bound
`insulin by charcoal separation. The assay sensitivity is 1 -
`2 uU/mL.
`In order to measure the release of IRI into the
`15 cell culture medium of cells grown in tissue culture , one
`preferably incorporates radioactive label into proinsulin.
`Although any radioactive label capable of labeling a
`polypeptide can be used, it is preferable to use 3H leucine
`in order to obtain labeled proinsulin.
`To determine whether a GLP- 1 analog has
`insulinotropic proper ties may also be determined by
`pancreatic infusion. The in situ isolated perfused rat
`pancreas assay is a modification of the method of Penhos,
`J.C., et al., Diabetes, 18:733-738 (1969). Fasted male
`25 Charles River strain albino rats, weighing 350-600 g, are
`anesthetized with an intraperitoneal injection of Arnytal
`Sodium (Eli Lilly and Co.: 160 ng/kg). Renal, adrenal,
`gastric, and lower colonic blood vessels are ligated. The
`entire intestine is resected except for about four cm of
`duodenum and the descending colon and rectum. Therefore,
`only a small part of the intestine is perfused, minimizing
`possible interference by enteric substances with glucagon(cid:173)
`like imrnunoreactivity. The perfusate is a modified Krebs(cid:173)
`Ringer bicarbonate buffer with 4% dextran T70 and 0.2%
`bovine serum albumi n (f raction V), and is bubbled with 95%
`0 2 and 5% CO2 • A nonpulsatile flow, 4 - channel roller
`bearing pump (Buchler polystatic, Buchler Instruments
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`Division, Nuclear-Chicago Corp.) is used, and a switch from
`one perfusate source to another is accomplished by switching
`a 3-way stopcock. The manner in which perfusion is
`performed, monitored, and analyzed follow the method of
`5 We ir, G.C., et al., J. Clin. Inestigat . 54:1403-1412 (197 4),
`which is hereby incorporated by reference.
`The present invention also provides pharmaceutical
`compositions comprising a GLP - 1 analog of the present
`invent ion in combination with a pharmaceutically acceptable
`carrier , diluent, or excipient. Such pharmaceutical
`compositions are prepared in a manner wel l known in the
`pharmaceutical art, and are administered individual l y or in
`combination with other therapeutic agents, preferably via
`parenteral routes. Especially preferred r outes include
`intramuscular and subcutaneous administration.
`Parenteral daily dosages, preferably a single,
`daily dose, are in the range from about 1 pg/kg to about
`1,000 µg/kg of body weight, although lower or higher dosages
`may be administered. The required dosage will depend upon
`the severity of the condition of the patient and upon such
`criteria as the patient's height, weight, sex, age, and
`medical history.
`In making the compositions of the present
`invention, the active ingredien t , which comprises at least
`one protein of the present invention, is usually mixed with
`an excipient or diluted by an excipient. When an excipient
`is used as a diluent, it may be a solid, semi-solid, or
`liquid material which acts as a vehicle, carrier, or medium
`for the active ingredient.
`In preparing a formulation, it may be n ecessary to
`mil l the active compound to provide the appropriate particle
`size prior to combining with the other ingredients.
`If the
`active protein is substantially insoluble, i t ordinari ly is
`milled to particle size of less than ab out 200 mesh .
`If the
`active compound is substantially water soluble, the particle
`size is normally adjusted by milling to provide a
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`s ubstantially uniform dis t ribut ion in the f ormula tion, e.g.,
`about 40 mesh.
`Some examples of suitable excipients include
`lactose, dextrose, sucrose, trehalose, sorbitol, mannitol,
`starches, gum acacia, calcium silicate, microcrystalline
`cellulose, polyvinylpyrrolidone, cellulose, water, syrup,
`and methyl cell ulose. The formulations can additionall y
`i nclude lubricating agents such as talc, magnesium stearate(cid:173)
`and mineral oil, wetting agents, emulsifying and suspending
`agents, preserving agents such as methyl- and
`propylhydroxybenzoates, sweetening agents or flavoring
`agents. The compositions of the invention can be formulated
`so as to provide quick, sustained or delayed release of the
`active ingredient after administration to the patient by
`employing procedures well known in t he art.
`The compositions are preferably formulated in a
`unit dosage fo