`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`
`(11) International Publication Number:
`wo 98/43658
`(51) International Patent Classification 6;
`AG61K 38/00
`
` (43) International Publication Date:
`8 October 1998 (08.10.98)
`
`
`
`
`PCT/US98/05945|(81) Designated States: AL, AM, AU, AZ, BA, BB, BG,BR,BY,
`(21) International Application Number:
`CA, CN, CU, CZ, EE, GE, GH, GM, GW, HU,ID,IL,IS,
`
`JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LV, MD,
`(22) International Filing Date:
`MG, MK, MN, MW, MX, NO, NZ, PL, RU, SD, SG, SI,
`SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW,
`ARIPOpatent (GH, GM, KE, LS, MW, SD, SZ, UG, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU,TJ, TM),
`OAPIpatent (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR,
`NE, SN, TD, TG).
`
`PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`25 March 1998 (25.03.98)
`
`
`
`
`
`
`
`
`(30) Priority Data:
`60/041,167
`
`31 March 1997 (31.03.97)
`
`US
`
`(71) Applicant (for all designated States except US): ELI LILLY
`AND COMPANY[US/US]; Lilly Corporate Center, Indi-
`anapolis, IN 46285 (US).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): HOFFMANN, James, A.
`[US/US]; 4272 Woodland Streams Drive, Greenwood, IN
`46143 (US).
`
`(74) Agents: MACIAK,Ronald, S. et al.; Eli Lilly and Company,
`Lilly Corporate Center, Indianapolis, IN 46285 (US).
`
`Published
`With international search report.
`
`-
`
`
`
`
`
`
`
`(54) Title: GLUCAGON-LIKE PEPTIDE-1 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.
`
`
`
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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.
`
`
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Céte 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
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`Japan
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`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 former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`Slovenia
`SI
`Slovakia
`SK
`Senegal
`SN
`Swaziland
`8Z
`Chad
`TD
`Togo
`TG
`‘Tajikistan
`TJ
`™ Turkmenistan
`TR
`Turkey
`TT
`Trinidad and Tobago
`UA
`Ukraine
`UG
`Uganda
`us
`United States of America
`UZ
`Uzbekistan
`VN
`Viet Nam
`yU
`Yugoslavia
`ZW
`Zimbabwe
`
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`WO 98/43658
`
`PCT/US98/05945
`
`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.
`
`Background of the Invention
`
`The World Health Organization estimates that there
`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 (non-insulin
`dependent diabetes mellitus - NIDDM)
`is characterized by a
`resistance to insulin action in peripheral tissues such as
`muscle, adipose and liver and by a progressive failure in
`the ability of the islet B-cell to secrete insulin. Because
`current therapeutics do not halt the progression of B-cell
`failure, virtually all NIDDM patients eventually require
`insulin to control blood glucose levels.
`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,
`
`insulin has
`
`a narrow therapeutic index. This leads 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 diabetic patient should administer
`is dependent on the amount of food consumed,
`the time
`between meals,
`the amount of physical exercise, and the
`prevailing blood glucose level which requires blood glucose
`monitoring to determine.
`The general diabetic population is
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`ill-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
`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
`Giabetes to control blood glucose levels tightly.
`In
`addition, 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 intestine after being proteolytically
`processed from the 160 amino acid precursor protein,
`preproglucagon. Cleavage of preproglucagon first yields
`GLP-1, a 37 amino acid peptide, GLP-1(1-37)0H,
`that is
`poorly active.
`A subsequent cleavage at the 7-position
`yields biologically active GLP-1(7-37)0H. Approximately 80%
`of the GLP-1(7-37)0OH that is synthesized is amidated at the
`C-terminal after removal of the terminal glycine residue in
`the L-cells.
`The biological effects and metabolic turnover
`of the free acid GLP-1(7-37)0H , and the amide, GLP-1
`(7-36)NH2, are indistinguishable.
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`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.,
`
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`WO 98/43658
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`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-Lys9-GLP-1(7-37), Thr+®-Lys18-GLP-1(7-37), and
`Lys18-GLP-1(7-37). Derivatives of GLP-1 include, for
`example, acid addition salts, carboxylate salts,
`lower alkyl
`esters, and amides (see, e.g., WO91/11457 (1991);
`EP 0
`-
`733,644 (1996); and US Patent No: 5,512,549 (1996)).
`It has
`also been demonstrated that the N-terminal histidine residue
`
`is very important to insulinotropic activity of GLP-
`(His 7)
`1
`(Suzuki, S., et al. Diabetes Res.; Clinical Practice 5
`(Supp. 1):S30 (1988).
`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)
`[see, e.g., Nauck, M.A., et al., Diabetologia,
`36:741-744 (1993); Gutniak, M., et al., New England J. of
`Medicine, 326(20) :1316-1322 (1992); Nauck, M.A., et al., J.
`Clin. Invest., 91:301-307 (1993); and Thorens, B., et al.,
`Diabetes, 42:1219-1225 (1993)].
`GLP-1 based peptides hold great promise as
`alternatives to insulin therapy for patients with diabetes
`who have failed on sulfonylureas. 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)
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`in addition
`GLP-1 suppresses glucagon secretion, and this,
`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 shown to be a
`potential regulator of appetite.
`Meal-time use of GLP-1 based peptides offers
`
`Insulin therapy
`several advantages over 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,
`particularly 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 gastric emptying. These observations
`indicate that GLP-1 is may be useful as a treatment in IDDM
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`as well as in NIDDM.
`
`the biological half-life of native GLP-1
`However,
`molecules which are affected by the activity of dipeptidyl-
`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
`minutes (U.S. Pat. No. 5,118,666). Therefore extended-
`action GLP-1 based peptides are needed to enhance glycemic
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`
`control during the night while minimizing the risk of
`
`hypoglycemia.
`the present invention provides
`In one embodiment,
`GLP-1 analogs that have extended time action relative to
`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
`
`Ri -X-Glu-Gly-Thr- Phe -Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-
`
`Gly-Gln-Ala-Ala-Lys-Z-Phe-Tle-Ala-Trp-Leu-Val-Lys-Gly-
`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, desamino-histidine, 2-amino-histidine,
`&-hydroxy-histidine, homohistidine, alpha-fluoromethyl-
`histidine, and alpha-methyl-histidine;
`
`X is selected from the group consisting of Met, Asp, Lys,
`
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`
`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;
`provided that, if R, is His, X is Val, Y is Glu, and Z is
`Glu,
`then R, is NH,;
`
`Also provided by the present invention are
`pharmaceutical compositions comprising a GLP-1 compound of
`the present invention in combination with one or more
`pharmaceutically acceptable carriers, diluents, or
`excipients.
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`The present invention further provides a method of
`treating diabetes which comprises administering 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-active GLP-1 based peptides.
`It should -
`be noted that this specification uses the nomenclature
`scheme that has developed around processed forms of GLP-1.
`In this scheme,
`the amino terminus of native GLP-1(7-37) 0H
`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)0H. Likewise KX in SEQ ID NO:1 corresponds to
`residue 8 of GLP-1(7-37)OH and Y corresponds to residue 21
`and so forth. Moreover, all amino acids referred to in this
`specification are in the L form, unless otherwise specified.
`In a preferred embodiment, R1 is His, and Y and 2
`are Glu. Another preferred group is when Rj is His, R2 is
`Gly-OH, and any one or more of X, Y, and Z differ from the
`residues present in native GLP-1(7-37)OH. Another preferred
`group is when Rj is His, X is 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 disclosed
`and the state of the art in solid phase protein synthesis,
`GLP-1 analogs can be obtained via chemical synthesis.
`However, it also is possible to obtain a GLP-1 analog by
`fragmenting proglucagon using,
`for example, proteolytic
`enzymes. Moreover,
`recombinant DNA techniques may be used
`to express GLP-1 analogs of the invention.
`The principles of solid phase chemical synthesis
`of polypeptides are well known in the art and may be found
`in general texts in the area such as Dugas, H. and Penney,
`C., Bioorganic Chemistry (1981) Springer-Verlag, New York,
`pgs. 54-92, Merrifield, J.M., Chem. Soc., 85:2149 (1962),
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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
`Applied Biosystems 430A peptide synthesizer (Applied
`Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA
`94404) and synthesis cycles supplied by Applied Biosystems.
`Boc amino acids and other reagents are commercially
`available from Applied Biosystems and other chemical supply
`houses. Sequential Boc chemistry using double couple
`protocols are applied to the starting p-methyl benzhydryl
`amine resins for the production of C-terminal carboxamides.
`For the production of C-terminal acids,
`the corresponding
`PAM resin is used. Asp, Gln, and Arg are coupled using
`preformed hydroxy benzotriazole esters.
`The following side
`chain protecting groups may be used:
`
`Arg, Tosyl
`Asp, cyclohexyl
`Glu, cyclohexyl
`Ser, Benzyl
`Thr, Benzyl
`Tyr, 4-bromo carbobenzoxy
`
`Boc deprotection may be accomplished with
`trifluoroacetic acid in methylene chloride.
`Following
`completion of the synthesis the peptides may be deprotected
`and cleaved from the resin with anhydrous hydrogen fluoride
`(HF) containing 10% meta-cresol. Cleavage of the side chain
`protecting group(s) and of the peptide from the resin is
`carried out at zero degrees centigrade or below, preferably
`-20°C for thirty minutes followed by thirty minutes at 0°C.
`After removal of the HF,
`the peptide/resin is washed with
`ether, and the peptide extracted with glacial acetic acid
`and 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
`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:
`
`a)
`
`b)
`
`c)
`
`d)
`
`e)
`
`isolating a natural DNA sequence encoding
`GLP-1 or constructing a synthetic or semi-
`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
`fusion 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.
`
`the coding sequences for
`As previously stated,
`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
`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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`the skilled artisan will
`degeneracy of the genetic code,
`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.
`Once designed,
`the sequence itself may be generated using
`conventional DNA synthesizing apparatus such as the Applied
`Biosystems Model 380A or 380B DNA synthesizers (Applied
`Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA
`94404).
`
`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.
`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
`
`The
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`expressed.
`
`To achieve efficient transcription of the coding
`region, it must be operably associated with a promoter-
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`-~ 10 -
`
`the promoter-operator region of
`operator region. Therefore,
`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
`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:
`
`(a)
`
`(b)
`
`(c)
`
`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;
`
`stabilize maintenance of the plasmid in host cell
`populations;
`
`indicative of
`(ad) direct synthesis of a protein prod.
`plasmid maintenance in a host cell population;
`
`(e)
`
`provide in series restriction endonuclease
`recognition sites unique to the plasmid; and
`
`(£)
`
`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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`lil
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`-
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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
`Publication No. 89-18S, 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 herein.
`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
`organic acids,
`to forma salt. Acids commonly employed
`to form acid addition salts are inorganic acids such as
`hydrochloric acid, hydrobromic acid, hydroiodic acid,
`sulfuric acid, phosphoric acid, and the like, and organic
`acids such as p-toluenesulfonic acid, methanesulfonic
`acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic
`acid, succinic acid, citric acid, benzoic acid, acetic
`acid, and the like. Examples of such salts include the
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`sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,
`phosphate, monohydrogenphosphate, dihydrogenphosphate,
`metaphosphate, pyrophosphate, chloride, bromide,
`iodide,
`acetate, propionate, decanoate, caprylate, acrylate,
`formate,
`isobutyrate, caproate, heptanoate, propiolate,
`oxalate, malonate, succinate, suberate, sebacate,
`fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,
`benzoate, chlorobenzoate, methylbenzoate,
`dinitrobenzoate, hydroxybenzoate, methoxybenzoate,
`phthalate, sulfonate, xylenesulfonate, phenylacetate,
`phenylpropionate, phenylbutyrate, citrate,
`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 alkali or alkaline
`earth metal hydroxides, carbonates, bicarbonates, and the
`like.
`Such bases useful in preparing the salts of this
`invention thus include sodium hydroxide, potassium
`hydroxide, ammonium hydroxide, potassium carbonate, and the
`like. Salt forms of GLP-1 analogs are particularly
`preferred. Of course, when the compounds of this invention
`are used for therapeutic purposes,
`those compounds may also
`be in the form of a salt, but the salt must be
`
`pharmaceutically acceptable.
`The ability of a GLP-1 analog to stimulate insulin
`secretion may be determined by providing a GLP-1 analog to
`cultured animal cells, such as the RIN-38 rat insulinoma
`cell line, and monitoring the release of immunoreactive
`insulin (IRI)
`into the media. Alternatively one can inject
`a GLP-1 analog into an animal and monitor plasma levels of
`immunoreactive 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
`The
`phosphate/albumin buffer with a pH of 7.4 is employed.
`incubation is prepared with the consecutive addition of 500
`ul of phosphate buffer, 50 wl of perfusate sample or rat
`insulin standard in perfusate, 100 pl of anti-insulin
`antiserum (Wellcome Laboratories; 1:40,000 dilution), and
`
`insulin, giving a total volume of 750 pl in
`[125I)
`100 pl of
`a 10x75 mm disposable glass tube. After incubation for 2-3
`days at 4° C,
`free insulin is separated from antibody-bound
`insulin by charcoal separation.
`The assay sensitivity is 1-
`2 uU/mL.
`In order to measure the release of IRI into the
`cell culture medium of cells grown in tissue culture, one
`preferably incorporates radioactive label into proinsulin.
`Although any radioactive label capable of labeling a
`polypeptide can be used, it is preferable to use 3H leucine
`in order to obtain labeled proinsulin.
`
`To determine whether a GLP-1 analog has
`insulinotropic properties may also be determined by
`pancreatic infusion.
`The in situ isolated perfused rat
`pancreas assay is a modification of the method of Penhos,
`J.C., et al., Diabetes, 18:733-738 (1969). Fasted male
`Charles River strain albino rats, weighing 350-600 g, are
`
`anesthetized with an intraperitoneal injection of Amytal
`Sodium (Eli Lilly and Co.: 160 ng/kg). Renal, adrenal,
`The
`gastric, and lower colonic blood vessels are ligated.
`entire intestine is resected except for about four cm of
`
`duodenum and the descending colon and rectum. Therefore,
`only a small part of the intestine is perfused, minimizing
`possible interference by enteric substances with glucagon-
`like immunoreactivity.
`The perfusate is a modified Krebs-
`
`Ringer bicarbonate buffer with 4% dextran T70 and 0.2%
`bovine serum albumin (fraction V), and is bubbled with 95%
`Op and 5% CO2.
`A nonpulsatile flow, 4-channel roller
`bearing pump (Buchler polystatic, Buchler Instruments
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`
`is used, and a switch from
`Division, Nuclear-Chicago Corp.)
`one perfusate source to another is accomplished by switching
`a 3-way stopcock.
`The manner in which perfusion is
`performed, monitored, and analyzed follow the method of
`Weir, G.Cc., et al., J. Clin. Inestigat. 54:1403-1412 (1974),
`which is hereby incorporated by reference.
`The present invention also provides pharmaceutical
`compositions comprising a GLP-1 analog 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. Especially preferred routes 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 ywg/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
`the active ingredient, which comprises at least
`invention,
`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 necessary to
`mill the active compound to provide the appropriate particle
`size prior to combining with the other ingredients.
`If the
`active protein is substantially insoluble, it ordinarily is
`milled to particle size of less than about 200 mesh.
`If the
`active compound is substantially water soluble,
`the particle
`size is normally adjusted by milling to provide a
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`substantially uniform distribution in the formulation, 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 cellulose.
`The formulations can additionally
`include lubricating agents such as talc, magnesium stearate”
`and mineral oil, wetting agents, emulsifying and suspending
`agents, preserving agents such as methyl- and
`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 the art.
`The compositions are preferably formulated in a
`unit dosage form with each dosage normally containing from
`about 50 ng to about 100 mg, more usually from about 1 mg to
`about 10 mg of the active ingredient.
`The term "unit dosage
`form" refers to physically discrete units suitable as
`unitary dosages for human subjects and other mammals, each
`unit containing a predetermined quantity of active material
`calculated to produce the desired therapeutic effect in
`association with a suitable pharmaceutical excipient.
`For the purpose of parenteral administration,
`compositions containing a protein of the present invention
`preferably are combined with distilled water and t