`.,
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`Intematlonal Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6 i
`A61K 38/00
`
`(11) International Publication Number:
`
`WO‘ 98/43658
`
`(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 (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).
`
`(81) Designated States: AL, AM, AU, AZ, BA, BB, BG, BR, BY,
`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,
`MG, MK, MN, MW, MX, NO, NZ, PL, RU, SD, SG, SI,
`SK, SL, TJ, TM, TR, Tl‘, 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.
`
`the risk of hypoglycemia.
`
`(54) Title: GLUCAGON—LIKE PEPTIDE—1 ANALOGS
`
`(57) Abstract
`
`The invention provides extended—action GLP—l based peptides and compositions that are useful for treating diabetes and minimize
`
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`MYLAN INST. EXHIBIT 1042 PAGE 1
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`
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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.
`
`ES
`FI
`FR
`GA
`GB
`GE
`GI-I
`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
`N0
`NZ
`PL
`PT
`R0
`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
`
`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
`
`Singapore
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Gate d’Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`
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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
`
`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 (non—insulin
`
`dependent diabetes mellitus — NIDDM)
`
`is characterized by a
`
`resistance to insulin action in peripheral tissues such as
`
`15
`
`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
`
`20
`
`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.
`
`25
`
`3O
`
`35
`
`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
`
`MYLAN INST. EXHIBIT 1042 PAGE 3
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`WO 98/43658
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`PCT/US98/05945
`
`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
`
`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
`
`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
`
`20
`
`glucose levels in the normal range.
`
`Glucagon—like peptide—l
`
`(GLP—l) 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
`
`25
`
`processed from the 160 amino acid precursor protein,
`
`preproglucagon. Cleavage of preproglucagon first yields
`
`GLP—l, a 37 amino acid peptide, GLP—l(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—l(7—37)OH 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—l(7—37)OH , and the amide, GLP—l
`
`(7—36)NH2, are indistinguishable.
`
`GLP—1 is known to stimulate insulin secretion
`
`30
`
`35
`
`(insulinotropic action) causing glucose uptake by cells
`
`which decreases serum glucose levels (see, e g., Mojsov, S.,
`
`MYLAN INST. EXHIBIT 1042 PAGE 4
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`MYLAN INST. EXHIBIT 1042 PAGE 4
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`
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`WO 98/43658
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`PCT/US98/05945
`
`Int. J. Peptide Protein Research, 49:333—343 (1992)).
`
`Numerous GLP—l 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—l(7-
`
`37), acetyl-Lys9—GLP—1(7—37), ThrlG—Lysls—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 0
`
`'
`
`733,644 (1996); and US Patent NO: 5,512,549 (1996)).
`
`It has
`
`10
`
`also been demonstrated that the N—terminal histidine residue
`
`(His 7)
`
`is very important to insulinotropic activity of GLP—
`
`1
`
`(Suzuki, 8., et al. Diabetes Res.; Clinical Practice g
`
`(Supp.
`
`l):S3O (1988).
`
`15
`
`between laboratory experimentation and mammalian,
`
`Multiple authors have demonstrated the nexus
`
`insulinotropic responses to exogenous
`particularly human,
`administration of GLP—1, particularly GLP—l
`(7-36)NH2 and
`
`GLP—1
`
`(7—37)
`
`(see, e.g., Nauck, M.A., et al., Diabetologia,
`
`§§:741-744 (1993); Gutniak, M., et al., New England J. of
`
`20
`
`MEdicine, §2§(20):1316—1322 (1992); Nauck, M.A., et al., J.
`
`Clin. Invest., 21:301—307 (1993); and Thorens, B., et al.,
`
`Diabetes, 42:1219—1225 (1993)].
`
`GLP—l based peptides hold great promise as
`
`alternatives to insulin therapy for patients with diabetes
`
`25
`
`who have failed on sulfonylureas.
`
`GLP—l 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—l stimulates insulin secretion, but only
`
`30
`
`35
`
`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—l 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 subcutanebus insulin injections.
`
`2)
`
`MYLAN INST. EXHIBIT 1042 PAGE 5
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`MYLAN INST. EXHIBIT 1042 PAGE 5
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`MYLAN INST. EXHIBIT 1042 PAGE 5
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`WO 98/43658
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`PCT/US98/05945
`
`GLP—l 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-l 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-l may
`
`enhance insulin sensitivity in peripheral tissues such as
`
`'
`
`muscle and fat.
`
`5) Finally, GLP—l has been shown 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,
`
`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—l in the treatment of insulin
`
`15
`
`20
`
`dependent diabetes mellitus (IDDM). Similar to NIDDM
`
`patients, GLP—1 is effective in reducing fasting
`
`25
`
`hyperglycemia through its glucagonostatic properties.
`
`Additional studies have indicated that GLP—l 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
`
`3O
`
`as well as in NIDDM.
`
`However,
`
`the biological half—life of native GLP—l
`
`molecules which are affected by the activity of dipeptidyl—
`
`peptidase IV (DPP IV)
`
`is quite short.
`
`For example,
`
`the
`
`biological half—life of GLP—l(7—37)OH is a mere 3 to 5
`
`35
`
`minutes (U.S. Pat. No. 5,118,666). Therefore extended—
`
`action GLP—l based peptides are needed to enhance glycemic
`
`MYLAN INST. EXHIBIT 1042 PAGE 6
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`MYLAN INST. EXHIBIT 1042 PAGE 6
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`MYLAN INST. EXHIBIT 1042 PAGE 6
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`
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`WO 98/43658
`
`PCT/US98/05945
`
`control during the night while minimizing the risk of
`
`hypoglycemia.
`
`In one embodiment,
`
`the present invention provides
`
`GLP—l analogs that have extended time action relative to
`
`native GLP—l,
`
`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—l 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—
`
`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,
`
`fi—hydroxy—histidine, homohistidine, alpha—fluoromethyl—
`
`histidine, and alpha—methyl—histidine;
`
`X is 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;
`
`R2 is selected from the group consisting of NH2, and Gly—OH;
`
`provided that, if R1 is His, X is Val, Y is Glu, and Z is
`
`25
`
`Glu,
`
`then R2 is NHfi
`
`Also provided by the present invention are
`
`pharmaceutical compositions comprising a GLP—l compound of
`
`the present invention in combination with one or more
`
`pharmaceutically acceptable carriers, diluents, or
`
`30
`
`excipients.
`
`MYLAN INST. EXHIBIT 1042 PAGE 7
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`MYLAN INST. EXHIBIT 1042 PAGE 7
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`MYLAN INST. EXHIBIT 1042 PAGE 7
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`
`
`WO 98/43658
`
`PCT/US98/05945
`
`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—l
`
`compound of the present invention.
`
`Detailed Description of the Invention
`
`In one embodiment,
`the present invention provides
`novel, biologically—active GLP—l based peptides.
`It should-
`
`be noted that this specification uses the nomenclature
`
`scheme that has developed around processed forms of GLP—1.
`
`10
`
`In this scheme,
`
`the amino terminus of native GLP—l(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—l(7—37)OH. Likewise X in SEQ ID NO:1 corresponds to
`
`residue 8 of GLP—l(7—37)OH and Y corresponds to residue 21
`
`15
`
`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 Z
`
`are Glu. Another preferred group is when R1 is His, R2 is
`
`Gly—OH, and any one or more of X, Y, and Z differ from the
`
`20
`
`residues present in native GLP—l(7—37)OH. Another preferred
`
`group is when R1 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,
`
`25
`
`GLP—l analogs can be obtained via chemical synthesis.
`
`However, it also is possible to obtain a GLP—l analog by
`
`fragmenting proglucagon using, for example, proteolytic
`
`enzymes. Moreover,
`
`recombinant DNA techniques may be used
`
`to express GLP—l analogs of the invention.
`
`30
`
`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., §§:2149 (1962),
`
`MYLAN INST. EXHIBIT 1042 PAGE 8
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`MYLAN INST. EXHIBIT 1042 PAGE 8
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`MYLAN INST. EXHIBIT 1042 PAGE 8
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`
`
`WO 98/43658
`
`PCT/US98/05945
`
`and Stewart and Young, Solid Phase Peptide Synthesis, pp.
`
`24—66, Freeman (San Francisco, 1969).
`
`For example, a GLP-l 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
`
`10
`
`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
`
`15
`
`20
`
`25
`
`trifluoroacetic acid in methylene chloride.
`
`Following
`
`Boc deprotection may be accomplished with
`
`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
`
`30
`
`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.
`
`35
`
`The preparation of protected, unprotected, and
`
`partially protected GLP—l has been described in the art.
`
`See U.S. Pat. No. 5,120,712 and 5,118,666, herein
`
`incorporated by reference, and Qrskov, C., et al., J. Biol.
`
`MYLAN INST. EXHIBIT 1042 PAGE 9
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`MYLAN INST. EXHIBIT 1042 PAGE 9
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`MYLAN INST. EXHIBIT 1042 PAGE 9
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`
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`WO 98/43658
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`PCT/US98/05945
`
`
`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—l analogs can be obtained. Although GLP—
`
`1 analogs may be produced by solid phase peptide synthesis,
`
`recombinant methods, or by fragmenting glucagon,
`
`recbmbinant
`
`methods may be preferable because higher yields are
`
`possible.
`
`The basic steps in the recombinant production of
`
`10
`
`a GLP—l analog are:
`
`a)
`
`b)
`
`c)
`
`d)
`
`e)
`
`isolating a natural DNA sequence encoding
`GLP—l or constructing a synthetic or semi-
`synthetic DNA coding sequence for GLP—l,
`
`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.
`
`15
`
`20
`
`25
`
`30
`
`As previously stated,
`
`the coding sequences for
`
`GLP—l 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. UlS.A. 19:345—349
`
`(1982) and may be used as starting material in the
`
`recombinant production of a GLP—l analog by altering the
`
`native sequence to achieve the desired results.
`
`Synthetic genes,
`
`the in Vitro or in vivo
`
`40
`
`transcription and translation of which results in the
`
`production of a GLP—l 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-l
`
`intermediates.
`
`The methodology of synthetic gene construction is
`
`well known in the art.
`
`See Brown, et a1.
`
`(1979) Methods in
`
`Enzymology, Academic Press, N.Y., Vol. 68, pgs. 109—151.
`
`DNA sequences that encode GLP—l intermediates can be
`
`designed based on the amino acid sequences herein disclosed.
`
`10
`
`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).
`
`15
`
`To effect the expression of a biologically—active
`
`GPL—l 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.
`
`20
`
`(1989) Molecular Cloning; A Laboratory Manual, Cold Springs
`
`25
`
`30
`
`Harbor Laboratory Press, N.Y., Vol. 1—3. Restriction
`
`endonuclease cleavage sites are engineered into either end
`
`of the DNA encoding the GLP-l 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 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
`
`35
`
`expressed.
`
`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
`
`20
`
`25
`
`30
`
`(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)
`
`stabilize maintenance of the plasmid in host cell
`populations;
`
`indicative of
`(d) 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
`
`(f)
`
`terminate mRNA transcription.
`
`These circular DNA plasmids are useful as vectors in
`
`recombinant DNA procedures for securing high levels of
`
`35
`
`expression of exogenous genes.
`
`Having constructed an expression vector for a
`
`GLP-l 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—l analog. Techniques for
`
`40
`
`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
`
`MYLAN INST. EXHIBIT 1042 PAGE 12
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`-11-
`
`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
`
`10
`
`levels of the overexpressed protein.
`
`Such protein
`
`aggregates must be solubilized, denatured and refolded using
`
`techniques well known in the art.
`
`See Kreuger, et a1.
`
`(1990)
`
`in Protein Folding, Gierasch and King, eds., pgs 136—
`
`142, American Association for the Advancement of Science
`
`15
`
`Publication No. 89—188, Washington, D.C.; and U.S. Patent
`
`No. 4,923,967.
`
`Regardless of the methods used to produce a GLP—l
`
`analog, purification of the protein generally will be
`
`required. Methods for purifying proteins are well known in
`
`20
`
`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
`
`25
`
`skilled artisan to purify GLP-l analogs claimed herein.
`
`The present invention also includes salt forms
`
`of GLP—1 analogs. A GLP—l analog of the invention may be
`
`sufficiently acidic or sufficiently basic to react with
`
`any of a number of inorganic bases, and inorganic and
`
`3O
`
`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—toluenesulfonic acid, methanesulfonic
`
`35
`
`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
`
`MYLAN INST. EXHIBIT 1042 PAGE 13
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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—l,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—z—sulfonate, mandelate, and the
`
`15
`
`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
`
`20
`
`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
`
`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—l analog to stimulate insulin
`
`3O
`
`secretion may be determined by providing a GLP—l 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-l analog into an animal and monitor plasma levels of
`
`35
`
`immunoreactive insulin (IRI).
`
`The presence of IRI is detected through the use of
`
`a radioimmunoassay which can specifically detect insulin.
`
`MYLAN INST. EXHIBIT 1042 PAGE 14
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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.,
`
`19:487—509 (1972).
`
`In this modification, a
`
`5
`
`phosphate/albumin buffer with a pH of 7.4 is employed.
`
`The
`
`incubation is prepared with the consecutive addition of 500
`
`pl of phosphate buffer, 50 pl of perfusate sample or rat
`
`insulin standard in perfusate, 100 pl of anti—insulin
`
`antiserum (Wellcome Laboratories;
`
`l:40,000 dilution), and
`
`10
`
`100 pl of
`
`[1251)
`
`insulin, giving a total volume of 750 pl 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 l—
`
`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—l 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,
`
`l_8_: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,
`
`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—
`
`like immunoreactivity.
`
`The perfusate is a modified Krebs—
`
`15
`
`20
`
`25
`
`30
`
`Ringer bicarbonate buffer with 4% dextran T70 and 0.2%
`
`35
`
`bovine serum albumin (fraction V), and is bubbled with 95%
`
`02 and 5% C02.
`
`A nonpulsatile flow, 4—channel roller
`
`bearing pump (Buchler polystatic, Buchler Instruments
`
`MYLAN INST. EXHIBIT 1042 PAGE 15
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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
`
`Weir, G.C., et al., J. Clin. Inestigat. §g:l403—1412 (1974),
`
`which is hereby incorporated by reference.
`
`The present invention also provides pharmaCeutical
`
`compositions comprising a GLP—l analog of the present
`
`'
`
`invention in combination with a pharmaceutically acceptable
`
`10
`
`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
`
`15
`
`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 ug/kg of body weight, although lower or higher dosages
`
`may be administered.
`
`The required dosage will depend upon
`
`20
`
`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
`
`25
`
`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.
`
`30
`
`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
`
`35
`
`active compound is substantially water soluble,
`
`the particle
`
`size is normally adjusted by milling to provide a
`
`MYLAN INST. EXHIBIT 1042 PAGE 16
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`MYLAN INST. EXHIBIT 1042 PAGE 16
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`MYLAN INST. EXHIBIT 1042 PAGE 16
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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