(51) International Patent Classification 6;
`
`
`A61K 31/44, 38/00
`23 March 2000 (23.03.00) —
`(43) International Publication Date:
`
`(11) International Publication Number:
`
`WO 00/15224
`
`
`
`
`
`PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(21) International Application Number:
`
`PCT/US99/21055
`
`(22) International Filing Date:
`
`14 September 1999 (14.09.99)
`
`(30) Priority Data:
`60/100,687
`
`17 September 1998 (17.09.98)
`
`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): RINELLA,Joseph, Vincent,
`Jr. [US/US]; 3640 Romar Drive, Brownsburg,
`IN 46112
`(US).
`
`(74) Agent: MACIAK,Ronald, S.; Eli Lilly and Company,Lilly
`Corporate Center, Indianapolis, IN 46285 (US).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, .CN, CR, CU, CZ, DE, DK, DM, EE,
`ES, FI, GB, GD, GE, GH, GM, HR,HU,ID,IL,IN, IS, JP,
`KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD,
`MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD,
`SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ,
`VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE, LS, MW,
`SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM, AZ, BY,
`KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, CH,
`CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
`GW, ML, MR, NE, SN, TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: PROTEIN FORMULATIONS
`
`(57) Abstract
`
`The present invention discloses a stable, soluble formulation comprising a medically useful peptide or protein, a hydrophobic
`preservative, and nicotinamide. Said storage-stable, soluble formulation is useful as a multi-use pharmaceutical product.
`
`Pe
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`Codesused to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`SI
`LS
`Slovenia
`Lesotho
`ES
`LT
`Slovakia
`Lithuania
`SK
`FI
`LU
`SN
`FR
`Senegal
`Luxembourg
`LV
`Latvia
`SZ
`Swaziland
`GA
`TD
`Monaco
`Chad
`MC
`GB
`TG
`MD
`GE
`Togo
`MG
`GH
`TJ
`Tajikistan
`T™
`MK
`Turkmenistan
`GN
`TR
`GR
`Turkey
`TT
`HU
`Trinidad and Tobago
`UA
`Ukraine
`IE
`UG
`IL
`Uganda
`US
`United States of America
`IS
`UZ
`Uzbekistan
`IT
`VN
`Viet Nam
`JP
`YU
`KE
`Yugoslavia
`ZW
`Zimbabwe
`KG
`KP
`
` 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
`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
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Treland
`Tsrae}
`Tceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongotia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`
`
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`PROTEIN FORMULATIONS
`
`Field of Invention
`
`The present invention is in the field of peptide
`and protein chemistry as it applies to human medicine. In
`particular,
`the invention relates to the preparation of
`soluble stabile peptide and protein formulations that
`include nicotinamide and hydrophobic preservatives.
`
`Background of the Invention
`Nicotinamide is not a widely-recognized excipient
`in pharmaceutical formulations.
`For example, it is not
`mentioned as an excipient in the Handbook of Pharmaceutical
`Excipients, 2nd ed., A. Wade & P. Weller, Eds.
`(1994).
`However, nicotinamide is known to increase the solubility of
`sparingly-soluble, non-protein,
`low molecular weight
`compounds, such as, certain piperazido and piperazino
`compounds
`[Fawzi, et al., J. Pharmaceut. Sci. 69:104-106
`(1980)], anti-cancer nucleoside analogs [Truelove, et al.,
`Int. J. Pharmaceutics 19:17-25 (1984)], paracetamol
`[Hamza,
`et al., Drug Dev. Industr. Pharmacy 11:1577-1536 (1985)],
`
`diazepam, griseofulvin, progesterone, 17B-estradiol, and
`testosterone [Rasool, et al., J. Pharmaceut. Sci. 80:387-393
`(1991)],
`the phenothiazine derivative, moricizine [Hussain,
`et al., J. Pharmaceut. Sci. 82:77-79 (1993)], and riboflavin
`[Coffman, et al., J. Pharmaceut. Sci. 85:951-954 (1996)].
`In the above cited formulations, nicotinamide
`apparently operates as a hydrotropic agent to increase the
`solubility of another solute when nicotinamide is added at a
`high concentration. This hydrotropic phenomenon is in
`direct opposition to normal solution behavior where addition
`of a second solute to a solution of a sparingly soluble
`
`substance will cause the less soluble substance to
`
`precipitate.
`A combination of insulin and nicotinamide,
`optionally containing a preservative, was previously
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`The
`described by Jorgensen in U.S. Patent No. 5,382,574.
`formulation was reported to promote faster absorption of
`insulin from an injection site.
`Jorgensen does not discuss
`any effect of nicotinamide on formulation stability.
`Moreover, it is likely that the effect of nicotinamide was
`not observerd or appreciated because the specification
`specifically recommends that known stabilizing agents such
`as phospholipids be added to stabilize the formulations.
`Also, it fails to mention any effect on insulin stability
`produced by nicotinamide alone.
`The molecular interactions in peptide and protein
`formulations are complex because a variety of factors such
`as choice of preservative, buffer,
`ionic strength, pH,
`temperature, and other excipients must be balanced to
`produce a relatively stable formulation suitable for
`manufacturing, shipping, and storage that meets regulatory
`requirement for such products.
`The role that each factor
`contributes to aggregation is uncertain in view of the
`complexity of the given peptide or protein molecule as well
`as the propensity for that peptide or protein to aggregate
`and precipitate in formulations containing preservatives.
`In view of this complexity and tendency to aggregate,
`the
`effect of nicotinamide on the stability of peptides and
`protein forumulations containing a hydrophobic preservative
`could not have been predicted from the art describing
`nicotinamide's effect as a hydrotropic agent for relatively
`small molecules, nor from its apparent ability to facilitate
`absorption of insulin from a subcutaneous injection.
`Thus,
`the present invention provides conditions
`that increase the physical stability of medically useful
`peptides and proteins in the presence of hydrophobic
`preservatives and makes possible commercially-viable, multi-
`use soluble pharmaceutical products to treat a variety of
`human diseases.
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`Summary of the Invention
`This invention provides a stable soluble
`formulation comprising a medically useful peptide or
`protein, a hydrophobic preservative, and nicotinamide.
`The invention further provides a process for
`preparing said formulation which comprises combining a
`peptide or protein, a hydrophobic preservative, and
`nicotinamide to produce said formulation.
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`Detailed Description and Preferred Embodiments
`For purposes of the present invention, as
`disclosed and claimed herein,
`the following terms and
`abbreviations are defined as follows:
`Administering -- an act whose effect is to
`transfer a formulation of the present invention into the
`body of a mammal in need thereof. Administration may be via
`any route known to be effective by the physician of ordinary
`skill. Parenteral administration is commonly understood in
`the medical literature as the injection of a dosage form
`into the body by a sterile syringe or some other mechanical
`device such as an infusion pump. Peripheral parenteral
`routes of administration include, without limitation,
`intravenous,
`intramuscular, subcutaneous, and
`intraperitoneal routes of administration.
`Alkylparaben -- refers to a C; to C, alkyl
`paraben, or mixtures thereof. Preferably, alkylparaben is
`methylparaben, ethylparaben, propylparaben, or butylparaben.
`Cresol
`- refers to meta-cresol, ortho-cresol,
`para-cresol, chloro-cresol, or mixtures thereof.
`Hydrophobic preservative -- refers toa
`hydrophobic compound that is added to a pharmaceutical
`formulation to act as an anti-microbial agent.
`A parenteral
`formulation must meet guidelines for preservative
`effectiveness to be a commercially viable multi-use product.
`Among hydrophobic preservatives known in the art as being
`effective and acceptable in parenteral formulations are the
`alkylparabens,
`the phenolic preservatives, benzyl alcohol,
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`chlorobutanol, benzoic acid and various mixtures thereof.
`See, e.g., WALLHAUSER, K.-H., DEVELOP. BrIoL.
`STANDARD. 24, Pp.
`9-28 (Basel, S. Krager, 1974).
`Isotonicity agent -- refers to a compound that is
`tolerated physiologically and imparts a suitable tonicity to
`the formulation to prevent the net flow of water across cell
`membranes. Compounds, such as glycerin, are commonly used
`for such purposes at known concentrations. Other possible
`isotonicity agents include salts, e.g., NaCl, and sugars,
`e.g., dextrose, mannitol, and sucrose.
`Nicotinamide -- refers to a compound of the
`
`formula:
`
`~ _ CONH2
`
`|N
`
`Pharmaceutically acceptable buffer -- The pH of
`the formulation may be buffered with a pharmaceutically
`acceptable buffer, such as, without limitation,
`sodium
`acetate, sodium phosphate, sodium citrate,
`sodium tartarate,
`TRIS or a basic amino acid, such as, histidine,
`lysine or
`arginine. Other pharmaceutically acceptable buffers are
`known in the art.
`The selection and concentration of buffer
`is known in the art.
`Phenolic preservative-- refers to phenol and
`
`cresol.
`
`Soluble -- refers to the relative absence of
`aggregated protein that is visually perceivable.
`The degree
`of aggregation of proteins ina formulation may be inferred
`by measuring the turbidity of the formulation.
`The greater
`the turbidity of the formulation,
`the greater the extent of
`aggregation of the protein in the formulation. Turbidity is
`commonly determined by nephelometry, and measured in
`Nephalometric Turbidity Units (NTU).
`Stable -- A "stable" formulation is one in which
`the protein or peptide remains soluble for an extended
`period of time under the conditions of storage.
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`Treating -- as used herein, describes the
`management and care of a patient for the purpose of
`combating a disease, condition, or disorder and includes the
`administration of a formulation of the present invention to
`prevent the onset of the symptoms or complications,
`alleviating the symptoms or complications, or eliminating
`the disease, condition, or disorder. Treating, as used
`herein,
`includes the administration of the protein for
`cosmetic purposes.
`A cosmetic purpose seeks to control the
`weight of a mammal to improve bodily appearance.
`In one embodiment,
`the invention relates to
`formulations containing a hydrophobic preservative,
`nicotinamide and a medically useful peptide or protein.
`which are defined to include native hormones and functional
`analogs (excluding insulin and insulin analogs and leptin
`and leptin analogs), native cytokines and functional
`analogs, soluble protein vaccines, and antibodies and
`antibody fragments of all forms.
`The following list of medically useful peptides
`and protiens is provided for illustrative purposes and is in
`no way meant to limit the scope of the medically useful
`peptides and proteins that are consistent with the
`invention:
`ocytocin, vasopressin, adrenocorticotropin
`hormone and analogs, epidermal growth factor, platelet-
`derived growth factor, prolactin,
`luteinising hormone
`releasing hormone, growth hormone, growth hormone releasing
`hormone, somatostatin, glucagon, GLP-1 related compounds,
`IL-2,
`IL-10,
`IL-15,
`interferon-a,8,Y, gastrin tetragastrin,
`pentagastrin, urogastrin, secretin, calcitonin, enkaphalins,
`endorphins, angiotensins,
`thyrotropin releasing hormone,
`tumor necrosis factor, nerve growth factor, granulocyte
`colony stimulating factor, granulocyte macrophage colony
`stimulating factor, macrophage colony stimulating factor,
`renin, bradykinin, bacitracins, polymixins, colistins,
`tyrocidin, gramicidins.
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`A more preferred group of medically useful
`peptides and proteins, defined as “ Group I polypeptide”
`for purposes of this specification, consists of acylated
`insulins, particularly C,-C2o acylations of the epsilon amino
`group of Lys on the insulin B-chain, especially C14 acylated
`Lys®** pro’? human insulin, mammalian growth hormone, growth
`hormone releasing hormone, GLP-1 related compounds,
`erythrocyte progenitor hormone (EPO), parathyroid hormone
`and fragments, especially as disclosed in US Pat NOS:
`4,086,196 and 5,208,041, §-lipotropin, fibroblast growth
`factor-8 and analogs, osteoprotegrin-2 and 3,
`interleukin-10
`and 15 and their analogs, vascular endothelial growth
`factor, and follicle stimulating hormone (FSH) and variants.
`Follicle stimulating hormone “ FSH” , whether
`produced recombinantly or isolated, and follicle stimulating
`hormone variants “ FSH variants” as defined herein are
`well-known in the art.
`FSH as used herein refers to the FSH
`produced as a full length mature protein which includes, but
`are not limited to human FSH or “ hFSH” , whether produced
`recombinantly ofr isolated from human sources (see Shome B.,
`et al., J. Prot. Chem., 7:325-339, 1988; Saxena B.B. and
`Rathnam P., J. Biol. Chem., 251:993-1005, 1976; Watkins, et
`al., DNA, 6:205-212, 1987; Shome B. and Parlow A.F., J.
`Clin. Endocrinol. Metab., 39(1):203-205, 1974; and Beck, et
`al., DNA, 4:76, 1985; U.S. 5,405,945, and U.S. 5,639,640) -
`each citation incorporated by reference. Furthermore,
`various FSH variants are known or are understood from the
`art
`(see Shome, J. Clin. Endocrin. Metab 39:187 (1974);
`Saxena, J. Biol Chem 251 (4) :993-1005 (1976); 1978; Sairam et
`al., Biochem J 197:541 (1981); additionally see Closset Eur.
`J. Biochem. 86:115-120; Fujiki, J. Biol. Chem. 253:5363-5368
`(1978); Sairam, Biochem. J. 197:541-552 (1981)
`- each
`citation independently incorporated by reference). Prior-
`art FSH beta subunits would include the Saxena sequence as
`well as a genus of sequences implicated in Sairam's
`discussion of
`(a) evolutionarily conserved amino acids and
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`(b) well-known and characterized errors in sequencing.
`Further,
`those of skill in the art recognize that the
`substitution of a prior art identified amino acid with (i)
`a chemically similar amino acid or (ii) an evolutionarily
`conserved amino acid would have no appreciable affect on
`the biological activity of an FSH heterodimer comprised of
`an hFSH beta subunit,
`thus modified.
`In particular, Sairam's commentary on the Saxena
`hFSH sequence, as well as his discussion of amino acid
`substitutions identified between functional FSH molecules,
`defines a genus of FSH beta chain sequences in the prior
`art. More specifically,
`the 1981 Sairam publication
`identifies conserved amino acid sequences referring to
`publications by Saxena et al., Shome et al., Closset et
`al., and Fujiki et al.
`Sairam, Biochem J 197:541, 551
`(1981).
`The prior art
`(1) evidences a preference for the
`FSH beta-chain sequence of Saxena over that of Shome;
`(2)
`addresses the issue of carboxy-terminal heterogeneity;
`(3)
`states that portions of the molecule affected by
`interspecies differences that are not essential for
`activity of the hormone and (4) highlights the guidance
`drawn from homologies between species and between the beta
`chains of the three, human glycoprotein hormones, FSH, LH
`
`and TSH.
`
`C-terminal heterogeneity is reported for all the
`published sequences except for that of the porcine FSH-s,
`in which glutamic acid was the only C-terminal residue.
`For position 27, Saxena assigned one tryptophan residue to
`this position also found support in the evolutionary
`conservation demonstrated for a tryptophan at position 24
`for FSH-B, among all prior art species.
`For positions 44
`and 46, Saxena shows that, at position 44,
`the residue
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`should be arginine instead of lysine and, at position 46,
`lysine instead of arginine.
`The porcine, equine and ovine
`sequences also reflected an evolutionary pressure to
`conserve the arginine at the position 44.
`The variations
`at three positions, 21, 22 and 44 involve a structurally
`conservative or evolutionarily-conserved ("homologous")
`substitutions, each of which possess bio-activity.
`Fach of the Sairam, Shome, and Closset references
`disclose residues isoleucine, serine at positions 21-22,
`while Saxena discloses leucine,
`threonine and Fujiki
`discloses isoleucine,
`threonine at these positions. Each
`of these disclosures is not only an evolutionarily
`conservative substitution, but also a structurally
`conservative substitution.
`The variation at position 41
`between the aspartic acid disclosed by each of Sairam,
`Shome, Closset, and Fujiki and the asparagine disclosed by
`Saxena, Closset and Sairam involves two evolutionarily
`conserved residues, each of which provide bio-activity.
`These disclosures of conservative substitutions and
`evolutionarily conserved substitutions guide the skilled
`artisan to distinct FSH beta chain variants, within the
`
`hFSH-B chain genus.
`A more preferred group of medically useful
`peptides and proteins consists of GLP-1 related compounds
`and native mammalian growth hormone.
`A more highly preferred group of peptides
`consistent with the present invention is glucagon-like
`peptide-1, its analogs and derivatives as defined herein.
`Water soluble copolymers and polymer conjugates of
`the aforementioned peptides and proteins are also consistent
`with the present invention and include for example
`polyethylene glycol, copolymers of ethylene glycol/propylene
`glycol, carboxymethylcellulose, dextran, polyvinyl alcohol,
`polyvinyl pyrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane,
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`ethylene/maleic anhydride copolymer, polyaminoacids (either
`homopolymers or random or nonrandom copolymers copolymers) ,
`and dextran or poly(n-vinyl pyrolidone) polyethylene glycol,
`propylene glycol homopolymers polypropylene oxide/ethylene
`oxide co-polymers, polyoxyethylated polyols,
`polystyrenemaleate and polyvinyl alcohol. Polyethylene
`glycol propionaldenhyde is preferred.
`The term "GLP-1" refers to human glucagon-like
`peptide-1 whose sequences and structures are known in the
`art.
`See US Patent No. 5,120,712, herein incorporated by
`reference. There are two native forms of human GLP-1, GLP-
`1(7-37)OH and GLP-1(7-36)NH2 which will be distinguished
`
`only when necessary.
`The term "GLP-1 analog" is defined as a molecule
`having one or more amino acid substitutions, deletions,
`inversions, or additions compared with GLP-1. Many GLP-1
`analogs are known in the art and include,
`for example, GLP-
`(7-34) and GLP-1(7-35), GLP-1(7-36), Val'-GLP-1(7-37),
`Gin?-GhP-1(7-37), D-Gln’-GLP-1(7-37), Thr-Lys -GLP-1(7-
`37), and Lys-GLP-1 (7-37) . Preferred GLP-1 analogs are
`GLP-1(7-34) and GLP-1(7-35), which are disclosed in U.S.
`Patent No: 5,118,666, herein incorporated by reference.
`The term "GLP-1 derivative" is defined as a
`molecule having the amino acid sequence of GLP-1 or a GLP-1
`analog, but additionally having chemical modification of
`one or more of its amino acid side groups, a-carbon atoms,
`terminal amino group, or terminal carboxylic acid group.
`chemical modification includes, but is not limited to,
`adding chemical moieties, creating new bonds, and removing
`chemical moieties. Modifications at amino acid side groups
`include, without limitation, acylation of lysine e-amino
`groups, N-alkylation of arginine, histidine, or lysine,
`alkylation of glutamic or aspartic carboxylic acid groups,
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`
`- 10 -
`
`and deamidation of glutamine or asparagine. Modifications
`of the terminal amino include, without limitation,
`the des-
`amino, N-lower alkyl, N-di-lower alkyl, and N-acyl
`modifications. Modifications of the terminal carboxy group
`include, without limitation,
`the amide,
`lower alkyl amide,
`dialkyl amide, and lower alkyl ester modifications.
`Lower
`alkyl is C1-C4 alkyl.
`Furthermore, one or more side
`groups, or terminal groups, may be protected by protective
`groups known to the ordinarily-skilled protein chemist.
`The a-carbon of an amino acid may be mono- or di-
`
`methylated.
`The term "GLP-1 molecule" means GLP-1, GLP-1
`
`analog, or GLP-1 derivative.
`Another preferred group of GLP-1 analogs is
`
`defined by the formula:
`R,-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-R,
`SEQ ID NO:1
`
`and the pharmaceutically-acceptable salts thereof, wherein:
`R, is selected from the group consisting of L-histidine, D-
`histidine, desamino-histidine, 2-amino-histidine, b-
`hydroxy-histidine, homohistidine, alpha-fluoromethyl-
`histidine, and alpha-methyl-histidine;
`X is selected from
`the group consisting of Ala, Gly, Val, Thr, Ile, and alpha-
`methyl-Ala;
`Y is selected from the group consisting of
`Glu, Glin, Ala, Thr, Ser, and Gly;
`4 is selected from the
`group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; and R,
`is selected from the group consisting of NH2, and Gly-OH;
`providing that when R, is His, X is Ala, Y is Glu, and Z is
`
`Glu, R, must be NH,.
`
`10
`
`15
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`20
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`25
`
`30
`
`MYLANINST. EXHIBIT 1126 PAGE 12
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`MYLAN INST. EXHIBIT 1126 PAGE 12
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`

`

`WO 00/15224
`
`PCT/US99/21055
`
`-11-
`
`Yet another preferred group of GLP-1 compounds
`consistent with the present invention is disclosed in WO
`91/11457 (U.S. Patent No. 5,545,618, herein incorporated by
`reference) and consists essentially of GLP-1(7-34), GLP-
`1(7-35), GLP-1(7-36), or GLP-1(7-37), or the amide forms
`thereof, and pharmaceutically-acceptable salts thereof,
`having at least one modification selected from the group
`
`consisting of:
`(a) substitution of glycine, serine, cysteine,
`threonine, asparagine, glutamine,
`tyrosine, alanine,
`valine,
`isoleucine,
`leucine, methionine, phenylalanine,
`arginine, or D-lysine for lysine at position 26 and/or
`position 34; or substitution of glycine, serine, cysteine,
`threonine, asparagine, glutamine,
`tyrosine, alanine,
`valine,
`isoleucine,
`leucine, methionine, phenylalanine,
`lysine, or a D-arginine for arginine at position 36;
`(b) substitution of an oxidation-resistant amino acid
`for tryptophan at position 31;
`tyrosine for
`(c) substitution of at least one of:
`valine at position 16;
`lysine for serine at position 18;
`aspartic acid for glutamic acid at position 21; serine for
`glycine at position 22; arginine for glutamine at position
`23; arginine for alanine at position 24; and glutamine for
`lysine at position 26; and
`(a) substitution of at least one of: glycine, serine,
`or cysteine for alanine at position 8; aspartic acid,
`glycine, serine, cysteine,
`threonine, asparagine,
`leucine,
`glutamine,
`tyrosine, alanine, valine,
`isoleucine,
`methionine, or phenylalanine for glutamic acid at position
`9; serine, cysteine,
`threonine, asparagine, glutamine,
`tyrosine, alanine, valine,
`isoleucine,
`leucine, methionine,
`
`10
`
`15
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`20
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`25
`
`30
`
`MYLANINST. EXHIBIT 1126 PAGE 13
`
`MYLAN INST. EXHIBIT 1126 PAGE 13
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`MYLAN INST. EXHIBIT 1126 PAGE 13
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`

`

`WO 00/15224
`
`PCT/US99/21055
`
`-12-
`
`or phenylalanine for glycine at position 10; and glutamic
`acid for aspartic acid at position 15; and
`(e) substitution of glycine, serine, cysteine,
`threonine, asparagine, glutamine,
`tyrosine, alanine,
`valine,
`isoleucine,
`leucine, methionine, or phenylalanine,
`or the D- or N-acylated or alkylated form of histidine for
`histidine at position 7; wherein,
`in the substitutions is
`(a),
`(b),
`(ad), and (e),
`the substituted amino acids can
`optionally be in the D-form and the amino acids substituted
`at position 7 can optionally be in the N-acylated or N-
`
`alkylated form.
`Still other GLP-1 molecules consistent with the
`present invention have also been described in WO 98/08871,
`and include a lipophilic substituent attached to the N-
`terminal or to the C-terminal amino acid residue wherein
`
`the substituent is an alkyl group or a group which has an
`
`omega carboxylic group.
`Because the enzyme, dipeptidyl-peptidase IV (DPP
`Iv), may be responsible for the observed rapid in vivo
`inactivation of administered GLP-1,
`[see, e.g., Mentlein,
`
`R., et al., Eur. J. Biochem., 214:829-835 (1993)],
`administration of GLP-1 analogs and derivatives that are
`
`protected from the activity of DPP IV is preferred, and the
`8
`administration of Gly -GLP-1(7-36)NH,, Val-GLP-1(7-37) 0H,
`o-methy1-Ala’-GLP-1(7-36)NH2, and Gly-Gln’-GLP-1(7-37)0H,
`or pharmaceutically-acceptable salts thereof,
`is more
`
`preferred.
`
`The use in the present invention of a molecule
`
`Claimed in U.S. Patent No. 5,188,666, herein incorporated
`by reference,
`is preferred.
`Such molecule is selected from
`the group consisting of a peptide having the amino acid
`
`sequence:
`
`10
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`15
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`20
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`25
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`30
`
`MYLANINST. EXHIBIT 1126 PAGE 14
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`MYLAN INST. EXHIBIT 1126 PAGE 14
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`MYLAN INST. EXHIBIT 1126 PAGE 14
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`

`

`WO 00/15224
`
`PCT/US99/21055
`
`-~ 13 -
`
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-
`Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-
`Val-xX
`SEQ ID NO:2
`
`wherein X is selected from the group consisting of Lys and
`Lys-Gly; and a derivative of said peptide, wherein said
`peptide is selected from the group consisting of: a
`pharmaceutically-acceptable acid addition salt of said
`peptide; a pharmaceutically-acceptable carboxylate salt of
`said peptide; a pharmaceutically-acceptable lower
`alkylester of said peptide; and a pharmaceutically-
`acceptable amide of said peptide selected from the group
`consisting of amide,
`lower alkyl amide, and lower dialkyl
`
`10
`
`amide.
`
`Another preferred group of GLP-1 molecules for
`
`15
`
`use in the present invention consists of compounds
`disclosed in U.S. Patent No. 5,512,549, herein incorporated
`
`by reference, having the general formula 1:
`R'-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-
`Leu-Glu-Gly-Gln-Ala-Ala-Xaa-Glu-Phe-Ile-Ala-Trp-
`Leu-Val-Lys-Gly-Arg-R°
`
`| R
`
`’
`SEQ ID NO:3
`and pharmaceutically-acceptable salts thereof, wherein R is
`selected from the group consisting of 4-imidazopropionyl,
`2
`4-imidazoacetyl, or 4-imidazo-a, a dimethyl-acetyl; R is
`selected from the group consisting of C,-C,, unbranched acyl,
`
`20
`
`25
`
`or is absent; R is selected from the group consisting of
`Gly-OH or NH2; and, Xaa is Lys or Arg, may be used in
`
`present invention.
`
`MYLANINST. EXHIBIT 1126 PAGE 15
`
`MYLAN INST. EXHIBIT 1126 PAGE 15
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`

`

`WO00/15224
`
`PCT/US99/21055
`
`- 14 -
`
`More preferred compounds of formula 1 for use in
`,
`:
`.
`2
`the present invention are those in which Xaa is Arg and R
`
`is C,-C,, unbranched acyl.
`
`Highly preferred compounds of formula 1 for use
`in the present invention are those in which Xaa is Arg, R.
`is C,-C,, umbranched acyl, and R is Gly-OH.
`More highly preferred compounds of formula 1 for
`use in the present invention are those in which Xaa is Arg,
`R is C,-C,, unbranched acyl, R is Gly-OH, and R is 4-
`imidazopropionyl .
`The most preferred compound of formula 1 for use
`in the present invention is that in which Xaa is Arg, R is
`Cg unbranched acyl, R is Gly-OH, and R is 4-
`
`10
`
`imidazopropionyl .
`The use of Val-GLP-1(7-37)OH or a
`
`15
`
`pharmaceutically-acceptable salt thereof, as claimed in US
`Patent Number 5,705,483, herein incorporated by reference,
`
`in the present invention is highly preferred.
`Methods for preparing the GLP-1, GLP-1 analogs,
`
`20
`
`or GLP-1 derivatives useful in the present invention are
`
`well-known in the art and are easily within the grasp of
`
`ordinarily skilled protein chemists or biochemists.
`amino acid portion of the active compound used in the
`present invention, or a precursor thereto, can be made
`either by solid-phase synthetic chemistry, purification of
`GLP-1 molecules from natural sources, or
`recombinant DNA
`
`The
`
`25
`
`technology. Routine synthetic organic techniques enable
`the alkylation and acylation of the GLP-1 derivatives.
`The term “GLP-1 related compound” refers to any
`
`30
`
`compound falling within the GLP-1, GLP-1 analog, or GLP-1
`
`derivative definition.
`
`MYLANINST. EXHIBIT 1126 PAGE 16
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`

`

`WO 00/15224
`
`PCT/US99/21055
`
`- 15 -
`
`The unexpected effect of nicotinamide on
`formulation stability was demonstrated by preparing
`formulations of the present invention, and comparing their
`turbidity with the turbidity of controls lacking
`nicotinamide.
`Parenteral formulations of the present invention
`can be prepared using conventional dissolution and mixing
`procedures. One ordinarily skilled in the formulation
`sciences will recognize that the order of addition of a
`medically useful peptide or protein, hydrophobic
`preservative, and nicotinamide could be varied without
`compromising the stability of the resulting formulations.
`In one embodiment of the invention, nicotinamide
`may be added to a purified peptide or protein solution and
`then lyophilized without adversely affecting chemical or
`physical stability while in the solid state. Upon
`reconstitution with a diluent which contains a hydrophobic
`preservative and not containing nicotinamide,
`the protein
`formulation exhibits superior physical stability
`attributable to the presence of nicotinamide in the
`formulation. Conversely, a stable formulation of the
`present invention may be prepared by dissolving a measured
`mass of pure lyophilized protein in water, and then adding
`measured volumes of nicotinamide and hydrophobic
`preservative solutions in quantities sufficient to provide
`the desired concentrations. Optional compounds may also be
`added, such as an isotonicity agent or a pharmaceutically-
`acceptable buffer.
`The pH of the formulation may be
`adjusted using, for example, hydrochloric acid or sodium
`hydroxide solutions. Once prepared,
`the formulations of the
`present invention are generally sterile-filtered prior to
`administration.
`Formulations of the present invention may
`be prepared by many other processes which are readily
`apparent to one of ordinary skill in the art.
`For example,
`the manner and conditions under which the components are
`combined,
`the type of acid or base used to adjust pH, and
`
`10
`
`15
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`20
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`25
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`30
`
`35
`
`MYLANINST. EXHIBIT 1126 PAGE 17
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`

`

`WO 00/15224
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`PCT/US99/21055
`
`- 16 -
`
`the method for sterilizing the formulations may be optimized
`by one of ordinary skill.
`The hydrophobic preservative and nicotinamide used
`in the formulations of the present invention are readily
`available from commercial suppliers in sufficient quality to
`meet regulatory requirements for administration to humans.
`The formulations of the present invention
`optionally may contain other compounds in addition to the
`medically useful peptide or protein, hydrophobic
`preservative, and nicotinamide.
`For example,
`pharmaceutically acceptable solubilizers like Tween 20
`(polyoxyethylene (20) sorbitan monolaurate), Tween 40
`(polyoxyethylene (20) sorbitan monopalmitate), Tween 80
`(polyoxyethylene (20) sorbitan monooleate), Pluronic F68
`(polyoxyethylene polyoxypropylene block copolymers), a