`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`Intematlonal Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6 :
`
`(11) International Publication Number:
`
`WO 00/15224
`
`
`A61K 31/44, 38/00
`
`23 March 2000 (23.03.00) r—‘
`(43) International Publication Date:
`
`
`
`(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).
`
`(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, Fl, 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).
`
`(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, 8.; Eli Lilly and Company, Lilly
`Corporate Center, Indianapolis, IN 46285 (US).
`
`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.
`
`L__________________.J
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`MYLAN INST. EXHIBIT 1126 PAGE 1
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`MYLAN INST. EXHIBIT 1126 PAGE 1
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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.
`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
`TM
`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’lvoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`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
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`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
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`
`
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`WO 00/15224
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`PCT/US99/21055
`
`PROTEIN FORMULATIONS
`
`Field of Invention
`
`The present invention is in the field of peptide
`
`and protein chemistry as it applies to human medicine. In
`
`5
`
`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
`
`10
`
`15
`
`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—1596 (1985)],
`
`20
`
`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)].
`
`25
`
`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
`
`30
`
`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
`
`MYLAN INST. EXHIBIT 1126 PAGE 3
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`PCT/US99/21055
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`described by Jorgensen in U.S. Patent No. 5,382,574.
`
`The
`
`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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`MYLAN INST. EXHIBIT 1126 PAGE 4
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`PCT/US99/21055
`
`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.
`
`Detailed Description and Preferred Embodiments
`
`10
`
`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
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`15
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`20
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`25
`
`30
`
`35
`
`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 C1 to C4 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 to a
`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,
`
`MYLAN INST. EXHIBIT 1126 PAGE 5
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`chlorobutanol, benzoic acid and various mixtures thereof.
`See, e.g.,
`“mLLmumER, K.—H., DEWflDP. BIOL. SmmmARD. 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
`
`5
`
`lo
`
`formula:
`
`| \ /CONH2
`
`N/
`
`15
`
`20
`
`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.
`
`25
`
`30
`
`35
`
`Soluble —— refers to the relative absence of
`aggregated protein that is visually perceivable.
`The degree
`of aggregation of proteins in a 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.
`
`MYLAN INST. EXHIBIT 1126 PAGE 6
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`
`Treating —— as used herein, describes the
`management and care of a patient for the pur
`condition, or disorder and includes the
`combating a disease,
`nt invention to
`administration of a formulation of the prese
`event the onset of the symptoms or complications,
`pr
`or eliminating
`alleviating the symptoms or complications,
`or disorder. Treating, as used
`the disease, condition,
`protein'for
`includes the administration of the
`A cosmetic purpose seeks to control the
`
`herein,
`
`cosmetic purposes.
`ance.
`weight of a mammal to improve bodily appear
`In one embodiment,
`the invention relates to
`ervative,
`formulations containing a hydrophobic pres
`nicotinamide and a medically useful peptid
`es and functional
`which are defined to include native hormon
`(excluding insulin and insulin analogs and leptin
`native cytokines and functional
`and leptin analogs),
`and antibodies and
`analogs, soluble protein vaccines,
`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
`ocytocin, vasopressin, adrenocorticotropin
`epidermal growth factor, platelet—
`hormone and analogs,
`luteinising hormone
`derived growth factor, prolactin,
`growth hormone, growth hormone releasing
`releasing hormone,
`related compounds,
`hormone, somatostatin, glucagon, GLP-l
`IL—2,
`IL—10,
`IL—15,
`interferon—a,B,y, gastrin tetragastrin,
`pentagastrin, urogastrin, secretin, calcitonin, enkaphalins,
`thyrotropin releasing hormone,
`endorphins, angiotensins,
`tumor necrosis factor, nerve growth factor, granulocyte
`colony stimulating factor, granulocyte macrophage colony
`phage colony stimulating factor,
`stimulating factor, macro
`renin, bradykinin, bacitracins, polymixins, colistins,
`tyrocidin, gramicidins.
`
`e or protein.
`
`analogs
`
`invention:
`
`5
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`15
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`20
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`30
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`35
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`MYLAN INST. EXHIBIT 1126 PAGE 7
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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 C6—C20 acylations of the epsilon amino
`group of Lys on the insulin B—chain, especially C14 acylated
`Lys’328 ProB29 human insulin, mammalian growth hormone, growth
`hormone releasing hormone, GLP—l related compounds,
`erythrocyte progenitor hormone (EPO), parathyroid hormone
`and fragments, especially as disclosed in US Pat NOS:
`4,086,196 and 5,208,041, B—lipotropin, fibroblast growth
`factor-8 and analogs, osteoprotegrin—Z 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 or 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
`(a) evolutionarily conserved amino acids and
`
`discussion of
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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.
`Each 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.
`
`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,
`
`MYLAN INST. EXHIBIT 1126 PAGE 10
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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-l" refers to human glucagon-like
`
`10
`
`peptide—1 whose sequences and structures are known in the
`art.
`gee 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—l(7—36)NH2 which will be distinguished
`
`only when necessary.
`The term "GLP—l analog" is defined as a molecule
`
`15
`
`having one or more amino acid substitutions, deletions,
`inversions, or additions compared with GLP—l. Many GLP—l
`
`analogs are known in the art and include,
`
`8
`1(7—34) and GLP—l(7—35), GLP—l(7—36), Val —GLP—1(7—37),
`9
`9
`16
`18
`Gln -GLP—1(7—37) , D-Gln —GLP—l (7—37) , Thr
`-LyS -GLP-l (7—
`
`for example, GLP—
`
`20
`
`37), and Lysm-GLP—l(7—37). Preferred GLP—l 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—l derivative" is defined as a
`
`molecule having the amino acid sequence of GLP—1 or a GLP-l
`
`25
`
`analog, but additionally having chemical modification of
`
`one or more of its amino acid side groups, d—carbon atoms,
`
`terminal amino group, or terminal carboxylic acid group.
`
`A
`
`Chemical modification includes, but is not limited to,
`adding chemical moieties, creating new bonds, and removing
`chemical moieties. Modifications at amino acid side groups
`
`30
`
`include, without limitation, acylation of lysine e—amino
`groups, N—alkylation of arginine, histidine, or lysine,
`alkylation of glutamic or aspartic carboxylic acid groups,
`
`MYLAN INST. EXHIBIT 1126 PAGE 11
`
`MYLAN INST. EXHIBIT 1126 PAGE 11
`
`MYLAN INST. EXHIBIT 1126 PAGE 11
`
`
`
`WO 00/15224
`
`PCT/US99/21055
`
`-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.
`
`10
`
`The a-carbon of an amino acid may be mono— or di—
`
`methylated.
`
`The term "GLP—l molecule" means GLP—l, GLP-l
`
`analog, or GLP—l derivative.
`
`Another preferred group of GLP—1 analogs is
`
`15
`
`defined by the formula:
`
`Rl—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
`
`20
`
`and the pharmaceutically—acceptable salts thereof, wherein:
`
`R1 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
`
`25
`
`the group consisting of Ala, Gly, Val, Thr, Ile, and alpha—
`
`Y is selected from the group consisting of
`methyl-Ala;
`Glu, Gln, Ala, Thr, Ser, and Gly;
`Z is 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;
`
`30
`
`providing that when R1 is His, X is Ala, Y is Glu, and Z is
`
`Glu,
`
`R.2 must be NH2_
`
`MYLAN INST. EXHIBIT 1126 PAGE 12
`
`MYLAN INST. EXHIBIT 1126 PAGE 12
`
`MYLAN INST. EXHIBIT 1126 PAGE 12
`
`
`
`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,
`
`10
`
`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,
`
`15
`
`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;
`
`(c) substitution of at least one of:
`
`tyrosine for
`
`20
`
`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
`
`25
`
`(d) substitution of at least one of: glycine, serine,
`
`or cysteine for alanine at position 8; aspartic acid,
`
`glycine, serine, cysteine,
`
`threonine, asparagine,
`
`glutamine,
`
`tyrosine, alanine, valine,
`
`isoleucine,
`
`leucine,
`
`methionine, or phenylalanine for glutamic acid at position
`
`30
`
`9; serine, cysteine,
`
`threonine, asparagine, glutamine,
`
`tyrosine, alanine, valine,
`
`isoleucine,
`
`leucine, methionine,
`
`MYLAN INST. EXHIBIT 1126 PAGE 13
`
`MYLAN INST. EXHIBIT 1126 PAGE 13
`
`MYLAN INST. EXHIBIT 1126 PAGE 13
`
`
`
`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,
`
`5
`
`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),
`
`(d), and (e),
`
`the substituted amino acids can
`
`optionally be in the D—form and the amino acids substituted
`
`10
`
`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-
`
`15
`
`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
`
`20
`
`inactivation of administered GLP-l,
`
`[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—l(7—36)NH2, Vale—GLP—1(7—37)OH,
`
`25
`
`oc—methyl—AlaB—GLP—l(7—36)NH2, and GlyB—Gln21-GLP—1(7—37)OH,
`
`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
`
`30
`
`by reference,
`
`is preferred.
`
`Such molecule is selected from
`
`the group consisting of a peptide having the amino acid
`
`sequence :
`
`MYLAN INST. EXHIBIT 1126 PAGE 14
`
`MYLAN INST. EXHIBIT 1126 PAGE 14
`
`MYLAN INST. EXHIBIT 1126 PAGE 14
`
`
`
`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—X
`
`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
`
`10
`
`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
`
`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:
`
`I6—Ala—Glu-Gly—Thr—Phe—Thr—Ser—Asp-Val—Ser—Ser—Tyr-
`
`Leu—Glu—Gly-Gln—Ala—Ala—Xaa—Glu-Phe—Ile—Ala—Trp—
`
`20
`
`Leu—Val-Lys—Gly—Arg—R3
`
`SEQ ID NO : 3
`
`I R
`
`2
`
`and pharmaceutically—acceptable salts thereof, wherein R1 is
`
`selected from the group consisting of 4-imidazopropionyl,
`2
`4—imidazoacetyl, or 4-imidazo—a, a dimethyl—acetyl; R is
`
`25
`
`selected from the group consisting of C6—C10 unbranched acyl,
`3
`.
`.
`.
`or is absent; R is selected from the group con31st1ng of
`
`Gly—OH or NH2; and, Xaa is Lys or Arg, may be used in
`
`present invention.
`
`MYLAN INST. EXHIBIT 1126 PAGE 15
`
`MYLAN INST. EXHIBIT 1126 PAGE 15
`
`MYLAN INST. EXHIBIT 1126 PAGE 15
`
`
`
`WO 00/15224
`
`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 C6—C10 unbranched acyl.
`
`Highly preferred compounds of formula 1 for use
`.
`_
`.
`.
`.
`2
`in the present invention are those in which Xaa is Arg, R
`3
`is C6—Clo unbranched 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,
`
`R2 is C6—C10 unbranched acyl, R3 is Gly—OH, and R1 is 4—
`
`10
`
`imidazopropionyl.
`
`The most preferred compound of formula 1 for use
`
`in the present invention is that in which Xaa is Arg, R2 is
`
`C3 unbranched acyl, R3 is Gly—OH, and R1 is 4—
`
`imidazopropionyl.
`
`15
`
`The use of Vale—GLP—l(7—37)OH or a
`
`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—l, GLP—l analogs,
`
`20
`
`or GLP—l 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.
`
`The
`
`amino acid portion of the active compound used in the
`
`present invention, or a precursor thereto, can be made
`
`25
`
`either by solid—phase synthetic chemistry, purification of
`
`GLP—1 molecules from natural sources, or
`
`recombinant DNA
`
`technology. Routine synthetic organic techniques enable
`
`the alkylation and acylation of the GLP—l derivatives.
`
`The term “GLP—l related compound” refers to any
`
`30
`
`compound falling within the GLP—l, GLP—l analog, or GLP-l
`
`derivative definition.
`
`MYLAN INST. EXHIBIT 1126 PAGE 16
`
`MYLAN INST. EXHIBIT 1126 PAGE 16
`
`MYLAN INST. EXHIBIT 1126 PAGE 16
`
`
`
`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
`
`10
`
`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
`
`15
`
`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
`
`20
`
`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
`
`25
`
`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
`
`3O
`
`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,
`
`35
`
`the manner and conditions under which the components are
`
`combined,
`
`the type of acid or base used to adjust pH, and
`
`MYLAN INST. EXHIBIT 1126 PAGE 17
`
`MYLAN INST. EXHIBIT 1126 PAGE 17
`
`MYLAN INST. EXHIBIT 1126 PAGE 17
`
`
`
`WO 00/15224
`
`PCT/US99/21055
`
`-l6—
`
`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
`
`10
`
`preservative, and nicotinamide.
`
`For example,
`
`pharmaceutically acceptable solubilizers like Tween 20
`
`(polyoxye