PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 7;
`A61K 38/26, 38/28, 47/10, 47/26, A61P
`3/10 / (A61K 38/28, 38:26)
`
`(11) International Publication Number:
`
`WO 00/37098
`
`(43) International Publication Date:
`
`29 June 2000 (29.06.00)
`
`(21) International Application Number:
`
`PCT/US99/30395
`
`(22) International Filing Date:
`
`21 December 1999 (21.12.99)
`
`(30) Priority Data:
`60/113,499
`
`22 December 1998 (22.12.98)
`
`US
`
`(71) Applicant (for all designated States except US): ELI LILLY
`AND COMPANY[US/US]; Lilly Corporate Center, Indi-
`anapolis, IN 46285 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): BRADER, Mark, L.
`[NZ/US]; 6465 North Park Avenue, Indianapolis, IN 46220
`(US). PEKAR,Allen, H. [US/US]; 5354 North Park Avenue,
`Indianapolis, IN 46220 (US).
`
`(74) Agent: MACIAK,Ronald, S.; Eli Lilly and Company, Lilly
`Corporate Center, Drop Code1501, 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.
`
`(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, HR, HU,ID,IL, IN, IS, JP, KE, KG, KP,
`KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG,
`MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE,
`SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ,
`VN, YU, ZA, ZW, ARIPOpatent (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).
`
`modifier, and that has a pH between about 8.2 to about 8.8.
`
`(54) Title: SHELF-STABLE FORMULATION OF GLUCAGON-LIKE PEPTIDE-1
`
`(57) Abstract
`
`Glucagon-like peptide~1 (GLP-1) has been shown to be useful in the treatment of diabetes. The invention encompasses a shelf
`stable formulation that comprises a therapeutically effective amount of GLP-1, a pharmaceutically acceptable preservative, and a tonicity
`
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`MPI EXHIBIT 1092 PAGE 1
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`

`

`Zimbabwe
`
`Codes used to identify States party to the PCT onthe front pages of pamphlets publishing international applications under the PCT.
`Albania
`ES
`LS
`Lesotho
`SI
`Armenia
`FI
`LT
`SK
`Lithuania
`Austria
`FR
`LU
`SN
`Luxembourg
`Australia
`GA
`LV
`Latvia
`SZ
`GB
`MC
`Monaco
`TD
`Azerbaijan
`GE
`MD
`TG
`Bosnia and Herzegovina
`Republic of Moldova
`Barbados
`GH
`MG
`TJ
`Madagascar
`MK
`GN
`Belgium
`The former Yugoslav
`Burkina Faso
`GR
`Republic of Macedonia
`HU
`Mali
`Bulgaria
`Benin
`TE
`Mongolia
`Brazil
`IL
`Mauritania
`Belarus
`IS
`Malawi
`Canada
`IT
`Mexico
`JP
`Central African Republic
`Niger
`KE
`Netherlands
`Congo
`Switzerland
`KG
`Norway
`Céte dIvoire
`KP
`New Zealand
`Cameroon
`Poland
`China
`Portugal
`Cuba
`Romania
`Russian Federation
`Czech Republic
`Sudan
`Germany
`Denmark
`Sweden
`Estonia
`Singapore
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`
`™T
`
`R
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`Zw
`
`MPI EXHIBIT 1092 PAGE 2
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Treland
`Israel
`Iceland
`Ttaly
`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
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`8G
`
`MPI EXHIBIT 1092 PAGE 2
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`WO 00/37098
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`PCT/US99/30395
`
`SHELF-STABLE FORMULATION OF GLUCAGON-LIKE PEPTIDE-1
`
`Backgroundofthe Invention
`
`Glucagon-like peptide-1 (7-37)-OH (GLP-1) is a 31 amino acid hormonethatis
`produced by post-translational processing of the preglucagon gene productin the brain,
`stomach, intestine, and pancreas. The main physiological function of GLP-1 is to regulate
`insulin secretion in response to glucose, andthusit has the ability to normalize blood
`glucose levels. As such, there has been interest in GLP-1, its analogs and derivatives as
`potential therapeutic agents for the treatmentof diabetes. A particular advantageto the use
`of GLP-1 over other drugs in the treatment ofdiabetes is that administration of GLP-1 at
`doses in the 1-5 nmole range exhibit few adverse side effects, such as hypoglycemia.
`Unexpectedly, GLP-1 also has been shownto workin patients that have secondary failure
`to sulfonylurea drugs, the most commondrug type for the treatmentoftype II diabetes.
`GLP-1 also is a potent inhibitor of gastric acid secretion and gastric emptying.
`In general, effective therapeutic administration of peptides can be problematic since
`peptides often are degradedin the gastrointestinal tract by various peptidases. Additionally,
`certain peptide treatmentprotocols require either continuous or repeated administration of
`the peptide agent over an extended period of time. Repeated injections cause both
`inconvenience and discomfort to the user. Thus, chronic use ofthe peptide agent, which
`would be required for patients afflicted with diabetes, would result in inconvenience and
`
`discomfort to the user.
`
`The long-term stability of peptides, particularly GLP-1, as components ofa
`pharmaceutical composition for administration to mammals, is questionable. Such a lack of
`stability adversely affects bioavailability. In fact, when stored at low temperatures of4° C,
`by-products of GLP-1(7-37) have been found as early as eleven monthsafter sample
`Preparation (see Mojsov, Int. J. Peptide Protein Res., Vol. 40, pages 333-343 (1992)).
`Additionally, the biological half-life of GLP-1 molecules, particularly those molecules
`affected by the activity of dipeptidyl-peptidase IV (DPPIV), is quite short. For example,
`
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`the biological half-life of GLP-1(7-37) is only 3 to 5 minutes (see U.S. Patent No.
`5,118,666), which is further augmented byits rapid absorption following parenteral
`administration to a mammal.
`
`Another factor decreasing the bioavailability of GLP-1 is the solubility of GLP-1
`when incorporated into an aqueous solution. The solubility of GLP-1 is highly dependent
`on the environment, such as the choice of buffering system, and the treatmentthat the
`peptide has undergone. For example, conversionof a peptide into its salt form plays a role
`in its solubility. In this regard, synthetic GLP-1 is highly soluble in neutral phosphate
`buffered saline. Becausethe solubility of the peptideis high in such aqueoussolutions,
`slow release of the peptide can bedifficult to attain unless the peptideis incorporated into a
`system for slow release.
`Stable formulations of therapeutic agents are particularly required for use in delivery
`devices that expose these agents to elevated temperatures and/or mechanical stress. For
`example, stable GLP-1 formulations are required for use in continuous infusion systems
`and pen delivery devices. Current formulations provide only limited stability in these types
`of delivery devices.
`In continuousinfusion systems,a fluid containing a therapeutic agent is pumped
`from a reservoir, usually to a subcutaneous, intravenous, or intraperitoneal depot. The
`reservoir, which mustberefilled periodically, is attached to the patient’s body,or is
`implanted in the patient’s body.
`In either case, the patient’s body heat and body motion,
`and turbulencein the tubing and pump impart a relatively high amount of thermo-
`mechanical energy to the formulation.
`In the interest of minimizing the frequency with
`which the reservoir is refilled, and of minimizing the size of the reservoir, formulations
`havingarelatively high concentration of the therapeutic agent are advantageous.
`Injector pens also have been developedto allow diabetic patients to accurately
`measure and administer controlled doses of insulinotropic agents. Generally, these pens are
`secured onto a cartridge having a particular quantity of liquid medication sealed therein.
`The cartridge includes a plunger and a mechanism for advancing the plunger in the
`cartridge in such a mannerto dispense the medication.
`Injector pens may be reusable or
`disposable. In reusable pens, a user can change a spent cartridge andreset the leadscrew of
`the pen backtoits initial position. In a disposable pen, the cartridge is permanently
`
`-2-
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`embodiment, the GLP-1 molecule of the formulation is a derivative of GLP-1 and is
`
`selected from the group consisting of a peptide having the amino acidsequence:
`NH,-His’-Ala-Glu-Gly'°-Thr-Phe-Thr-Ser-Asp'*-Val-Ser-Ser-Tyr-Leu”-Glu-Gly-Gin-Ala-
`Ala®-Lys-Glu-Phe-Ile-Ala®’-Trp-Leu-Val-X
`(SEQ ID NO:3)
`and a pharmaceutically-acceptable salt thereof, wherein X is selected from the group
`consisting of Lys and Lys-Gly; a pharmaceutically-acceptable loweralkylester of the peptide;
`and a pharmaceutically-acceptable amide ofthe peptide selected from the group consisting of
`amide, lower alkyl amide, and lower dialkyl amide.
`In another preferred embodiment, the
`formulation also comprises a long-acting insulin agent.
`
`The present invention also provides a method of enhancing the expressionof insulin
`in a mammalian pancreatic B-typeislet cell in need of such enhancement, comprising
`administering to the cell, an effective amountof a shelf-stable pharmaceutical formulation,
`wherein the formulation comprises a therapeutically effective amount of a GLP-1 molecule,
`a pharmaceutically acceptable preservative, and a tonicity modifier, and wherein the
`formulation has a pHthat is about 8.2 to about 8.8.
`In a preferred embodiment, the
`formulation used in the therapeutic method comprises a buffer, such as TRIS.
`In another
`preferred embodiment, the formulation used in the therapeutic method further comprises a
`surfactant, such as Brij-35.
`In an additional preferred embodiment, the GLP-1 molecule of
`the formulation thus administered is an analog of GLP-1 andis selected from the group
`consisting of a peptide having the amino acid sequence:
`R,-X-Glu-Gly°-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu””-Y-Gly-Gin-Ala-Ala®-
`Lys-Z-Phe-Ile-Ala*-Trp-Leu-Val-Lys-Gly*-Arg-R,
`(SEQ ID NO:2)
`and a pharmaceutically-acceptable salt thereof, wherein R,
`is His or desamino-histidine, X
`is Ala, Gly or Val, Y is Glu or Gln, Z is Glu or Gin and R, is Gly-OH.
`In an especially
`preferred embodiment, the GLP-1 molecule administered is according to SEQ ID NO:2,
`wherein R,is L-histidine, X is Val, Y is Glu, Z is Glu, and R, is Gly-OH. In an alternative
`preferred embodiment, the GLP-1 molecule administered is a derivative of GLP-1 and is
`selectedfrom the group consisting of a peptide having the amino acid sequence:
`NH,-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:3)
`and a pharmaceutically-acceptable salt thereof, wherein X is selected from the group
`consisting of Lys and Lys-Gly; a pharmaceutically-acceptable lower alkylester of the peptide;
`
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`and a pharmaceutically-acceptable amide ofthe peptide selected from the group consisting of
`amide, lower alkyl amide, and lower dialkyl amide.
`
`The present invention also provides a methodof treating diabetes comprising
`administering to a patient in need of such treatmentan effective amountofa shelf-stable
`
`pharmaceutical formulation, wherein the formulation comprises a therapeutically effective
`amount of a GLP-1 molecule, a pharmaceutically acceptable preservative, and a tonicity
`modifier, and wherein the formulation has a pH that is about 8.2 to about 8.8. Ina
`
`preferred embodiment, the formulation used in the therapeutic method comprises a buffer,
`such as TRIS.
`In another preferred embodiment, the formulation used in the therapeutic
`method further comprises a surfactant, such as Brij-35.
`In an additional preferred
`
`embodiment, the GLP-1 moleculeof the formulation thus administered is an analog of GLP-
`1 and is selected from the group consisting of a peptide having the amino acid sequence:
`R,-X-Glu-Gly°-Thr-Phe-Thr-Ser-Asp’*-Val-Ser-Ser-Tyr-Leu”-Y-Gly-Gin-Ala-Ala™-
`Lys-Z-Phe-Ile-Ala”’-Trp-Leu-Val-Lys-Gly**-Arg-R,
`(SEQ ID NO:2)
`and a pharmaceutically-acceptable salt thereof, wherein R,
`is His or desamino-histidine, X
`is Ala, Gly or Val, Y is Glu or Gln,Z is Glu or Gln and R, is Gly-OH.
`In an especially
`preferred embodiment, the GLP-1 molecule administered is according to SEQ ID NO: 2,
`
`wherein R, is L-histidine, X is Val, Y is Glu, Z is Glu, and R,is Gly-OH. In an alternative
`
`preferred embodiment, the GLP-1 molecule administered is a derivative of GLP-1 andis
`
`selected from the group consisting of a peptide having the aminoacid sequence:
`NH,-His’-Ala-Glu-Gly*-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gin-
`Ala-Ala®-Lys-Glu-Phe-Ile-Ala*®-Trp-Leu-Val-X
`-
`(SEQ ID NO:3)
`and a pharmaceutically-acceptable salt thereof, wherein X is selected from the group
`consisting of Lys and Lys-Gly; a pharmaceutically-acceptable lower alkylester of the peptide;
`and a pharmaceutically-acceptable amide of the peptide selected from the group consisting of
`amide, lower alkyl amide, and lower dialkyl amide.
`
`Anadditional embodiment encompasses a method of providing meal-time glycemic
`control and basal glycemic control with a single injection comprising administering to a
`patient in need thereof an effective amountof a shelf-stable pharmaceutical formulation,
`
`wherein the formulation comprisesa therapeutically effective amount of a GLP-1 molecule,
`a long acting insulin agent, a pharmaceutically acceptable preservative, and a tonicity
`modifier, wherein said formulation has a pH that is about 8.2 to about 8.8.
`
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`captured in the pen which is disposed ofafter the contents of the cartridge have been
`exhausted.
`
`With the development of GLP-1 as well as analogs and derivatives thereof for the
`
`treatmentof diabetes, there exists a need to improve treatment regimes that can balance
`chemical and physical stability with chronic use requirements of diabetic patients.
`
`Summaryof the Invention
`
`In order to overcome the problems of chemical and physical stability of GLP-1
`formulations, the present inventors have developed a shelf-stable formulation of GLP-1. In
`particular, the inventors have discovered that when certain physiologically tolerated buffers
`are used in formulations of GLP-1, the physical stability of such formulationsis
`
`unexpectedly and considerably greater than when compared to GLP-1 formulations made
`with a phosphate buffer.
`In addition, maintaining the pH in a rangeof about 8.2 to about
`8.8 unexpectedly improves the chemicalstability of the formulations. The shelf-stable
`formulation of GLP-1 of the invention comprises a therapeutically effective amount of a
`GLP-1 molecule, a pharmaceutically acceptable preservative, and a tonicity modifier,
`wherein the pH of said formulation is maintained in the range from about 8.2 to about8.8.
`In accordance with the chemical and physical stability needs of GLP-1 formulations,
`the present invention provides a shelf-stable pharmaceutical formulation comprising a
`therapeutically effective amount of a GLP-1 molecule, a pharmaceutically acceptable
`preservative, and a tonicity modifier, wherein the formulation has a pH that is about 8.2 to
`about 8.8.
`In a preferred embodiment, the formulation further includes a buffer, such as
`TRIS.
`In another preferred embodiment, the formulation comprises a surfactant, such as
`Brij-35.
`In an additional preferred embodiment, the GLP-1 molecule of the formulation is
`an analog of GLP-1 andis selected from the groupconsisting ofa peptide having the amino
`acid sequence:
`R,-X-Glu-Gly*-Thr-Phe-Thr-Ser-Asp'’-Val-Ser-Ser-Tyr-Leu”-Y-Gly-Gin-Ala-Ala®-
`Lys-Z-Phe-Ile-Ala”-Trp-Leu-Val-Lys-Gly**-Arg-R,
`(SEQ ID NO:2)
`and a pharmaceutically-acceptable salt thereof, wherein R, is His or desamino-histidine, X
`is Ala, Gly or Val, Y is Glu or Gin, Z is Glu or Gln and R, is Gly-OH. Inan especially
`preferred embodiement, the GLP-1 molecule is according to SEQ ID NO: 2, wherein R,is L-
`histidine, X is Val, Y is Glu, Z is Glu, and R, is Gly-OH.
`In an alternative preferred
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`Detailed Description of the Preferred Embodiments
`The present invention provides a shelf-stable formulation of GLP-1, GLP-1 analogs,
`and GLP-1 derivatives. Becauseit is knownthat there are problems with long term
`stability of GLP-1 as a componentin a pharmaceutical composition, the present inventors
`developed a pharmaceutical formulation whichstabilizes GLP-1, its derivatives and
`analogs. This developmentled to the shelf-stable GLP-1 formulations ofthe invention.
`In another embodiment,the present invention encompasses a GLP-1 formulation
`that also comprises a long acting diabetic agent.
`It has long been a goalof insulin therapy
`to mimic the pattern of endogenousinsulin secretion in normal individuals. The daily
`physiological demand for insulin fluctuates and can be separated into two phases: (a) the
`absorptive phase requiring a pulse ofinsulin to dispose ofthe meal-related blood glucose
`surge, and (b) the post-absorptive phase requiring a sustained delivery of insulin to regulate
`hepatic glucose output for maintaining optimal fasting blood glucose. Accordingly,
`effective therapy for people with diabetes generally involves the combineduseof two types
`of exogenousinsulin formulations: a fast-acting mealtimeinsulin provided by bolus
`injections and a long-acting, so-called, basal insulin, administered by injection once or
`twice daily to control blood glucose levels between meals. Examples of commercialbasal
`insulin preparations include NPH (Neutral Protamine Hagedorn)insulin, protamine zinc
`insulin (PZI), and ultralente (UL).
`The term “stability” is used to mean chemical as well as physical stability. Physical
`stability refers to properties such as protein aggregation, which can be measured bya
`sample’s attenuation of light. The measurementrelates to the turbidity of a formulation.
`Turbidity is produced by aggregation or precipitation of proteins or complexes in the
`formulation and is indicative of decreased stability of a solution formulation. The more
`turbid a protein preparation, the less stable the preparationis. Stability also refers to the
`chemicalstability of the formulation such as the propensity of the proteins to form high
`order polymers whichis indicative of decreased stability.
`One factor that plays a role in the stability of GLP-1 formulations is the maintenance
`of pH at a prescribed level. Specifically, the present inventors have found that achieving
`and maintaining the pH of the formulation at about 8.2 to about8.8 is advantageous.
`Typical peptide formulations have a more neutral pH of7 to about 7.8 or an acidic pH.
`Furthermore, a composition containing a GLP-1 molecule that has a pH in the range of
`
`-6-
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`about 6.8 to about 7.5 exhibits less physical stability than a composition of a GLP-1
`molecule containing a preservative and having a pH in the range of about 8.2 to about 8.8.
`A preserved formulation which has a pH ofless than about 8.0 tends to exhibit turbidity, a
`telltale sign of decreased physicalstability of the peptide formulation. Conversely, a
`formulation which has a pH greater than about 8.8 tends to have decreased chemical
`stability.
`Therefore, the invention contemplates GLP-1 formulations having a pH range of
`about 8.2 to about 8.8, which preserves optimal chemical and physical stability of the GLP-
`I molecule. A particularly preferred range for the inventive GLP-1 formulations is about
`8.3 to about 8.6, and a mostparticularly preferred pH range is about 8.4 to about 8.5. As
`used in this specification with respect to pH, the term “about” means plus or minus 0.1 pH
`units. Thus, a pH of “about 8.5” denotes a PH of 8.4 to 8.6.
`GLP-1 molecules themselves exhibit a buffering capacity. However, to maintain the
`pH of the composition for long term storage and stability, it is preferable to add a buffer.
`The choice of buffer affects the chemical and physical stability of the formulation
`because it influences pH. There are very few pharmaceutically acceptable buffers in the
`alkaline range. Phosphate buffers, whichare typically used in peptide pharmaceutical
`formulations, cannot maintain a pH rangeof 8.2 to about 8.8. However, the present
`inventors have discovered that certain other amine-containing buffers are capable of
`imparting chemical as well as physical stability to formulations of GLP-1 molecules.
`The buffers used in the present invention preferably provide a buffering capacity in
`the range of about 8.2 to about 8.8. The buffers which are used may be tromethane
`(TRIS), and amino-acid based buffers such as lysine and hydroxy-lysine. Although any
`non-phosphate buffer which has a buffering capacity in the range of about 8.2 to about 8.8
`may be used, TRISis the preferred buffer for the formulations of the present invention.
`The term “TRIS” refersto 2-amino-2-hydroxymethy1-1,3-propanediol ( also knownin the
`art as tromethane, trimethylol aminomethane or tris(hydroxymethyl) aminomethane), and to
`any pharmacologically acceptable salt thereof. The free base and the hydrochloride form
`are two common forms of TRIS. TRIS is one of the few buffers which is capable of
`maintaining the pHat the alkaline level desired, thereby stabilizing the formulation.
`A second factor that playsa role in the stability of GLP-1 formulationsis the
`concentration of the GLP-1 moleculethat is used in the inventive formulation. The present
`
`_
`
`-7-
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`inventors determined that a concentration of about 0.30 to about 0.65 mg/ml of the GLP-1
`molecule was stable in the inventive formulations. However, a concentration which was
`about equal to or greater than 1 mg/ml was unstable. This stability was evidenced by the
`developmentof turbidity in the formulation. A particularly stable formulation includes
`about 0.5 mg/ml of a GLP-1 molecule.
`Anadditional factor which contributes to the overall stability of the GLP-1
`formulations of the present invention is the choice of preservative. The preservative is an
`essential component in the formulation because it enables multiple uses of the formulation.
`Whileit is typical that most preservatives would be capable of stabilizing a pharmaceutical
`formulation, some pharmaceutically acceptable preservatives act to promote physical
`instability of the formulation. The present inventors have found that a phenolic
`preservative is preferred. Specifically, the preservative may be m-cresol, phenol, benzyl
`alcohol, or methyl paraben and is present in an amount from about 2 mg/ml to about 6
`mg/ml, Ultimately, the concentration of preservative necessary for effective preservation
`depends on the preservative used, the pH ofthe formulation, andwhether substancesthat
`bind or sequester the preservative are also present. Preferably, m-cresol is used in the
`|
`formulations as a preservative.
`
`While a buffer and a preservative are most preferably included in the formulation,
`other additional excipients may be included, suchas a tonicity modifier and/or a surfactant
`as well as distilled water for injections.
`The tonicity modifier may be included to make the formulation approximately
`isotonic with bodily fluid depending on the modeof administration. The concentration of
`the tonicity modifier is in accordance with the known concentration of a tonicity modifier in
`a peptide formulation. A preferable tonicity modifier used in the present inventionis
`glycerol.
`
`A surfactant, which may be included in the formulation of the present invention, can
`be cationic, anionic, or non-ionic. A preferable class of surfactants is polyoxyethylene
`ethers. A preferred surfactant useful in the present invention is Brij-35, a polyoxyethylene
`23 lauryl ether, available from ICI United States, Inc.
`The present invention contemplates use of not only natural GLP-1, but also analogs,
`derivatives and salts of GLP-1. As used herein, the term “a GLP-1 molecule”refers (1) to
`the naturally-occurring GLP-1, which is GLP-1 (7-37)-OH; (2) GLP-1(7-36)NH,, as well
`
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`as (3) GLP-1 (7-37); (4) natural and synthetic functional GLP-1 analogs; (5) derivatives of
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`GLP-1 and (6) salts of any of the aforementioned molecules.
`
`A “GLP-1 analog” is defined as a molecule having one or more amino acid
`
`substitutions, deletions, inversions, or additions relative to GLP-1(7-37) and may includethe
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`D-amino acid forms. Numerous GLP-1 analogs are knownin theart and include, but are not
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`limited to, GLP-1(7-34), GLP-1(7-35), GLP-1(7-36)NH,, Gin’-GLP-1(7-37), d-Gln’-GLP-
`1(7-37), Thr'*-Lys'*-GLP-1(7-37), and Lys'*-GLP-1(7-37), Gly*-GLP-1(7-36)NH,, Gly*-GLP-
`1(7-37)OH, Val*-GLP-1(7-37)OH, Met’-GLP-1(7-37)OH, acetyl-Lys°-GLP-1(7-37), Thr?-
`GLP-1(7-37), D-Thr’-GLP-1(7-37), Asn’-GLP-1(7-37), D-Asn°-GLP-1(7-37), Ser?-Arg?-
`Arg’*-Gin®-GLP-1(7-37), Arg”-GLP-1(7-37), Arg’*~-GLP-1(7-37), o-methyl-Ala®-GLP-1(7-
`36)NH,, and Gly®-Gin”'-GLP-1(7-37)OH,andthelike.
`|
`Other GLP-1 analogs consistent with the present invention are described by the
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`formula:
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`R,-X-Glu-Gly'°-Thr-Phe-Thr-Ser-Asp°-Val-Ser-Ser-Tyr-Leu””-Y-Gly-Gin-Ala-Ala”-
`Lys-Z-Phe-Ile-Ala*”-Trp-Leu-Val-Lys-Gly®-Arg-R,
`(SEQ ID NO:2)
`
`wherein: R, is selected from the group consisting of L-histidine, D-histidine, desamino-
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`histidine, 2-amino-histidine, beta-hydroxy-histidine, homohistidine, alpha-fluoromethy]-
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`histidine, and alpha-methyl-histidine; X is selected from the group consisting of Ala, Gly,
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`Val, Thr,Ile, and alpha-methyl-Ala; Y is selected from the group consisting of Glu, Gin,
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`Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu, Gln, Ala, Thr, Ser,
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`and Gly; and R,is selected from the group consisting of NH,, and Gly-OH.
`GLP-1 analogs also have been described in WO 91/11457, and include GLP-1(7-34),
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`GLP-1(7-35), GLP-1(7-36), or GLP-1(7-37), or the amide form thereof, and
`| pharmaceutically-acceptable salts thereof, having at least one modification selected from the
`group consisting of:
`,
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`(a) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine,tyrosine,
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`alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine, or D-lysine for
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`lysine at position 26 and/or position 34; or substitution of glycine, serine, cysteine, threonine,
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`asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,
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`phenylalanine, lysine, or a D-arginine for arginineat position 36;
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`(b) substitution of an oxidation-resistant amino acid for tryptophan atposition 31;
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`(c) substitution of at least one of: tyrosine for 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 glutaminefor
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`lysine at position 26; and
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`(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 9;
`serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine,
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`leucine, methionine, or phenylalanine for glycine at position 10; and glutamic acid for
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`aspartic acid at position 15; and
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`(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;
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`wherein, in the substitutions described in (a), (b), (d), and (e), the substituted amino acids can
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`optionally be in the D-form and the aminoacids substituted at position 7 can optionally be in
`the N-acylated or N-alkylated form.
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`Preferred GLP-1 molecules used in the present inventive formulation also include
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`analogs of GLP-1 (7-37)NH, and GLP-1 (7-37) in which one or more amino acids which are
`
`not present in the original sequence are added or deleted, and derivatives thereof.
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`Specifically, His and desamino-histidine are preferred for R,. Ala, Gly and Val are preferred
`at the “X” position. Also, Glu and Ginare preferred for at the “Y” position. Glu and Gin
`
`are preferred at the “Z” position and Gly-OHis preferred for R,.
`A particularly preferred GLP-1 analog is known as Val(8)GLP-1 (V8GLP-1) and has
`a formula according to SEQ ID NO:2, wherein R,is L-histidine, X is Val, Y is Glu, Z is Glu
`
`and R, is Gly-OH.
`
`A "GLP-1 derivative" is defined as a molecule having the aminoacid sequence of
`GLP-1(7-37) or of a GLP-1 analog, but additionally comprises chemical modification of one
`
`or more ofits aminoacid side groups, a-carbon atoms, terminal amino group, or terminal
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`carboxylic acid group. A chemical modification includes, butis not limited to, adding
`chemical moieties, creating new bonds, and removing chemical moieties. Modifications at
`amino acid side groups include, withoutlimitation, acylation of lysine s-amino groups, N-
`alkylation of arginine,histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid
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`groups, and deamidution 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 loweralkyl] ester modifications. Lower alkyl is
`C,-C, alkyl. Furthermore, one or more side groups, or terminal groups, maybeprotected by
`protective groups knownto the ordinarily-skilled protein chemist. The a-carbon of an amino
`acid may. be mono- or dimethylated.
`Other GLP-1 derivatives include molecules which are selected from the group
`consisting of a peptide having the amino acid sequence:
`
`NH,-His’-Ala-Glu-Gly”°-Thr-Phe-Thr-Ser-Asp'*-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
`Gin-Ala-Ala”-Lys-Glu-Phe-Ile-Ala”-Trp-Leu-Val-X
`(SEQ ID NO:3)
`and pharmaceutically-acceptable salts thereof, 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 lower alkylester of said peptide; and a
`pharmaceutically-acceptable amideofsaid peptide, selected from the group consisting of amide,
`lower alkyl amide, and lowerdialkyl amide.
`Yet other GLP-1 derivatives appropriate for use in the present invention include
`compounds claimed in U.S. Patent No. 5,512,549 described by the formula:
`R'-Ala-Glu-Gly'°-Thr-Phe-Thr-Ser-Asp'°-Val-Ser-Ser-Tyr-Lew2°-Glu-Gly-Gln-Ala-Ala-Xaa-
`SepiemanVASNe
`(SEQIDNO:3)
`wherein R'is selected from the group consisting of4-imidazopropionyl, 4-imidazoacetyl, or
`4-imidazo-a,, « dimethyl-acetyl; R? is selected from the group consisting of C,-C,,
`unbranchedacyl, or is absent; R? is selected from the group consisting of Gly-OH or NH,;
`and, Xaa is Lys or Arg, may be usedin present invention.
`“DPP-IV protected GLPs” refers to GLP-1 analogs which areresistant to the action of
`DPP-IV. These include analogs having a modified or D amino acid residue in position 8 and
`includes biosynthetic GLP-1 analogs having Gly, Val, Thr, Met, Ser, Cys, or Aspin position
`8. Other DPP-IV protected GLPs include desamino His’ derivatives.
`“GLP-1 peptide analogs” are defined as GLP-1 analogs or derivatives which exclude
`acylated forms.
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`2
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`“Biosynthetic GLP-1 analogs” are defined as any GLP-1 analogsor derivatives which
`contain only naturally occurring aminoacid residues and are thus capable of being expressed
`by living cells, including recombinant cells and organisms.
`Methods for preparing the GLP-1 molecules which are incorporated into the shelf
`stable formulations of the invention are well known to the skilled artisan.
`In one method,
`GLP-1 molecules are prepared by well-known methods of peptide synthesis, such as those
`described in Merrifield, (Chem. Soc. Vol. 85, page 2149, 1962).
`It is also contemplate