PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 7 :
`
`(11) International Publication Number:
`
`WO 00/37098
`
`A61K 38/26, 38/28, 47/10, 47/26, A61P
`3/10 // (A61K 38/28, 38:26)
`
`(43) International Publication Date:
`
`29 June 2000 (29.06.00)
`
`(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, TI‘, TZ, 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.
`
`(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.
`[NZJUS]; 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, 8.; Eli Lilly and Company, Lilly
`Corporate Center, Drop Code1501, Indianapolis, IN 46285
`(US).
`
`
`
`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—l) 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
`
`MYLAN INST. EXHIBIT 1021 PAGE 1
`
`MYLAN INST. EXHIBIT 1021 PAGE 1
`
`MYLAN INST. EXHIBIT 1021 PAGE 1
`
`

`

`Zimbabwe
`
`Codes used to identify States party to the PCT on the 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
`TM
`Belgium
`The former Yugoslav
`Burkina Faso
`GR
`TR
`Republic of Macedonia
`HU
`Mali
`TT
`Bulgaria
`Benin
`IE
`UA
`Mongolia
`Brazil
`IL
`Mauritania
`UG
`Belarus
`IS
`Malawi
`US
`Canada
`IT
`Mexico
`UZ
`JP
`VN
`Central African Republic
`Niger
`KE
`Netherlands
`YU
`Congo
`Switzerland
`KG
`ZW
`Norway
`Céte d’Ivoire
`KP
`New Zealand
`Cameroon
`Poland
`China
`Portugal
`Cuba
`Romania
`Russian Federation
`Czech Republic
`Sudan
`Germany
`Denmark
`Sweden
`Estonia
`Singapore
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`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
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`MYLAN INST. EXHIBIT 1021 PAGE 2
`
`MYLAN INST. EXHIBIT 1021 PAGE 2
`
`MYLAN INST. EXHIBIT 1021 PAGE 2
`
`

`

`WO 00/37098
`
`PCT/US99/30395
`
`SHELF-STABLE FORMULATION OF GLUCAGON-LIKE PEPTIDE-1
`
`Background of the Invention
`
`Glucagon—like peptide-1 (7-37)-OH (GLP-1) is a 31 amino acid hormone that is
`produced by post-translational processing of the preglucagon gene product in the brain,
`
`stomach, intestine, and pancreas. The main physiological function of GLP-1 is to regulate
`
`insulin secretion in response to glucose, and thus it has the ability to normalize blood
`
`glucose levels. As such, there has been interest in GLP-l, its analogs and derivatives as
`
`potential therapeutic agents for the treatment of diabetes. A particular advantage to the use
`I of GLP-1 over other drugs in the treatment of diabetes is that administration of GLP-1 at
`
`doses in the 1-5 nmole range exhibit few adverse side effects, such as hypoglycemia.
`
`Unexpectedly, GLP-l also has been shown to work in patients that have secondary failure
`to sulfonylurea drugs, the most common drug type for the treatment of type H diabetes.
`
`GLP—l 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 degraded in the gastrointestinal tract by various peptidases. Additionally,
`
`certain peptide treatment protocols 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 of the 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-l, as components of a
`
`pharmaceutical composition for administration to mammals, is questionable. Such a lack of
`
`stability adversely affects bioavailability. In fact, when stored at low temperatures of 4° C,
`
`by-products of GLP-1(7-37) have been found as early as eleven months after 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,
`
`MYLAN INST. EXHIBIT 1021 PAGE 3
`
`MYLAN INST. EXHIBIT 1021 PAGE 3
`
`MYLAN INST. EXHIBIT 1021 PAGE 3
`
`

`

`WO 00/37098
`
`PCT/US99/30395
`
`the biological half-life of GLP-1(7-37) is only 3 to 5 minutes (see US. Patent No.
`
`5,118,666), which is further augmented by its 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 treatment that the
`
`peptide has undergone. For example, conversion of 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. Because the solubility of the peptide is high in such aqueous solutions,
`
`slow release of the peptide can be difficult to attain unless the peptide is 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-l 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 continuous infusion systems, a fluid containing a therapeutic agent is pumped
`from a reservoir, usually to a subcutaneous, intravenous, or intraperitoneal depot. The
`
`reservoir, which must be refilled 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 turbulence in the tubing and pump impart a relatively high amount of thermo-
`
`In the interest of minimizing the frequency with
`mechanical energy to the formulation.
`which the reservoir is refilled, and of minimizing the size of the reservoir, formulations
`
`having a relatively high concentration of the therapeutic agent are advantageous.
`
`Injector pens also have been developed to 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 manner to dispense the medication.
`
`Injector pens may be reusable or
`
`disposable. In reusable pens, a user can change a spent cartridge and reset the leadscrew of
`
`the pen back to its initial position. In a disposable pen, the cartridge is permanently
`
`-2-
`
`MYLAN INST. EXHIBIT 1021 PAGE 4
`
`MYLAN INST. EXHIBIT 1021 PAGE 4
`
`MYLAN INST. EXHIBIT 1021 PAGE 4
`
`

`

`WO 00/37098
`
`PCT/US99/30395
`
`embodiment, the GLP—l molecule of the formulation is a derivative of GLP-1 and is
`
`selected from the group consisting of a peptide having the amino acid'sequence:
`
`NHz-His7-Ala-Glu-Glyl°—Thr-Phe—Thr-Ser-Asp‘5-Val-Ser-Ser-Tyr-Leum-Glu—Gly-Gln-Ala-
`
`Ala”-Lys~Glu-Phe—Ile-A1a3°-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 phannaceutically-acceptable lower alkylester of the peptide;
`
`and a phannaceutically—acceptable amide of the 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 expression of insulin
`
`in a mammalian pancreatic B—type islet cell in need of such enhancement, comprising
`
`administering to the cell, an effective amount of a shelf-stable pharmaceutical formulation,
`
`wherein the formulation comprises a therapeutically effective amount of a GLP-l 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.
`
`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-l molecule of
`
`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:
`
`Rl-X—Glu-Glylo-Thr-Phe-Thr-Ser—Asp’S-Val-Ser-Ser-Tyr—LeuZ°-Y-Gly—Gln-Ala-Ala”-
`
`(SEQ II) NO:2)
`Lys-Z—Phe-Ile—Ala3O-Trp-Leu-Val-Lys-Gly35-Arg-R2
`and a pharmaceutically-acceptab1e salt thereof, wherein R1 is His or desamino—histidine, X
`
`is Ala, Gly or Val, Y is Glu or‘Gln, Z is Glu or Gln and R2 is Gly-OH.
`
`In an especially
`
`preferred embodiment, the GLP-l molecule administered is according to SEQ ID NO: 2,
`
`wherein R1 is L—histidine, X is Val, Y is Glu, Z is Glu, and R2 is Gly-OH. In an alternative
`
`preferred embodiment, the GLP-l molecule administered is a derivative of GLP-1 and is
`selected from the group consisting of a peptide having the amino acid sequence:
`NHz-His7-Ala-G1u-Glym—Thr—Phe-Thr-Ser-Asp15-Val—Ser-Ser—Tyr—Leuzo-Glu-Gly-Glu-
`
`Ala-Alazs-Lys-Glu-Phe-Ile—A1a30-Trp-Leu-Val-X
`
`(SEQ ID NO:3)
`
`and a phannaceutically-acceptable salt thereof, wherein X is selected from the group
`
`consisting of Lys and Lys-Gly; a pharmaceutically-acceptable lower alkylester of the peptide;
`
`.4-
`
`MYLAN INST. EXHIBIT 1021 PAGE 5
`
`MYLAN INST. EXHIBIT 1021 PAGE 5
`
`MYLAN INST. EXHIBIT 1021 PAGE 5
`
`

`

`WO 00/37098
`
`PCT/US99/3039S
`
`and a pharmaceutically-acceptable amide of the peptide selected from the group consisting of
`
`amide, lower alkyl amide, and lower dialkyl amide.
`
`The present invention also provides a method of treating diabetes comprising
`
`administering to a patient in need of such treatment an effective amount of a shelf-stable
`
`pharmaceutical formulation, wherein the formulation comprises a therapeutically effective
`
`amount of a GLP-l 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.
`
`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—l 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-Glylo-Thr—Phe-ThI-Ser-Asp15-Val—Ser-Ser-Tyr-Leum—Y—G1y-G1n-Ala-Ala25-
`
`Lys—Z-Phe-Ile-Ala3O-Trp-Leu-Val—Lys-Gly35—Arg-R2
`
`(SEQ ID NO:2)
`
`and a pharmaceutically-acceptable salt thereof, wherein R1 is His or desamino-histidine, X
`
`is Ala, Gly or Val, Y is Glu or G111, Z is Glu or Glu and R2 is Gly-OH.
`
`In an especially
`
`preferred embodiment, the GLP-l molecule administered is according to SEQ ID NO: 2,
`
`wherein R1 is L—histidine, X is Val, Y is Glu, Z is Glu, and R2 is Gly-OH. In an alternative
`
`preferred embodiment, the GLP-l molecule administered is a derivative of GLP-1 and is
`
`selected from the group consisting of a peptide having the amino acid sequence:
`
`NHz-His7—Ala—Glu-Glym—Thr-Phe—Thr-Ser-Asp15-Va1-Ser-Ser—Tyr-LeuZO-Glu-Gly-Gln—
`
`Ala-Alazj-Lys-Glu-Phe-Ile-Ala3°-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.
`
`An additional 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 amount of a shelf-stable pharmaceutical formulation,
`
`wherein the formulation comprises a therapeutically effective amount of a GLP—l 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.
`
`-5-
`
`MYLAN INST. EXHIBIT 1021 PAGE 6
`
`MYLAN INST. EXHIBIT 1021 PAGE 6
`
`MYLAN INST. EXHIBIT 1021 PAGE 6
`
`

`

`WO 00/37098
`
`PCT/US99/30395
`
`captured in the pen which is disposed of after the contents of the cartridge have been
`
`exhausted.
`
`With the development of GLP-1 as well as analogs and derivatives thereof for the
`
`treatment of diabetes, there exists a need to improve treatment regimes that can balance
`
`chemical and physical stability with chronic use requirements of diabetic patients.
`
`Summary of 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 formulations is
`
`unexpectedly and considerably greater than when compared to GLP-l formulations made
`
`with a phosphate buffer.
`
`In addition, maintaining the pH in a range of about 8.2 to about
`
`8.8 unexpectedly improves the chemical stability of the formulations. The shelf-stable
`
`formulation of GLP-1 of the invention comprises a therapeutically effective amount of a
`
`GLP—l 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 about 8.8.
`
`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-l molecule, a pharrnaceutically 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—l molecule of the formulation is
`
`an analog of GLP-1 and is selected from the group consisting of a peptide having the amino
`
`acid sequence:
`
`Rl-X-Glu-Glylo-Thr-Phe-Thr-Ser-Asp‘5-Val-Ser-Ser-Tyr-Leuzo-Y-Gly-Gln-Ala-Alafi-
`
`Lys-Z-Phe-Ile-A1a3°—Trp-Leu-Val-I_.ys-Gly35-Arg-R2
`
`(SEQ II) NO:2)
`
`and a pharmaceuticalIy-acceptable salt thereof, wherein R1 is His or desamino—histidine, X
`
`is Ala, Gly or Val, Y is Glu or Gln, Z is Glu or Glu and R2 is Gly-OH. In an especially
`
`preferred embodiement, the GLP-l molecule is according to SEQ ID NO: 2, wherein R1 is L-
`
`histidine, X is Val, Y is Glu, Z is Glu, and R2 is Gly—OH.
`
`In an alternative preferred
`
`-3-
`
`MYLAN INST. EXHIBIT 1021 PAGE 7
`
`MYLAN INST. EXHIBIT 1021 PAGE 7
`
`MYLAN INST. EXHIBIT 1021 PAGE 7
`
`

`

`W0 00/37 098
`
`PCT/USQ9/30395
`
`Detailed Description of the Preferred Embodiments
`
`The present invention provides a shelf-stable formulation of GLP—1, GLP-l analogs,
`
`and GLP—1 derivatives. Because it is known that there are problems with long term
`
`stability of GLP-1 as a component in a pharmaceutical composition, the present inventors
`
`developed a pharmaceutical formulation which stabilizes GLP—l, its derivatives and
`
`analogs. This development led to the shelf-stable GLP—l formulations of the invention.
`
`In. another embodiment, the present invention encompasses a GLP—l formulation
`
`It has long been a goal of insulin therapy
`that also comprises a long acting diabetic agent.
`to mimic the pattern of endogenous insulin 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 of insulin to dispose of the 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 combined use of two types
`of exogenous insulin formulations: a fast-acting meal time insulin 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 commercial basal
`
`insulin preparations include NPH (Neutral Protamine Hagedorn) insulin, protamine zinc
`
`insulin (P21), 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 by a
`
`sample’s attenuation of light. The measurement relates 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 preparation is. Stability also refers to the
`
`chemical stability of the formulation such as the propensity of the proteins to form high
`order polymers which is 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 about 8.8 is advantageous.
`
`Typical peptide formulations have a more neutral pH of 7 to about 7.8 or an acidic pH.
`Furthermore, a composition containing a GLP-l molecule that has a pH in the range of
`
`-6—
`
`MYLAN INST. EXHIBIT 1021 PAGE 8
`
`MYLAN INST. EXHIBIT 1021 PAGE 8
`
`MYLAN INST. EXHIBIT 1021 PAGE 8
`
`

`

`WO 00/37098
`
`PCT/US99/30395
`
`about 6.8 to about 7.5 exhibits less physical stability than a composition of a GLP-l
`
`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 of less than about 8.0 tends to exhibit turbidity, a
`telltale sign of decreased physical stability 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-l formulations having a pH range of
`about 8.2 to about 8.8, which preserves optimal chemical and physical stability of the GLP-
`1 molecule. A particularly preferred range for the inventive GLP—l formulations is about
`
`8.3 to about 8.6, and a most particularly 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-l 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, which are typically used in peptide pharmaceutical
`
`'
`
`formulations, cannot maintain a pH range of 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, TRIS is the preferred buffer for the formulations of the present invention.
`
`The term “TRIS” refers to 2—amino—2—hydroxyrnethyl-1,3-propanediol ( also known in 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 pH at the alkaline level desired, thereby stabilizing the formulation.
`
`A second factor that plays a role in the stability of GLP-1 formulations is the
`
`concentration of the GLP—l molecule that is used in the inventive formulation. The present
`
`-7-
`
`MYLAN INST. EXHIBIT 1021 PAGE 9
`
`MYLAN INST. EXHIBIT 1021 PAGE 9
`
`MYLAN INST. EXHIBIT 1021 PAGE 9
`
`

`

`WO 00/370923
`
`'
`
`PCT/US99/30395
`
`inventors determined that a concentration of about 0.30 to about 0.65 mg/ml of the GLP-l
`
`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
`
`development of turbidity in the formulation. A particularly stable formulation includes
`
`about 0.5 mg/ml of a GLP-l molecule.
`
`An additional factor which contributes to the overall stability of the GLP—l
`
`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.
`
`While it 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 of the formulation, and‘whether substances that
`
`bind or sequester the preservative are also present. Preferably, m-cresol is used in the
`
`L
`
`formulations as a preservative.
`
`While a buffer and a preservative are most preferably included in the formulation,
`
`other additional excipients may be included, such as 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 mode of 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 invention is
`
`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-l, but also analogs,
`
`derivatives and salts of GLP-1. As used herein, the term “a GLP-l molecule” refers (1) to
`
`the naturally-occurring GLP-l, which is GLP—l (7-37)—OH; (2) GLP-l(7-36)NH2, as well
`
`-8—
`
`MYLAN INST. EXHIBIT 1021 PAGE 10
`
`MYLAN INST. EXHIBIT 1021 PAGE 10
`
`MYLAN INST. EXHIBIT 1021 PAGE 10
`
`

`

`wo 00/37098
`
`PCT/US99/30395
`
`as (3) GLP-l (7-37); (4) natural and synthetic functional GLP-l analogs; (5) derivatives of
`
`GLP-1 and (6) salts of any of the aforementioned molecules.
`
`A “GLP—l 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 include the
`
`D-amino acid forms. Numerous GLP-l analogs are known in the art and include, but are not
`
`limited to, GLP—1(7-34), GLP-1(7-35), GLP-1(7-36)NH2, Gln"-GLP—1(7—37), d—Glng-GLP-
`
`1(7-37), Thr‘fi-Lysls-GLP-l(7—37), and Lysm-GLP—1(7-37), Gly“-GLP-1(7-36)NH2, Glys—GLP—
`
`1(7-37)OH, Val“-GLP-1(7-37)OH, Met“-GLP—1(7—37)OH, acetyl—Lys9—GLP-1(7—37), Thr9—
`
`GLP-1(7-37), D-Thr9—GLP-1(7-37), Asng-GLP—1(7—37), D-Asn9-GLP-1(7-37), Ser’l-ArgB-
`
`ArgM-GanG-GLP—1(7-37), Arg“-GLP—1(7-37), Arg24-GLP—1(7-37), a-methyl—Alas-GLP-IU-
`36)NH2, and GlyS—Gln21-GLP-l(7-37)OH, and the like.
`‘
`
`Other GLP—l analogs consistent with the present invention are described by the
`
`formula:
`
`R1-X-Glu-G1y‘°—Thr-Phe-Thr—Ser—Asp’5-Val-Ser~Ser-Tyr—Leu2°-Y-G1y-G1n-Ala-Ala”-
`
`Lys-Z—Phe—I1e-Ala30—Trp-Leu-Val-Lys-Gly35-Arg-R2
`
`(SEQ ID NO:2)
`
`wherein: R1 is selected from the group consisting of L—histidine, D—histidine, desarnino-
`
`histidine, 2—amino-histidine, beta-hydroxy—histidine, homohistidine, alpha-fluoromethyl-
`
`histidine, and alpha-methyl-histidine; X is selected from the group consisting of Ala, Gly,
`
`Val, Thr, He, and alpha-methyl-Ala; Y is selected from the group consisting of Glu, Gln,
`
`Ala, Thr, Set, 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.
`
`GLP-l analogs also have been described in WO 91/11457, and include GLP—1(7-34),
`
`GLP—1(7-35), GLP—1(7—36), or GLP-1(7-37), or the amide form thereof, and
`I 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, glutarnine, 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;
`
`-9-
`
`MYLAN INST. EXHIBIT 1021 PAGE 11
`
`MYLAN INST. EXHIBIT 1021 PAGE 11
`
`MYLAN INST. EXHIBIT 1021 PAGE 11
`
`

`

`WO 00/37098
`
`PCT/US99/30395
`
`(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 glutamine for
`
`lysine at position 26; and
`
`(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,
`
`leucine, methionine, 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 described in (a), (b), (d), 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.
`
`Preferred GLP—l molecules used in the present inventive formulation also include
`
`analogs of GLP-1 (7—37)NH2 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.
`
`Specifically, His and desamino-histidine are preferred for R1. Ala, Gly and Val are preferred
`
`at the “X” position. Also, Glu and Gln are preferred for at the “Y” position. Glu and Gln
`
`are preferred at the “Z” position and Gly-OH is preferred for R2.
`
`A particularly preferred GLP-l analog is known as Val(8)GLP-1 (V8GLP—l) and has
`
`a formula according to SEQ ID NO:2, wherein R1 is L-histidine, X is Val, Y is Glu, Z is Glu
`
`and R2 is Gly-OH.
`
`A "GLP—l derivative" is defined as a molecule having the amino acid sequence of
`
`GLP-1(7-37) or of a GLP-l analog, but additionally comprises chemical modification of one
`
`or more of its amino acid side groups, a-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 include, without limitation, acylation of lysine a-amino groups, N-
`
`alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid
`
`-10-
`
`MYLAN INST. EXHIBIT 1021 PAGE 12
`
`MYLAN INST. EXHIBIT 1021 PAGE 12
`
`MYLAN INST. EXHIBIT 1021 PAGE 12
`
`

`

`WO 00/37098
`
`PCT/US99/30395
`
`groups, 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 oc-carbon of an amino
`
`acid maybe mono- or dimethylated.
`
`Other GLP-l derivatives include molecules which are selected from the group
`consisting of a peptide having the amino acid sequence:
`
`NHz-His7-Ala-Glu-Gly‘o-Thr-Phe-Thr-Ser~Asp1s-Val-Ser-Ser—Tyr—Leu2°—Glu-Gly—
`Gln-Ala-Ala25-Lys-Glu-Phe—Ile—Ala3°-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 amide of said peptide, selected from the group consisting of amide,
`lower alkyl amide, and lower dialkyl amide.
`
`Yet other GLP—l derivatives appropriate for use in the present invention include
`
`compounds claimed in U.S. Patent No. 5,512,549 described by the formula:
`R1—Ala-G1u-G1y1O—Thr—Phe-Thr-Ser—Aspl5—Val—Ser-Ser-Tyr-Leuzo-Glu—Gly-Gln—Ala-Ala—Xaa-
`Glu-Phe-Ile~A1a30-Trp—Leu-Val-Lrs-Gly35-Arg-R3
`(SEQ ID NO: 3)
`wherein R1 is selected from the griup consisting of4-imida20propionyl, 4-imidazoacetyl, or
`4-imidazo—0t, on dimethyl-acetyl; R2 is selected from the group consisting of Cé-Cm
`
`2
`
`unbranched acyl, or is absent; R3 is selected from the group consisting of Gly-OH or NHZ;
`and, Xaa is Lys or Arg, may be used in present invention.
`
`“DPP—IV protected