`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`4 September 2003 (04.09.2003)
`
`
`
`PCT
`
`(10) International Publication Number
`W0 03/072195 A2
`
`(51) International Patent Classificati0n7:
`
`A61P
`
`SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG).
`
`(21) International Application Number:
`
`PCT/USO3/03111
`
`Declarations under Rule 4.17:
`
`(22) International Filing Date: 7 February 2003 (07.02.2003)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/358,184
`
`20 February 2002 (20.02.2002)
`
`US
`
`(71) Applicant (for all designated States except US): ELI
`LILLY AND COMPANY [US/US]; Lilly Corporate
`Center, Indianapolis, IN 46285 (US).
`
`(72) Inventor; and
`(7S) Inventor/Applicant (for US only): KHAN, Mohammed,
`Amin [US/US]; 5163 Sue Drive, Carmel, IN 46033 (US).
`
`(74) Agents: DAVIS, Paula, K. et a1.; Eli Lilly And Company,
`P. O. Box 6288, Indianapolis, IN 46206—6288 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT (util—
`ity model), AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ (utility model), CZ, DE (util—
`ity model), DE, DK (utility model), DK, DM, DZ, EC, EE
`(utility model), EE, ES, FI (utility model), FI, GB, GD, GE,
`GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ,
`LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN,
`MW, MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SC,
`SD, SE, SG, SK (utility model), SK, SL, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HU, IE, IT, LU, MC, NL, PT, SE, SI,
`
`as to applicant’s entitlement to apply for and be granted
`a patent (Rule 4.I7(ii)) for the following designations AE,
`AG, AL, AM AT AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM DZ, EC, EE, ES, FI,
`GB, GD, GE, GH, GM HR, HU, ID, IL, IN, IS, JP, KE, KG,
`KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK,
`MN, MW, MX, MZ, NO, NZ, OM PH, PL, PT RO, RU, SC,
`SD, SE, SG, SK, SL, TJ, TM TN, TR, TT TZ, UA, UG, UZ,
`VC, VN, YU, ZA, ZM, ZW, ARIPO patent (GH, GM, KE, LS,
`MW, MZ, SD, SL, SZ, TZ, UG, ZM ZW), Eurasian patent
`(AM AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent
`(AT BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB,
`GR, HU, IE, IT LU, MC, NL, PT SE, SI, SK, TR), OAPI
`patent (BF, BJ, CF, CG, CI, CM GA, GN, GQ, GW, ML,
`MR, NE, SN, TD, TG)
`as to the applicant’s entitlement to claim the priority ofthe
`earlier application (Rule 4. I 7(iii)) for the following desig-
`nations AE, AG, AL, AM AT AU, AZ, BA, BB, BG, BR, BY,
`BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM DZ, EC,
`EE, ES, FI, GB, GD, GE, GH, GM HR, HU, ID, IL, IN, IS,
`JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT LU, LV, MA,
`MD, MG, MK, MN, MW, MX, MZ, NO, NZ, OM PH, PL,
`PT R0, RU, SC, SD, SE, SG, SK, SL, TJ, TM TN, TR, TT
`TZ, UA, UG, UZ, VC, VN, YU, ZA, ZM ZW, ARIPOpatent
`(GH, GM KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM ZW),
`Eurasian patent (AM AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HU, IE, IT LU, MC, NL, PT SE, SI,
`SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG)
`of inventorship (Rule 4.] 7(iv)) for US only
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report
`
`For two-letter codes and other abbreviations, refer to the ”Guid-
`ance Notes on Codes andAbbreviations ” appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
`
`WO03/072195A2
`
`(54) Title: METHOD FOR ADMINISTERING GLP—1 MOLECULES
`
`(57) Abstract: The invention encompasses formulations that demonstrate the feasibility of oral absorption comprising GLP—1 com—
`pounds and specified delivery agents.
`
`MYLAN INST. EXHIBIT 1014 PAGE 1
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`MYLAN INST. EXHIBIT 1014 PAGE 1
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`MYLAN INST. EXHIBIT 1014 PAGE 1
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`
`
`W0 03/072195
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`PCT/US03/03111
`
`METHOD FOR ADMINISTERING GLP-l MOLECULES
`
`FIELD OF THE INVENTION
`
`The present invention relates to a formulation useful for the oral administration
`
`comprising a glucagon—like peptide-1 (GLP-l) compound and a specified delivery agent.
`
`Oral administration of the formulations can be used to treat type 2 diabetes as well as a
`
`variety of other conditions.
`
`BACKGROUND OF THE INVENTION
`
`10
`
`Over the past several decades, continuous strides have been made to improve the
`
`treatment of diabetes mellitus. Approximately 90% of people with diabetes have type 2
`
`diabetes, also known as non-insulin dependent diabetes mellitus (NIDDM). Type 2
`
`diabetics generally still make insulin, but the insulin cannot be used effectively by the
`
`body’s cells. This is primarily because the amount of insulin produced in response to
`
`15
`
`rising blood sugar levels is not sufficient to allow cells to efficiently take up glucose and
`
`thus, reduce blood sugar levels.
`
`A large body of pre-clinical and clinical research data suggests that glucagon—like
`
`peptide-1 (GLP~1) compounds show great promise as a treatment for type 2 diabetes and
`
`other conditions. GLP-l induces numerous biological effects such as stimulating insulin
`
`20
`
`secretion, inhibiting glucagon secretion, inhibiting gastric emptying, enhancing glucose
`
`utilization, and inducing weight loss. Further, pre-clinical studies suggest that GLP-l
`
`may also act to prevent the B cell deterioration that occurs as the disease progresses.
`
`Perhaps the most salient characteristic of GLP-1 is its ability to stimulate insulin secretion
`
`without the associated risk of hypoglycemia that is seen when using insulin therapy or
`
`25
`
`some types of oral therapies that act by increasing insulin expression.
`
`However, development of a GLP-l therapeutic has been extremely difficult. This
`
`is primarily due to the instability of the peptide during manufacturing processes, in
`
`solution formulations, and in vivo. The only published clinical studies employing GLP-l
`
`compounds to treat hyperglycemia or other conditions involve formulating GLP—l
`
`30
`
`compounds such that they can be delivered by subcutaneous injection or through
`
`continuous subcutaneous infusion or continuous intravenous administration. Many type 2
`
`MYLAN INST. EXHIBIT 1014 PAGE 2
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`MYLAN INST. EXHIBIT 1014 PAGE 2
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`MYLAN INST. EXHIBIT 1014 PAGE 2
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`
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`W0 03/072195
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`PCT/US03/03111
`
`-2-
`
`diabetics or obese patients desiring to lose weight will not be willing to undertake a
`
`treatment regimen that may involve several injections per day. Thus, there is a need to
`
`develop GLP-l compound therapeutics that can be delivered by an alternative non-
`
`invasive means such as by oral delivery.
`
`Unfortunately, there are numerous barriers to effective oral delivery of peptides.
`
`The high acid content and ubiquitous digestive enzymes of the digestive tract will often
`
`degrade proteins and peptides before they reach the site of absorption. Further, many
`
`peptides cannot effectively traverse the cells of the epithelial membrane in the small
`
`intestine to reach the bloodstream. Finally, many drugs become insoluble at the low pH
`
`10
`
`levels encountered in the digestive tract and, thus, are not absorbed effectively.
`
`The fact that GLP—l compounds are relatively unstable in solution formulations,
`
`only remain in solution under a fairly narrow set of conditions, and have a relatively short
`
`in vivo half-life when administered as a solution formulation, suggested that these
`
`compounds could not be effectively delivered through the oral route. Thus, it was
`
`15
`
`surprising that GLP-l compounds could be formulated such that biologically active
`
`molecules were absorbed into the blood stream after oral administration.
`
`The present invention involves the use of specific delivery agent molecules that
`
`interact with GLP-l compounds in a non-covalent fashion to allow the compounds to
`
`cross gut membranes and yet remain therapeutically active. Although the delivery agents
`
`20
`
`employed in the present invention have been disclosed in a series of US. Patents (see
`
`US. Patent Nos. 5,541,155; 5,693,338; 5,976,569; 5,643,957; 5,955,503; 6,100,298;
`
`5,650,386; 5,866,536; 5,965,121; 5,989,539; 6,001,347; 6,071,510; 5,820,881; and
`
`6,242,495; see also W0 02/02509; W0 01/51454; WO 01/44199; WO 01/32130;
`
`W0 00/59863; WO 00/50386; W0 00/47188; and WO 00/40203), oral administration of
`
`25
`
`formulations comprising GLP—l compounds with these delivery agents has not been
`
`disclosed or suggested. Further, numerous parameters impact whether a particular class
`
`of compounds can be effectively delivered in combination with one or more classes of
`
`delivery agents. For example, the conformation of the peptide, the surface charges on the
`
`molecule under certain formulation conditions, the solubility profile, the stability as a
`
`30
`
`formulated component, as well as susceptibility to protease digestion and in viva stability
`
`all influence the ability to deliver a compound orally.
`
`MYLAN INST. EXHIBIT 1014 PAGE 3
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`MYLAN INST. EXHIBIT 1014 PAGE 3
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`MYLAN INST. EXHIBIT 1014 PAGE 3
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`
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`W0 03/072195
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`PCT/US03/03111
`
`SUMMARY OF THE INVENTION
`
`The present invention encompasses the development of novel formulations
`
`comprising GLP—l compounds and delivery agents that can be administered orally. The
`
`present invention provides a formulation Which can be administered orally comprising a
`
`GLP-l compound and a specified delivery agent. The GLP—l compound can be native
`
`GLP—l; GLP~1 fragments; GLP-l analogs; GLP-l derivatives of native, fragments, or
`
`analogs of GLP-1 ; and Exendin—3 and Exendin—4I The delivery agent is selected from
`
`delivery agents described in US. Patents 5,541,155; 5,693,338; 5,976,569; 5,643,957;
`
`10
`
`5,955,503; 6,100,298; 5,650,386; 5,866,536; 5,965,121; 5,989,539, 6,001,347;
`
`6,071,510; 5,820,881; and 6,242,495; and W0 02/02509; W0 01/51454;
`
`W0 01/44199; W0 01/32130; W0 00/59863; W0 00/50386; W0 00/47188; and
`
`WO 00/40203.
`
`Preferred GLP-l compounds are analogs or derivatives of analogs having
`
`15
`
`modifications at one or more of the following positions: 8, 12, 16, 18, 19, 20, 22, 25, 27,
`
`30, 33, and 37 and show increased potency compared With ValS-GLP-l(7-37)0H.
`
`Preferred GLP—l compounds are also described in SEQ ID N0:1, SEQ ID N0:2, SEQ 1D
`
`N023, SEQ ID N024, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ
`
`ID N0z9, SEQ ID N0:10, SEQ ID N0:11, SEQ ID N0:12, SEQ ID N0:13, or SEQ ID
`
`20
`
`N0:14. More preferred GLP—l compounds are described in compounds of SEQ ID N0:2,
`
`SEQ ID N0:12, SEQ ID N0:13, and SEQ ID N0:14.
`
`Preferred delivery agents are described in Table 1. More preferred delivery agents
`
`are delivery agents corresponding to numbers of Table 1 selected fiom the group
`
`consisting of 1, 2, 4, 5, 6, 9,10,11,13,14,15, 20, 21, 22, 23, 24, 26, 28, 30, 31, 35, 36,
`
`25
`
`38, 39, 40, 41, 42, 43, 44, 46, 51, 52, and 54.
`
`The present invention also encompasses a method of stimulating the GLP—1
`
`receptor in a subject in need of such stimulation, said method comprising the step of
`
`administering to the subject an effective amount of the oral formulation described herein.
`
`Subjects in need of GLP—1 receptor stimulation include those with non-insulin dependent
`
`30
`
`diabetes and obesity.
`
`MYLAN INST. EXHIBIT 1014 PAGE 4
`
`MYLAN INST. EXHIBIT 1014 PAGE 4
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`MYLAN INST. EXHIBIT 1014 PAGE 4
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`
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`W0 03/072195
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`PCT/US03/03111
`
`_4_
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The three—letter abbreviation code for amino acids used in this specification
`
`conforms with the list contained in Table 3 of Annex C, Appendix 2 of the PCT
`
`Administrative Instructions and with 37 CPR. § l.822(d)(1)(2000).
`
`For purposes of the present invention as disclosed and described herein, the
`
`following terms and abbreviations are defined as follows.
`
`The term “formulation” as used herein refers to a GLP-l compound and a
`
`specified delivery agent combined together which can be administered orally such that
`
`'
`
`GLP-l compound passes through the gut into the systemic circulation and has the ability
`
`10
`
`to bind to the GLP—1 receptor and initiate a signal transduction pathway resulting in
`insulinotropic activity. The formulation can optionally comprise other agents so long as
`
`the GLP-l retains the ability to bind the GLP—1 receptor.
`
`The term “ora ” as used herein refers to delivery of a compound by mouth such
`
`that the compound passes through the stomach, small intestine, or large intestine into the
`
`15
`
`systemic circulation.
`
`The term “GLP-l compound” as used herein refers to polypeptides that include
`
`naturally occurring GLP-l polypeptides (GLP-1(7-37)OH and GLP-l(7—36)NH2), GLP-l
`
`fragments, GLP-l analogs, GLP-l derivatives of naturally occurring GLP-l polypeptides,
`
`GLP—l fragments, or GLP-l analogs, and Exendin—3 and Exendin—4 that have the ability
`
`20
`
`to bind to the GLP-1 receptor and initiate a signal transduction pathway resulting in
`
`insulinotropic activity.
`
`The term “insulinotropic activity” refers to the ability to stimulate insulin secretion
`
`in response to elevated glucose levels, thereby causing glucose uptake by cells and
`
`decreased plasma glucose levels. For example, insulinotropic activity can be determined
`
`25
`
`using the method described in Example 1. A GLP-l molecule has insulinotropic activity
`
`if islet cells seerete insulin levels in the presence of the GLP-l molecule above
`
`background levels.
`
`The term “DPP IV resistant” refers to GLP-l molecules that have extended
`
`metabolic stability and improved biological activity. For example, DPP IV resistance can
`
`30
`
`be determined using the method described in Example 2. A GLP-l molecule is DPP IV
`
`resistant if in the presence of DPP IV the GLP-l molecule has extended metabolic
`
`MYLAN INST. EXHIBIT 1014 PAGE 5
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`MYLAN INST. EXHIBIT 1014 PAGE 5
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`MYLAN INST. EXHIBIT 1014 PAGE 5
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`-5-
`
`stability above that of native GLP-l. DPP IV resistant GLP-l molecules can have an
`
`amino acid change at the DPP IV recognition site (position 8), or DPP IV resistant
`
`peptides can have an attached group that restricts the accessibility of the DPP IV to the
`
`recognition site, or both.
`
`A "GLP-l fragment" is a polypeptide obtained after truncation of one or more
`
`amino acids from the N-terminus and/or C—terminus of GLP-l(7-37)OH or an analog or
`
`derivative thereof. The nomenclature used to describe GLP-l (7-37)OH is also applicable
`
`to GLP—l fragments. For example, GLP-l(9-36)OH denotes a GLP-l fragment obtained
`by truncating two amino acids from the N—terminus and one amino acid from the CL
`
`10
`
`terminus. The amino acids in the fragment are denoted by the same number as the
`
`correspondingamino acid in GLP—l(7-37)OH. For example, the N—terminal glutamic acid
`
`in GLP—1(9-3 6)OH is at position 9; position 12 is occupied by phenylalanine; and position
`
`22 is occupied by glycine, as in GLP—l(7—37)OH. For GLP-l(7-36)OH, the glycine at
`
`position 37 of GLP-l(7-37)OH is deleted.
`
`15
`
`A “GLP-l analog” has sufficient homology to GLP-l(7—37)OH or a fragment of
`
`GLP-l(7-37)OH such that the analog has insulinotropic activity. Preferably, a GLP-l
`
`analog has the amino acid sequence of GLP-1(7-37)OH or a fragment thereof, modified
`
`so that from one, two, three, four or five amino acids differ from the amino acid in
`
`corresponding position of GLP-l(7—37)OH or a fragment of GLP-l(7—37)OH. In the
`
`20
`
`nomenclature used herein to designate GLP—l compounds, the substituting amino acid and
`
`its position is indicated prior to the parent structure. For example, GluZZ-GLP-1(7-37)OH
`
`designates a GLP-l compound in which the glycine normally found at position 22 of
`
`GLP—l(7-3 7)OH has been replaced with glutamic acid; Valg-GluZZ-GLP-l(7-37)OH
`
`designates a GLP-l compound in which alanine normally found at position 8 and glycine
`
`25
`
`normally found at position 22 of GLP-1(7-37)OH have been replaced with valine and
`
`glutamic acid, respectively.
`
`GLP-l molecules also include polypeptides in which one or more amino acids
`
`have been added to the N—terminus and/or C—terminus of GLP-l(7—37)OI-I, or fragments or
`
`analogs thereof. It is preferred that GLP—l molecules of this type have up to about thirty-
`
`30
`
`nine amino acids. The amino acids in the “extended” GLP-l molecule are denoted by the
`
`same number as the corresponding amino acid in GLP-1(7—37)OH. For example, for a
`
`MYLAN INST. EXHIBIT 1014 PAGE 6
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`MYLAN INST. EXHIBIT 1014 PAGE 6
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`MYLAN INST. EXHIBIT 1014 PAGE 6
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`-5-
`
`GLP—l molecule obtained by adding two amino acids to the N—terminus of
`
`GLP-1(7-3 7)OH, the N-tenninal amino acid is located at position 5; and for a GLP—l
`
`molecule obtained by adding one amino acid to the C—terminus of GLP—l(7—37)OH, the C-
`
`terminal amino acid is located at position 38. Thus, position 12 is occupied by
`
`phenylalanine and position 22 is occupied by glycine in both of these “extended” GLP-l
`
`compounds, as in GLP-l(7-37)OH. Amino acids 1-6 of an extended GLP-l molecule are
`
`preferably the same as or a conservative substitution of the amino acid at the
`
`corresponding position of GLP-l(l~37)OH. Amino acids 38—45 of an extended GLP—l
`
`molecule are preferably the same as or a conservative substitution of the amino acid at the
`
`10
`
`corresponding position of glucagon or Exendin—4.
`
`A "GLP-l derivative" refers to a molecule having the amino acid sequence of
`
`GLP-1, a GLP-l fragment, or a GLP—l analog, but additionally having chemical
`
`modification of one or more of its amino acid side groups, a-carbon atoms, terminal
`
`amino group, or terminal carboxylic acid group. A chemical modification includes, but is
`
`15
`
`not limited to, adding chemical moieties, creating new bonds, and removing chemical
`
`moieties. Modifications at amino acid side groups include, without limitation, acylation
`
`of lysine e—amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of
`
`glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine.
`
`Modifications of the terminal amino group include, without limitation, the des-amino,
`
`20
`
`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 (it-carbon of an amino acid may be mono- or
`
`25
`
`dimethylated.
`
`For the purposes of the present invention, an in vitro GLP-l receptor—signaling
`
`assay is used to determine whether a particular extended GLP-l peptide will exhibit
`
`insulinotropic activity in viva. Extended GLP-l peptides encompassed by the present
`
`invention have an in vitro potenCy that is not less than one-tenth the in vitro potency of
`
`30
`
`the DPP IV resistant GLP-l analog known as ValS-GLP-1(7—37)OH. More preferably, the
`
`MYLAN INST. EXHIBIT 1014 PAGE 7
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`MYLAN INST. EXHIBIT 1014 PAGE 7
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`MYLAN INST. EXHIBIT 1014 PAGE 7
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`
`extended GLP—l peptides of the present invention are as potent or more potent than
`
`ValS—GLP-1(7-37)OH.
`
`“In vitro potency” as used herein is the measure of the ability of a peptide to
`
`activate the GLP-1 receptor in a cell-based assay. In vitro potency is expressed as the
`
`“EC50” which is the effective concentration of compound that results in 50% activity in a
`
`single dose—response experiment. For the purposes of the present invention, in vitro
`
`potency is determined using a fluorescence assay that employs HEK-293 Aurora CRE-
`
`BLAM cells that stably express the human GLP-1 receptor. These HEK—293 cells have
`
`stably integrated a DNA vector having a CAMP response element (CRE) driving
`
`10
`
`expression of the B-lactamase (BLAM) gene. The interaction of a GLP—l agonist with the
`
`receptor initiates a signal that results in activation of the CAMP response element and
`
`subsequent expression of B-lactamase. The B-lactamase CCFZ/AM substrate that emits
`
`fluorescence when it is cleaved by B-lactamase (Aurora Biosciences Corp.) can then be
`
`added to cells that have been exposed to a specific amount of GLP-1 agonist to provide a
`
`15
`
`measure of GLP-1 agonist potency. The assay is further described in Zlokarnik, er al.
`
`(1998) Science 279:84-88 (See also Example 1). The EC50 values for the compounds
`
`listed in example 1 were determined using the BLAM assay described above by
`
`generating a dose response curve using dilutions ranging from 0.00003 nanomolar to 30
`
`nanomolar. Relative in vitro potency values are established by running
`
`20
`
`Valg-GLP-1(7—37)OH as a control and assigning the control a reference value of 1.
`
`The term “delivery agent” refers to molecules in US. Patents 5,541,155;
`
`5,693,338; 5,976,569; 5,643,957; 5,955,503; 6,100,298; 5,650,386; 5,866,536;
`
`5,965,121; 5,989,539; 6,001,347; 6,071,510; 5,820,881; and 6,242,495; and
`
`WO 02/02509; WO 01/51454; W0 01/44199; WO 01/32130; WO 00/59863;
`
`25
`
`WO 00/503 86; WO 00/47188; and WO 00/40203. The delivery agents are generally
`
`derived from amino acids and are useful in the oral formulations of the present invention.
`
`The derived amino acids can also be in the form of poly amino acids, and peptides. An
`
`amino acid is any carboxylic acid having at least one free amine group and includes
`naturally occurring and synthetic amino acids. Poly amino acids are either peptides or
`
`30
`
`two or more amino acids linked by a bond formed by other groups which can be linked,
`
`e.g., an ester, anhydride, or an anhydride linkage. Peptides are two or more amino acids
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`joined by a peptide bond. Peptides can vary in length from dipeptides with two amino
`
`acids to polypeptides with several hundred amino acids. Preferred peptides include di—
`
`peptides, tri-peptides, tetra-peptides, and penta—peptides.
`
`Furthermore, the delivery agents of the present invention are optionally in a salt
`
`form. Examples of salts include sodium, hydrochloric acid, sulfuric acid, phosphoric
`
`acid, citric acid, acetic acid, sulfate, phosphate, chloride, bromide, iodide, acetate,
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`propionate, hydrobromic acid, sodium hydroxide, potassium hydroxide, ammonium
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`hydroxide, and potassium carbonate.
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`10'
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`The various oral formulations of the present invention may optionally encompass
`
`a pharmaceutically acceptable buffer. Examples of pharmaceutically acceptable buffers
`
`include phosphate buffers such as dibasic sodium phosphate, TRIS, glycylglycine,
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`maleate, sodium acetate, sodium citrate, sodium tartrate, or an amino acid such as glycine,
`
`histidine, lysine or arginine. Other pharmaceutically acceptable buffers are known in the
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`15
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`art. Preferably, the buffer is selected from the group consisting of phosphate, TRIS,
`
`maleate, and glycine. Even more preferably the buffer is TRIS.
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`Preferably, the TRIS concentration is between about 1 mM and 100 mM. Even
`
`more preferably, the concentration is between about 10 mM and about 50 mM, most
`
`preferably the buffer is about 20 mM.
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`20
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`The pH of the oral formulations is adjusted to provide stability and to be
`
`acceptable for oral administration. Preferably, the pH is adjusted to between about 7.0
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`and about 9.0, more preferably the pH is between about 7.4 and 8.4. Even more
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`preferably the pH is between about 7.8 and 8.4. Most preferably, the pH is between about
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`7.8 and 8.1.
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`25
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`The various oral formulations of the present invention may optionally encompass
`
`a suspending agent. Some delivery agents require a suspending agent due to their
`
`solubility characteristics. An example of a suspending agent is hydroxypropyl—
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`methylcellulose. Preferably, the final concentration of hydroxypropylmethylcellulose is
`
`between about 2% and about 10% (weight/volume). Even more preferably, the
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`30
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`concentration is between about 2% and about 5% (w/v). Most preferably the
`
`concentration is about 3.9% (w/v).
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`The oral formulations of the present invention may optionally comprise a
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`cosolvent. Some delivery agents require cosolvents due to their solubility characteristics.
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`Examples of cosolvents include ethanol, N—methylpyrrolidone, N,N-dimethylacetamide,
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`N,N-dimethy1formamide, glycofurol, ethoxydiol, propylene glycol, polyethylene glycol
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`300 and polyvinylpyrrolidone. Preferably, the final concentration of the cosolvents is
`
`between about 5% and about 30% (volume/volume). Even more preferably, the
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`concentration is between about 10% and about 25% (v/v). Most preferably the
`
`concentration is about 20% (v/v).
`
`The oral formulations of the present invention may optionally comprise a
`
`preservative. Preservative refers to a compound that is added to the formulation to act as
`
`an antimicrobial agent. Among preservatives known in the art as being effective and
`
`acceptable in parenteral formulations are phenolic preservatives, alkylparabens, benzyl
`
`alcohol, chlorobutanol, resorcinol, and other similar preservatives, and various mixtures
`
`thereof. Examples of phenolic derivatives include cresols and phenol or a mixture of
`
`cresols and phenol. Examples of cresols include meta-cresol, ortho-cresol, para—cresol,
`
`chlorocresol, or mixtures thereof. Alkylparaben refers to a C1 to C4 alkylparaben, or
`
`mixtures thereof. Examples of alkylparabens include methylparaben, ethylparaben,
`
`‘ propylparaben, or butylparaben. The concentrations must be sufficient to maintain
`
`preservative effectiveness by retarding microbial growth. Preferably, the preservative is a
`
`phenol derivative. More preferably the preservative is a cresol. Even more preferably the
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`10
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`15
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`20
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`preservative is meta—cresol.
`
`A preferred concentration of a preservative in the final mixture is about
`
`1.0 mg/mL to about 20.0 mg/mL. More preferred ranges of concentration of preservative
`
`in the final mixture are about 2.0 mg/mL to about 8.0 mg/mL, about 2.5 mg/mL to about
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`25
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`4.5 mg/mL and about 2.0 mg/mL to about 4.0 mg/mL. A most preferred concentration of
`
`preservative in the final mixture is about 3.0 mg/mL.
`
`The oral formulations of the present invention may optionally comprise an
`
`isotonicity agent. Isotonicity agents refer to compounds that are tolerated physiologically
`
`and impart a suitable tonicity to the formulation to prevent the net flow of water across
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`30
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`cell membranes. Examples of such compounds include glycerin, salts, e.g., NaCl, and
`
`sugars, e.g., dextrose, mannitol, and sucrose. These compounds are commonly used for
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`such purposes at known concentrations. One or more isotonicity agents may be added to
`
`adjust the ionic strength or tonicity. The preferred isotonicity agent is NaCl. The
`
`concentration of the NaCl is preferably between about 10 mM and 200 mM, more
`
`preferred is between about 50 mM and 150 mM, and most preferred is about 100 mM.
`
`The administration compositions may alternatively be in the form of a solid, such
`
`as a tablet, capsule or particle, such as a powder. Solid dosage forms may be prepared by
`
`‘ mixing the solid form of the compound with the solid form of the active agent.
`
`Alternatively, a solid may be obtained from a solution of compound and active agent by
`
`methods known in the art, such as freeze drying, precipitation, crystallization ad solid
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`10
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`dispersion.
`
`GLP—J compounds appropriatefor use in the present invention:
`
`The GLP-l compounds of the present invention can be made by a variety of
`
`methods known in the art such as solid—phase synthetic chemistry, purification of GLP—1
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`15
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`molecules from natural sources, recombinant DNA technology, or a combination of these
`
`methods. For example, methods for preparing GLP-l peptides are described in United
`
`States Patent Nos. 5,118,666; 5,120,712; 5,512,549; 5,977,071; and 6,191,102.
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`By custom in the art, the amino terminus of GLP-1(7-37)OH has been assigned
`
`number residue 7, and the carboxy—terminus has been assigned number 37. The other
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`20
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`amino acids in the polypeptide are numbered consecutively, as shown in SEQ ID NO:1.
`
`For example, position 12 is phenylalanine and position 22 is glycine.
`
`The two naturally occurring truncated GLP-l peptides are represented in
`
`Formula I, SEQ ID NO:1.
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`25
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`His7—A1a-Glu-Gly1O-Thr-Phe-Thr—Ser—Asp—Val-Ser—Ser—Tyr-LeuZO-Glu-
`
`G1y—Gln—Ala-Ala25-Lys-G1u—Phe-Ile-A1a30-Trp~Leu-Val—Lys-Gly35—
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`Arg—Xaa37
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`Formula I, SEQ ID N011
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`30
`
`wherein:
`
`Xaa37 is Gly, or -NH2.
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`Preferably, a GLP—l compound has the amino acid sequence of SEQ ID NO:1 or is
`
`modified so that from one, two, three, four or five amino acids differ from SEQ ID N021.
`
`A preferred group of GLP-1 compounds is composed of GLP-1 analogs of
`
`5
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`Formula I (SEQ ID NO:2).
`
`His-Xaag-Xaag-Gly—Xaa1 1-Phe-Thr-Xaa14-Asp-Xaa1 6—Xaa17—Xaa1 8-Xaa1 9—
`
`XaazO-XaaZI-Xaa22-Xaa23~Xaa24-Xaa25-Xaa26-Xaa27-Phe-Ile-Xaa3O-Xaa31—
`
`Xaa32-Xaa33-Xaa34-Xaa35—Xaa36-Xaa37—Xaa3 8-Xaa39—Xaa40-Xaa41—Xaa42-
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`10
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`Xaa43 —Xaa44-Xaa45
`
`Formula I (SEQ ID NO:2)
`
`wherein:
`
`Xaa8 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
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`15
`
`Xaa9 is Glu, Asp, or Lys;
`
`Xaa11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
`
`Xaa14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`
`Xaa16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp, or Lys;
`
`Xaa17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`
`20
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`Xaa18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr, or Lys;
`
`Xaa19 is Tyr, Phe, Trp, Glu, Asp, Gln, or Lys;
`
`Xaa2O is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp, Tyr, or Lys;
`
`Xaa21 is Glu, Asp, or Lys;
`
`Xaa22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`
`25
`
`Xaa23 is Gln, Asn, Arg, Glu, Asp, or Lys;
`
`Xaa24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, or Lys;
`
`Xaa25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`
`Xaa26 is Lys, Arg, Gln, Glu, Asp, or His;
`
`Xaa27 is Leu, Glu, Asp, or Lys;
`
`30
`
`Xaa3O is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`
`Xaa31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
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`Xaa32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
`
`Xaa33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, or Lys;
`
`Xaa34 is Asn, Lys, Arg, Glu, Asp, or His;
`
`Xaa35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`
`Xaa36 is Gly, Arg, Lys, Glu, Asp, or His;
`
`Xaa37 is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys, or is deleted;
`
`Xaa38 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted;
`
`Xaa39 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted;
`
`Xaa40 is Gly, Asp, Glu, or Lys, or is deleted;
`
`10
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`Xaa41 is Ala, Phe, Trp, Tyr, Glu, Asp, or Lys, or is deleted;
`
`Xaa42 is Ser, Pro, Lys, Glu, or Asp, or is deleted;
`
`Xaa43tis Ser, Pro, Glu, Asp, or Lys, or is deleted;
`
`Xaa44 is Gly, Pro, Glu, Asp, or Lys, or is deleted; and
`
`Xaa45 is Ala, Ser, Val, Glu, Asp, or Lys, Ala—NHZ, Ser—NHZ, Val-NHz, Glu—NHZ,
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`15
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`Asp-NHZ, or Lys-NHz, or is deleted;
`
`provided that when the amino acid at position 37, 38, 39, 40, 41, 42, 43, or 44 is deleted,
`
`then each amino acid downstream of that amino acid is also deleted.
`
`It is preferred that the GLP-l compound of formula I contain less than six amino
`
`acids that differ from the corresponding amino acid in GLP-1(7-37)OH or Exendin—4. It
`
`20
`
`is more preferred that less than five amino acids differ from the corresponding amino acid
`
`in GLP—1(7-3 7)OH or Exendin-4. It is even more preferred that less than four amino
`
`acids differ from the corresponding amino acid in GLP-1(7-37)OH or Exendin-4.
`
`GLP-l compounds of the present invention include derivatives of formula I such
`
`as a C-1-6-ester, or amide, or C-1-6-alkylamide, or C-1-6-dialky1amide thereof.
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`25
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`WO99/43706 describes derivatives of GLP—1 compounds of formula I and is incorporated
`
`by reference herein in its entirety. The compounds of formula I derivatized as described
`
`in WO 99/43706 and underivatized are encompassed by the present invention.
`
`Another preferred group of GLP-1 compounds is composed of GLP—1 analogs of
`
`formula II (SEQ ID NO:3):
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`MYLAN INS