(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`4 September 2003 (04.09.2003)
`
`
`
`PCT
`
`(10) International Publication Number
`WO 03/072195 A2
`
`(51) International Patent Classification’:
`
`A6IP
`
`SK, TR), OAPIpatent (BF, BJ, CF, CG, CI, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG).
`
`(21) International Application Number:=PCT/US03/03111
`Declarations under Rule 4.17:
`as to applicant’s entitlement to apply for and be granted
`a patent (Rule 4.17(ii)) for the following designations AF,
`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. 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, IU, IE, ff LU, MC, NL, PT, SE, SI, SK, TR), OAPI
`patent (BF. BJ. CF CG, Cl, CM, GA, GN, GO, GW, MT,
`MR, NE, SN, TD, TG)
`as to the applicant’s entitlement to claim the priority ofthe
`earlier application (Rule 4.17(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,
`Ek, 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, 14, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HU, IE, IT, LU, MC, NEI, PT, SE, SE
`SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
`GO, GW, ML, MR, NE, SN, TD, TG)
`of inventorship (Rule 4.17(iv)) for US only
`
`(22) InternationalFiling 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
`(75) Inventor/Applicant (for US only): KHAN, Mohammed,
`Amin [US/US]; 5163 Sue Drive, Carmel, IN 46033 (US).
`
`(74) Agents: DAVIS,Paula, K.ct al.; 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, KS, 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,
`
`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.
`
`(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-
`poundsandspecified delivery agents.
`
`WO03/072195A2
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`WO 03/072195
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`PCT/US03/03111
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`METHOD FOR ADMINISTERING GLP-1 MOLECULES
`
`FIELD OF THE INVENTION
`
`The present invention relates to a formulation useful for the oral administration
`comprising a glucagon-like peptide-1 (GLP-1) compoundand 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, continuousstrides have been madeto 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
`rising blood sugarlevels is not sufficient to allow cells to efficiently take up glucose and
`
`15
`
`thus, reduce blood sugar levels.
`A large body ofpre-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-1 induces numerousbiological 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-1
`
`mayalso act to prevent the 8 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 whenusing insulin therapy or
`sometypes oforal therapies that act by increasing insulin expression.
`|
`However, development of a GLP-1 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-1
`
`compoundsto treat hyperglycemia or other conditions involve formulating GLP-1
`compounds such that they can be delivered by subcutaneousinjection or through
`continuous subcutaneous infusion or continuous intravenous administration, Many type 2
`
`25
`
`30
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`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-1 compoundtherapeutics that can be delivered by an alternative non-
`
`invasive means suchas byoral delivery.
`
`Unfortunately, there are numerousbarriers to effective oral delivery of peptides.
`
`The high acid content and ubiquitous digestive enzymesof 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 becomeinsoluble at the low pH
`
`10
`
`levels encountered in the digestive tract and, thus, are not absorbed effectively.
`
`The fact that GLP-1 compoundsarerelatively unstable in solution formulations,
`
`only remain in solution undera fairly narrow set of conditions, and havea relatively short
`
`in vivo half-life when administered as a solution formulation, suggested that these
`
`compounds could notbe effectively delivered through the oral route. Thus, it was
`surprising that GLP-1 compounds could be formulated such that biologically active
`
`15
`
`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-1 compoundsin a non-covalent fashion to allow the compoundsto
`
`cross gut membranesand yet remain therapeutically active. Although the delivery agents
`
`20
`
`employed in the present invention have been disclosedin a series of U.S. Patents (see
`
`U.S. 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 WO 02/02509; WO 01/51454; WO 01/44199; WO 01/32130;
`
`WO 00/59863; WO 00/50386; WO 00/47188; and WO 00/40203), oral administration of
`
`25
`
`formulations comprising GLP-1 compoundswith these delivery agents has not been
`
`disclosed or suggested. Further, numerous parameters impact whether a particular class
`
`of compoundscanbeeffectively delivered in combination with one or more classes of
`
`delivery agents. For example, the conformationofthe 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 andin vivo stability
`
`all influence the ability to deliver a compoundorally.
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`SUMMARYOF THE INVENTION
`
`The present invention encompasses the development of novel formulations
`
`comprising GLP-1 compoundsand delivery agents that can be administered orally. The
`
`present invention provides a formulation which can be administered orally comprising a
`
`GLP-1 compound anda specified delivery agent. The GLP-1 compound canbe native
`
`GLP-1; GLP-1 fragments; GLP-1 analogs; GLP-1 derivatives of native, fragments, or
`
`analogs of GLP-1; and Exendin-3 and Exendin-4. The delivery agent is selected from
`
`delivery agents described in U.S. 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 WO 02/02509; WO 01/51454;
`
`WO 01/44199; WO 01/32130; WO 00/59863; WO 00/50386; WO 00/47188; and
`
`WO 00/40203.
`
`Preferred GLP-1 compoundsare analogsor 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 Val®-GLP-1(7-37)OH.
`
`Preferred GLP-1 compoundsare also described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID
`
`NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ
`
`ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID
`
`20
`
`NO:14. More preferred GLP-1 compoundsare described in compounds of SEQ ID NO:2,
`
`SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.
`
`Preferred delivery agents are described in Table 1. More preferred delivery agents
`
`are delivery agents corresponding to numbers of Table 1 selected from 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 methodofstimulating the GLP-1
`
`receptor in a subject in need of such stimulation, said method comprising the step of
`
`administering to the subject an effective amountof the oral formulation described herein.
`
`Subjects in need of GLP-1 receptor stimulation include those with non-insulin dependent
`
`30
`
`diabetes and obesity.
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`-4-
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The three-letter abbreviation code for amino acids usedin this specification
`
`conforms with the list contained in Table 3 of Annex C, Appendix 2 of the PCT
`
`Administrative Instructions and with 37 C.F.R. § 1.822(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-1 compound and a
`
`specified delivery agent combined together which can be administered orally such that -
`
`GLP-1 compoundpasses through the gut into the systemic circulation and has theability
`
`10
`
`to bind to the GLP-1 receptor andinitiate a signal transduction pathwayresulting in
`insulinotropic activity. The formulation can optionally comprise other agents so long as
`the GLP-1 retains the ability to bind the GLP-1 receptor.
`
`The term “oral” as used herein refers to delivery of a compound by mouth such
`
`that the compoundpasses through the stomach, small intestine, or large intestine into the
`
`15
`
`systemic circulation.
`
`The term “GLP-1 compound”as used herein refers to polypeptides that include
`
`naturally occurring GLP-1 polypeptides (GLP-1(7-37)OH and GLP-1(7-36)NH9), GLP-1
`
`fragments, GLP-1 analogs, GLP-1 derivatives of naturally occurring GLP-1 polypeptides,
`
`GLP-1 fragments, or GLP-1 analogs, and Exendin-3 and Exendin-4 that have the ability
`
`20
`
`to bind to the GLP-1 receptor andinitiate a signal transduction pathway resulting in
`
`insulinotropic activity.
`
`The term “insulinotropic activity” refers to the ability to stimulate insulin secretion
`
`in responseto 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-1 molecule has insulinotropicactivity
`
`if islet cells secrete insulin levels in the presence of the GLP-1 molecule above
`
`backgroundlevels.
`
`The term “DPPIV resistant”refers to GLP-1 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-1 molecule is DPP IV
`
`resistant if in the presence of DPP IV the GLP-1 molecule has extended metabolic
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`stability above that of native GLP-1. DPP IVresistant GLP-1 molecules can have an
`amino acid change at the DPP IV recognitionsite (position 8), or DPP IV resistant
`peptides can have an attached groupthat restricts the accessibility of the DPP IV to the
`
`recognition site, or both.
`
`A"GLP-1 fragment" is a polypeptide obtained after truncation of one or more
`
`amino acids from the N-terminus and/or C-terminus of GLP-1(7-37)OHor an analog or
`
`derivative thereof. The nomenclature used to describe GLP-1 (7-37)OHis also applicable
`
`to GLP-1 fragments. For example, GLP-1(9-36)OH denotes a GLP-1 fragment obtained
`by truncating two amino acids from the N-terminus and one aminoacid from the C-
`terminus. The amino acids in the fragment are denoted by the same numberas the
`
`10
`
`correspondingaminoacid in GLP-1(7-37)OH. For example, the N-terminal glutamic acid
`in GLP-1(9-36)OHis at position 9; position 12 is occupied by phenylalanine; and position
`
`22 is occupied by glycine, as in GLP-1(7-37)OH. For GLP-1(7-36)OH,the glycine at
`
`position 37 of GLP-1(7-37)OHis deleted.
`A “GLP-1 analog”hassufficient homology to GLP-1(7-37)OH or a fragment of
`
`15
`
`GLP-1(7-37)OHsuchthat the analog has insulinotropicactivity. Preferably, a GLP-1
`analog has the amino acid sequence of GLP-1(7-37)OHora fragment thereof, modified
`
`so that from one, two, three, four or five amino acids differ from the amino acid in
`
`20
`
`corresponding position of GLP-1(7-37)OHor a fragment of GLP-1(7-37)OH. In the
`nomenclature used herein to designate GLP-1 compounds,the substituting amino acid and
`its position is indicated prior to the parent structure. For example, Glu’?-GLP-1(7-37)OH
`designates a GLP-1 compoundin which the glycine normally found at position 22 of
`GLP-1(7-37)OH has beenreplaced with glutamic acid; Val°-Glu’?-GLP-1(7-37)OH
`designates a GLP-1 compoundin which alanine normally found atposition 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-1 molecules also include polypeptides in which one or more amino acids
`
`have been added to the N-terminus and/or C-terminus of GLP-1(7-37)OH,or fragments or
`
`analogs thereof. It is preferred that GLP-1 molecules of this type have up to aboutthirty-
`
`30
`
`nine amino acids. The aminoacids in the “extended” GLP-1 molecule are denoted by the
`
`game numberas the corresponding amino acid in GLP-1(7-37)OH. For example, for a
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`
`GLP-1 molecule obtained by adding two amino acids to the N-terminus of
`
`GLP-1(7-37)OH,the N-terminal amino acidis located at position 5; and for a GLP-1
`
`molecule obtained by adding one aminoacid to the C-terminus of GLP-1(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 ofthese “extended” GLP-1
`compounds, as in GLP-1(7-37)OH. Amino acids 1-6 of an extended GLP-1 molecule are
`
`preferably the sameas or a conservative substitution of the aminoacid at the
`corresponding position of GLP-1(1-37)OH. Amino acids 38-45 of an extended GLP-1
`molecule are preferably the sameas or a conservative substitution of the amino acid at the
`
`10
`
`corresponding position of glucagon or Exendin-4.
`
`A"GLP-1 derivative" refers to a molecule having the amino acid sequence of
`
`GLP-1, a GLP-1 fragment, or a GLP-1 analog, but additionally having chemical
`
`modification of one or more of its amino acid side groups, o-carbon atoms, terminal :
`
`amino group, or terminal carboxylic acid group. A chemical modification includes,butis
`not limited to, adding chemical moieties, creating new bonds, and removing chemical
`
`15
`
`moieties. Modifications at aminoacid side groups include, withoutlimitation, acylation
`
`of lysine e-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of
`glutamic or aspartic carboxylic acid groups, and deamidation of glutamineor asparagine.
`Modifications of the terminal amino group include, without limitation, the des-amino,
`
`20
`
`N-loweralkyl, N-di-loweralkyl, and N-acyl modifications. Modificationsof the terminal
`
`carboxy group include, withoutlimitation, the amide, lower alkyl amide, dialkyl amide,
`
`and lower alkyl ester modifications. Lower alkyl is C;-C, alkyl. Furthermore, one or
`
`more side groups, or terminal groups, may be protected by protective groups known to the
`
`ordinarily-skilled protein chemist. The a-carbon of an amino acid may be mono- or
`
`25
`
`dimethylated.
`
`For the purposesof the present invention, an in vitro GLP-1 receptor-signaling
`
`assay is used to determine whether a particular extended GLP-1 peptide will exhibit
`insulinotropic activity in vivo. Extended GLP-1 peptides encompassedby the present
`
`invention have an in vitro potencythatis not less than one-tenth the in vitro potency of
`the DPP IV resistant GLP-1 analog known as Val°-GLP-1(7-37)OH. Morepreferably, the
`
`30
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`extended GLP-1 peptides of the present invention are as potent or more potent than
`Val®-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
`
`“ECs” whichis the effective concentration of compoundthat results in 50% activity in a
`
`single dose-response experiment. For the purposesof the present invention, in vitro
`
`potencyis determined using a fluorescence assay that employs HEK-293 Aurora CRE-
`
`BLAMcells that stably express the human GLP-1 receptor. These HEK-293cells have
`
`stably integrated a DNA vector having a cAMPresponse element (CRE) driving
`
`10
`
`expression ofthe B-lactamase (BLAM)gene. The interaction of a GLP-1 agonist with the
`
`receptor initiates a signal that results in activation of the cAMP response element and
`
`subsequent expression of 8-lactamase. The B-lactamase CCF2/AM substrate that emits
`
`fluorescence whenit is cleaved by B-lactamase (Aurora Biosciences Corp.) can then be
`
`added to cells that have been exposedto a specific amount of GLP-1 agonist to provide a
`
`15
`
`measure of GLP-1 agonist potency. The assay is further described in Zlokarnil, et al.
`
`(1998) Science 279:84-88 (See also Example 1). The ECsp 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
`Val®-GLP-1(7-37)OHasa control and assigning the control a reference value of1.
`
`20
`
`The term “delivery agent” refers to molecules in U.S. 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; WO 01/44199; WO 01/32130; WO 00/59863;
`
`25
`
`WO 00/50386; WO 00/47188; and WO 00/40203. Thedelivery 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 andincludes
`naturally occurring and synthetic amino acids. Poly amino acids are either peptides or
`two or more amino acids linked by a bond formed by other groups which can be linked,
`
`30
`
`e.g., an ester, anhydride, or an anhydride linkage. Peptides are two or more amino acids
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`-8-
`
`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. Examplesofsalts include sodium, hydrochloric acid, sulfuric acid, phosphoric
`
`acid, citric acid, acetic acid, sulfate, phosphate, chloride, bromide, iodide, acetate,
`
`propionate, hydrobromicacid, sodium hydroxide, potassium hydroxide, ammonium
`hydroxide, and potassium carbonate.
`
`10°
`
`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,
`
`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
`
`15
`
`art. Preferably, the buffer is selected from the group consisting of phosphate, TRIS,
`
`maleate, and glycine. Even more preferably the buffer is TRIS.
`
`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.
`
`20
`
`The pH oftheoral formulations is adjusted to provide stability and to be
`
`acceptable for oral administration. Preferably, the pH is adjusted to between about 7.0
`
`and about 9.0, more preferably the pH is between about 7.4 and 8.4. Even more
`
`preferably the pH is between about 7.8 and 8.4. Mostpreferably, the pH is between about
`
`7.8 and 8.1.
`
`25
`
`The various oral formulations of the present invention may optionally encompass
`
`a suspending agent. Somedelivery agents require a suspending agent dueto their
`solubility characteristics. An example of a suspending agent is hydroxypropyl-
`methylcellulose. Preferably, the final concentration of hydroxypropylmethylcelluloseis
`between about 2% and about 10% (weight/volume). Even more preferably, the
`
`30
`
`concentration is between about 2% and about 5% (w/v). Mostpreferably the
`
`concentration is about 3.9% (w/v).
`
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`The oral formulations of the present invention may optionally comprise a
`
`cosolvent. Somedelivery agents require cosolvents due to their solubility characteristics.
`
`Examples of cosolvents include ethanol, N-methylpyrrolidone, N,N-dimethylacetamide,
`
`N,N-dimethylformamide, glycofurol, ethoxydiol, propylene glycol, polyethylene glycol
`
`300 and polyvinylpyrrolidone. Preferably, the final concentration ofthe cosolvents is
`
`between about 5% and about 30% (volume/volume). Even morepreferably, the
`
`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
`
`10
`
`preservative. Preservative refers to a compoundthat is added to the formulation to act as
`
`an antimicrobial agent. Amongpreservatives knownin 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 pheno! or a mixture of
`
`15
`
`cresols and phenol. Examplesof cresols include meta-cresol, ortho-cresol, para-cresol,
`
`chlorocresol, or mixtures thereof. Alkylparaben refers to a C; 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
`
`20
`
`phenol derivative. More preferably the preservative is a cresol. Even more preferably the
`
`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. Morepreferred 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
`
`25
`
`4.5 mg/mL and about 2.0 mg/mL to about 4.0 mg/mL. A most preferred concentration of
`
`preservativein the final mixture is about 3.0 mg/mL.
`
`The oral formulations of the present invention may optionally comprise an
`
`isotonicity agent. Jsotonicity agents refer to compoundsthat are tolerated physiologically
`
`and impart a suitable tonicity to the formulation to prevent the net flow of water across
`
`30
`
`cell membranes. Examples of such compoundsinclude 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 addedto
`adjust the ionic strength or tonicity. The preferred isotonicity agent is NaCl. The
`concentration of the NaClis preferably between about 10 mM and 200 mM, more
`
`preferred is between about 50 mM and 150 mM,and mostpreferred is about 100 mM.
`The administration compositions mayalternatively be in the form ofa 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 compoundandactive agent by
`methods knowninthe art, such as freeze drying, precipitation, crystallization ad solid
`
`10
`
`dispersion.
`
`GLP-1 compounds appropriatefor use in the present invention:
`The GLP-1 compoundsofthe present invention can be made byavariety of
`
`methods knownin the art suchas solid-phase synthetic chemistry, purification of GLP-1
`
`15
`
`molecules from natural sources, recombinant DNAtechnology, or a combination of these
`
`methods. For example, methods for preparing GLP-1 peptides are described in United
`
`States Patent Nos. 5,118,666; 5,120,712; 5,512,549; 5,977,071; and 6,191,102.
`
`By custom in the art, the amino terminus of GLP-1(7-37)OH has been assigned
`numberresidue 7, and the carboxy-terminus has been assigned number 37. The other
`
`20
`
`amino acidsin the polypeptide are numbered consecutively, as shown in SEQ ID NO:1.
`
`For example, position 12 is phenylalanine andposition 22 is glycine.
`
`The two naturally occurring truncated GLP-1 peptides are represented in
`
`Formula J, SEQ ID NO:1.
`
`25
`
`His’-Ala-Glu-Gly' °_Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Ty:-Leu”’-Glu-
`Gly-Gln-Ala-Ala”*-Lys-Glu-Phe-Ile-Ala*”-Trp-Leu-Val-Lys-Gly**-
`Arg-Xaa”’
`Formula I, SEQ ID NO:1
`
`30
`
`wherein:
`
`Xaa?’is Gly, or -NH)p,
`
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`Preferably, a GLP-1 compound has the amino acid sequence of SEQ ID NO:1 oris
`
`modified so that from one, two, three, four or five amino acids differ from SEQ ID NO:1.
`
`A preferred group of GLP-1 compounds is composed of GLP-1 analogs of
`
`5
`
`Formula I (SEQ ID NO:2).
`
`His-Xaa®-Xaa’-Gly-Xaa' !_Phe-Thr-Xaa!*-Asp-Xaa!°Xaa!7-Xaa'8-Xaa’?-
`Xaa”-Xaa?!-Xaa’?-Kaa>-Xaa*-Xaa”>-Xaa”*-Xaa”’-Phe-Ile-Xaa’’-Xaa’!-
`
`Xaa??-Xaa®?-Kaa**-Kaa*>-Xaa*’-Xaa>’-Xaa*®-Xaa*’-Kaa’-Xaa’/-Kaa‘’-
`
`Xaa-Xaa*-Xaa
`
`Formula I (SEQ ID NO:2)
`
`wherein:
`
`Xaa® is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`Xaa’ is Glu, Asp, or Lys;
`Xaa!! is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
`Xaa"* is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`Xaa!®is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp, or Lys;
`Xaa!” is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`Xaa’® is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr, or Lys;
`Xaal’ is Tyr, Phe, Trp, Glu, Asp, Gln, or Lys;
`Xaa”is Leu, Ala, Gly, Ser, Thr, Ne, Val, Glu, Asp, Met, Trp, Tyr, or Lys;
`Xaa”’ is Glu, Asp,or Lys;
`Xaa”’ is Gly, Ala, Ser, Thr, Leu, Mle, Val, Glu, Asp, or Lys;
`Xaa”™is Gln, Asn, Arg, Glu, Asp, or Lys;
`Xaa™ is Ala, Gly, Ser, Thr, Leu,Ile, Val, Arg, Glu, Asp, or Lys;
`Xaa”is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`Xaa”®is Lys, Arg, Gln, Glu, Asp, or His;
`Xaa’’ is Leu, Glu, Asp, or Lys;
`Xaa””is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`Xaa*! is Trp, Phe, Tyr, Glu, Asp, or Lys;
`
`10
`
`15
`
`20
`
`25
`
`30
`
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`Xaa™is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
`Xaa’’ is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, or Lys;
`Xaa* is Asn, Lys, Arg, Glu, Asp, or His;
`Xaa*> is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
`Xaa’® is Gly, Arg, Lys, Glu, Asp,or His;
`Xaa’’ is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys, or is deleted;
`Xaa’®is Ser, Arg, Lys, Glu, Asp, or His, or is deleted;
`Xaa>’ is Ser, Arg, Lys, Glu, Asp, or His, or is deleted;
`Xaa”is Gly, Asp, Glu, or Lys, or is deleted;
`Xaa"! is Ala, Phe, Trp; Tyr, Glu, Asp,or Lys, or is deleted;
`Xaa”is Ser, Pro, Lys, Glu, or Asp,oris deleted;
`Xaa™is Ser, Pro, Glu, Asp, or Lys,or is deleted;
`Xaa™is Gly, Pro, Glu, Asp, or Lys,or is deleted; and
`Xaa‘} is Ala, Ser, Val, Glu, Asp, or Lys, Ala-NH>, Ser-NH2, Val-NH2, Glu-NHp,
`
`Asp-NHb2, or Lys-NH), 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-1 compoundof formula I contain less than six amino
`acids that differ from the corresponding amino acid in GLP-1(7-37)OHor Exendin-4. It
`is more preferred that less than five amino acids differ from the corresponding aminoacid
`in GLP-1(7-37)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-1 compoundsofthe 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-dialkylamide thereof.
`
`W099/43706 describes derivatives of GLP-1 compoundsof formula I and is incorporated
`by reference herein in its entirety. The compoundsof formula I derivatized as described
`in WO 99/43706 and underivatized are encompassed by the present invention.
`
`Anotherpreferred group of GLP-1 compounds is composed of GLP-1 analogs of
`
`formula II (SEQ ID NO:3):
`
`10
`
`15
`
`20
`
`25
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`Xaa’-Xaa®-Xaa?-Gly-Xaa!!-Xaa!?-Thr-Ser-Asp-Xaa'°-Ser-Xaa' 8
`Xaa!?-Leu-Glu-Gly-Xaa™-Xaa"*-Ala-Xaa”®-Xaa’’-Phe-Ile-Xaa’”-
`Xaa*!-Leu-Kaa**-Xaa™*Xaa’’-Kaa’’-R””
`
`Formula Il (SEQ ID NO:3)
`
`10
`
`15
`
`wherein:
`
`Xaa’ is: L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, 8-hydroxy-
`
`histidine, homohistidine, a-fluoromethyl-histidine or a-methyl-histidine;
`Xaa’is: Gly, Ala, Val, Leu,Ile, Ser, or Thr;
`Xaa’ is: Thr, Ser, Arg, Lys, Trp, Phe, Tyr, Glu, or His;
`Xaal!! is:
`
`Asp, Glu, Arg, Thr, Ala, Lys, or His;
`
`Xaa’