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`US 20050148497Al
`
`(19) United States
`(12) Patent Application Publication
`Khan
`
`(10) Pub. No.: US 2005/0148497 Al
`Jul. 7, 2005
`(43) Pub. Date:
`
`(54) METHOD FOR ADMINISTERING GLP-1
`MOLECULES
`
`(76)
`
`Inventor: Mohammed Amin Khan, Carmel, IN
`(US)
`
`Correspondence Address:
`ELI LILLY AND COMPANY
`PATENT DIVISION
`P.O. BOX 6288
`INDIANAPOLIS, IN 46206-6288 (US)
`
`(21) Appl. No.:
`
`10/504,717
`
`(22) PCT Filed:
`
`Feb. 7, 2003
`
`(86) PCT No.:
`
`PCT/US03/03111
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/358,184, filed on Feb.
`20, 2002.
`
`Publication Classification
`
`Int. Cl.7
`(51)
`(52) U.S. Cl .
`
`.......................... A61K 38/26; A61K 38/17
`.................................................................. 514/8
`
`(57)
`
`ABSTRACT
`
`The invention encompasses formulations that demonstrate
`the feasibility of oral absorption comprising GLP-1 com(cid:173)
`pounds and specified delivery agents.
`
`FRESENIUS EXHIBIT 1018
`Page 1 of 47
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`US 2005/0148497 A l
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`Jul. 7, 2005
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`1
`
`METHOD FOR ADl\HNISTERING GLP-1
`MOLECULES
`
`FIELD OF THE INVENTION
`
`[0001) The present invention relates to a formulation
`useful for the oral administration comprising a glucagon-like
`peptide-1 ( GLP-1) 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
`
`[0002) Over the past several decades, continuous strides
`have been made to improve the treatment of diabetes mel(cid:173)
`litus. 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 sugar levels is not
`sufficient to allow cells to efficiently take up glucose and
`thus, reduce blood sugar levels.
`
`[0003) A large body of pre-clinical and clinical research
`data suggests that glucagon-Iike peptide-1 (GLP-1) com(cid:173)
`pounds show great promise as a treatment for type 2 diabetes
`and other conditions. GLP-1 induces numerous biological
`effects such as stimulating insulin secretion, inhibiting glu(cid:173)
`cagon secretion, inhibiting gastric emptying, enhancing glu(cid:173)
`cose utilization, and inducing weight loss. Further, pre(cid:173)
`clinical studies suggest that GLP-1 may also act to prevent
`the ~ cell deterioration that occurs as the disease progresses.
`Perhaps the most salient characteristic of GLP-1 is iLs ability
`to stimulate insulin secretion without the associated risk of
`hypoglycemia that is seen when using insulin therapy or
`some types of oral therapies that act by increasing insulin
`expression.
`[0004) However, development of a GLP-1 therapeutic bas
`been extremely difficult. This is primarily due to the insta(cid:173)
`bility of the peptide during manufacturing proce.sses, in
`solution formulations, and in vivo. The only published
`clinical studies employing GLP-1 compounds to treat hyper(cid:173)
`glycemia or other conditions involve formulating GLP-1
`compounds such that they can be delivered by subcutaneous
`injection or through continuous subcutaneous infusion or
`continuous intravenous administration. Many type 2 diabet(cid:173)
`ics or obese patients desiring to Jose weight will not be
`willing to undertake a treatment regimen that may involve
`several injections per clay. Thus, there is a need to develop
`GLP-1 compound therapeutics that can be delivered by an
`alternative non-invasive means such as by oral delivery.
`
`[0005) Unfortunately, there are numerous barriers to effec(cid:173)
`tive 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 levels encountered in the
`digestive tract and, thus, are not absorbed effectively.
`
`[0006) The fact that GLP-1 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
`surprising that GLP-1 compounds could be formulated such
`that biologically active molecules were absorbed into the
`blood stream after oral administration.
`[0007) The present invention involves the use of specific
`delivery agent molecules that interact with GLP-1 com(cid:173)
`pounds in a non-covalent fashion to allow the compounds to
`cross gut membranes and yet remain therapeutically active.
`Although the delivery agents employed in the present inven(cid:173)
`tion have been disclosed in a series of U.S . patents (see U.S.
`Pat. 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 formulations
`comprising GLP-1 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 formulated component, as well as susceptibility
`to protease digestion and in vivo stability all influence the
`ability to deliver a compound orally.
`
`SUMMARY OF THE INVENTION
`[0008) The present invention encompasses the develop(cid:173)
`ment of novel formulations comprising GLP-1 compounds
`and delivery agenLs that can be administered orally. The
`present invention provides a formulation which can be
`administered orally comprising a GLP-1 compound and a
`specified delivery agent. The GLP-1 compound can be
`native GLP-1; GLP-1 fragments; GLP-1 analogs; GLP-1
`derivatives of native, fragmenLs, or analogs of GLP-1; and
`Exendin-3 and Exendin-4. The delivery agent is selected
`from delivery agents described in U.S. Pat. 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; and WO 02/02509;
`WO 01/51454; WO0l/44199; WO0l /32130; WO00/59863;
`WO00/50386; WO00/47188; and WO 00/40203.
`[0009) Preferred GLP-1 compounds are analogs or deriva(cid:173)
`tives of analogs having 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 Val8
`-
`GLP-1(7-37) OH. Preferred GLP-1 compounds are also
`described in SEQ ID NO:l, 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 NO: 14.
`More preferred GLP-1 compounds are described in com(cid:173)
`pounds of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:13,
`and SEQ ID NO: 14
`[0010) Preferred delivery agents are described in Table l.
`More preferred delivery agents are delivery agents corre(cid:173)
`sponding 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, 38, 39, 40, 41, 42, 43, 44, 46,
`51, 52, and 54.
`
`FRESENIUS EXHIBIT 1018
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`US 2005/0148497 Al
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`Jul. 7, 2005
`
`2
`
`[0011] 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 admin(cid:173)
`istering 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 depen(cid:173)
`dent diabetes and obesity.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0012] 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 C.F.R. § 1.822(d)(1)(2000).
`[0013] For purposes of the present invention as disclosed
`and described herein, the following terms and abbreviations
`are defined as follows.
`[0014] 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
`compound passes through the gut into the systemic circu(cid:173)
`lation and has the ability to bind to the GLP-1 receptor and
`initiate a signal transduction pathway resulting in insulino(cid:173)
`tropic activity. The formulation can optionally comprise
`other agents so long as the GLP-1 retains the ability to bind
`the GLP-1 receptor.
`[0015] The term "oral" 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 systemic circulation.
`[0016] The term "OLP-I compound" as used herein refers
`to polypeptides that include naturally occurring GLP-1
`polypeptides (GLP-1(7-37)OH and GLP-1(7-36)NH:z),
`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 to bind to the GLP-1 receptor and initiate a signal
`transduction pathway resulting in insulinotropic activity.
`[0017] 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, insulinotro(cid:173)
`pic activity can be determined using the method described in
`Example 1. A GLP-1 molecule has insulinotropic activity if
`islet cells secrete insulin levels in the presence of the GLP-1
`molecule above background levels.
`[0018] The term " OPP IV resistant" refers to GLP-1
`molecules that have extended metabolic stability and
`improved biological activity. For example, DPP IV resis(cid:173)
`tance can be determined using the method described in
`Example 2. A GLP-1 molecule is OPP Iv resistant if in the
`presence of OPP IV the GLP-1 molecule bas extended
`metabolic stability above that of native GLP-1. OPP IV
`resistant GLP-1 molecules can bave an amino acid change at
`the OPP IV recognition site (position 8), or OPP IV resistant
`peptides can have an attached group that restricts the acces(cid:173)
`sibility of tbe OPP IV to tbe recognition site, or both.
`[0019] 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)OH or an
`
`analog or derivative thereof. The nomenclature used to
`describe GLP-1 (7-37)OH is 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 amino acid from the C-terminus. The
`amino acids in the fragment are denoted by the same number
`as the corresponding amino acid in GLP-1(7-37)OH. For
`example, the N-terminal glutamic acid in GLP-1(9-36)OH is
`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)OH is deleted.
`[0020] A "GLP-1 analog" bas sufficient homology to
`GLP-1(7-37)OH or a fragment of GLP-1(7-37)OH such that
`the analog has insulinotropic activity. Preferably, a GLP-1
`analog bas 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-1(7-37)OH or a fragment of
`GLP-1(7-37)OH. In the nomenclature used herein to desig(cid:173)
`nate GLP-1 compounds, the substituting amino acid and its
`position is indicated prior to the parent structure. For
`example, Glu22-GLP-1(7-37)OH designates a GLP-1 com(cid:173)
`pound in which the glycine normally found at position 22 of
`GLP-1(7-37)OH has been replaced with glutamic acid;
`Val8-Glu22-GLP-1(7-37)OH designates a GLP-1 compound
`in which alanine normally found at position 8 and glycine
`normally found at position 22 of GLP-1(7-37)OH have been
`replaced with valine and glutamic acid, respectively.
`[0021] 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 frag(cid:173)
`ments or analogs thereof. It is preferred that GLP-1 mol(cid:173)
`ecules of this type have up to about thirty-nine amino acids.
`The amino acids in the "extended" GLP-1 molecule are
`denoted by the same number as the corresponding amino
`acid in GLP-1(7-37)OH. For example, for a GLP-1 molecule
`obtained by adding two amino acids to the N-terminus of
`GLP-1(7-37)OH, the N-terminal amino acid is located at
`position 5; and for a GLP-1 molecule obtained by adding
`one amino acid 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 of these "extended'' GLP-1
`compounds, as in GLP-1(7-37)OH. Amino acids 1-6 of an
`extended GLP-1 molecule are preferably the same as or a
`conservative substitution of the amino acid at the corre(cid:173)
`sponding position of GLP-1(1-37)OH. Amino acids 38-45 of
`an extended GLP-1 molecule are preferably the same as or
`a conservative substitution of the amino acid at the corre(cid:173)
`sponding position of glucagon or Exendin-4.
`[0022] 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 modifica(cid:173)
`tion 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
`~-amino groups, N-alk-ylation of arginine, histidine, or
`lysine, alkylation of glutamic or aspartic carboxylic acid
`groups, and deamidation of glutamine or asparagine. Modi-
`
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`US 2005/0148497 A l
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`Jul. 7, 2005
`
`3
`
`fications of the terminal amino group 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
`al1.-yl amide, dial1.-yl amide, and lower alkyl ester modifica(cid:173)
`tions. Lower allyl is C1-C4 alkyl. Furthermore, one or more
`side groups, or terminal groups, may be protected by pro(cid:173)
`tective groups known to the ordinarily-skilled protein chem(cid:173)
`ist. The o.-carbon of an amino acid may be mono- or
`dimethylatecl
`[0023] For the purposes of 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
`encompassed by the present invention have an in vitro
`potency that is not less than one-tenth the in vitro potency of
`the OPP IV resistant GLP-1 analog known as Val8-GLP-
`1(7-37)OH. More preferably, the extended GLP-1 peptides
`of the present invention are as potent or more potent than
`Val8-GLP-1 (7-37)OH.
`[0024] " 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 expression of the 3-lactamase (BLAM) gene. The
`interaction of a GLP-1 agoni5t with the receptor initiates a
`signal that results in activation of the cAMP response
`element and subsequent expression of ~-lactamase. The
`~-lactama5e CCF2/AM substrate that emits fluorescence
`when it is cleaved by f3-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 measure of
`GLP-1 agonist potency. The assay is further described in
`Zlokarnik, et 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
`Val8-GLP-1(7-37)OH as a control and assigning the control
`a reference value of 1.
`[0025] The term "delivery agent" refers to molecules in
`U.S. Pat. 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;
`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. The delivery agents are generally
`derived from amino acids and are useful in the oral formu(cid:173)
`lations 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 l\VO or
`more amino acids linked by a bond formed by other groups
`which can be linked, e.g., an ester, anhydride, or an anhy(cid:173)
`dride linkage. Peptides are two or more amino acids joined
`
`by a peptide bond. Peptides can vary in length from dipep(cid:173)
`tides with two amino acids to polypeptides with several
`hundred amino acids. Preferred peptides include di-peptides,
`tri-peptides, tetra-peptides, and penta-peptides.
`[0026] 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, propionate, hydrobromic acid,
`sodium hydroxide, potassium hydroxide, ammonium
`hydroxide, and potassium carbonate.
`[0027] The various oral formulations of the present inven(cid:173)
`tion may optionally encompa5s a pharmaceutically accept(cid:173)
`able buffer. Examples of pharmaceutically acceptable buff(cid:173)
`ers 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 art. Preferably, the buffer
`is selected from the group consisting of phosphate, TRIS,
`maleate, and glycine. Even more preferably the buffer is
`TRIS.
`[0028] 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 i5 about 20 mM.
`[0029] The pH of the oral formulations is adjusted to
`provide stability and to be acceptable for oral administra(cid:173)
`tion. 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. Most preferably, the pH is between about 7.8 and 8.1.
`[0030] The various oral formulations of the present inven(cid:173)
`tion may optionally encompass a suspending agent. Some
`delivery agents require a suspending agent due to their
`solubility characteri5tics. An example of a suspending agent
`is hydroxypropylmethylcellulose. Preferably, the final con(cid:173)
`centration of hydroxypropylmethylcellulose is between
`about 2% and about 10% (weight/volume). Even more
`preferably, the concentration is between about 2% and about
`5% (w/v). Most preferably the concentration is about 3.9%
`(w/v).
`[0031] The oral formulations of the present invention may
`optionally comprise a cosolvent. Some delivery agents
`require cosolvents due to their solubility characteristics.
`Examples of cosolvents include ethanol, N-methylpyrroli(cid:173)
`done, N,N-dimethylacetamide, N,N-dimethylformamide,
`glycofurol, ethoxydiol, propylene glycol, polyethylene gly(cid:173)
`col 300 and polyvinylpyrrolidone. Preferably, the final con(cid:173)
`centration of the cosolvents is between about 5% and about
`30% (volume/volume). Even more preferably, the concen(cid:173)
`tration is between about 10% and about 25% (v/v). Most
`preferably tbe concentration is about 20% (v/v).
`[0032] 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 deriva-
`
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`
`lives include cresols and phenol or a mixture of cresols and
`phenol. Examples of cresols include meta-cresol, ortho(cid:173)
`cresol, para-cresol, chlorocresol, or mixtures thereof. Alky(cid:173)
`lparaben refers lo a C1 to C4 alkylparaben, or mixtures
`thereof. Examples of alkylparabens include methylparaben,
`etbylparaben, propylparaben, or butylparaben. The concen(cid:173)
`trations must be sufficient to maintain preservative effec(cid:173)
`tiveness by retarding microbial growth. Preferably, the pre(cid:173)
`servative is a phenol derivative. More preferably the
`preservative is a cresol. Even more preferably the preserva(cid:173)
`tive is meta-cresol.
`
`[0033] A preferred concentration of a preservative in the
`final mixture is about 1.0 rng!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 4.5 mgfmL and about 2.0 mg!mL
`to about 4 .0 rng/rnL. A most preferred concentration of
`preservative in the final mixture is about 3.0 mg/mL.
`
`[0034] 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 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 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.
`
`[0035] 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 dispersion.
`
`[0036] GLP-1 Compounds Appropriate for use in the
`Present Invention:
`
`[0037] The GLP-1 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
`molecules from natural sources, recombinant DNA technol(cid:173)
`ogy, or a combination of these methods. For example,
`methods for preparing GLP-1 peptides are described in U.S.
`Pat. Nos. 5,118,666; 5,120,712; 5,512,549; 5,977,071; and
`6,191,102.
`
`[0038] By custom in the art, the amino terminus of GLP-
`1(7-37)OH bas been assigned number residue 7, and the
`carboxy-terminus bas been assigned number 37. The other
`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.
`
`[0039] The two naturally occurring truncated GLP-1 pep(cid:173)
`tides are represented in Formula I, SEQ ID NO:1.
`
`FORMULA I
`
`SEQ ID NO: 1
`
`Hi s 7 -Al a-Glu-Gly'0 - Thr-Phe-Thr-Ser-Asp-Val -Ser(cid:173)
`
`Ser - Tyr-Leu20-Glu-Gly-Gln-Ala-Ala25-Lys-G l u-Phe(cid:173)
`
`I l e-Ala 3 0 -Trp-Leu-Va l -Lys-Gly35-Arg-xaa37
`
`[0040] wherein:
`[0041] Xaa37 is Gly, or - NHz.
`[0042] Preferably, a GLP-1 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 NO:
`L.
`[0043] A preferred group of GLP-1 compounds is com(cid:173)
`posed of GLP-1 analogs of Formula I (SEQ ID NO:2).
`
`FORMULA I
`
`(SEQ ID NO: 2)
`
`His-Xaa 8-Xaa9 -Gl y-Xaa 11- Phe-Thr-Xaa 14-Asp-Xaa 16
`
`-
`
`Xaa 17- Xaa 18 - Xaa 1 9 - Xaa20- xaa 21 - Xaa 22 - Xaa2 3-Xaa2 4 -
`
`Xaa2 5- Xaa 26- Xaa 27 -Phe-Ile-Xaa30- xaa3 1 - Xaa 3 2- Xaa 33 -
`
`Xaa4 2 - Xaa 43- Xaa 4 4 -Xaa4 s
`
`[0044] wherein:
`
`[0045] Xaa8 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
`Asp, or Lys;
`
`[0046] Xaa9 is Glu, Asp, or Lys;
`
`[0047] Xaa11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu,
`Asp, or Lys;
`
`[0048] Xaa14 is Ser, Ala, Gly, Tor, Leu, Ile, Val, Glu,
`Asp, or Lys;
`[0049] Xaa16 is Val, Ala, Gly, Ser, Tor, Leu, Ile, Tyr,
`Glu, Asp, Trp, or Lys;
`
`[0050] Xaa17 is Ser, Ala, Gly, Tor, Leu, Ile, Val, Glu,
`Asp, or Lys;
`
`[0051] Xaa18 is Ser, Ala, Gly, Tor, Leu, Ile, Val, Glu,
`Asp, Trp, Tyr, or Lys;
`
`[0052] Xaa19 is Tyr, Phe, Trp, Glu, Asp, Gin, or Lys;
`
`[0053] Xaa20 is Leu, Ala, Gly, Ser, Tur, Ile, Val, Glu,
`Asp, Met, Trp, Tyr, or Lys;
`
`[0054] Xaa21 is Glu, Asp, or Lys;
`
`[0055] Xaa22 is Gly, Ala, Ser, Tor, Leu, Ile, Val, Glu,
`Asp, or Lys;
`
`[0056] Xaa23 is Gin, Asn, Arg, Glu, Asp, or Lys;
`
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`5
`
`[0057] Xaa2 4 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg,
`Glu, Asp, or Lys;
`
`[0058] Xaa25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
`Asp, or Lys;
`
`[0059] Xaa2 6 is Lys, Arg, Gin, Glu, Asp, or His;
`[0060] Xaa27 is Leu, Glu, Asp, or Lys;
`[0061] Xaa30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,
`Asp, or Lys;
`[0062] Xaa31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
`[0063] Xaa32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu,
`Asp, or Lys;
`[0064] Xaa33 is Val, Gly, Ala, Ser, Tbr, Leu, Ile, Glu,
`Asp, or Lys;
`[0065] Xaa34 is Asn, Lys, Arg, Glu, Asp, or His;
`
`[0066] Xaa35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,
`Asp, or Lys;
`
`[0067] Xaa36 is Gly, Arg, Lys, Glu, Asp, or His;
`
`[0068] Xaa37 is Pro, Gly, Ala, Ser, Tor, Leu, Ile, Val,
`Glu, Asp, or Lys, or is deleted;
`[0069] Xaa38 is Ser, Arg, Lys, Glu, Asp, or His, or is
`deleted;
`[0070] Xaa39 is Ser, Arg, Lys, Glu, Asp, or His, or is
`deleted;
`
`[0071] Xaa4 0 is Gly, Asp, Glu, or Lys, or is deleted;
`[0072] Xaa41 is Ala, Phe, Trp, Tyr, Glu, Asp, or Lys,
`or is deleted;
`
`[0073] Xaa42 is Ser, Pro, Lys, Glu, or Asp, or is
`deleted;
`
`[0074] Xaa43 is Ser, Pro, Glu, Asp, or Lys, or is
`deleted;
`
`[0075] Xaa4 4 is Gly, Pro, Glu, Asp, or Lys, or is
`deleted; and
`[0076] Xaa45 is Ala, Ser, Val, Glu, Asp, or Lys,
`Ala-NH2 , Ser-NH2 , Val-NH2 , Glu-NHv
`[0077] Asp-NH2 , or Lys-NH2, or is deleted; provided that
`when the amino acid al position 37, 38, 39, 40, 41, 42, 43,
`or 44 is deleted, then each amino acid downstream of that
`amino acid is also deleted.
`[0078]
`It is preferred that the GLP-1 compound of formula
`I contain Jess than six amino acids that differ from the
`corresponding amino acid in GLP-1(7-37)OH or Exendin-4.
`It ic; more preferred that Jess than five amino acids differ
`from the corresponding amino acid in GLP-1(7-37)OH or
`Exendin-4. It is even more preferred that Jess than four
`amino acids differ from the corresponding amino acid in
`GLP-1(7-37)OH or Exendin-4.
`[0079] GLP-1 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-dialkylamide thereof. WO99/
`43706 describes derivatives of GLP-1 compounds of for(cid:173)
`mula I and is incorporated by reference herein in its entirety.
`
`The compounds of formula I derivalized as described in WO
`99/43706 and underivatized are encompassed by the present
`invention.
`[0080] Another preferred group of GLP-1 compounds ic;
`composed of GLP-1 analogs of formula II (SEQ ID NO:3):
`
`FORMULA II
`
`(SEQ ID NO: 3)
`
`xaa27 -Phe-I le-xa a 30-xaa31- Leu-xaa 33 -xaa3 4 - xaa35-
`
`[0081] wherein:
`
`[0082] Xaa 7 is: L-histidine, D-histidine, desamino(cid:173)
`histidine, 2-amino-bistidine, ~-hydroxy-histidine,
`homohistidine, a-fluoromethyl-histidine or a-me(cid:173)
`thyl-histidine;
`
`[0083] Xaa8 is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;
`
`[0084] Xaa9 is: Tor, Ser, Arg, Lys, Trp, Phe, Tyr, Glu,
`or His;
`
`[0085] Xaa11 is: Asp, Glu, Arg, Tor, Ala, Lys, or His;
`
`[0086] Xaa12 is: His, Trp, Phe, or Tyr;
`
`[0087] Xaa16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
`Val, Tyr, Glu, or Ala;
`
`[0088] Xaa18 is: His, Pro, Asp, Glu, Arg, Ser, Ala, or
`Lys;
`
`[0089] Xaa19 is: Gly,Asp, Glu, Gln,Asn, Lys,Arg, or
`Cys;
`
`[0090] Xaa23 is: His, Asp, Lys, Glu, Gin, or Arg;
`
`[0091] Xaa24 is: Glu, Arg, Ala, or Lys;
`
`[0092] Xaa26 is: Trp, Tyr, Phe, Asp, Lys, Glu, or His;
`
`[0093] Xaa27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or
`Lys;
`
`[0094] Xaa30 is: Ala, Glu, Asp, Ser, or His;
`
`[0095] Xaa31 is: Asp, Glu, Ser, Tbr, Arg, Trp, or Lys;
`
`[0096] Xaa33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu;
`
`[0097] Xaa34 is: Glu, Lys, or Asp;
`
`[0098] Xaa35 is: Tor, Ser, Lys, Arg, Trp, Tyr, Phe,
`Asp, Gly, Pro, His, or Glu;
`
`[0099] Xaa36 is: Thr, Ser, Asp, Tip, Tyr, Phe, Arg,
`Glu, or His;
`
`[0100] R37 is: Lys, Arg, Tor, Ser, Glu, Asp, Trp, Tyr,
`Phe, His, Gly, Gly-Pro, or is deleted.
`
`[0101] Another preferred group of GLP-1 compounds is
`composed of GLP-1 analog.s of Formula III (SEQ ID NO:4):
`
`FRESENIUS EXHIBIT 1018
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`
`6
`
`FORMULA III
`
`(SEQ ID NO: 4 )
`
`Xaa 7 - Xaa 8 -G l u-G ly- Xaa 1 1- Xaa 1 2-Thr-Ser -Asp-Xaa 16 -
`Ser-Se r -Tyr-Leu-G lu-Xaa 22-Xaa23-Xa a 24 -Xaa 25-Lys(cid:173)
`
`Xaa 2 7 - P he -I l e - Xaa 30- Trp-Leu- Xa a 33-Xaa 34- Xa a 3 5 -
`
`Xaa 36 _R37
`
`[0102] wherein:
`[0103] Xaa7 is: L-histidine, D-histidine, desamino(cid:173)
`histidine, 2-amino-histidine, ~-hydroxy-histidine,
`hornobistidine, a-fluorornethylhistidine or a-rneth(cid:173)
`ylhistidine;
`[OH14] Xaa8 is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;
`[0105] Xaa11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His;
`[0106] Xaa12 is: His, Trp, Phe, or Tyr;
`[0107] Xaa16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
`Val, Glu, or Ala;
`[0108] Xaaz2 is: Gly, Asp, Glu, Gin, Asn, Lys, Arg, or
`Cys;
`[0109] Xaa23 is: His, Asp, Lys, Glu, or Gin;
`[0110] Xaa2 4 is: Glu, His, Ala, or Lys;
`[0111] Xaazs is: Asp, Lys, Glu, or His;
`[0112] Xaa27 is: Al.a, Glu, His, Phe, Tyr, Trp, Arg, or
`Lys;
`[0113] Xaa30 is: Ala, Glu, Asp, Ser, or His;
`[0114] Xaa33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu;
`[0115] Xaa34 is: Glu, Lys, or Asp;
`[0116] Xaa35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
`Asp, Gly, Pro, His, or Glu;
`[0117] Xaa36 is: Arg, Glu, or His;
`[0118] R37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr,
`Phe, His, Gly, Gly-Pro, or is deleted.
`[0119] Another preferred group of GLP-1 compounds is
`composed of GLP-1 analogs of Formula IV (SEQ ID NO:5):
`
`FORMULA IV
`
`(SEQ ID NO: 5)
`Xaa7- Xaa8-Gu-G l y-Thr-Xaa 12-Thr-Ser-Asp -Xaa16-Ser(cid:173)
`
`Ser - Tyr-Le u-Gl u-xaa 22-Xaa23-Ala-Ala -Z aa 26-Gl u-Phe(cid:173)
`I le- Xa a30-Trp-Leu-Va l-Lys-Xa a35-Arg-R37
`
`[0120] wherein:
`[0121] Xaa