Title of Invention	A STABLE GLP-1Fc FUSION FORMULATION
Abstract	The invention provides a stable solution formulation comprising a GLP-1-Fc fusion at a pH between about pH 6 and about pH 8.5 analogs fused to specific IgG4-Fc derivatives. These formulations provide unexpected and considerably greater chemical stability than when compared to GLP-1-Fc fusions at a pH outside the described ranges. The formulations comprising a GLP-1-Fc fusion are useful in treating diabetes, obesity, irritable bowel syndrome and other conditions that would be benefited by lowering plasma glucose, inhibiting gastric and/or intestinal motility and inhibiting gastric and/or intestinal emptying, or inhibiting food intake.

Title of Invention

A STABLE GLP-1Fc FUSION FORMULATION

Abstract

The invention provides a stable solution formulation comprising a GLP-1-Fc fusion at a pH between about pH 6 and about pH 8.5 analogs fused to specific IgG4-Fc derivatives. These formulations provide unexpected and considerably greater chemical stability than when compared to GLP-1-Fc fusions at a pH outside the described ranges. The formulations comprising a GLP-1-Fc fusion are useful in treating diabetes, obesity, irritable bowel syndrome and other conditions that would be benefited by lowering plasma glucose, inhibiting gastric and/or intestinal motility and inhibiting gastric and/or intestinal emptying, or inhibiting food intake.

Full Text	FIELD OF THE INVENTION The present invention relates to formulations of glucagon-like peptide analogs fused to proteins that have the effect of stabilizing the fusion proteins. These formulations can be used to treat diabetes as well as a variety of other conditions or disorders. BACKGROUND OF THE INVENTION Glucagon-like peptide-1 (GLP-1) analogs and derivatives show promise in clinical trials for the treatment of type 2 diabetes. GLP-1 induces numerous biological effects such as stimulating insulin secretion, inhibiting glucagon secretion, inhibiting gastric emptying, inhibiting gastric molility or intestinal motility, and inducing weight loss. A significant 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 some types of oral therapies that act by increasing insulin expression. The usefulness of therapy involving GLP-1 peptides has been limited by the fact that GLP-l(l-37) is poorly active, and the two naturally occurring truncated peptides, GLP-1 (7-37)OH and GLP-1 (7-36)NH2, are rapidly cleared in vivo and have extremely short in vivo half lives. It is known that endogenously produced dipeptidyl-peptidase IV (DPP-IV) inactivates circulating GLP-1 peptides by removing the N-terminal histidine and alanine residues and is a major reason for the short in vivo half-life. Various approaches have been undertaken to extend the elimination half-life of a GLP-1 peptide or reduce clearance of the peptide from the body while maintaining biological activity. One approach involves fusing a GLP-1 peptide to the Fc portion of an immunoglobulin. Immunoglobulins typically have long circulating half-lives in vivo. For example, IgG molecules can have a half-life in humans of up to 23 days. The Fc portion of the immunoglobulin is responsible, in part, for this in vivo stability. GLP-1 - Fc fusion proteins take advantage of the stability provided by the Fc portion of an immunoglobulin while preserving the biological activity of the GLP-1 molecule. Although this approach is feasible for GLP-1 therapeutics (See WO 02/46227), there is a general concern regarding the antigenicity of various fusion proteins when administered repeatedly over prolonged periods of time. This is especially a concern for -2- GLP-l-Fc fusion therapeutics as a patient with diabetes must be treated for her entire life once diagnosed with the disease. In addition, Fc fusion protein therapeutics can be a concern if the Fc portion retains unwanted effector functions. This approach is the focus of PCT/US 04/15595 (WO2005/000892), in which problems associated with the potential immunogenicity and effector activity associated with administration of GLP-l-Fc fusions are overcome by identifying specific GLP-l-Fc fusion proteins that have a reduced risk of inducing an immune response after repeated and prolonged administration and no longer have effector function. The fusion proteins of this nature are technically too large and complex to produce synthetically or recombinantly in bacterial cells. These fusion proteins are typically produced in mammalian cells, such as CHO, 293, or NSO. It was observed that the fusion proteins produced in mammalian cells where more readily susceptible to degradation by endogenous proteases and chemical alteration than non-fusion proteins produced in bacterial cells. This problem was sought to be overcome in the present invention. It is discovered that a formulation comprising a GLP-l-Fc fusion buffered between about pH 6 and about pH 8.5 provided increased chemical stability. SUMMARY OF THE INVENTION In order to overcome the problem of chemical stability of GLP-l-Fc fusions, the present inventors have developed a stable solution formulation. In particular, the inventors have discovered that a formulation comprising a therapeutically effective amount of a GLP-l-Fc fusion at a pH between about pH 6 and about pH 8.5, preferably between about pH 6 and about pH 7.5, between about pH 6 and about pH 7, between about pH 6.5 and about pH 7.5, or between about pH 6 and about pH 6.5, and even more preferably, about pH 6 or about 6.5, provided unexpectedly and considerably greater chemical stability than when compared to GLP-l-Fc fusions at a pH outside the described ranges. The present invention also includes methods of treating patients suffering from non-insulin dependent as well as insulin dependent diabetes mellitus, obesity, and various other disorders and conditions comprising administering the formulations of GLP-l-Fc fusions. -3- DETAILED DESCRIPTION OF THE INVENTION The fusion proteins comprise a GLP-1 compound fused to an Fc portion of an immunoglobulin, an analog of an Fc portion of an immunoglobulin, or a fragment of an Fc portion of an immunoglobulin. The C-terminus of the GLP-1 compound may be fused directly, or fused via a peptide linker, to the N-terminus of an Fc protein. These fusion proteins are biologically active and have an increased half-life compared to native GLP-1. The GLP-1 compounds that make up part of the fusion protein encompass polypeptides having from about twenty-five to about thirty-nine naturally occurring or non-naturally occurring amino acids that have sufficient homology to native GLP-1 (7-37)OH such that they exhibit insulinotropic activity by binding to the GLP-1 receptor on P-cells in the pancreas. A GLP-1 compound typically comprises a polypeptide having the amino acid sequence of GLP-1 (7-37)OH, an analog of GLP-1 (7-37)OH, a fragment of GLP-1 (7- 37)OH or a fragment of a GLP-1 (7-37)OH analog. Examples of fusion proteins useful in the formulations include fusion proteins comprising a GLP-1 analog comprising a sequence selected from the group consisting of a) (SEQ ED NO: 1) His-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly wherein Xaas is selected from Gly and Val; b) (SEQ ID NO:2) His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Gly wherein Xaas is selected from Gly and Val; c) (SEQ ID NO:3) His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro wherein Xaas is selected from Gly and Val; d) (SEQ ID NO:4) His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu- Gln- Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Pro wherein Xaas is selected from Gly and Val; e) (SEQ ID NO:5) -4- His-XaarGlu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-AIa-AIa-Lys-Glu-Phe-Ee-Ala-Trp-Leu-VaJ-Lys-Gly-Gly wherein Xaag is selected from Gly and Val; f) (SEQ ID NO:6) His-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-VaJ-Ser-Ser-Tyr-Leu-GJu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ue-Ala-Trp-Leu-Lys-Asn-Gly-Gly wherein Xaas is selected from Gly and Val; fused to the Fc portion of an immunoglobulin comprising the sequence of SEQ ID NO:7 Ala-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro-Cys-Pro-Ala-Pro- Xaai6-Xaai7-Xaa,8-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro- Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val- Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp- Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Aia-Lys-Thr-Lys-Pro-Arg- Glu-Glu-Gln-Phe-Xaago-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Uu-Thr- Val-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys- Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser- Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro- Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys- Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu- Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val- Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Axg-Leu-Thr-Val- Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val- Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser- Leu-Ser-Leu-Gly-Xaa23o (SEQ ID NO:7) wherein: Xaa at position 16 is Pro or Glu; Xaa at position 17 is Phe, Val, or Ala; Xaa at position 18 is Leu, Glu, or Ala; Xaa at position 80 is Asn or Ala; and Xaa at position 230 is Lys or is absent. -5- The C-terminus of the GLP-1 analog portion and the N-terminus of the Fc portion of the fusion proteins are preferably fused together via 1, 1.5 or 2 repeats of a G-rich peptide linker having the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly- Gly-Gly-Ser (SEQ ID NO:8). The fusion proteins useful in the formulations of the present invention comprise a GLP-1 analog portion and an Fc portion. The GLP-1 analog portion and the Fc portion comprise substitutions to the native GLP-1 sequence and the human IgG4 sequence respectively that provide the protein with increased potency and in vivo stability compared to native GLP-1 or GLP-1 analogs not fused to an Fc sequence while decreasing the potential for inducing antibody formation after prolonged and repeated administration in humans. Native GLP-1 is processed in vivo such that the first 6 amino acids are cleaved from the molecule. Thus, by custom in the art, the amino terminus of GLP-1 has been assigned the number 7 and the carboxy-terminus, number 37. The other amino acids in the polypeptide are numbered consecutively as shown in SEQ ID NO:9. For example, position 8 is alanine and position 22 is glycine. The processed peptide may be further modified in vivo such that the C-terminal glycine residue is removed and replaced with an amide group. Thus, GLP-1(7-37)QH and GLP-1 (7-36)amide represent the two native forms of the molecule. GLP-1 (7-37)OH has the amino acid sequence of SEQ ID NO:9: 7His-Ala-Glu-10Gly-Thr-Phe-Thr-Ser-l5Asp-Val-Ser-Ser-Tyr-20Leu-Glu-Gly- Gln-Ala-25Ala-Lys-Glu-Phe-De-3OAla-Trp-Leu-Val-Lys-35Gly-Arg-37Gly (SEQ ID NO:9) The GLP-1 analog portion of the fusion protein comprises three primary substitutions at positions 8, 22, and 36 relative to native GLP-1 (7-37). The substitution at position 8 reduces the rate at which the endogenous enzyme dipeptidyl-peptidase IV (DPP-IV) inactivates the analog. DPP-IV cleaves native GLP-1 between the 2nd and 3rd amino acids (between position 8 and 9) and the resulting molecule is less active. Thus, the fusion proteins useful in the formulations of the present invention are DPP-IV resistant. The substitution at position 22 reduces the potential of the molecule to aggregate and increases the -6- potency of the molecule. The substitution at position 36 in the context of the analog with changes at 8 and 22 as well as in the context of the entire fusion protein reduces the risk that the fusion protein will induce a neutralizing immune response after repeated and prolonged administration in humans. The GLP-1 analog C-terminus is preferably one of the following sequences: Trp-Leu-Val-Lys-Gly-Gly-Gly (SEQ ID NO: 10); Trp-Leu-Lys-Asn- Gly-Gly-Gly (SEQ ID NO:11); Trp-Leu-Val-Lys-Gly-Gly-Pro (SEQ ID NO: 12); Trp-Leu-Lys-Asn-Gly-Gly-Pro (SEQ ID NO: 13); Trp-Leu-Val-Lys-Gly-Gly (SEQ ID NO: 14); and Trp-Leu-Lys-Asn-Gly-Gly (SEQ ID NO: 15). The fusion proteins useful in the formulations of the present invention contain an Fc portion which is derived from human IgG4, but comprises one or more substitutions compared to the wild-type human sequence. As used herein, the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, this term refers to an antibody fragment which does not contain the two antigen binding regions (the Fab fragments) from the antibody. The Fc portion consists of the constant region of an antibody from both heavy chains, which associate through non-covalent interactions and disulfide bonds. The Fc portion can include the hinge regions and extend through the CH2 and CH3 domains to the c-terminus of the antibody. The Fc portion can further include one or more glycosylation sites. There are five types of human immunoglobulins with different effector functions and pharmcokinetic properties. IgG is the most stable of the five types having a serum half-life in humans of about 23 days. There are four IgG subclasses (Gl, G2, G3, and G4) each of which have different biological functions known as effector functions. These effector functions are generally mediated through interaction with the Fc receptor (FcyR) or by binding Clq and fixing complement. Binding to FcyR can lead to antibody dependent cell mediated cytolysis, whereas binding to complement factors can lead to complement mediated cell lysis. In designing Fc fusion proteins wherein the Fc portion is being utilized solely for its ability to extend half-life, it is important to minimize any effector function. Thus, the fusion proteins useful in the formulations of the present invention are derived from the human IgG4 Fc region because of its reduced ability to bind FeyR and complement factors compared to other IgG sub-types. IgG4, however, has been shown to deplete target cells in humans [Issacs et al., (1996) CHn. Exp. Immunol. 106:427-433]. Because the fusion proteins target beta cells in the pancreas to induce insuJin expression, using an IgG4 derived region in an Fc fusion protein could initiate an immune response against the pancreateic beta cell through interaction of the fusion protein with the GLP-1 receptor present on pancreatic beta cells. Thus, the IgG4 Fc region which is part of the fusion proteins contains substitutions that eliminate effector function. The lgG4 Fc portion of the fusion proteins may contain one or more of the following substitutions: substitution of proline for glutamate at residue 233, alanine or valine for phenylalanine at residue 234 and alanine or glutamate for leucine at residue 235 (EU numbering, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5' Ed. U.S. Dept. of Health and Human Services, Bethesda, MD, NIH Publication no. 91-3242). These residues corresponds to positions 16, 17 and 18 in SEQ ID NO:7. Further, removing the N-linked glycosylation site in the IgG4 Fc region by substituting Ala for Asn at residue 297 (EU numbering) which corresponds to position 80 of SEQ ID NO:7 is another way to ensure that residual effector activity is eliminated in the context of a fusion protein. In addition, the IgG4 Fc portion of the fusion proteins contain a substitution that stabilizes heavy chain dimer formation and prevents the formation of half-IgG4 Fc chains. The fusion proteins preferably exist as dimers joined together by disulfide bonds and various non-covalent interactions. Wild- type IgG4 contains a Pro-Pro-Cys-Pro-Ser-Cys (SEQ ID NO: 16) motif beginning at residue 224 (EU numbering). This motif in a single GLP-1 analog-Fc chain forms disulfide bonds with the corresponding motif in another GLP-1 analog-Fc chain. However, the presence of serine in the motif causes the formation of single chain fusion proteins. The present invention encompasses Fc fusion proteins wherein the IgG4 sequence is further modified such that serine at position at 228 (EU numbering) is substituted with proline (amino acid residue llinSEQIDNO:7). The C-termina] lysine residue present in the native molecule may be -8- deleted in the IgG4 derivative Fc portion of the fusion proteins discussed herein (position 230 of SEQ ID NO:7; deleted lysine referred to as des-K). Fusion proteins expressed in some cell types (such as NSO cells) wherein lysine is encoded by the C-terminal codon are heterogeneous in that a portion of the molecules have lysine as the C-terminal amino acid and a portion have lysine deleted. The deletion is due to protease action during expression in some types of mammalian cells. Thus, to avoid this heterogeneity, it is preferred that Fc fusion expression constructs lack a C-terminal codon for lysine. It is preferred that the C-terminal amino acid of the GLP-1 analog portion discussed herein is fused to the N-terminus of the IgG4 Fc analog portion via a glycine-rich linker. The in vivo function and stability of the fusion proteins can be optimized by adding small peptide linkers to prevent potentially unwanted domain interactions. Further, a glycine-rich linker provides some structural flexibility such that the GLP-1 analog portion can interact productively with the GLP-1 receptor on target cells such as the beta cells of the pancreas. These linkers, however, can significantly increase the risk that the fusion protein will be immunogenic in vivo. Thus, it is preferred that the length be no longer than necessary to prevent unwanted domain interactions and/or optimize biological activity and/or stability. The preferred glycine-rich linker comprises the sequence: Gly-Gly-GJy-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:8). Although more copies of this linker may be used in the fusion proteins, it is preferred that a single copy of this linker be used to minimize the risk of immunogenicity associated with prolonged and repeated administration. Preferred GLP-1-Fc fusions include the following proteins: Gly8-Glu22-Gly36- GLP-l(7-37)-lL-IgG4 (S228P), Gly8-Glu22-Gly36-GLP-l(7-37)-lL-IgG4 (S228P, F234A, L235A), Gly8-Glu22-Gly36-GLP-l(7-37)-lL-IgG4 (S228P, N297A), Gly8-Glu22-Gly36- GLP-l(7-37)-lL-IgG4 (S228P, F234A, L235A, N297A), Gly8-Glu22-Gly36-GLP-1(7-37)- 1.5L-IgG4 (S228P), Gly8-Glu22-Gly36-GLP-](7-37)-1.5L-IgG4 (S228P, F234A, L235A), Gly8-Glu22-Gly36-GLP-l(7-37)-1.5L-IgG4 (S228P, N297A), Gly8-Glu22-Gly36-GLP-1(7- 37)-1.5L-IgG4 (S228P, F234A, L235A, N297A), Gly8-Glu22-GIy36-GLP-l(7-37)-2L- IgG4 (S228P), Gly8-Glu22-Gly36-GLP-l(7-37)-2L-IgG4 (S228P, F234A, L235A), Gly8- Glu22-Gly36-GLP-l(7-37)-2L-IgG4 (S228P, N297A), and Gly8-Glu22-Gly36-GLP-1(7-37)- 2L-IgG4 (S228P, F234A, L235A, N297A), and the Val8 and des-K forms of all of the above. The nomenclature used herein to refer to specific fusion proteins is defined as follows: Specific substitutions to the GLP-1 portion of the fusion protein are indicated using the specific arnino acid being substituted followed by the residue number. GLP- 1(7-37) indicates that the GLP-1 portion of the mature fusion protein begins with His at position 7 and ends with Gly at position 37. L refers to a linker with the sequence Gly- Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:8). The number immediately preceding the L refers to the number of linkers separating the GLP-1 portion from the Fc portion. A linker specified as 1.5L refers to the sequence Gly-Ser- Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 17). IgG4 refers to an analog of the human IgG4 Fc sequence specified as SEQ ED NO:7. Substitutions in the lgG4 Fc portion of the fusion protein are indicated in parenthesis. The wild-type amino acid is specified by its common abbreviation followed by the position number in the context of the entire IgG4 sequence using the EU numbering system followed by the amino acid being substituted at that position specified by its common abbreviation. The fusion proteins have biological activity. Biological activity refers to the ability cf the fusion protein to bind to and activate the GLP-1 receptor in vivo and elicit a response. Responses include, but are not limited to, secretion of insulin, suppression of glucagon, inhibition of appetite, weight loss, induction of satiety, inhibition of apoptosis, induction of pancreatic beta cell proliferation, and differentiation of pancreatic beta cells. A representative number of GLP-1 fusion proteins were tested for in vitro as well as in vivo activity. In vitro activity based on the ability of the fusion protein to interact with and activate the human GLP-1 receptor have been shown. (See PCTAJS 04/15595 (WO2005/000892)). HEK293 cells over-expressing the human GLP-1 receptor are used. Activation of the GLP-1 receptor in these cells causes adenylyl cyclase activation which in turn induces expression of a reporter gene driven by a cyclic AMP response element (CRE). The pH of the GLP-1-Fc fusion formulations is adjusted to provide acceptable stability, to maintain the solubility and insulinotropic activity of the GLP-1-Fc fusion and be acceptable for parenteral administration. The pH of the GLP-1 -Fc fusion formulations is preferably adjusted to between about pH 6 and about pH 8.5, preferably between about -10- pH 6 and about pH 7.5, between about pH 6 and about pH 7, between about pH 6.5 and about pH 7.5, or between about pH 6 and about pH 6.5, and even more preferably, about pH 6 or about 6.5. The formulations comprising a GLP-l-Fc fusion of the present invention may optionally encompass a pharmaceutically acceptable buffer. However, the selection and concentration of the buffer shall be such that the formulation can be adjusted to the described ranges that provide acceptable stability and insulinotropic activity. Examples of pharmaceutically acceptable buffers include phosphate buffers like dibasic sodium phosphate, TRIS, acetate, such as sodium acetate, citrate, such as sodium citrate, sodium tartarate, basic amino acids such as histidine, lysine or arginine, or neutral amino acids such as glycine and glycyl-glycine. Other pharmaceutically acceptable buffers are known in the art. Preferably, the buffer is selected from the group consisting of citrate, phosphate and TRIS. The skilled artisan will recognize that the selection of the buffer is dependent upon the described pH ranges and the pKa of the buffer. Preferably, the concentration of a buffer is between about 1 mM and 30 mM. Even more preferably, the concentration is between about 4 mM and 14 mM or between about 5 mM and 20 mM. Even more preferably, the concentration is between about 10 mM and 20 mM. Even more preferably, the concentration is about 10 mM or about 20 mM. The formulations of the present invention may optionally encompass a preservative. However, the selection and concentration of the preservative shall be such that the formulation can be adjusted to the described ranges that provide acceptable stability and insulinotropic activity. Preservative refers to a compound that is added to a pharmaceutical formulation to act as an anti-microbial agent. A parenteral formulation must meet guidelines for preservative effectiveness to be a commercially viable multi-use product. 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, chloro-cresol, or mixtures thereof. Alkylparaben refers to a Ci to C4 alkyl paraben, or mixtures thereof. Examples of alkylparabens include methylparaben, ethylparaben, propylparaben, or butylparaben. The concentration of the preservative is known to one skilled in the art. -11- The concentrations must be sufficient to maintain preservative effectiveness by retarding microbial growth. The preferred preservative is meta-cresol or phenol. In general, the concentration of meta-cresol is between about 2.0 to about 8.0 mg/mL, about 2.5 mg/mL to about 4.5 mg/mL, and about 2.0 mg/mL to about 4.0 mg/mL. A most preferred concentration of preservative in the formulation is about 2.7 mg/mL. In another embodiment, the concentration of phenol is between about 2.0 to about 10.0 mg/mL, and about 4.0 to about 8.0 mg/mL. A most preferred concentration of preservative in the formulation is about 5.0 mg/mL. The formulations of the present invention may optionally encompass an isotonicity agent. However, the selection and concentration of the isotonicity agent shall be such that the formulation can be adjusted to the described ranges that provide acceptable stability and insulinotropic activity. 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 (or glycerol), 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 NaCl is preferably between about 10 mM and 500 mM, more preferred is between about 50 mM and 200 mM, and most preferred is about 150 mM. In another embodiment, the preferred isotonicity agent is mannitol. The concentration of the mannitol is preferably between about 1% (weight (w)/volume (v)) and 10% (w/v), and more preferred is between about 2% (w/v) and 8% (w/v). In another embodiment, the preferred isotonicity agent is glycerin. The concentration of the glycerin is preferably between about 12 mg/mL and 25 mg/ml, preferably between about 12 mg/mL and 20 mg/ml, and more preferred is about 17 mg/ml. The formulations of the present invention may optionally encompass a solubility enhancer. However, the selection and concentration of the solubility enhancer shall be such that the formulation can be adjusted to the described ranges that provide acceptable stability and insulinotropic activity. Solubility enhancers provide stability such that the GLP-l-Fc fusion remains soluble for an extended period of time under the conditions of -12- storage. Preferably the solubility enhancer is nicotinamide. In general, the concentration of nicotinamide is between 0.01 and 2 molar. Other preferred ranges of nicotinamide concentration are: between 0.05 molar and 1.5 molar; between 0.1 molar and 1.0 molar; between 0.1 molar and 0.5 molar; between 0.5 molar and 1.0 molar; and between 0.15 molar and 0.25 molar. Other additives, such as a pharmaceutically acceptable solubilizers like Tween 20® (polyoxyethylene (20) sorbitan monolaurate), Tween 40® (polyoxyethylene (20) sorbitan monopalmitate), Tween 80® (polyoxyethylene (20) sorbitan monooleate), Pluronic F68® (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) may optionally be added to the formulation. Preferably the solubilizer is Tween 20® or TweenSO®. In general, the concentration of Tween 20® or Tween80® is between 0.001% and 0.05%. Other preferred ranges of Tween 20® or Tween80® concentrations are: between 0.005% and 0.05%; between 0.0075% and 0.05%; and between 0.01% and 0.05%. Administration of the formulations may be via any route known to be effective by the physician of ordinary skill. Peripheral parenteral is one such method. Parenteral administration is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump. Peripheral parenteral routes can include intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration. The formulations may also be amenable to administration by oral, rectal, nasal, or lower respiratory routes, which are non-parenteral routes. Of these non-parenteral routes, the lower respiratory route and the oral route are preferred. The formulations comprising a GLP-l-Fc fusion can be used to treat a wide variety of diseases and conditions. The GLP-l-Fc fusions primarily exert their biological effects by acting at a receptor referred to as the "GLP-1 receptor." Subjects with diseases and/or conditions that respond favorably to GLP-1 receptor stimulation or to the administration of GLP-1 compounds can therefore be treated with the GLP-l-Fc fusions. These subjects are said to "be in need of treatment with GLP-1 compounds" or "in need of GLP-1 receptor stimulation". Included are subjects with non-insulin dependent diabetes, insulin dependent diabetes, stroke (see WO 00/16797), myocardial infarction (see WO 98/08531), obesity (see WO 98/19698), catabolic changes after surgery (see U.S. Patent No. 6,006,753), functional -13- dyspepsia and irritable bowel syndrome (see WO 99/64060). Also included are subjects requiring prophylactic treatment with a GLP-1 compound, e.g., subjects at risk for developing non-insulin dependent diabetes (see WO 00/07617). Subjects with impaired glucose tolerance or impaired fasting glucose, subjects whose body weight is about 25% above normal body weight for the subject's height and body build, subjects with a partial pancreatectomy, subjects having one or more parents with non-insulin dependent diabetes, subjects who have had gestational diabetes and subjects who have had acute or chronic pancreatitis are at risk for developing non-insulin dependent diabetes. An effective amount of the GLP-1-Fc fusions in the context of the described formulations is the quantity which results in a desired therapeutic and/or prophylactic effect without causing unacceptable side-effects when administered to a subject in need of GLP-1 receptor stimulation. A "desired therapeutic effect" includes one or more of the following: 1) an amelioration of the symptom(s) associated with the disease or condition; 2) a delay in the onset of symptoms associated with the disease or condition; 3) increased longevity compared with the absence of the treatment; and 4) greater quality of life compared with the absence of the treatment. For example, an "effective amount" of a GLP-l-Fc fusion for the treatment of diabetes is the quantity that would result in greater control of blood glucose concentration than in the absence of treatment, thereby resulting in a delay in the onset of diabetic complications such as retinopathy, neuropathy or kidney disease. An "effective amount" of a GLP-1-Fc fusion for the prevention of diabetes is the quantity that would delay, compared with the absence of treatment, the onset of elevated blood glucose levels that require treatment with anti-hypoglycaemic drugs such as sulfonyl ureas, thiazolidinediones, insulin and/or bisguanidines. The dose of fusion protein effective to normalize a patient's blood glucose will depend on a number of factors, among which are included, without limitation, the subject's sex, weight and age, the severity of inability to regulate blood glucose, the route of administration and bioavailability, the pharmacokinetic profile of the fusion protein, the potency, and the formulation. Doses may be in the range of 0.01 to 1 mg/kg body weight, preferably in the range of 0.05 to 0.5 mg/kg body weight. It is preferable that the fusion proteins be administered either once every two weeks or once a week. Depending on the disease being treated, it may be necessary to administer the fusion protein more frequently such as two to three time per week. The present invention will now be described only by way of non-limiting example with reference to the following Examples. EXAMPLES In vitro GLP-1 receptor activation assay: HEK-293 cells expressing the human GLP-1 receptor, using a CRE-BLAM system, are seeded at 20,000 to 40,000 cells/well/100 fxl DMEM medium with 10%FBS into a poly-d-lysine coated 96 well black, clear-bottom plate. The day after seeding, the medium is flicked off and 80 jil plasma-free DMEM medium is added. On the third day after seeding, 20 \l\ of plasma-free DMEM medium with 0.5% BSA containing different concentrations of various GLP-1 -Fc fusion protein is added to each well to generate a dose response curve. Generally, fourteen dilutions containing from 3 nanomolar to 30 nanomolar or GLP-1 Fc fusion protein are used to generate a dose response curve from which EC50 values can be determined. After 5 hours of incubation with the fusion protein, 20 Μ1 of P-lactamase substrate (CCF2/AM, PanVera LLC) is added and incubation continued for 1 hour at which time fluorescence is determined on a cytofluor. The assay is further described in Zlokarnik, et al. (1998), Science, 278:84-88. In vitro GLP-1 receptor activation assay: HEK-293 cells stably expressing the human GLP-1 receptor, using a CRE- Luciferase system, are seeded at 30,000 cells/well/80 fil low serum DMEM F12 medium into 96 well plates. The day after seeding, 20 μl aliquots of test protein dissolved in 0.5% BSA are mixed and incubated with the cells for 5 hours. Generally 12 dilutions containing from 3 pM to 3 nM are prepared at a 5X concentration for each test protein before addition to the cells to generate a dose response curve from which EC50 values are determined. After incubation, 100 p.1 of Luciferase reagent is added directly to each plate and mixed gently for 2 minutes. Plates are placed in a Tri-lux luminometer and light output resulting from luciferase expression is calculated. Effect of Tween. NaCl. and pH on stability of Glv8-Glu22-GLP-1(7-37)-!L-IgG4 (S228P, F234A. L235A): The effects of tween-20 (± 0.01%), NaCl (0,150, and 500mM), and pH (6.5, 7.5, and 8.5) on the chemical stability of 0.5mg/mL Gly8-Glu22-GLP-l(7-37)-lL-IgG4 (S228P, F234A, L235A) are determined. A full matrix of these conditions are prepared by mixing a respective 2x buffer stock with a lmg/mL Gly8-GIu22-GLP-l(7-37)-lL-IgG4 (S228P, F234A, L235A) solution dissolved in 20mM phosphate, 20mM Tris, pH 7.5. The final solution conditions are terminally filtered through 0.2micron Millex-GV filters into HPLC vials and incubated at 37°C. Periodically time points are removed and analyzed by size-exclusion chromatography, reversed-phase chromatography, and gel electrophores. Identification of formulation components for samples A-R (all contained 0.5mg/mL Gly8- Glu22-GLP-l(7-37)-lL-IgG4 (S228P, F234A, L235A), 20mM phosphate, and 20mM Tris) Size-Exclusion HPLC method: Samples are injected on a Tosohas TSK3000-SW-XL (5 micron, 7.8x3OOmm) column pre-equilibrated in lx PBS, pH 7.4 plus 10% acetonitrile (v/v) mobile buffer (A- pump buffer). A-buffer is flowing at 0.5mL/min isocratic and protein was detected at 214nm. Reversed-Phase HPLC method: Samples are injected on a Zorbax 300SB-C8 (4.6x50mm) column pre-equilibrated in 0.1% TFA (v/v) mobile buffer (A-pump buffer). B-pump buffer consists of 0.085% TFA in acetonitrile (v/v). The following gradient is run and material was detected at 214nm. -18- lime % B-pump buffer 0 30 5 30 35 45 40 90 45 90 50 25 65 25 Gel Electrophoresis method: Time points are diluted with 4x NuPage LDS sample buffer (lx final cone) and heated at 95°C for 5 minutes. The cooled samples were loaded on a 4-12% bis-tris NuPage gel. The gel is run at constant voltage (200 volts) for 50-60min in MOPS buffer and stained with SimplyBlue SafeStain. Stability of Glv8-Glu"-GLP-1(7-37)- IL-IgG4 (S228P, F234A, L235A) and effect of Tween-20: Stability of Gly8-Glu22-GLP-1(7-37)- lL-IgG4 (S228P, F234A, L235A) at 0.3mg/mL in lx PBS at pH 7.5 is evaluated by monitoring the increase in turbidity at 35Onm under accelerated stress conditions (stirring with hydrophobic stir bar in PBS at 37oC). The effect of Tween-20 on reducing the aggregation Solution Additive Time for turbidity to increase > 0.05 OD at 350nm None 0.001% Tween-20 16-hr 0.005% Tween-20 27-hr 0.01% Tween-20 > 65-hr 0.02% Tween-20 > 65-hr 0.05% Tween-20 > 65-hr -19- Solubility of Glv8-Glu"-GLP-1(7-37)- !L-IgG4 (S228P. F234A. L235A) as a function of solution pH: Solubility of Gly8-Glu22-GLP-1(7-37)-lL-IgG4 (S228P, F234A, L235A) as a function of pH is determined by diluting a stock solution with various buffered solution at differ pH values, allowing the solution to equilibrate, centrifugating away any precipitated material, then analyzing the supernant for remaining soluble protein concentration. Stock protein frozen in PBS, is dialyzed against water at approximately pH 8. After dialysis the protein stock was filtered through a 0.22micron Millex-GV filter and concentrated to ~12mg/mL using a rinsed Ultrafree centrifugal device (4ml, 5000 dalton MWCO, at 7000xG). This concentrated solution is diluted 1:1 with 2x stock buffer solutions (final volume of 50uL in 0.5mL eppendorf tubes) and is allowed to equilibrate for 15-30 minutes at room temperature. The solution is centrifuged (14,000xG for 5 minutes) to pellet any insoluble material. lOuL of the supernant is removed and combined with 40uL of the pH 8 water. Two replicates are performed for each sample. The concentration of soluble GLP-Fc is determined by reversed-phase HPLC against a standard curve. Effect of solution conditions on solubility 20mM Citrate, 3mg/mL m-Cresol, pH 5 1.3 0.0 20mM Citrate, 3mg/mL m-Cresol, pH 6 5.5 0.1 20mM Phosphate, 3mg/mL m-Cresol, pH 7 6.2 0.1 20mM Tris, 3mg/mL m-Cresol, pH 8 6.0 0.0 Effect of solution pH on stability as monitored by reversed-phase HPLC: Gly8-Glu22-GLP-l(7-37)-lL-IgG4 (S228P, F234A, L235A) is dialyzed against water adjusted to approximately pH 8. After dialysis this protein stock is filtered (0.2 micron Millex-GV) and concentrated to >2mg/mL using a rinsed Ultrafree centrifugal device. The final protein concentration is determined by UV and various formulations are made by diluted 2x formulation buffer stocks to lx keeping the final protein concentration at lmg/mL. The solution pH is checked and adjusted if needed. These solutions are terminally filtered (0.22micon Millex-GV, 4mm) in sterilized 1.8mLHPLC vials with screw caps. The vials are placed at 37°C and time points are pulled and analyzed by reversed-phase, size-exclusion, and gel electrophoresis. The change in percent main-peak as monitored by size-exclusion chromatography and reversed-phase HPLC are presented. The linear degradation rates obtained by fitting a plot of incubation time at 37oC with main peak purity for the size-exclusion and reversed-phase HPLC data is tabulated. Identification of formulation parameters. Formulation code PH Buffer (l0mM) Additive 3C 3.0 Citrate none 4C 4.0 Citrate none 5C 5.0 Citrate none 6C 6.0 Citrate none 6CN 6.0 Citrate 150mM NaCl 6CC 6.0 Citrate 3mg/mL m-Cresol 6P 6.0 Phosphate none 7P-R1 7.0 Phosphate None, replicate #1 7P-R2 7.0 Phosphate None, replicate #2 7P-R3 7.0 Phosphate None, replicate #3 7PN 7.0 Phosphate 150mM NaCl 7PC 7.0 Phosphate 3mg/mL m-Cresol 8P 8.0 Phosphate none 8T 8.0 Tris none -21- The change in percent main-peak upon storage at 37°C. Data is analyzed by size- exclusion chromatography. 8T 0.003 -2.2 6CN -0.85 -2.1 6CC -1.6 -2.7 7PN -0.87 -4.3 7PC -0.55 -3.0 -23- We Claim: 1. A stable solution formulation comprising a therapeutically effective amount of a GLP- 1-Fc fusion at a pH of between about pH 6 and about pH 8.5 wherein the GLP-l-Fc fusion comprises a GLP-1 analog comprising a sequence selected from the group consisting of: a)(SEQDDNO:l) His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ee-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly wherein Xaa8 is selected from Gly and Val; b) (SEQ ED NO:2) His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Gly wherein Xaa8 is selected from Gly and Val; c) (SEQ ID NO:3) His-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-VaJ-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro wherein Xaa8 is selected from Gly and Val; d) (SEQ ID NO:4) His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ue-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Pro wherein Xaag is selected from Gly and Val; e) (SEQ ID NO:5) His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ue-Ala-Trp-Leu-Val-Lys-Gly-Gly wherein Xaa8 is selected from Gly and Val; f) (SEQ ID NO:6) His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Va]-Ser-Ser-Tyr-Leu-Glu-Glu- Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly wherein Xaa8 is selected from Gly and Val; fused to the Fc portion of an immunoglobulin comprising the sequence of SEQ ID NO:7 -24- la-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro-Cys-Pro-Ala-Pro- Xaa,6-Xaai7-Xaa,8-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro- Lys-Asp-Thr-Leu-Met-Le-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val- Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp- Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg- Glu-Glu-Gln-Phe-Xaago-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Leu-Thr- Val-Leu-His-Gln-Asp-Trp-Lcu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys- Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-De-Glu-Lys-Thr-Ile-Ser- Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro- Pro-Ser-Gln-GIu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys- Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-IJc-Ala-Val-GIu-Trp-Glu- Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val- Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val- Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val- Met-His-Glu-Ala-Leu'-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser- Leu-Ser-Leu-Gly-Xaa23o (SEQ ID NO:7) wherein: Xaa at position 16 is Pro or Glu; Xaa at position 17 is Phe, Val, or Ala; Xaa at position 18 is Leu, Glu, or Ala; Xaa at position 80 is Asn or Ala; and Xaa at position 230 is Lys or is absent.. 2. The stable solution formulation of claim 1, wherein the pH is between about pH 6 and about pH 7.5. 3. The stable solution formulation of claim 2, wherein the pH is between about pH 6 and about pH 7. 4. The stable solution formulation of claim 3, wherein the pH is between about pH 6 and about pH 6.5. 5. The stable solution formulation of claim 4, wherein the pH is about pH 6. 6. The stable solution formulation of claim 4, wherein the pH is about pH 6.5. 7. The stable solution formulation of claim 1, wherein the formulation further comprises Tween 20®. 8. The stable solution formulation of claim 1, wherein the formulation further comprises Tween 80®. 9. The stable solution formulation of claim 1, wherein the formulation further comprises NaCl. 10. The stable solution formulation of claim 1, wherein the formulation further comprises m-Cresol. 11. The stable solution formulation of claim 1, wherein the formulation further comprises Tween 20®, NaCl, and m-Cresol. 12. The stable solution formulation of claim 1, wherein the formulation further comprises Tween 80®, NaCl, and m-Cresol. -26- 13. A stable solution formulation substantially as herein described with reference to forgoing description, examples and sequence listings. The invention provides a stable solution formulation comprising a GLP-1-Fc fusion at a pH between about pH 6 and about pH 8.5 analogs fused to specific IgG4-Fc derivatives. These formulations provide unexpected and considerably greater chemical stability than when compared to GLP-1-Fc fusions at a pH outside the described ranges. The formulations comprising a GLP-1-Fc fusion are useful in treating diabetes, obesity, irritable bowel syndrome and other conditions that would be benefited by lowering plasma glucose, inhibiting gastric and/or intestinal motility and inhibiting gastric and/or intestinal emptying, or inhibiting food intake.

Full Text

FIELD OF THE INVENTION
The present invention relates to formulations of glucagon-like peptide analogs
fused to proteins that have the effect of stabilizing the fusion proteins. These
formulations can be used to treat diabetes as well as a variety of other conditions or
disorders.
BACKGROUND OF THE INVENTION
Glucagon-like peptide-1 (GLP-1) analogs and derivatives show promise in clinical
trials for the treatment of type 2 diabetes. GLP-1 induces numerous biological effects
such as stimulating insulin secretion, inhibiting glucagon secretion, inhibiting gastric
emptying, inhibiting gastric molility or intestinal motility, and inducing weight loss. A
significant 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 some types of
oral therapies that act by increasing insulin expression.
The usefulness of therapy involving GLP-1 peptides has been limited by the fact
that GLP-l(l-37) is poorly active, and the two naturally occurring truncated peptides,
GLP-1 (7-37)OH and GLP-1 (7-36)NH2, are rapidly cleared in vivo and have extremely
short in vivo half lives. It is known that endogenously produced dipeptidyl-peptidase IV
(DPP-IV) inactivates circulating GLP-1 peptides by removing the N-terminal histidine
and alanine residues and is a major reason for the short in vivo half-life.
Various approaches have been undertaken to extend the elimination half-life of a
GLP-1 peptide or reduce clearance of the peptide from the body while maintaining
biological activity. One approach involves fusing a GLP-1 peptide to the Fc portion of an
immunoglobulin. Immunoglobulins typically have long circulating half-lives in vivo.
For example, IgG molecules can have a half-life in humans of up to 23 days. The
Fc portion of the immunoglobulin is responsible, in part, for this in vivo stability. GLP-1 -
Fc fusion proteins take advantage of the stability provided by the Fc portion of an
immunoglobulin while preserving the biological activity of the GLP-1 molecule.
Although this approach is feasible for GLP-1 therapeutics (See WO 02/46227),
there is a general concern regarding the antigenicity of various fusion proteins when
administered repeatedly over prolonged periods of time. This is especially a concern for
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GLP-l-Fc fusion therapeutics as a patient with diabetes must be treated for her entire life
once diagnosed with the disease. In addition, Fc fusion protein therapeutics can be a
concern if the Fc portion retains unwanted effector functions. This approach is the focus
of PCT/US 04/15595 (WO2005/000892), in which problems associated with the potential
immunogenicity and effector activity associated with administration of GLP-l-Fc fusions
are overcome by identifying specific GLP-l-Fc fusion proteins that have a reduced risk of
inducing an immune response after repeated and prolonged administration and no longer
have effector function.
The fusion proteins of this nature are technically too large and complex to produce
synthetically or recombinantly in bacterial cells. These fusion proteins are typically
produced in mammalian cells, such as CHO, 293, or NSO. It was observed that the fusion
proteins produced in mammalian cells where more readily susceptible to degradation by
endogenous proteases and chemical alteration than non-fusion proteins produced in
bacterial cells. This problem was sought to be overcome in the present invention. It is
discovered that a formulation comprising a GLP-l-Fc fusion buffered between about pH
6 and about pH 8.5 provided increased chemical stability.
SUMMARY OF THE INVENTION
In order to overcome the problem of chemical stability of GLP-l-Fc fusions, the
present inventors have developed a stable solution formulation. In particular, the
inventors have discovered that a formulation comprising a therapeutically effective
amount of a GLP-l-Fc fusion at a pH between about pH 6 and about pH 8.5, preferably
between about pH 6 and about pH 7.5, between about pH 6 and about pH 7, between
about pH 6.5 and about pH 7.5, or between about pH 6 and about pH 6.5, and even more
preferably, about pH 6 or about 6.5, provided unexpectedly and considerably greater
chemical stability than when compared to GLP-l-Fc fusions at a pH outside the described
ranges.
The present invention also includes methods of treating patients suffering from
non-insulin dependent as well as insulin dependent diabetes mellitus, obesity, and various
other disorders and conditions comprising administering the formulations of GLP-l-Fc
fusions.
-3-

DETAILED DESCRIPTION OF THE INVENTION
The fusion proteins comprise a GLP-1 compound fused to an Fc portion of an
immunoglobulin, an analog of an Fc portion of an immunoglobulin, or a fragment of an
Fc portion of an immunoglobulin. The C-terminus of the GLP-1 compound may be fused
directly, or fused via a peptide linker, to the N-terminus of an Fc protein. These fusion
proteins are biologically active and have an increased half-life compared to native GLP-1.
The GLP-1 compounds that make up part of the fusion protein encompass polypeptides
having from about twenty-five to about thirty-nine naturally occurring or non-naturally
occurring amino acids that have sufficient homology to native GLP-1 (7-37)OH such that
they exhibit insulinotropic activity by binding to the GLP-1 receptor on P-cells in the
pancreas. A GLP-1 compound typically comprises a polypeptide having the amino acid
sequence of GLP-1 (7-37)OH, an analog of GLP-1 (7-37)OH, a fragment of GLP-1 (7-
37)OH or a fragment of a GLP-1 (7-37)OH analog.
Examples of fusion proteins useful in the formulations include fusion proteins
comprising a GLP-1 analog comprising a sequence selected from the group consisting of
a) (SEQ ED NO: 1)
His-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly
wherein Xaas is selected from Gly and Val;
b) (SEQ ID NO:2)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Gly
wherein Xaas is selected from Gly and Val;
c) (SEQ ID NO:3)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro
wherein Xaas is selected from Gly and Val;
d) (SEQ ID NO:4)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln- Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Pro
wherein Xaas is selected from Gly and Val;
e) (SEQ ID NO:5)
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His-XaarGlu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-AIa-AIa-Lys-Glu-Phe-Ee-Ala-Trp-Leu-VaJ-Lys-Gly-Gly
wherein Xaag is selected from Gly and Val;
f) (SEQ ID NO:6)
His-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-VaJ-Ser-Ser-Tyr-Leu-GJu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ue-Ala-Trp-Leu-Lys-Asn-Gly-Gly
wherein Xaas is selected from Gly and Val;
fused to the Fc portion of an immunoglobulin comprising the sequence of SEQ ID
NO:7
Ala-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro-Cys-Pro-Ala-Pro-
Xaai6-Xaai7-Xaa,8-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-
Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-
Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-
Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Aia-Lys-Thr-Lys-Pro-Arg-
Glu-Glu-Gln-Phe-Xaago-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Uu-Thr-
Val-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-
Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser-
Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-
Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-
Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-
Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-
Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Axg-Leu-Thr-Val-
Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-
Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-
Leu-Ser-Leu-Gly-Xaa23o (SEQ ID NO:7)
wherein:
Xaa at position 16 is Pro or Glu;
Xaa at position 17 is Phe, Val, or Ala;
Xaa at position 18 is Leu, Glu, or Ala;
Xaa at position 80 is Asn or Ala; and
Xaa at position 230 is Lys or is absent.
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The C-terminus of the GLP-1 analog portion and the N-terminus of the Fc portion
of the fusion proteins are preferably fused together via 1, 1.5 or 2 repeats of a G-rich
peptide linker having the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-
Gly-Gly-Ser (SEQ ID NO:8).
The fusion proteins useful in the formulations of the present invention
comprise a GLP-1 analog portion and an Fc portion. The GLP-1 analog portion
and the Fc portion comprise substitutions to the native GLP-1 sequence and the
human IgG4 sequence respectively that provide the protein with increased
potency and in vivo stability compared to native GLP-1 or GLP-1 analogs not
fused to an Fc sequence while decreasing the potential for inducing antibody
formation after prolonged and repeated administration in humans.
Native GLP-1 is processed in vivo such that the first 6 amino acids are cleaved
from the molecule. Thus, by custom in the art, the amino terminus of GLP-1 has been
assigned the number 7 and the carboxy-terminus, number 37. The other amino acids in
the polypeptide are numbered consecutively as shown in SEQ ID NO:9. For example,
position 8 is alanine and position 22 is glycine. The processed peptide may be further
modified in vivo such that the C-terminal glycine residue is removed and replaced with an
amide group. Thus, GLP-1(7-37)QH and GLP-1 (7-36)amide represent the two native
forms of the molecule. GLP-1 (7-37)OH has the amino acid sequence of SEQ ID NO:9:
7His-Ala-Glu-10Gly-Thr-Phe-Thr-Ser-l5Asp-Val-Ser-Ser-Tyr-20Leu-Glu-Gly-
Gln-Ala-25Ala-Lys-Glu-Phe-De-3OAla-Trp-Leu-Val-Lys-35Gly-Arg-37Gly
(SEQ ID NO:9)
The GLP-1 analog portion of the fusion protein comprises three primary
substitutions at positions 8, 22, and 36 relative to native GLP-1 (7-37). The
substitution at position 8 reduces the rate at which the endogenous enzyme
dipeptidyl-peptidase IV (DPP-IV) inactivates the analog. DPP-IV cleaves native
GLP-1 between the 2nd and 3rd amino acids (between position 8 and 9) and the
resulting molecule is less active. Thus, the fusion proteins useful in the
formulations of the present invention are DPP-IV resistant. The substitution at
position 22 reduces the potential of the molecule to aggregate and increases the
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potency of the molecule. The substitution at position 36 in the context of the
analog with changes at 8 and 22 as well as in the context of the entire fusion
protein reduces the risk that the fusion protein will induce a neutralizing immune
response after repeated and prolonged administration in humans.
The GLP-1 analog C-terminus is preferably one of the following
sequences: Trp-Leu-Val-Lys-Gly-Gly-Gly (SEQ ID NO: 10); Trp-Leu-Lys-Asn-
Gly-Gly-Gly (SEQ ID NO:11); Trp-Leu-Val-Lys-Gly-Gly-Pro (SEQ ID NO: 12);
Trp-Leu-Lys-Asn-Gly-Gly-Pro (SEQ ID NO: 13); Trp-Leu-Val-Lys-Gly-Gly
(SEQ ID NO: 14); and Trp-Leu-Lys-Asn-Gly-Gly (SEQ ID NO: 15).
The fusion proteins useful in the formulations of the present invention
contain an Fc portion which is derived from human IgG4, but comprises one or
more substitutions compared to the wild-type human sequence. As used herein,
the Fc portion of an immunoglobulin has the meaning commonly given to the
term in the field of immunology. Specifically, this term refers to an antibody
fragment which does not contain the two antigen binding regions (the Fab
fragments) from the antibody. The Fc portion consists of the constant region of
an antibody from both heavy chains, which associate through non-covalent
interactions and disulfide bonds. The Fc portion can include the hinge regions
and extend through the CH2 and CH3 domains to the c-terminus of the antibody.
The Fc portion can further include one or more glycosylation sites.
There are five types of human immunoglobulins with different effector
functions and pharmcokinetic properties. IgG is the most stable of the five types
having a serum half-life in humans of about 23 days. There are four IgG
subclasses (Gl, G2, G3, and G4) each of which have different biological
functions known as effector functions. These effector functions are generally
mediated through interaction with the Fc receptor (FcyR) or by binding Clq and
fixing complement. Binding to FcyR can lead to antibody dependent cell
mediated cytolysis, whereas binding to complement factors can lead to
complement mediated cell lysis. In designing Fc fusion proteins wherein the Fc
portion is being utilized solely for its ability to extend half-life, it is important to
minimize any effector function. Thus, the fusion proteins useful in the
formulations of the present invention are derived from the human IgG4 Fc region

because of its reduced ability to bind FeyR and complement factors compared to
other IgG sub-types. IgG4, however, has been shown to deplete target cells in
humans [Issacs et al., (1996) CHn. Exp. Immunol. 106:427-433]. Because the
fusion proteins target beta cells in the pancreas to induce insuJin expression,
using an IgG4 derived region in an Fc fusion protein could initiate an immune
response against the pancreateic beta cell through interaction of the fusion
protein with the GLP-1 receptor present on pancreatic beta cells. Thus, the IgG4
Fc region which is part of the fusion proteins contains substitutions that eliminate
effector function. The lgG4 Fc portion of the fusion proteins may contain one or
more of the following substitutions: substitution of proline for glutamate at
residue 233, alanine or valine for phenylalanine at residue 234 and alanine or
glutamate for leucine at residue 235 (EU numbering, Kabat, E.A. et al. (1991)
Sequences of Proteins of Immunological Interest, 5' Ed. U.S. Dept. of Health
and Human Services, Bethesda, MD, NIH Publication no. 91-3242). These
residues corresponds to positions 16, 17 and 18 in SEQ ID NO:7. Further,
removing the N-linked glycosylation site in the IgG4 Fc region by substituting
Ala for Asn at residue 297 (EU numbering) which corresponds to position 80 of
SEQ ID NO:7 is another way to ensure that residual effector activity is
eliminated in the context of a fusion protein.
In addition, the IgG4 Fc portion of the fusion proteins contain a
substitution that stabilizes heavy chain dimer formation and prevents the
formation of half-IgG4 Fc chains. The fusion proteins preferably exist as dimers
joined together by disulfide bonds and various non-covalent interactions. Wild-
type IgG4 contains a Pro-Pro-Cys-Pro-Ser-Cys (SEQ ID NO: 16) motif beginning
at residue 224 (EU numbering). This motif in a single GLP-1 analog-Fc chain
forms disulfide bonds with the corresponding motif in another GLP-1 analog-Fc
chain. However, the presence of serine in the motif causes the formation of
single chain fusion proteins. The present invention encompasses Fc fusion
proteins wherein the IgG4 sequence is further modified such that serine at
position at 228 (EU numbering) is substituted with proline (amino acid residue
llinSEQIDNO:7).
The C-termina] lysine residue present in the native molecule may be
-8-

deleted in the IgG4 derivative Fc portion of the fusion proteins discussed herein
(position 230 of SEQ ID NO:7; deleted lysine referred to as des-K). Fusion
proteins expressed in some cell types (such as NSO cells) wherein lysine is
encoded by the C-terminal codon are heterogeneous in that a portion of the
molecules have lysine as the C-terminal amino acid and a portion have lysine
deleted. The deletion is due to protease action during expression in some types
of mammalian cells. Thus, to avoid this heterogeneity, it is preferred that Fc
fusion expression constructs lack a C-terminal codon for lysine.
It is preferred that the C-terminal amino acid of the GLP-1 analog portion
discussed herein is fused to the N-terminus of the IgG4 Fc analog portion via a
glycine-rich linker. The in vivo function and stability of the fusion proteins can
be optimized by adding small peptide linkers to prevent potentially unwanted
domain interactions. Further, a glycine-rich linker provides some structural
flexibility such that the GLP-1 analog portion can interact productively with the
GLP-1 receptor on target cells such as the beta cells of the pancreas. These
linkers, however, can significantly increase the risk that the fusion protein will be
immunogenic in vivo. Thus, it is preferred that the length be no longer than
necessary to prevent unwanted domain interactions and/or optimize biological
activity and/or stability. The preferred glycine-rich linker comprises the
sequence: Gly-Gly-GJy-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser
(SEQ ID NO:8). Although more copies of this linker may be used in the fusion
proteins, it is preferred that a single copy of this linker be used to minimize the
risk of immunogenicity associated with prolonged and repeated administration.
Preferred GLP-1-Fc fusions include the following proteins: Gly8-Glu22-Gly36-
GLP-l(7-37)-lL-IgG4 (S228P), Gly8-Glu22-Gly36-GLP-l(7-37)-lL-IgG4 (S228P, F234A,
L235A), Gly8-Glu22-Gly36-GLP-l(7-37)-lL-IgG4 (S228P, N297A), Gly8-Glu22-Gly36-
GLP-l(7-37)-lL-IgG4 (S228P, F234A, L235A, N297A), Gly8-Glu22-Gly36-GLP-1(7-37)-
1.5L-IgG4 (S228P), Gly8-Glu22-Gly36-GLP-](7-37)-1.5L-IgG4 (S228P, F234A, L235A),
Gly8-Glu22-Gly36-GLP-l(7-37)-1.5L-IgG4 (S228P, N297A), Gly8-Glu22-Gly36-GLP-1(7-
37)-1.5L-IgG4 (S228P, F234A, L235A, N297A), Gly8-Glu22-GIy36-GLP-l(7-37)-2L-
IgG4 (S228P), Gly8-Glu22-Gly36-GLP-l(7-37)-2L-IgG4 (S228P, F234A, L235A), Gly8-
Glu22-Gly36-GLP-l(7-37)-2L-IgG4 (S228P, N297A), and Gly8-Glu22-Gly36-GLP-1(7-37)-

2L-IgG4 (S228P, F234A, L235A, N297A), and the Val8 and des-K forms of all of the
above.
The nomenclature used herein to refer to specific fusion proteins is defined as
follows: Specific substitutions to the GLP-1 portion of the fusion protein are indicated
using the specific arnino acid being substituted followed by the residue number. GLP-
1(7-37) indicates that the GLP-1 portion of the mature fusion protein begins with His at
position 7 and ends with Gly at position 37. L refers to a linker with the sequence Gly-
Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:8). The
number immediately preceding the L refers to the number of linkers separating the GLP-1
portion from the Fc portion. A linker specified as 1.5L refers to the sequence Gly-Ser-
Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser
(SEQ ID NO: 17). IgG4 refers to an analog of the human IgG4 Fc sequence specified as
SEQ ED NO:7. Substitutions in the lgG4 Fc portion of the fusion protein are indicated in
parenthesis. The wild-type amino acid is specified by its common abbreviation followed
by the position number in the context of the entire IgG4 sequence using the EU
numbering system followed by the amino acid being substituted at that position specified
by its common abbreviation.
The fusion proteins have biological activity. Biological activity refers to the ability cf
the fusion protein to bind to and activate the GLP-1 receptor in vivo and elicit a response.
Responses include, but are not limited to, secretion of insulin, suppression of glucagon,
inhibition of appetite, weight loss, induction of satiety, inhibition of apoptosis, induction of
pancreatic beta cell proliferation, and differentiation of pancreatic beta cells. A
representative number of GLP-1 fusion proteins were tested for in vitro as well as in vivo
activity. In vitro activity based on the ability of the fusion protein to interact with and
activate the human GLP-1 receptor have been shown. (See PCTAJS 04/15595
(WO2005/000892)). HEK293 cells over-expressing the human GLP-1 receptor are used.
Activation of the GLP-1 receptor in these cells causes adenylyl cyclase activation which in
turn induces expression of a reporter gene driven by a cyclic AMP response element (CRE).
The pH of the GLP-1-Fc fusion formulations is adjusted to provide acceptable
stability, to maintain the solubility and insulinotropic activity of the GLP-1-Fc fusion and
be acceptable for parenteral administration. The pH of the GLP-1 -Fc fusion formulations
is preferably adjusted to between about pH 6 and about pH 8.5, preferably between about
-10-

pH 6 and about pH 7.5, between about pH 6 and about pH 7, between about pH 6.5 and
about pH 7.5, or between about pH 6 and about pH 6.5, and even more preferably, about
pH 6 or about 6.5.
The formulations comprising a GLP-l-Fc fusion of the present invention may
optionally encompass a pharmaceutically acceptable buffer. However, the selection and
concentration of the buffer shall be such that the formulation can be adjusted to the
described ranges that provide acceptable stability and insulinotropic activity. Examples of
pharmaceutically acceptable buffers include phosphate buffers like dibasic sodium
phosphate, TRIS, acetate, such as sodium acetate, citrate, such as sodium citrate, sodium
tartarate, basic amino acids such as histidine, lysine or arginine, or neutral amino acids
such as glycine and glycyl-glycine. Other pharmaceutically acceptable buffers are known
in the art. Preferably, the buffer is selected from the group consisting of citrate,
phosphate and TRIS. The skilled artisan will recognize that the selection of the buffer is
dependent upon the described pH ranges and the pKa of the buffer. Preferably, the
concentration of a buffer is between about 1 mM and 30 mM. Even more preferably, the
concentration is between about 4 mM and 14 mM or between about 5 mM and 20 mM.
Even more preferably, the concentration is between about 10 mM and 20 mM. Even
more preferably, the concentration is about 10 mM or about 20 mM.
The formulations of the present invention may optionally encompass a
preservative. However, the selection and concentration of the preservative shall be such
that the formulation can be adjusted to the described ranges that provide acceptable
stability and insulinotropic activity. Preservative refers to a compound that is added to a
pharmaceutical formulation to act as an anti-microbial agent. A parenteral formulation
must meet guidelines for preservative effectiveness to be a commercially viable multi-use
product. 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, chloro-cresol,
or mixtures thereof. Alkylparaben refers to a Ci to C4 alkyl paraben, or mixtures thereof.
Examples of alkylparabens include methylparaben, ethylparaben, propylparaben, or
butylparaben. The concentration of the preservative is known to one skilled in the art.
-11-

The concentrations must be sufficient to maintain preservative effectiveness by retarding
microbial growth.
The preferred preservative is meta-cresol or phenol. In general, the concentration
of meta-cresol is between about 2.0 to about 8.0 mg/mL, about 2.5 mg/mL to about 4.5
mg/mL, and about 2.0 mg/mL to about 4.0 mg/mL. A most preferred concentration of
preservative in the formulation is about 2.7 mg/mL. In another embodiment, the
concentration of phenol is between about 2.0 to about 10.0 mg/mL, and about 4.0 to about
8.0 mg/mL. A most preferred concentration of preservative in the formulation is about
5.0 mg/mL.
The formulations of the present invention may optionally encompass an
isotonicity agent. However, the selection and concentration of the isotonicity agent shall
be such that the formulation can be adjusted to the described ranges that provide
acceptable stability and insulinotropic activity. 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 (or glycerol), 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 NaCl is preferably
between about 10 mM and 500 mM, more preferred is between about 50 mM and 200
mM, and most preferred is about 150 mM. In another embodiment, the preferred
isotonicity agent is mannitol. The concentration of the mannitol is preferably between
about 1% (weight (w)/volume (v)) and 10% (w/v), and more preferred is between about
2% (w/v) and 8% (w/v). In another embodiment, the preferred isotonicity agent is
glycerin. The concentration of the glycerin is preferably between about 12 mg/mL and 25
mg/ml, preferably between about 12 mg/mL and 20 mg/ml, and more preferred is about
17 mg/ml.
The formulations of the present invention may optionally encompass a solubility
enhancer. However, the selection and concentration of the solubility enhancer shall be
such that the formulation can be adjusted to the described ranges that provide acceptable
stability and insulinotropic activity. Solubility enhancers provide stability such that the
GLP-l-Fc fusion remains soluble for an extended period of time under the conditions of
-12-

storage. Preferably the solubility enhancer is nicotinamide. In general, the concentration
of nicotinamide is between 0.01 and 2 molar. Other preferred ranges of nicotinamide
concentration are: between 0.05 molar and 1.5 molar; between 0.1 molar and 1.0 molar;
between 0.1 molar and 0.5 molar; between 0.5 molar and 1.0 molar; and between 0.15
molar and 0.25 molar.
Other additives, such as a pharmaceutically acceptable solubilizers like Tween
20® (polyoxyethylene (20) sorbitan monolaurate), Tween 40® (polyoxyethylene (20)
sorbitan monopalmitate), Tween 80® (polyoxyethylene (20) sorbitan monooleate),
Pluronic F68® (polyoxyethylene polyoxypropylene block copolymers), and PEG
(polyethylene glycol) may optionally be added to the formulation. Preferably the
solubilizer is Tween 20® or TweenSO®. In general, the concentration of Tween 20® or
Tween80® is between 0.001% and 0.05%. Other preferred ranges of Tween 20® or
Tween80® concentrations are: between 0.005% and 0.05%; between 0.0075% and 0.05%;
and between 0.01% and 0.05%.
Administration of the formulations may be via any route known to be effective by the
physician of ordinary skill. Peripheral parenteral is one such method. Parenteral
administration is commonly understood in the medical literature as the injection of a dosage
form into the body by a sterile syringe or some other mechanical device such as an infusion
pump. Peripheral parenteral routes can include intravenous, intramuscular, subcutaneous,
and intraperitoneal routes of administration.
The formulations may also be amenable to administration by oral, rectal, nasal, or
lower respiratory routes, which are non-parenteral routes. Of these non-parenteral routes, the
lower respiratory route and the oral route are preferred.
The formulations comprising a GLP-l-Fc fusion can be used to treat a wide variety of
diseases and conditions. The GLP-l-Fc fusions primarily exert their biological effects by
acting at a receptor referred to as the "GLP-1 receptor." Subjects with diseases and/or
conditions that respond favorably to GLP-1 receptor stimulation or to the administration of
GLP-1 compounds can therefore be treated with the GLP-l-Fc fusions. These subjects are
said to "be in need of treatment with GLP-1 compounds" or "in need of GLP-1 receptor
stimulation". Included are subjects with non-insulin dependent diabetes, insulin dependent
diabetes, stroke (see WO 00/16797), myocardial infarction (see WO 98/08531), obesity (see
WO 98/19698), catabolic changes after surgery (see U.S. Patent No. 6,006,753), functional
-13-

dyspepsia and irritable bowel syndrome (see WO 99/64060). Also included are subjects
requiring prophylactic treatment with a GLP-1 compound, e.g., subjects at risk for
developing non-insulin dependent diabetes (see WO 00/07617). Subjects with impaired
glucose tolerance or impaired fasting glucose, subjects whose body weight is about 25%
above normal body weight for the subject's height and body build, subjects with a partial
pancreatectomy, subjects having one or more parents with non-insulin dependent diabetes,
subjects who have had gestational diabetes and subjects who have had acute or chronic
pancreatitis are at risk for developing non-insulin dependent diabetes.
An effective amount of the GLP-1-Fc fusions in the context of the described
formulations is the quantity which results in a desired therapeutic and/or prophylactic
effect without causing unacceptable side-effects when administered to a subject in need of
GLP-1 receptor stimulation. A "desired therapeutic effect" includes one or more of the
following: 1) an amelioration of the symptom(s) associated with the disease or condition;
2) a delay in the onset of symptoms associated with the disease or condition; 3) increased
longevity compared with the absence of the treatment; and 4) greater quality of life
compared with the absence of the treatment. For example, an "effective amount" of a
GLP-l-Fc fusion for the treatment of diabetes is the quantity that would result in greater
control of blood glucose concentration than in the absence of treatment, thereby resulting
in a delay in the onset of diabetic complications such as retinopathy, neuropathy or
kidney disease. An "effective amount" of a GLP-1-Fc fusion for the prevention of
diabetes is the quantity that would delay, compared with the absence of treatment, the
onset of elevated blood glucose levels that require treatment with anti-hypoglycaemic
drugs such as sulfonyl ureas, thiazolidinediones, insulin and/or bisguanidines.
The dose of fusion protein effective to normalize a patient's blood glucose will
depend on a number of factors, among which are included, without limitation, the subject's
sex, weight and age, the severity of inability to regulate blood glucose, the route of
administration and bioavailability, the pharmacokinetic profile of the fusion protein, the
potency, and the formulation. Doses may be in the range of 0.01 to 1 mg/kg body weight,
preferably in the range of 0.05 to 0.5 mg/kg body weight.
It is preferable that the fusion proteins be administered either once every two
weeks or once a week. Depending on the disease being treated, it may be necessary to
administer the fusion protein more frequently such as two to three time per week.

The present invention will now be described only by way of non-limiting example
with reference to the following Examples.
EXAMPLES
In vitro GLP-1 receptor activation assay:
HEK-293 cells expressing the human GLP-1 receptor, using a CRE-BLAM
system, are seeded at 20,000 to 40,000 cells/well/100 fxl DMEM medium with 10%FBS
into a poly-d-lysine coated 96 well black, clear-bottom plate. The day after seeding, the
medium is flicked off and 80 jil plasma-free DMEM medium is added. On the third day
after seeding, 20 \l\ of plasma-free DMEM medium with 0.5% BSA containing different
concentrations of various GLP-1 -Fc fusion protein is added to each well to generate a
dose response curve. Generally, fourteen dilutions containing from 3 nanomolar to 30
nanomolar or GLP-1 Fc fusion protein are used to generate a dose response curve from
which EC50 values can be determined. After 5 hours of incubation with the fusion protein,
20 Μ1 of P-lactamase substrate (CCF2/AM, PanVera LLC) is added and incubation
continued for 1 hour at which time fluorescence is determined on a cytofluor. The assay
is further described in Zlokarnik, et al. (1998), Science, 278:84-88.
In vitro GLP-1 receptor activation assay:
HEK-293 cells stably expressing the human GLP-1 receptor, using a CRE-
Luciferase system, are seeded at 30,000 cells/well/80 fil low serum DMEM F12 medium
into 96 well plates. The day after seeding, 20 μl aliquots of test protein dissolved in 0.5%
BSA are mixed and incubated with the cells for 5 hours. Generally 12 dilutions
containing from 3 pM to 3 nM are prepared at a 5X concentration for each test protein
before addition to the cells to generate a dose response curve from which EC50 values are
determined. After incubation, 100 p.1 of Luciferase reagent is added directly to each plate
and mixed gently for 2 minutes. Plates are placed in a Tri-lux luminometer and light
output resulting from luciferase expression is calculated.
Effect of Tween. NaCl. and pH on stability of Glv8-Glu22-GLP-1(7-37)-!L-IgG4 (S228P,
F234A. L235A):

The effects of tween-20 (± 0.01%), NaCl (0,150, and 500mM), and pH (6.5, 7.5,
and 8.5) on the chemical stability of 0.5mg/mL Gly8-Glu22-GLP-l(7-37)-lL-IgG4
(S228P, F234A, L235A) are determined. A full matrix of these conditions are prepared
by mixing a respective 2x buffer stock with a lmg/mL Gly8-GIu22-GLP-l(7-37)-lL-IgG4
(S228P, F234A, L235A) solution dissolved in 20mM phosphate, 20mM Tris, pH 7.5. The
final solution conditions are terminally filtered through 0.2micron Millex-GV filters into
HPLC vials and incubated at 37°C. Periodically time points are removed and analyzed by
size-exclusion chromatography, reversed-phase chromatography, and gel electrophores.
Identification of formulation components for samples A-R (all contained 0.5mg/mL Gly8-
Glu22-GLP-l(7-37)-lL-IgG4 (S228P, F234A, L235A), 20mM phosphate, and 20mM
Tris)

Size-Exclusion HPLC method:
Samples are injected on a Tosohas TSK3000-SW-XL (5 micron, 7.8x3OOmm)
column pre-equilibrated in lx PBS, pH 7.4 plus 10% acetonitrile (v/v) mobile buffer (A-
pump buffer). A-buffer is flowing at 0.5mL/min isocratic and protein was detected at
214nm.
Reversed-Phase HPLC method:
Samples are injected on a Zorbax 300SB-C8 (4.6x50mm) column pre-equilibrated
in 0.1% TFA (v/v) mobile buffer (A-pump buffer). B-pump buffer consists of 0.085%
TFA in acetonitrile (v/v). The following gradient is run and material was detected at
214nm.
-18-

lime % B-pump buffer
0 30
5 30
35 45
40 90
45 90
50 25
65 25
Gel Electrophoresis method:
Time points are diluted with 4x NuPage LDS sample buffer (lx final cone) and
heated at 95°C for 5 minutes. The cooled samples were loaded on a 4-12% bis-tris
NuPage gel. The gel is run at constant voltage (200 volts) for 50-60min in MOPS buffer
and stained with SimplyBlue SafeStain.
Stability of Glv8-Glu"-GLP-1(7-37)- IL-IgG4 (S228P, F234A, L235A) and effect of
Tween-20:
Stability of Gly8-Glu22-GLP-1(7-37)- lL-IgG4 (S228P, F234A, L235A) at
0.3mg/mL in lx PBS at pH 7.5 is evaluated by monitoring the increase in turbidity at
35Onm under accelerated stress conditions (stirring with hydrophobic stir bar in PBS at
37oC).
The effect of Tween-20 on reducing the aggregation
Solution Additive Time for turbidity to increase > 0.05 OD at 350nm
None 0.001% Tween-20 16-hr
0.005% Tween-20 27-hr
0.01% Tween-20 > 65-hr
0.02% Tween-20 > 65-hr
0.05% Tween-20 > 65-hr
-19-

Solubility of Glv8-Glu"-GLP-1(7-37)- !L-IgG4 (S228P. F234A. L235A) as a function of
solution pH:
Solubility of Gly8-Glu22-GLP-1(7-37)-lL-IgG4 (S228P, F234A, L235A) as a
function of pH is determined by diluting a stock solution with various buffered solution at
differ pH values, allowing the solution to equilibrate, centrifugating away any
precipitated material, then analyzing the supernant for remaining soluble protein
concentration. Stock protein frozen in PBS, is dialyzed against water at approximately pH
8. After dialysis the protein stock was filtered through a 0.22micron Millex-GV filter and
concentrated to ~12mg/mL using a rinsed Ultrafree centrifugal device (4ml, 5000 dalton
MWCO, at 7000xG). This concentrated solution is diluted 1:1 with 2x stock buffer
solutions (final volume of 50uL in 0.5mL eppendorf tubes) and is allowed to equilibrate
for 15-30 minutes at room temperature. The solution is centrifuged (14,000xG for 5
minutes) to pellet any insoluble material. lOuL of the supernant is removed and
combined with 40uL of the pH 8 water. Two replicates are performed for each sample.
The concentration of soluble GLP-Fc is determined by reversed-phase HPLC against a
standard curve.
Effect of solution conditions on solubility

20mM Citrate, 3mg/mL m-Cresol, pH 5 1.3 0.0
20mM Citrate, 3mg/mL m-Cresol, pH 6 5.5 0.1
20mM Phosphate, 3mg/mL m-Cresol, pH 7 6.2 0.1
20mM Tris, 3mg/mL m-Cresol, pH 8 6.0 0.0
Effect of solution pH on stability as monitored by reversed-phase HPLC:
Gly8-Glu22-GLP-l(7-37)-lL-IgG4 (S228P, F234A, L235A) is dialyzed against
water adjusted to approximately pH 8. After dialysis this protein stock is filtered (0.2
micron Millex-GV) and concentrated to >2mg/mL using a rinsed Ultrafree centrifugal
device. The final protein concentration is determined by UV and various formulations are
made by diluted 2x formulation buffer stocks to lx keeping the final protein
concentration at lmg/mL. The solution pH is checked and adjusted if needed. These
solutions are terminally filtered (0.22micon Millex-GV, 4mm) in sterilized 1.8mLHPLC
vials with screw caps. The vials are placed at 37°C and time points are pulled and
analyzed by reversed-phase, size-exclusion, and gel electrophoresis. The change in
percent main-peak as monitored by size-exclusion chromatography and reversed-phase
HPLC are presented. The linear degradation rates obtained by fitting a plot of incubation
time at 37oC with main peak purity for the size-exclusion and reversed-phase HPLC data
is tabulated.
Identification of formulation parameters.
Formulation code PH Buffer (l0mM) Additive
3C 3.0 Citrate none
4C 4.0 Citrate none
5C 5.0 Citrate none
6C 6.0 Citrate none
6CN 6.0 Citrate 150mM NaCl
6CC 6.0 Citrate 3mg/mL m-Cresol
6P 6.0 Phosphate none
7P-R1 7.0 Phosphate None, replicate #1
7P-R2 7.0 Phosphate None, replicate #2
7P-R3 7.0 Phosphate None, replicate #3
7PN 7.0 Phosphate 150mM NaCl
7PC 7.0 Phosphate 3mg/mL m-Cresol
8P 8.0 Phosphate none
8T 8.0 Tris none
-21-

The change in percent main-peak upon storage at 37°C. Data is analyzed by size-
exclusion chromatography.

8T 0.003 -2.2
6CN -0.85 -2.1
6CC -1.6 -2.7
7PN -0.87 -4.3
7PC -0.55 -3.0
-23-

We Claim:
1. A stable solution formulation comprising a therapeutically effective amount of a GLP-
1-Fc fusion at a pH of between about pH 6 and about pH 8.5
wherein the GLP-l-Fc fusion comprises a GLP-1 analog comprising a sequence
selected from the group consisting of:
a)(SEQDDNO:l)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ee-Ala-Trp-Leu-Val-Lys-Gly-Gly-Gly
wherein Xaa8 is selected from Gly and Val;
b) (SEQ ED NO:2)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Gly
wherein Xaa8 is selected from Gly and Val;
c) (SEQ ID NO:3)
His-Xaag-Glu-Gly-Thr-Phe-Thr-Ser-Asp-VaJ-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Gly-Pro
wherein Xaa8 is selected from Gly and Val;
d) (SEQ ID NO:4)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ue-Ala-Trp-Leu-Lys-Asn-Gly-Gly-Pro
wherein Xaag is selected from Gly and Val;
e) (SEQ ID NO:5)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ue-Ala-Trp-Leu-Val-Lys-Gly-Gly
wherein Xaa8 is selected from Gly and Val;
f) (SEQ ID NO:6)
His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Va]-Ser-Ser-Tyr-Leu-Glu-Glu-
Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Lys-Asn-Gly-Gly
wherein Xaa8 is selected from Gly and Val;
fused to the Fc portion of an immunoglobulin comprising the sequence of SEQ ID
NO:7
-24-

la-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro-Cys-Pro-Ala-Pro-
Xaa,6-Xaai7-Xaa,8-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-
Lys-Asp-Thr-Leu-Met-Le-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-
Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-
Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-
Glu-Glu-Gln-Phe-Xaago-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Leu-Thr-
Val-Leu-His-Gln-Asp-Trp-Lcu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-
Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-De-Glu-Lys-Thr-Ile-Ser-
Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-
Pro-Ser-Gln-GIu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-
Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-IJc-Ala-Val-GIu-Trp-Glu-
Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-
Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val-
Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-
Met-His-Glu-Ala-Leu'-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-
Leu-Ser-Leu-Gly-Xaa23o (SEQ ID NO:7)
wherein:
Xaa at position 16 is Pro or Glu;
Xaa at position 17 is Phe, Val, or Ala;
Xaa at position 18 is Leu, Glu, or Ala;
Xaa at position 80 is Asn or Ala; and
Xaa at position 230 is Lys or is absent..
2. The stable solution formulation of claim 1, wherein the pH is between about pH 6 and
about pH 7.5.
3. The stable solution formulation of claim 2, wherein the pH is between about pH 6 and
about pH 7.
4. The stable solution formulation of claim 3, wherein the pH is between about pH 6 and
about pH 6.5.
5. The stable solution formulation of claim 4, wherein the pH is about pH 6.
6. The stable solution formulation of claim 4, wherein the pH is about pH 6.5.

7. The stable solution formulation of claim 1, wherein the formulation further comprises
Tween 20®.
8. The stable solution formulation of claim 1, wherein the formulation further comprises
Tween 80®.
9. The stable solution formulation of claim 1, wherein the formulation further comprises
NaCl.
10. The stable solution formulation of claim 1, wherein the formulation further comprises
m-Cresol.
11. The stable solution formulation of claim 1, wherein the formulation further comprises
Tween 20®, NaCl, and m-Cresol.
12. The stable solution formulation of claim 1, wherein the formulation further comprises
Tween 80®, NaCl, and m-Cresol.
-26-
13. A stable solution formulation substantially as herein described with reference to
forgoing description, examples and sequence listings.

The invention provides a stable solution formulation comprising a GLP-1-Fc fusion at a pH between about pH 6 and about pH 8.5 analogs fused to specific IgG4-Fc derivatives. These formulations provide unexpected and considerably greater chemical stability than when compared to GLP-1-Fc fusions at a pH outside the described ranges. The formulations comprising a GLP-1-Fc fusion are useful in treating diabetes, obesity, irritable bowel syndrome and other conditions that would be benefited by lowering plasma glucose, inhibiting gastric and/or intestinal motility and inhibiting gastric and/or intestinal emptying, or inhibiting food intake.

Documents:

02275-kolnp-2007-abstract.pdf

02275-kolnp-2007-assignment.pdf

02275-kolnp-2007-claims.pdf

02275-kolnp-2007-correspondence others.pdf

02275-kolnp-2007-description complete.pdf

02275-kolnp-2007-form 1.pdf

02275-kolnp-2007-form 18.pdf

02275-kolnp-2007-form 2.pdf

02275-kolnp-2007-form 26.pdf

02275-kolnp-2007-form 3.pdf

02275-kolnp-2007-form 5.pdf

02275-kolnp-2007-gpa.pdf

02275-kolnp-2007-international publication.pdf

02275-kolnp-2007-international search report.pdf

02275-kolnp-2007-others.pdf

02275-kolnp-2007-pct request form.pdf

02275-kolnp-2007-priority document.pdf

02275-kolnp-2007-sequence listing.pdf

2275-KOLNP-2007-(04-09-2014)-CLAIMS.pdf

2275-KOLNP-2007-(04-09-2014)-CORRESPONDENCE.pdf

2275-KOLNP-2007-(04-09-2014)-OTHERS.pdf

2275-KOLNP-2007-ABSTRACT-1.1.pdf

2275-KOLNP-2007-AMENDED PAGES.pdf

2275-KOLNP-2007-CANCELLED PAGES.pdf

2275-KOLNP-2007-CLAIMS-1.1.pdf

2275-KOLNP-2007-CORRESPONDENCE 1.1.pdf

2275-KOLNP-2007-CORRESPONDENCE-1.2.pdf

2275-KOLNP-2007-DESCRIPTION (COMPLETED)-1.1.pdf

2275-KOLNP-2007-FORM 1-1.1.pdf

2275-KOLNP-2007-FORM 13.pdf

2275-KOLNP-2007-FORM 2-1.1.pdf

2275-KOLNP-2007-FORM 3.1.1.pdf

2275-KOLNP-2007-REPLY TO EXAMINATION REPORT.pdf

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Patent Number

263401

Indian Patent Application Number

2275/KOLNP/2007

PG Journal Number

44/2014

Publication Date

31-Oct-2014

Grant Date

27-Oct-2014

Date of Filing

20-Jun-2007

Name of Patentee

ELI LILLY AND COMPANY

Applicant Address

LILLY CORPORATE CENTER, CITY OF INDIANAPOLIS, INDIANA

Inventors:

#	Inventor's Name	Inventor's Address
1	GLAESNER, WOLFGANG	3808 CARWINION WAY, CARMEL, INDIANA 46032
2	MILLICAN, ROHN, LEE, JUNIOR	8145 GRASSY MEADOW COURT, INDIANAPOLIS, INDIANA 46259, USA

PCT International Classification Number

C07K 14/50

PCT International Application Number

PCT/US2005/045376

PCT International Filing date

2005-12-15

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/641,690	2004-12-22	U.S.A.