Glucagon-receptor selective polypeptides and methods of use thereof

Description

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable format. The Sequence Listing is provided as a file entitled 617297-102487-048CON-SEQ-TEXT.txt created Oct. 28, 2019 which 109,083 bytes in size. The information in the computer readable form of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD

This invention relates to isolated polypeptides that are glucagon-receptor selective analogs and peptide derivatives thereof. These analogs and peptide derivatives have improved solubility, thermal stability, and physicochemical properties as compared to native endogenous glucagon. This invention also relates to methods of using such polypeptides in a variety of therapeutic and diagnostic indications, as well as methods of producing such polypeptides. These analogs are useful in methods of treating obesity, diabetes, metabolic disorders, and other diseases or disorders.

BACKGROUND

Glucagon, a peptide hormone produced by the alpha cells of the pancreas, and glucagon-like peptide-1 (GLP-1), a neuropeptide, are derived from pre-proglucagon, a 158 amino acid precursor polypeptide that is processed in different tissues to form a number of different proglucagon-derived peptides. These proglucagon-derived peptides which include, for example, glucagon, GLP-1, glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM), are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the regulation of food intake.

Accordingly, there exists a need for therapeutics and therapies that mimic GLP-1 and/or glucagon activity.

SUMMARY

This invention relates to isolated polypeptides that are glucagon-receptor selective analogs and peptide derivatives thereof. Glucagon is a 29 amino acid peptide hormone that is produced by alpha cells in the pancreas and that interacts with the glucagon receptor (“GCGR”).

In some embodiments, an isolated polypeptide of the disclosure is a glucagon analog that binds to a glucagon receptor (GCGR) and is a selective glucagon receptor agonist. These isolated polypeptides of the disclosure are potent, stable, and soluble. The isolated polypeptides selectively bind the glucagon receptor as compared to native glucagon, for example, human glucagon, and as compared to the ability to bind to the GLP-1 receptor. The isolated polypeptides exhibit improved metabolic stability and clearance of the drug from the kidney at a rate close to glomerular filtration rate verses native glucagon, e.g., human glucagon. The isolated polypeptides exhibit an improved solubility as compared to native glucagon, for example, human glucagon. In preferred embodiments, the isolated polypeptides exhibit an improved solubility of at least 200 mg/ml. The isolated polypeptides exhibit an improved chemical stability at room temperature and at higher temperatures such as 37° C. or greater.

The isolated polypeptides of the disclosure are derived from a genus that imparts selectivity, solubility, and improved clearance of the molecule from the kidney. This genus is based on (a) a determination of the critical structure required for selectivity on the glucagon receptor, (b) the identification of critical amino acids and secondary structure motifs that provide for improved solubility while enhancing or at least maintaining the potency of human glucagon, and (c) the identification of amino acid substitutions for imparting chemical stability to the selective glucagon receptor agonists.

In some embodiments, an isolated polypeptide of the disclosure comprises a modified amino acid sequence based on the amino acid sequence of human glucagon: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH (SEQ ID NO: 140), where the modified amino acid sequence includes at least one amino acid substitution, at least two amino acid substitutions, at least three amino acid substitutions, at least four amino acid substitutions, at least five amino acid substitutions, at least six amino acid substitutions, at least seven amino acid substitutions, at least eight amino acid substitutions, at least nine amino acid substitutions, at least 10 amino acid substitutions, at least 11 amino acid substitutions, at least 12 amino acid substitutions, at least 13 amino acid substitutions, at least 14 amino acid substitutions, at least 15 amino acid substitutions, at least 16 amino acid substitutions, at least 17 amino acid substitutions, at least 18 amino acid substitutions, at least 19 amino acid substitutions, at least 20 amino acid substitutions, at least 21 amino acid substitutions, at least 22 amino acid substitutions, at least 23 amino acid substitutions, at least 24 amino acid substitutions, at least 25 amino acid substitutions, at least 26 amino acid substitutions, at least 27 amino acid substitutions, at least 28 amino acid substitutions, and/or at least 29 amino acid substitutions, provided that the isolated polypeptide having a modified amino acid sequence retains the ability to function as a selective glucagon analog.

(i) an amino acid substitution at position 1 selected from the group consisting of Y and W;

(ii) an amino acid substitution at position 2 selected from the group consisting of G and T;

(iii) an amino acid substitution at position 3 with H;

(iv) an amino acid substitution at position 10 with H;

(v) an amino acid substitution at position 11 with T;

(vi) an amino acid substitution at position 12 with R;

(vii) an amino acid substitution at position 13 selected from the group consisting of L and W;

(viii) an amino acid substitution at position 15 with E;

(ix) an amino acid substitution at position 16 selected from the group consisting of 2-Aminoisobutyric acid (Aib), A, E, I, K, L, and Q;

(x) an amino acid substitution at position 17 selected from the group consisting of A, E, K, S, and T;

(xi) an amino acid substitution at position 18 selected from the group consisting of A, E, L, and T;

(xii) an amino acid substitution at position 21 with E;

(xiii) an amino acid substitution at position 23 with T;

(xiv) an amino acid substitution at position 24 selected from the group consisting of 2-Aminoisobutyric acid (Aib), K, and L;

(xv) an amino acid substitution at position 25 with H; and

(xvi) an amino acid substitution at position 30 with a Z-tail selected from the group consisting of EEPSSGAPPPS-OH (SEQ ID NO: 4); EPSSGAPPPS-OH (SEQ ID NO: 5); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8); KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH (SEQ ID NO: 10); KRNKPPIA-OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13); PESGAPPPS-OH (SEQ ID NO: 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH₂(SEQ ID NO: 16); PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH₂(SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PKSKSPPPS-NH₂(SEQ ID NO: 21); PKSKSPPPS-OH (SEQ ID NO: 22); PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25); PSSGAPPPS-NH₂(SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27); PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH₂(SEQ ID NO: 31); PSSGKPPPS-OH (SEQ ID NO: 32); PSSGSPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS-OH (SEQ ID NO: 36); PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH(SEQ ID NO: 40); and

(xvii) combinations thereof,

provided that the isolated polypeptide having a modified amino acid sequence retains the ability to function as a selective glucagon receptor agonist.

In some embodiments, an isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by the consensus sequence of SEQ ID NO: 1:

(SEQ ID NO: 1)

X₁X₂X₃GTFTSDX₁₀X₁₁X₁₂X₁₃LX₁₅X₁₆X₁₇X₁₈AQEFX₂₃X₂₄X₂₅LEDE-Z-

tail-(OH/NH₂),

wherein:

- X₁is Y or W;
- X₂is S, G or T;
- X₃is Q or H;
- X₁₀is Y or H;
- X₁₁is S or T;
- X₁₂is K or R;
- X₁₃is Y, L or W;
- X₁₅is D or E;
- X₁₆is S, 2-Aminoisobutyric acid (Aib), A, E, L, Q, K, or I;
- X₁₇is K, E, S, T, or A;
- X₁₈is A, R, S, E, L, T or Y;
- X₂₃is T or V;
- X₂₄is K, I, L, or Aib;
- X₂₅is H or W; and
- Z-tail is absent or is selected from the group consisting of EEPSSGAPPPS-OH (SEQ ID NO: 4); EPSSGAPPPS-OH (SEQ ID NO: 5); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8); KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH (SEQ ID NO: 10); KRNKPPIA-OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13); PESGAPPPS-OH (SEQ ID NO: 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH₂(SEQ ID NO: 16); PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH₂(SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PKSKSPPPS-NH₂(SEQ ID NO: 21); PKSKSPPPS-OH (SEQ ID NO: 22); PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25); PSSGAPPPS-NH₂(SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27); PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH₂(SEQ ID NO: 31); PSSGKPPPS-OH (SEQ ID NO: 32); PSSGSPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS-OH (SEQ ID NO: 36); PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH(SEQ ID NO: 40).

(SEQ ID NO: 2)

X₁X₂X₃GTFTSDX₁₀X₁₁X₁₂X₁₃LX₁₅X₁₆X₁₇X₁₈AQEFVX24WLEDE-Z-

tail-(OHNH₂),

wherein:

- X₁is Y or W;
- X₂is S or G;
- X₃is Q or H;
- X₁₀is Y or H;
- X₁₁is S or T;
- X₁₂is K or R;
- X₁₃is Y, L or W;
- X₁₅is D or E;
- X₁₆is 2-Aminoisobutyric acid (Aib), A, or S;
- X₁₇is A or K;
- X₁₈is R, S, L, or Y;
- X₂₄is K, I, or Aib;
- X₂₅is H or W; and
- Z-tail is absent or is selected from the group consisting of PSSGAPPPS-NH₂(SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27); and PKSKSPPPS-NH₂(SEQ ID NO: 21).

(SEQ ID NO: 3)

YSX₃GTFTSDYSKYLDX₁₆X₁₇X₁₈AQEFVX₂₄WLEDE-Z-tail-(OH/NH₂),

wherein:

- X₃is Q or H;
- X₁₆is 2-Aminoisobutyric acid (Aib) or A;
- X₁₇is A or K;
- X₁₈is R, S, or Y;
- X₂₄is K or Aib;
- Z-tail is selected from the group consisting of PSSGAPPPS-OH (SEQ ID NO: 27) and PKSKSPPPS-NH₂(SEQ ID NO: 21).

In some embodiments, an isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 41), which is also referred to herein as Compound A1; YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 42), which is also referred to herein as Compound A2; YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH₂(SEQ ID NO: 43), which is also referred to herein as Compound A3; YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 44), which is also referred to herein as Compound A4; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS-NH₂(SEQ ID NO: 45), which is also referred to herein as Compound A5; and WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 46), which is also referred to herein as Compound A6.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 41). In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 42). In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH₂(SEQ ID NO: 43). In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 44). In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS-NH₂(SEQ ID NO: 45). In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 46).

In some embodiments, an isolated polypeptide of the disclosure consists of the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 41). In some embodiments, an isolated polypeptide of the disclosure consists of the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 42). In some embodiments, an isolated polypeptide of the disclosure consists of the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH₂(SEQ ID NO: 43). In some embodiments, an isolated polypeptide of the disclosure consists of the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 44). In some embodiments, an isolated polypeptide of the disclosure consists of the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS-NH₂(SEQ ID NO: 45). In some embodiments, an isolated polypeptide of the disclosure consists of the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 46).

In some embodiments, an isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of YSHGTFTSDYSKYLDAARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 143), which is also referred to herein as Compound A97; YSHGTFTSDYTRLLESKRAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 144), which is also referred to herein as Compound A98; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 145), which is also referred to herein as Compound A99; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDE-OH (SEQ ID NO: 146), which is also referred to herein as Compound A100; YGHGTFTSDHSKYLD(Aib)KRAQEFVKWLEDE-OH (SEQ ID NO: 147), which is also referred to herein as Compound A101; YSHGTFTSDYSKWLD(Aib)KRAQEFVKWLEDE-OH (SEQ ID NO: 148), which is also referred to herein as Compound A102; and YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 149), which is also referred to herein as Compound A103.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDAARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 143), which is also referred to herein as Compound A97.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYTRLLESKRAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 144), which is also referred to herein as Compound A98.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 145), which is also referred to herein as Compound A99.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDE-OH (SEQ ID NO: 146), which is also referred to herein as Compound A100.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YGHGTFTSDHSKYLD(Aib)KRAQEFVKWLEDE-OH (SEQ ID NO: 147), which is also referred to herein as Compound A101.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKWLD(Aib)KRAQEFVKWLEDE-OH (SEQ ID NO: 148), which is also referred to herein as Compound A102.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 149), which is also referred to herein as Compound A103.

In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 143-149.

In some embodiments, an isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 47); which is also referred to herein as Compound A7; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-NH₂(SEQ ID NO: 48); which is also referred to herein as Compound A8; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEEEPSSGAPPPS-OH (SEQ ID NO: 49); which is also referred to herein as Compound A9; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEEPSSGAPPPS-OH (SEQ ID NO: 50); which is also referred to herein as Compound A10; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEEEPSSGAPPPS-OH (SEQ ID NO: 51); which is also referred to herein as Compound A11; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEGAPPPS-OH (SEQ ID NO: 52); which is also referred to herein as Compound A12; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDESGAPPPS-OH (SEQ ID NO: 53); which is also referred to herein as Compound A13; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDESSGAPPPS-OH (SEQ ID NO: 54); which is also referred to herein as Compound A14; YSHGTFTSDYSKYLD(Aib)SRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 55); which is also referred to herein as Compound A15; YSHGTFTSDYSKYLD(Aib)TRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 56); which is also referred to herein as Compound A16; YSHGTFTSDYSKYLD(Aib)ERAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 57); which is also referred to herein as Compound A17; YSHGTFTSDYSKWLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 58); which is also referred to herein as Compound A18; YSHGTFTSDYSKWLD(Aib)SRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 59); which is also referred to herein as Compound A19; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 60); which is also referred to herein as Compound A20; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGEPPPS-OH (SEQ ID NO: 61); which is also referred to herein as Compound A21; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGSPPPS-OH (SEQ ID NO: 62); which is also referred to herein as Compound A22; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSKAPPPS-OH (SEQ ID NO: 63); which is also referred to herein as Compound A23; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSKGAPPPS-OH (SEQ ID NO: 64); which is also referred to herein as Compound A24; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPSS-OH (SEQ ID NO: 65); which is also referred to herein as Compound A25; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPSE-OH (SEQ ID NO: 66); which is also referred to herein as Compound A26; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEGGPSSGAPPPS-OH (SEQ ID NO: 67); which is also referred to herein as Compound A27; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEGPSSGAPPPS-OH (SEQ ID NO: 68); which is also referred to herein as Compound A28; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPKSGAPPPS-OH (SEQ ID NO: 69); which is also referred to herein as Compound A29; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSKGAPPPS-OH (SEQ ID NO: 70); which is also referred to herein as Compound A30; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPESGAPPPS-OH (SEQ ID NO: 71); which is also referred to herein as Compound A31; YTHGTFTSDYSKWLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 72); which is also referred to herein as Compound A32; YSHGTFTSDHSKWLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 73); which is also referred to herein as Compound A33; YTHGTFTSDHSKWLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 74); which is also referred to herein as Compound A34; YTHGTFTSDYSKWLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 75); which is also referred to herein as Compound A35; YSHGTFTSDHSKWLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 76); which is also referred to herein as Compound A36; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 77); which is also referred to herein as Compound A37; YSHGTFTSDYSKWLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 78); which is also referred to herein as Compound A38; YTHGTFTSDYSKWLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 79); which is also referred to herein as Compound A39; YSHGTFTSDHSKWLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 80); which is also referred to herein as Compound A40; YTHGTFTSDHSKWLDEARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 81); which is also referred to herein as Compound A41; YTHGTFTSDYSKWLDSKRAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 82); which is also referred to herein as Compound A42; YSHGTFTSDYSKYLDKARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 83); which is also referred to herein as Compound A43; YSHGTFTSDYSKYLDQARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 84); which is also referred to herein as Compound A44; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEKRNKPPPA-OH (SEQ ID NO: 85); which is also referred to herein as Compound A45; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEKRNKPPIA-OH (SEQ ID NO: 86); which is also referred to herein as Compound A46; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEKRNKNPPS-OH (SEQ ID NO: 87); which is also referred to herein as Compound A47; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEKRNKNPPPS-OH (SEQ ID NO: 88); which is also referred to herein as Compound A48; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPRNKNNPPS-OH (SEQ ID NO: 89); which is also referred to herein as Compound A49; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEKRNKPPPS-OH (SEQ ID NO: 90); which is also referred to herein as Compound A50; YSHGTFTSDYSKYLDLKRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 91); which is also referred to herein as Compound A51; YSHGTFTSDYSKYLDIKRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 92); which is also referred to herein as Compound A52; YSHGTFTSDYSKYLD(Aib)KRAQEFVLWLEDEPSSGAPPPS-OH (SEQ ID NO: 93); which is also referred to herein as Compound A53; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSKEPPPS-OH (SEQ ID NO: 94); which is also referred to herein as Compound A54; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSKSPPPS-OH (SEQ ID NO: 95); which is also referred to herein as Compound A55; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPKSKAPPPS-OH (SEQ ID NO: 96); which is also referred to herein as Compound A56; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPKSKQPPPS-OH (SEQ ID NO: 97); which is also referred to herein as Compound A57; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPKSKSPPPS-OH (SEQ ID NO: 98); which is also referred to herein as Compound A58; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSKQPPPS-OH (SEQ ID NO: 99); which is also referred to herein as Compound A59; YTHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 100); which is also referred to herein as Compound A60; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGKPPPS-OH (SEQ ID NO: 101); which is also referred to herein as Compound A61; YTHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 102); which is also referred to herein as Compound A62; YTHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGAPPPS-OH (SEQ ID NO: 103); which is also referred to herein as Compound A63; YTHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 104); which is also referred to herein as Compound A64; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPKSKEPPPS-NH₂(SEQ ID NO: 105); which is also referred to herein as Compound A65; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPKSKSPPPS-NH₂(SEQ ID NO: 106); which is also referred to herein as Compound A66; YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPKSKSPPPS-NH₂(SEQ ID NO: 107); which is also referred to herein as Compound A67; YSHGTFTSDYSKYLD(Aib)ARAQEFV(Aib)WLEDEPKSKEPPPS-NH₂(SEQ ID NO: 108); which is also referred to herein as Compound A68; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKEPPPS-OH (SEQ ID NO: 109); which is also referred to herein as Compound A69; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKSPPPS-OH (SEQ ID NO: 110); which is also referred to herein as Compound A70; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKAPPPS-OH (SEQ ID NO: 111); which is also referred to herein as Compound A71; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKEPPPS-NH₂(SEQ ID NO: 112); which is also referred to herein as Compound A72; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKSPPPS-NH₂(SEQ ID NO: 113); which is also referred to herein as Compound A73; YSHGTFTSDYSKYLDSARAQEFVKWLEDEPKSKAPPPS-NH₂(SEQ ID NO: 114); which is also referred to herein as Compound A74; WSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 115); which is also referred to herein as Compound A75; YSHGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 116); which is also referred to herein as Compound A76; YSHGTFTSDYSKYLD(Aib)KTAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 117); which is also referred to herein as Compound A77; YSHGTFTSDYSKYLD(Aib)KLAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 118); which is also referred to herein as Compound A78; YSHGTFTSDYSKYLD(Aib)KEAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 119); which is also referred to herein as Compound A79; YSHGTFTSDYSKYLDAARAQEFVKWLEDEPKSKSPPPS-NH₂(SEQ ID NO: 120); which is also referred to herein as Compound A80; YSQGTFTSDYSKYLDSARAQEFVKWLEDEPKSKSPPPS-OH (SEQ ID NO: 121); which is also referred to herein as Compound A81; YSQGTFTSDYSKYLDSARAQEFVKWLEDEPKSKAPPPS-OH (SEQ ID NO: 122); which is also referred to herein as Compound A82; YSHGTFTSDYSKYLDSARAQEFTKWLEDEPKSKSPPPS-OH (SEQ ID NO: 123); which is also referred to herein as Compound A83; YSHGTFTSDYSKYLDSARAQEFVKHLEDEPKSKSPPPS-OH (SEQ ID NO: 124); which is also referred to herein as Compound A84; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKPPPS-NH₂(SEQ ID NO: 125); which is also referred to herein as Compound A85; YSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 126); which is also referred to herein as Compound A86; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEKSSGKPPPS-OH (SEQ ID NO: 127); which is also referred to herein as Compound A87; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKKPPS-OH (SEQ ID NO: 128); which is also referred to herein as Compound A88; YSHGTFTSDYSKYLD(Aib)KRAQEFV(Aib)HLEDEPSSGKPPPS-OH (SEQ ID NO: 129); which is also referred to herein as Compound A89; YSHGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 130); which is also referred to herein as Compound A90; WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 131); which is also referred to herein as Compound A91; WSQGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 132); which is also referred to herein as Compound A92; WSQGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 133); which is also referred to herein as Compound A93; YSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 134); which is also referred to herein as Compound A94; YSQGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 135); which is also referred to herein as Compound A95; and YSQGTFTSDYSKYLD(Aib)KAAQEFV(Aib)WLEDEPSSGKPPPS-OH (SEQ ID NO: 136); which is referred to herein as Compound A96. In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-136.

(SEQ ID NO: 137)

YSQGTFTSDYSKYLDSX₁₇RAQX₂₁FVX₂₄WLX₂₇X₂₈T—OH,

wherein:

X₁₇is K*, where K* is in a lactam bridge with E* at X₂₁;

X₂₁is E*, where E* is in a lactam bridge with K* at X₁₇;

X₂₄is K or K**, where K** is in a lactam bridge with E** at X₂₈;

X₂₇is Q or D; and

X₂₈is E or E**, where E** is in a lactam bridge with K** at X₂₄

In some embodiments, an isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of YSQGTFTSDYSKYLDSK*RAQE*FVK**WLDE**T-OH (SEQ ID NO: 138), referred to herein as Compound A104 and YSQGTFTSDYSKYLDSK*RAQE*FVK**WLQE**T-OH (SEQ ID NO: 139), referred to herein as Compound A105. In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 138 and 139.

The isolated polypeptides of the disclosure contain amino acid motifs that allow for maintaining characteristics such as solubility, and/or stability, e.g., metabolic stability, of the glucagon-receptor selective analogs as compared to the native human glucagon molecule, as well as amino acid motifs that allow for maintaining additional characteristics such as glucagon selectivity over GLP-1.

In some embodiments, the C-terminus of the isolated polypeptide of the disclosure is extended with a sequence that binds to serum albumin, for example, to human serum albumin. In some embodiments, the C-terminus of the isolated polypeptide of the disclosure is extended with a sequence selected from the group consisting of EEPSSGAPPPS-OH (SEQ ID NO: 4); EPSSGAPPPS-OH (SEQ ID NO: 5); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8); KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH (SEQ ID NO: 10); KRNKPPIA-OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13); PESGAPPPS-OH (SEQ ID NO: 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH₂(SEQ ID NO: 16); PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH₂(SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PKSKSPPPS-NH₂(SEQ ID NO: 21); PKSKSPPPS-OH (SEQ ID NO: 22); PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25); PSSGAPPPS-NH₂(SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27); PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH₂(SEQ ID NO: 31); PSSGKPPPS-OH (SEQ ID NO: 32); PSSGSPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS-OH (SEQ ID NO: 36); PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH(SEQ ID NO: 40).

In some embodiments, the carboxyl group of the C-terminal amino acid residue of an isolated polypeptide of the disclosure is amidated. In some embodiments, the carboxyl group of the C-terminal amino acid residue of an isolated polypeptide of the disclosure unmodified.

In some embodiments, an isolated polypeptide provided herein is an agonist of glucagon activity. In some embodiments, an isolated polypeptide provided herein can bind to a glucagon receptor. In some embodiments, the glucagon receptor is a human glucagon receptor. In some embodiments, the isolated polypeptide of the disclosure binds to a human glucagon receptor with a pEC50 in the cAMP assay using an 11 point curve starting at 1 nM to 500 micromolar range (as described herein) in the range of greater than about 9.0. In some embodiments, the isolated polypeptide of the disclosure binds to a human glucagon receptor with a pEC50 in the cAMP assay using an 11 point curve starting at 1 nM to 500 micromolar range (as described herein) in the range of greater than about 11.0.

In some embodiments, an isolated polypeptide of the disclosure binds to a human glucagon receptor, but does not substantially bind to a human GLP-1 receptor. As used herein, the term “does not substantially bind” and variations thereof refer to polypeptides that exhibit low affinity to no affinity for a human GLP-1 receptor. In some embodiments, an isolated polypeptide of the disclosure binds to a human glucagon receptor at an affinity that is at least 100-fold greater than the affinity of the same isolated polypeptide for a human GLP-1 receptor. In preferred embodiments, an isolated polypeptide of the disclosure binds to a human glucagon receptor at an affinity that is at least 1,000-fold greater than the affinity of the same isolated polypeptide for a human GLP-1 receptor. In some embodiments, the isolated polypeptide of the disclosure binds to a human glucagon receptor with a pEC50 in the cAMP assay using an 11 point curve starting at 1 nM to 500 micromolar range in the range of greater than about 9.0, and the isolated polypeptide of the disclosure binds to human GLP-1 receptor with a pEC50 in the cAMP assay that is less than about 10.0. In some embodiments, the isolated polypeptide of the disclosure binds to a human glucagon receptor with a pEC50 in the cAMP assay using an 11 point curve starting at 1 nM to 500 micromolar range in the range of greater than about 11.0, and the isolated polypeptide of the disclosure binds to human GLP-1 receptor with a pEC50 in the cAMP assay that is less than about 9.0.

In some embodiments, an isolated polypeptide as provided herein can further comprise a heterologous moiety associated with the polypeptide. In some embodiments, the heterologous moiety is a protein, a peptide, a protein domain, a linker, an organic polymer, an inorganic polymer, a polyethylene glycol (PEG), biotin, an albumin, a human serum albumin (HSA), a HSA FcRn binding portion, an antibody, a domain of an antibody, an antibody fragment, a single chain antibody, a domain antibody, an albumin binding domain, an enzyme, a ligand, a receptor, a binding peptide, a non-FnIII scaffold, an epitope tag, a recombinant polypeptide polymer, a cytokine, or any combination of two or more of such moieties.

The isolated polypeptides provided herein exhibit glucagon receptor agonistic activity, for example by binding a glucagon receptor. The isolated polypeptides provided herein completely or partially agonize or otherwise stimulate glucagon activity upon binding to or otherwise interacting with a glucagon receptor. The stimulation or modulation of a biological function of glucagon is complete or partial upon interaction between the glucagon receptor agonist and the glucagon receptor.

These isolated polypeptides of the disclosure, which are selective glucagon receptor agonists, are useful alone or in combination with at least a second agent. In preferred embodiments, the second agent is a polypeptide. In preferred embodiments, the second polypeptide is an insulinotropic peptide. For example, the insulinotropic polypeptide is selected from the group consisting of exenatide, a derivative of exenatide, an analogue of exenatide, glucagon-like peptide-1 (GLP-1), a derivative of GLP-1, and an analogue of GLP-1.

In preferred embodiments, the insulinotropic polypeptide is exenatide, a derivative of exenatide, or an analog of exenatide. In preferred embodiments, the exenatide is synthetic exenatide. In preferred embodiments, the synthetic exenatide comprises the amino acid sequence H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂(SEQ ID NO: 142).

In combination therapies, the use of a selective glucagon analog allows for the titration of proper therapeutic doses of glucagon and any GLP-1 receptor agonist. This allows for the desired effects of glucagon/GLP-1 agonism (i.e., weight-loss, increase in energy expenditure), without a deleterious spike in blood glucose.

In some embodiments, an isolated polypeptide as provided herein and the additional agent are formulated into a single therapeutic composition, and the isolated polypeptide and additional agent are administered simultaneously. In some embodiments, the isolated polypeptide and additional agent are separate from each other, e.g., each is formulated into a separate therapeutic composition, and the isolated polypeptide and the additional agent are administered simultaneously, or the isolated polypeptide and the additional agent are administered at different times during a treatment regimen. For example, the isolated polypeptide is administered prior to the administration of the additional agent, the isolated polypeptide is administered subsequent to the administration of the additional agent, or the isolated polypeptide and the additional agent are administered in an alternating fashion. As described herein, the isolated polypeptide and additional agent are administered in single doses or in multiple doses.

Also provided herein are methods for treating, delaying the onset of, delaying the progression of, or otherwise ameliorating a symptom of a disease or condition caused, characterized by, or otherwise associated with aberrant glucagon activity. In some embodiments, the disease or condition is type 2 diabetes mellitus.

Also provided herein are methods of treating a metabolic disorder by administering an isolated polypeptide of the disclosure or any pharmaceutical composition described herein to a subject in need thereof.

Also provided herein are methods of treating obesity by administering an isolated polypeptide of the disclosure or any pharmaceutical composition described herein to a subject in need thereof.

Also provided herein are methods of treating, preventing, delaying the onset of, delaying the progression of, and/or otherwise ameliorating a symptom of a metabolic disease or disorder associated with elevated blood glucose in a patient by administering an isolated polypeptide of the disclosure or any pharmaceutical composition described herein to the patient in need thereof.

Also provided herein are methods of treating, preventing, delaying the onset of, delaying the progression of, and/or otherwise ameliorating a symptom of a disease or disorder in which agonism at the glucagon receptor is desired, such as, by way of non-limiting example, chronic pain, hemophilia and other blood disorders, endocrine disorders, metabolic disorders, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Alzheimer's disease, cardiovascular diseases, hypoglycemia unawareness, restrictive lung disease, chronic obstructive pulmonary disease, lipoatrophy, metabolic syndrome, leukemia, hepatitis, renal failure, autoimmune diseases (e.g., Grave's disease, systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis), shock and/or wasting disorders, pancreatitis, and neurological disorders and diseases such as Parkinson's disease.

Also provided herein are methods of treating, preventing, delaying the onset of, delaying the progression of, and/or otherwise ameliorating a symptom of an infectious disease requiring chronic treatment(s).

In the methods disclosed herein, the isolated polypeptides of the disclosure and/or pharmaceutical compositions described herein are administered alone or in combination with pharmaceutically acceptable carriers and/or excipients and/or polymers and/or organic solvent, in either single or multiple doses.

Pharmaceutical compositions according to the invention can include a polypeptide of the disclosure, along with a suitable carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a series of graphs depicting peptide-receptor activation profiles at the human glucagon receptor (GCGR) and the human GLP-1 receptor (GLP-1R) by human glucagon, GLP-1 (7-37), and two glucagon receptor selective peptide agonists. FIG. 1A demonstrates that the peptides referred to herein as Compound A2 and Compound A1 and glucagon are nearly equipotent on GCGR, while GLP-1 (7-37) activates GCGR at a much lower potency. FIG. 1B demonstrates that in contrast to the results in FIG. 1A, peptides Compound A2 and Compound A1 are inactive on GLP-1R demonstrating that peptides Compound A2 and Compound A1 profile as GCGR selective agonists.

FIGS. 2A and 2B are a series of graphs and tables depicting mean plasma concentration vs. time plot of various glucagon analogs in male rats after a 3-hr intravenous infusion a 0.3 mg/kg dose. FIG. 2A depicts the glucagon analogs referred to herein as Compound A99 (SEQ ID NO: 145), Compound A102 (SEQ ID NO: 148), Compound A98 (SEQ ID NO: 144), Compound A100 (SEQ ID NO: 146), and Compound A101 (SEQ ID NO: 147), where n=3 to 6, and the error bars in the graph represent standard error, and where the CL values in the table are shown as mean±standard error (n=3 to 6). FIG. 2B depicts the glucagon analogs referred to herein as Compound A2, Compound A1, Compound A5, Compound A6, Compound A4, and Compound A3, where n=3 to 5, and the error bars in the graph represent standard error, and where the CL values in the table are shown as mean±standard error (n=3 to 5).

FIGS. 3A, 3B, and 3C are a series of graphs depicting the efficacy of various glucagon analogs as measured by body weight change in DIO rats after 13 days. In particular, FIG. 3A depicts the efficacy of the glucagon analog referred to herein as Compound A104, when administered along (i.e., singly) or in combination with exendin-4.

FIG. 3B depicts the efficacy of the glucagon analog referred to herein as Compound A2, and FIG. 3C depicts the efficacy of the glucagon analog referred to herein as Compound A1. For all graphs, p<0.05 as compared to vehicle control.

FIGS. 4A and 4B are schematic representations of the chemical structure of the cyclical peptide glucagon analogs referred to herein as Compound A104 (SEQ ID NO: 138) (FIG. 4A) and Compound A105 (SEQ ID NO: 139) (FIG. 4B).

FIG. 5 is a graph that plots steady state plasma concentrations on the Y-axis against intravenous infusion rates on the X-axis of exenatide (circles) and GLP-1 (triangles) and compares these correlations against that for inulin (which is cleared by glomerular filtration). Data of FIG. 5 show that steady state plasma concentration of exenatide approaches that of inulin, consistent with exenatide being cleared mainly by glomerular filtration.

FIG. 6 is the graph of FIG. 5 that further plots steady state plasma concentrations on the Y-axis against intravenous infusion rates on the X-axis of glucagon (inverted triangles), and Compounds A1 and A2 (glucagon analogs, squares) in addition to exenatide (circles) and GLP-1 (triangles). These correlations are shown relative to that for inulin (as an estimate of glomerular filtration rate). Data of FIG. 6 show that steady state plasma concentrations of Compounds A1 and A2 approach that of inulin, consistent with these compounds being cleared mainly by glomerular filtration.

FIG. 7 is an illustration that plots clearance values for glucagon, Compounds A1, A2 and A3 (three points closest to GFR) and other glucagon analogs. Note that clearance values decrease along the y-axis. For the sake of comparison, the dashed upper line (closer to GFR) shows the clearance rate for exenatide and the dashed lower line (farther from GFR) shows the clearance rate for glucagon. FIG. 7 shows that clearance of certain glucagon analogs, including Compounds A1, A2 and A3 are less than that of exenatide and glucagon.

FIG. 8 is a graph that compares dose dependency of weight loss for male Sprague-Dawley rats upon subcutaneous administration of either exenatide or Compound A1.

FIG. 9 is a graph that compares dose dependency of weight loss for male Sprague-Dawley rats upon subcutaneous administration of Compounds A1, A2 or A3.

FIG. 10 is a graph that compares weight loss in DIO LE rats after administration of exenatide, a glucagon (“GCG”) analog, Compound A1, or a combination of exenatide and the GCG analog, for 27 days at various doses.

FIG. 11 is a bar graph that alternatively depicts that data shown in FIG. 10.

FIG. 12 is a graph that compares serum triglyceride levels in Zucker Diabetic Fatty (ZDF) rats after 14 days after administration of compound A104, alone or in combination with exenatide.

FIG. 13 is a graph that compares serum liver fat content and liver weight in Zucker Diabetic Fatty (ZDF) rats after 14 days after administration of different doses of compound A104. Note that liver weight is plotted on the left Y-axis as open circles. Liver fat is plotted on the right Y-axis as filled triangles.

FIG. 14 illustrates a 3-D depiction of weight loss in DIO LE rats upon continuous administration of exenatide or a glucagon analog.

FIG. 15 illustrates a 3-D evaluation of weight loss in DIO LE rats upon continuous administration of exenatide/glucagon analog combinations.

FIG. 16 illustrates a 3-D evaluation of weight loss in DIO LE rats upon continuous administration of fixed-ratio of exenatide/glucagon analog combinations.

FIG. 17 is a graphical evaluation of in vivo potency, defined as effect per unit total mass of drug, of different fixed-ratio of exenatide/glucagon analog combinations.

DETAILED DESCRIPTION

This invention relates to isolated polypeptides that are glucagon-receptor selective analogs and peptide derivatives thereof. Glucagon is produced by the pancreas and interacts with the glucagon receptor (“GCGR”). In some embodiments, an isolated polypeptide of the disclosure is a selective glucagon receptor agonist. In some embodiments, an isolated polypeptide of the disclosure binds to a glucagon receptor.

Definitions

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a solvent” includes a combination of two or more such solvents, reference to “a peptide” includes one or more peptides, or mixtures of peptides, reference to “a drug” includes one or more drugs, reference to “an osmotic delivery device” includes one or more osmotic delivery devices, and the like. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

Unless specifically stated or obvious from context, as used herein, the term “substantially” is understood as within a narrow range of variation or otherwise normal tolerance in the art. Substantially can be understood as within 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or 0.001% of the stated value.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although other methods and materials similar, or equivalent, to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

The terms “drug,” “therapeutic agent,” and “beneficial agent” are used interchangeably to refer to any therapeutically active substance that is delivered to a subject to produce a desired beneficial effect. In one embodiment of the present invention, the drug is a polypeptide. In another embodiment of the present invention, the drug is a small molecule, for example, hormones such as androgens or estrogens. The devices and methods of the present invention are well suited for the delivery of proteins, small molecules and combinations thereof.

The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and typically refer to a molecule comprising a chain of two or more amino acids (e.g., most typically L-amino acids, but also including, e.g., D-amino acids, modified amino acids, amino acid analogs, and amino acid mimetic). Peptides may be naturally occurring, synthetically produced, or recombinantly expressed. Peptides may also comprise additional groups modifying the amino acid chain, for example, functional groups added via post-translational modification. Examples of post-translation modifications include, but are not limited to, acetylation, alkylation (including, methylation), biotinylation, glutamylation, glycylation, glycosylation, isoprenylation, lipoylation, phosphopantetheinylation, phosphorylation, selenation, and C-terminal amidation. The term peptide also includes peptides comprising modifications of the amino terminus and/or the carboxy terminus. Modifications of the terminal amino group include, but are not limited to, des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, but are not limited to, amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications (e.g., wherein lower alkyl is C₁-C₄alkyl). The term peptide also includes modifications, such as but not limited to those described above, of amino acids falling between the amino and carboxy termini. In one embodiment, a peptide may be modified by addition of a small-molecule drug.

The terminal amino acid at one end of the peptide chain typically has a free amino group (i.e., the amino terminus). The terminal amino acid at the other end of the chain typically has a free carboxyl group (i.e., the carboxy terminus). Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminus and increasing in the direction of the carboxy terminus of the peptide.

The phrase “amino acid residue” as used herein refers to an amino acid that is incorporated into a peptide by an amide bond or an amide bond mimetic.

The term “insulinotropic” as used herein typically refers to the ability of a compound, e.g., a peptide, to stimulate or affect the production and/or activity of insulin (e.g., an insulinotropic hormone). Such compounds typically stimulate or otherwise affect the secretion or biosynthesis of insulin in a subject. Thus, an “insulinotropic peptide” is an amino acid-containing molecule capable of stimulating or otherwise affecting secretion or biosynthesis of insulin.

The term “insulinotropic peptide” as used herein includes, but is not limited to, glucagon-like peptide 1 (GLP-1), as well as derivatives and analogues thereof, exenatide, exenatide having the amino acid sequence of SEQ ID NO: 142, as well as derivatives and analogues thereof.

The phrase “incretin mimetics” as used herein includes, but is not limited to GLP-1 peptide, peptide derivatives of GLP-1, peptide analogs of GLP-1; exenatide, exenatide having the amino acid sequence of SEQ ID NO: 142, exenatide peptide, peptide derivatives of exenatide, and peptide analogs of exenatide. Examples of preferred incretin mimetics include exenatide, exenatide having the amino acid sequence of exendin-4 (the naturally-occurring form of exenatide, and has the amino acid sequence of SEQ ID NO: 142), exenatide-LAR, lixisenatide, GLP-1 (7-36), liraglutide, semaglutide, dulaglutide, albiglutide, and taspoglutide. Incretin mimetics are also referred to herein as “insulinotropic peptides.” Incretin mimetics which target the GLP-1 receptor are also known in the literature as “GLP-1 receptor agonists.”

The term “an exenatide” as used herein includes, but is not limited to exenatide, exenatide having the amino acid sequence of SEQ ID NO: 142, native exendin-4, exenatide peptides, exenatide peptide analogs, and exenatide peptide derivatives.

The term “GLP-1” refers to a polypeptide that is produced by the L-cell located mainly in the ileum and colon, and to a lesser extent by L-cells in the duodenum and jejunum. GLP-1 is a regulatory peptides to a G-coupled protein receptor on P cell and via adenyl cyclase activity and production of cAMP stimulates the insulin response to the nutrients that are absorbed from the gut [Baggio 2007, “Biology of incretins: GLP-1 and GIP,” Gastroenterology, vol. 132(6):2131-57; Holst 2008, “The incretin system and its role in type 2 diabetes mellitus,” Mol Cell Endocrinology, vol. 297(1-2):127-36]. The effects of GLP-1R agonism are multiple. GLP-1 maintains glucose homeostasis by enhancing endogenous glucose dependent insulin secretion, rendering the β cells glucose competent and sensitive to GLP-1, suppressing glucagon release, restoring first and second phase insulin secretion, slowing gastric emptying, decreasing food intake, and increasing satiety [Holst 2008 Mol. Cell Endocrinology; Kjems 2003 “The influence of GLP-1 on glucose-stimulated insulin secretion: effects on beta-cell sensitivity in type 2 and nondiabetic subjects,” Diabetes, vol. 52(2): 380-86; Holst 2013 “Incretin hormones and the satiation signal,” Int J Obes (Lond), vol. 37(9): 1161-69; Seufert 2014, “The extra-pancreatic effects of GLP-1 receptor agonists: a focus on the cardiovascular, gastrointestinal and central nervous systems,” Diabetes Obes Metab, vol. 16(8): 673-88]. The risk of hypoglycemia is minimal given the mode of action of GLP-1.

The term “glucagon” refers to a 29 amino acid peptide hormone that is produced by alpha cells in the pancreas and that interacts with GCGR. The amino acid sequence of glucagon is HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH (SEQ ID NO: 140).

As used herein, the term “glucagon analog” implies structural similarity to glucagon and the term “glucagon receptor agonist” describes a compound that is, functionally, an agonist of the glucagon receptor. The terms “glucagon analog” and “glucagon receptor agonist” are used alternatively to describe the peptides disclosed herein.

Glucagon originates from the 158 amino acid pre-proglucagon peptide which also acts as a precursor for the peptide hormones GLP-1, GLP-2, oxyntomodulin, glicentin, and glicentin-related pancreatic polypeptide via tissue-specific processing. Glucagon corresponds to the amino acid residues 33 to 61 of the precursor peptide and acts via interaction with a class B seven transmembrane G protein-coupled receptor located primarily in the liver. Immunostaining has also indicated the presence of glucagon receptors in the kidneys, gastrointestinal tract, heart, spleen, brain, adipocytes, and lymphoblasts. In response to low blood glucose levels, glucagon is released and stimulates hepatic glucose output via glycogenolysis and gluconeogenesis. Glucagon acts as a counter to the glucose-lowering effect of insulin and tight regulation via a feed-back system between the two hormones allows for effective glucose homeostasis.

In addition to its effect on blood glucose levels, glucagon is also known to increase energy expenditure and thermogenesis. Increased signaling has a direct effect on the regulation of triglycerides, free fatty acids, apolipoprotein, and bile acid metabolism. Current therapeutic uses for glucagon have primarily focused on its use as a rescue agent for hypoglycemic episodes. However, recent work has taken advantage of the hormone's ability to affect energy balance and lipid metabolism resulting in potential treatments for various metabolic disorders.

The poor solubility of glucagon (<1.0 mg/ml in aqueous or saline buffer) has prevented the opportunity for chronic study via continuous infusion. A summary of the solubility and the pEC50 value of glucagon with respect to glucagon receptor (GLUR) and GLP1, as determined in cAMP assays, is shown in Table 1 below:

TABLE 1

GLUR
GLP1

pEC50
pEC50
Solubility in

Sequence
(cAMP)
(cAMP)
Aqueous

Glucagon
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT—OH
10
9.9
0.03-10

(SEQ ID NO: 140)

μg/ml

The term “vehicle” as used herein refers to a medium used to carry a compound, e.g., a drug or a particle containing a drug. Vehicles of the present invention typically comprise components such as polymers and solvents. The suspension vehicles of the present invention typically comprise solvents and polymers that are used to prepare suspension formulations further comprising drug particle formulations.

The phrase “phase separation” as used herein refers to the formation of multiple phases (e.g., liquid and gel phases) in the suspension vehicle, such as when the suspension vehicle contacts the aqueous environment. In some embodiments of the present invention, the suspension vehicle is formulated to exhibit phase separation upon contact with an aqueous environment having less than approximately 10% water.

The phrase “single-phase” as used herein refers to a solid, semisolid, or liquid homogeneous system that is physically and chemically uniform throughout.

The term “dispersed” as used herein refers to dissolving, dispersing, suspending, or otherwise distributing a compound, for example, a drug particle formulation, in a suspension vehicle.

The phrase “chemically stable” as used herein refers to formation in a formulation of an acceptable percentage of degradation products produced over a defined period of time by chemical pathways, such as deamidation (usually by hydrolysis), aggregation, or oxidation.

The phrase “physically stable” as used herein refers to formation in a formulation of an acceptable percentage of aggregates (e.g., dimers and other higher molecular weight products). Further, a physically stable formulation does not change its physical state as, for example, from liquid to solid, or from amorphous to crystal form.

The term “viscosity” as used herein typically refers to a value determined from the ratio of shear stress to shear rate (see, e.g., Considine, D. M. & Considine, G. D., Encyclopedia of Chemistry, 4th Edition, Van Nostrand, Reinhold, N.Y., 1984) essentially as follows:

F/A=μ*V/L (Equation 1)

- where F/A=shear stress (force per unit area),
- μ=a proportionality constant (viscosity), and
- V/L=the velocity per layer thickness (shear rate).

From this relationship, the ratio of shear stress to shear rate defines viscosity. Measurements of shear stress and shear rate are typically determined using parallel plate rheometry performed under selected conditions (for example, a temperature of about 37° C.). Other methods for the determination of viscosity include, measurement of a kinematic viscosity using viscometers, for example, a Cannon-Fenske viscometer, a Ubbelohde viscometer for the Cannon-Fenske opaque solution, or a Ostwald viscometer. Generally, suspension vehicles of the present invention have a viscosity sufficient to prevent a particle formulation suspended therein from settling during storage and use in a method of delivery, for example, in an implantable, drug delivery device.

The term “non-aqueous” as used herein refers to an overall moisture content, for example, of a suspension formulation, typically of less than or equal to about 10 wt %, for example, less than or equal to about 7 wt %, less than or equal to about 5 wt %, and/or less than about 4 wt %. Also, a particle formulation of the present invention comprises less than about 10 wt %, for example, less than about 5 wt %, residual moisture.

The term “subject” as used herein refers to any member of the subphylum Chordata, including, without limitation, humans and other primates, including non-human primates such as rhesus macaques and other monkey species and chimpanzees and other ape species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age or gender. Thus, both adult and newborn individuals are intended to be covered.

The term “osmotic delivery device” as used herein typically refers to a device used for delivery of a drug (e.g., an isolated glucagon-specific agonist polypeptide) to a subject, wherein the device comprises, for example, a reservoir (made, e.g., from a titanium alloy) having a lumen that contains a suspension formulation comprising a drug (e.g., an isolated glucagon-specific agonist polypeptide) and an osmotic agent formulation. A piston assembly positioned in the lumen isolates the suspension formulation from the osmotic agent formulation. A semi-permeable membrane is positioned at a first distal end of the reservoir adjacent the osmotic agent formulation and a diffusion moderator (which defines a delivery orifice through which the suspension formulation exits the device) is positioned at a second distal end of the reservoir adjacent the suspension formulation. Typically, the osmotic delivery device is implanted within the subject, for example, subdermally or subcutaneously (e.g., in the inside, outside, or back of the upper arm and in the abdominal area). An exemplary osmotic delivery device is the DUROS® (ALZA Corporation, Mountain View, Calif.) delivery device. Examples of terms synonymous to “osmotic delivery device” include but are not limited to “osmotic drug delivery device”, “osmotic drug delivery system”, “osmotic device”, “osmotic delivery device”, “osmotic delivery system”, “osmotic pump”, “implantable drug delivery device”, “drug delivery system”, “drug delivery device”, “implantable osmotic pump”, “implantable drug delivery system”, and “implantable delivery system”. Other terms for “osmotic delivery device” are known in the art.

The term “continuous delivery” as used herein typically refers to a substantially continuous release of drug from an osmotic delivery device and into tissues near the implantation site, e.g., subdermal and subcutaneous tissues. For example, an osmotic delivery device releases drug essentially at a predetermined rate based on the principle of osmosis. Extracellular fluid enters the osmotic delivery device through the semi-permeable membrane directly into the osmotic engine that expands to drive the piston at a slow and consistent rate of travel. Movement of the piston forces the drug formulation to be released through the orifice of the diffusion moderator. Thus release of the drug from the osmotic delivery device is at a slow, controlled, consistent rate.

The term “substantial steady-state delivery” as used herein typically refers to delivery of a drug at or near a target concentration over a defined period of time, wherein the amount of the drug being delivered from an osmotic delivery device is substantially zero-order delivery. Substantial zero-order delivery of an active agent (e.g., an isolated glucagon-specific analog) means that the rate of drug delivered is constant and is independent of the drug available in the delivery system; for example, for zero-order delivery, if the rate of drug delivered is graphed against time and a line is fitted to the data the line has a slope of approximately zero, as determined by standard methods (e.g., linear regression).

The phrase “drug half-life” as used herein refers how long it takes a drug to be eliminated from blood plasma by one half of its concentration. A drug's half-life is usually measured by monitoring how a drug degrades when it is administered via injection or intravenously. A drug is usually detected using, for example, a radioimmunoassay (RIA), a chromatographic method, an electrochemiluminescent (ECL) assay, an enzyme linked immunosorbent assay (ELISA) or an immunoenzymatic sandwich assay (IEMA).

The terms “μg” and “mcg” and “ug” are understood to mean “micrograms”. Similarly, the terms “μl” and “uL” are understood to mean “microliter”, and the terms “μM” and “uM” are understood to mean “micromolar”.

The term “serum” is meant to mean any blood product from which a substance can be detected. Thus, the term serum includes at least whole blood, serum, and plasma. For example, “an amount of [a substance] in a subject's serum” would cover “an amount of [the substance] in a subject's plasma”.

Baseline is defined as the last assessment on or before the day of the initial placement of an osmotic delivery device (containing drug or placebo).

2.0.0 General Overview of the Invention

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular types of drug delivery devices, particular sources of drugs, particular solvents, particular polymers, and the like, as use of such particulars may be selected in view of the teachings of the present specification. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In a first aspect, the present invention relates to isolated polypeptides that are glucagon-receptor selective analogs and peptide derivatives thereof. In some embodiments, an isolated polypeptide of the disclosure is a selective glucagon receptor agonist. In some embodiments, an isolated polypeptide of the disclosure binds to a glucagon receptor (GCGR).

(i) an amino acid substitution at position 1 selected from the group consisting of Y and W;

(ii) an amino acid substitution at position 2 selected from the group consisting of G and T;

(iii) an amino acid substitution at position 3 with H;

(iv) an amino acid substitution at position 10 with H;

(v) an amino acid substitution at position 11 with T;

(vi) an amino acid substitution at position 12 with R;

(vii) an amino acid substitution at position 13 selected from the group consisting of L and W;

(viii) an amino acid substitution at position 15 with E;

(ix) an amino acid substitution at position 16 selected from the group consisting of 2-Aminoisobutyric acid (Aib), A, E, I, K, L, and Q;

(x) an amino acid substitution at position 17 selected from the group consisting of A, E, K, S, and T;

(xi) an amino acid substitution at position 18 selected from the group consisting of A, E, L, and T;

(xii) an amino acid substitution at position 21 with E;

(xiii) an amino acid substitution at position 23 with T;

(xiv) an amino acid substitution at position 24 selected from the group consisting of 2-Aminoisobutyric acid (Aib), K, and L;

(xv) an amino acid substitution at position 25 with H;

(xvii) combinations thereof,

provided that the isolated polypeptide having a modified amino acid sequence retains the ability to function as a selective glucagon receptor agonist.

(SEQ ID NO: 1)

X₁X₂X₃GTFTSDX₁₀X₁₁X₁₂X₁₃LX₁₅X₁₆X₁₇X₁₈AQEFX₂₃X₂₄X₂₅LEDE-Z-

tail-(OH/NH₂),

wherein:

- X₁is Y or W;
- X₂is S, G or T;
- X₃is Q or H;
- X₁₀is Y or H;
- X₁₁is S or T;
- X₁₂is K or R;
- X₁₃is Y, L or W;
- X₁₅is D or E;
- X₁₆is S, 2-Aminoisobutyric acid (Aib), A, E, L, Q, K, or I;
- X₁₇is K, E, S, T, or A;
- X₁₈is A, R, S, E, L, T or Y;
- X₂₃is T or V;
- X₂₄is K, I, L, or Aib;
- X₂₅is H or W; and
- Z-tail is absent; or is selected from the group consisting of EEPSSGAPPPS-OH (SEQ ID NO: 4); EPSSGAPPPS-OH (SEQ ID NO: 5); GAPPPS-OH (SEQ ID NO: 6); GGPSSGAPPPS-OH (SEQ ID NO: 7); GPSSGAPPPS-OH (SEQ ID NO: 8); KRNKNPPPS-OH (SEQ ID NO: 9); KRNKNPPS-OH (SEQ ID NO: 10); KRNKPPIA-OH (SEQ ID NO: 11); KRNKPPPA-OH (SEQ ID NO: 150); KRNKPPPS-OH (SEQ ID NO: 12); KSSGKPPPS-OH (SEQ ID NO: 13); PESGAPPPS-OH (SEQ ID NO: 14); PKSGAPPPS-OH (SEQ ID NO: 15); PKSKAPPPS-NH₂(SEQ ID NO: 16); PKSKAPPPS-OH (SEQ ID NO: 17); PKSKEPPPS-NH₂(SEQ ID NO: 18); PKSKEPPPS-OH (SEQ ID NO: 19); PKSKQPPPS-OH (SEQ ID NO: 20); PKSKSPPPS-NH₂(SEQ ID NO: 21); PKSKSPPPS-OH (SEQ ID NO: 22); PRNKNNPPS-OH (SEQ ID NO: 23); PSKGAPPPS-OH (SEQ ID NO: 24); PSSGAPPPSE-OH (SEQ ID NO: 25); PSSGAPPPS-NH₂(SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27); PSSGAPPPSS-OH (SEQ ID NO: 28); PSSGEPPPS-OH (SEQ ID NO: 29); PSSGKKPPS-OH (SEQ ID NO: 30); PSSGKPPPS-NH₂(SEQ ID NO: 31); PSSGKPPPS-OH (SEQ ID NO: 32); PSSGSPPPS-OH (SEQ ID NO: 33); PSSKAPPPS-OH (SEQ ID NO: 34); PSSKEPPPS-OH (SEQ ID NO: 35); PSSKGAPPPS-OH (SEQ ID NO: 36); PSSKQPPPS-OH (SEQ ID NO: 37); PSSKSPPPS-OH (SEQ ID NO: 38); SGAPPPS-OH (SEQ ID NO: 39); and SSGAPPPS-OH(SEQ ID NO: 40).

(SEQ ID NO: 2)

X₁X₂X₃GTFTSDX₁₀X₁₁X₁₂X₁₃LX₁₅X₁₆X₁₇X₁₈AQEFVX₂₄WLEDE-Z-

tail-(OHNH₂),

wherein:

- X₁is Y or W;
- X₂is S or G;
- X₃is Q or H;
- X₁₀is Y or H;
- X₁₁is S or T;
- X₁₂is K or R;
- X₁₃is Y, L or W;
- X₁₅is D or E;
- X₁₆is 2-Aminoisobutyric acid (Aib), A, or S;
- X₁₇is A or K;
- X₁₈is R, S, L, or Y;
- X₂₄is K, I, or Aib;
- X₂₅is H or W; and
- Z-tail is absent or is selected from the group consisting of PSSGAPPPS-NH₂(SEQ ID NO: 26); PSSGAPPPS-OH (SEQ ID NO: 27); and PKSKSPPPS-NH₂(SEQ ID NO: 21).

(SEQ ID NO: 3)

YSX₃GTFTSDYSKYLDX₁₆X₁₇X₁₈AQEFVX₂₄WLEDE-Z-tail-(OH/NH₂),

wherein:

- X₃is Q or H;
- X₁₆is 2-Aminoisobutyric acid (Aib) or A;
- X₁₇is A or K;
- X₁₈is R, S, or Y;
- X₂₄is K or Aib;
- Z-tail is selected from the group consisting of PSSGAPPPS-OH (SEQ ID NO: 27) and PKSKSPPPS-NH₂(SEQ ID NO: 21).

In some embodiments, the isolated polypeptide is selected from the group consisting of Compound A1, Compound A2, Compound A3, Compound A4, Compound A5 and Compound A6. In some embodiments, the isolated polypeptide is Compound A1. In some embodiments, the isolated polypeptide is Compound A2. In some embodiments, the isolated polypeptide is Compound A3. In some embodiments, the isolated polypeptide is Compound A4. In some embodiments, the isolated polypeptide is Compound A5. In some embodiments, the isolated polypeptide is Compound A6.

(SEQ ID NO: 137)

YSQGTFTSDYSKYLDSX₁₇RAQX₂₁FVX₂₄WLX₂₇X₂₈T-OH,

wherein:

X₁₇is K*, where K* is in a lactam bridge with E* at X₂₁;

X₂₁is E*, where E* is in a lactam bridge with K* at X₁₇;

X₂₄is K or K**, where K** is in a lactam bridge with E** at X₂₈;

X₂₇is Q or D; and

X₂₈is E or E**, where E** is in a lactam bridge with K** at X₂₄

Conjugation of a Lipophilic Substituent to any of the Peptides, Optionally Via a Spacer

In some embodiments, the disclosed peptide is optionally substituted with one or more lipophilic substituents each optionally via a bivalent spacer.

Conjugation of one or more lipophilic substituents, each optionally via a bivalent spacer, to a disclosed peptide is intended to prolong the action of the peptide by facilitating binding to serum albumin and delayed renal clearance of the conjugated peptide. Applicant has discovered that certain disclosed peptides, having affinity to albumin and prolonged elimination half-lives in humans, are particularly amenable to the disclosed methods of administration via an implantable osmotic drug delivery device.

In some embodiments, the disclosed peptide has an elimination half-life (t_1/2) in humans of at least about 5 hours following subcutaneous administration. In some embodiments, the disclosed peptide has an elimination half-life (t_1/2) in humans of at least about 8 hours, 10 hours, 12 hours, 16 hours, 24 hours or longer following subcutaneous administration.

As used herein, a “lipophilic substituent” comprises a substituent comprising 4 to 40 carbon atoms, 8 to 25 carbon atoms, or 12 to 22 carbon atoms. The lipophilic substituent may be attached to an amino group of the peptide (e.g., an ε-amino group of a lysine residue) by means of a carboxyl group of the lipophilic substituent, or optionally an amino group of the spacer, which spacer in turn forms an amide bond with an amino group of the amino acid (e.g., lysine) residue to which it is attached. In some embodiments, the peptide comprises three, two, or preferably one lipophilic substituent each with or without an optional spacer.

In some embodiments, the lipophilic substituent comprises a straight-chain or branched alkyl group. In some embodiments, the lipophilic substituent is an acyl group of a straight-chain or branched fatty acid. In some embodiments, the lipophilic substituent is the acyl group of the formula CH₃(CH₂)_nCO—, wherein n is an integer from 4 to 38, an integer from 4 to 24, such as CH₃(CH₂)₆CO—, CH₃(CH₂)₈CO—, CH₃(CH₂)₁₀CO—, CH₃(CH₂)₁₂CO—, CH₃(CH₂)₁₄CO—, CH₃(CH₂)₁₆CO—, CH₃(CH₂)₁₈CO—, CH₃(CH₂)₂₀CO— or CH₃(CH₂)₂₂CO—. In some embodiments, n is 6, 8, 10, 12, 14, 16, 18, 20 or 22. In some embodiments, the lipophilic substituent is an acyl group of the formula CH₃(CH₂)₁₄CO—.

In some embodiments, the lipophilic substituent is an acyl group of a straight-chain or branched fatty acid, further substituted with one or more carboxylic acid and/or hydroxamic acid groups. In some embodiments, the lipophilic substituent is an acyl group of the formula HOOC(CH₂)_mCO—, wherein m is an integer from 4 to 38, an integer from 4 to 24, such as HOOC(CH₂)₁₄CO—, HOOC(CH₂)₁₆CO—, HOOC(CH₂)₁₈CO—, HOOC(CH₂)₂₀CO— or HOOC(CH₂)₂₂CO—. In some embodiments, the lipophilic substituent is HOOC(CH₂)₁₆CO—. In some embodiments, m is 6, 8, 10, 12, 14, 16, 18, 20 or 22.

In some embodiments, the lipophilic substituent is attached to the parent peptide by means of a bivalent “spacer.” In some embodiments, the spacer comprises a bivalent group of Formula I:

—N(R₁)(CHR₂)_pCO—[N(R₃)((CH₂)₂O(CH₂)₂O)_q(CH₂)CO—]_r (Formula I)

wherein

each R₁and R₃is hydrogen or C₁-C₄alkyl;

each R₂is H or CO₂H;

p is 1, 2, 3, 4, 5 or 6;

q is 1, 2 or 3;

r is 0 or 1.

which spacer forms a bridge between an amino group of the disclosed peptide and a CO— group of the lipophilic substituent.

In some embodiments, each R₁is hydrogen. In some embodiments, each R₃is hydrogen. In some embodiments, each R₁and each R₃are hydrogen.

In some embodiments, at least one R₂is CO₂H. In some embodiments, one R₂is CO₂H.

In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6.

In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

In some embodiments, r is 0. In some embodiments, r is 1.

In some embodiments, the spacer is γ-glutamyl, i.e., —NH(CHCO₂H)(CH₂)₂CO—. In some embodiments, the spacer is γ-aminobutanoyl, i.e., —NH(CH₂)₃CO—. In some embodiments, the spacer is β-asparagyl, i.e., —NH(CHCO₂H)(CH₂)CO—. In some embodiments, the spacer is —NH(CH₂)₂CO—. In some embodiments, the spacer is glycyl. In some embodiments, the spacer is β-alanyl. In some embodiments, provided is the peptide of SEQ ID NO: 151, described below, wherein the lipophilic substituent is linked to the ε-amino group of a lysine via a spacer.

In some embodiments, the spacer is —NHCH(CO₂H)(CH₂)₂CO—[N(R₃)((CH₂)₂O(C)_q(CH₂)CO—]r. In some embodiments, the spacer is —NH(CH₂)₃CO—[N(R₃)((CH₂)₂O(CH₂)₂O)_q(CH₂)CO—]_r. In some embodiments, the spacer is —NHCH(CO₂H)(CH₂)₂CO—NH((CH₂)₂O(CH₂)₂O)₂(CH₂)CO—. In some embodiments, the spacer is —NH(CH₂)₃CO—NH((CH₂)₂O(CH₂)₂O)₂(CH₂)CO—. In some embodiments, the spacer is —NHCH(CO₂H)CH₂CO—[N(R₃)((CH₂)₂O(CH₂)₂O)_q(CH₂)CO—]_r. In some embodiments, the spacer is —NH(CH₂)₂CO—[N(R₃)((CH₂)₂O(CH₂)₂O)_q(CH₂)CO—]_r.

In some embodiments, the spacer is an amino acid, for example, Lys, Glu or Asp, or a dipeptide such as Gly-Lys. In some embodiments, when the spacer is Lys, Glu or Asp, one carboxyl group of the spacer may form an amide bond with an amino group of the disclosed peptide, and an amino group of the spacer may form an amide bond with a carboxyl group of the lipophilic substituent.

In some embodiments, the lipophilic substituent and spacer combine to form the structure of Formula II:

embedded image

In some embodiments, the lipophilic substituent and spacer combine to form the structure of Formula III:

embedded image

In some embodiments, each lipophilic substituent is attached, optionally via a spacer, to the ε-amino group of a lysine residue contained in the parent peptide.

For example, in some embodiments, provided is isolated polypeptide, wherein the isolated polypeptide is selected from the group consisting of Compounds A1-A105, and the isolated polypeptide is covalently joined to a lipophilic substituent, optionally via a spacer.

In some embodiments, the isolated polypeptide is selected from the group consisting of Compounds A1-A6, and the isolated polypeptide is covalently joined to a lipophilic substituent, optionally via a spacer.

In some embodiments, the isolated polypeptide is selected from the group consisting of Compounds A97-A103, and the isolated polypeptide is covalently joined to a lipophilic substituent, optionally via a spacer.

In some embodiments, the lipophilic substituents is covalently bound to any of the peptides of Compounds A1-A105, such as any of Compounds A1, A2, A3, A4, A5 or A6, via a spacer, and the lipophilic substituent and spacer are of Formula II.

In some embodiments, the lipophilic substituents is covalently bound to any of the peptides of Compounds A1-A105, such as any of Compounds A97, A98, A99, A100, A101, A102 or A103, via a spacer, and the lipophilic substituent and spacer are of Formula II.

In some embodiments, isolated polypeptide comprises a lysine, and the ε-amino group of the lysine is covalently bound to the lipophilic substituent, optionally via a spacer.

(SEQ ID NO: 151)

X₁SX₃GTFTSDX₁₀SKYLDX₁₆X₁₇X₁₈AQX₂₁FVX₂₄WLEDEPX₃₁

SX₃₃X₃₄PPPS-OH,

wherein:

X1=Y or W;

X3=H or Q;

X10=Y, K or K***;

X16=A, S, Aib, K or K***;

X17=A, K, Aib or K***;

X18=Y, S or R;

X21=E, K or K***;

X24=I, Aib, K or K***;

X31=S, K or K***;

X33=G, K or K***; and

X34=A or S;

where K*** is lysine, and the ε-amino group of the lysine sidechain is covalently joined to a lipophilic substituent, optionally via a spacer, both as defined herein.

In some embodiments, K*** is lysine, and the ε-amino group of the lysine sidechain is covalently joined to a lipophilic substituent via a spacer. In some embodiments, K*** is lysine, and the ε-amino group of the lysine sidechain is covalently joined to a lipophilic substituent in the absence of (i.e., without) a spacer.

X1=Y or W;

X3=H or Q;

X10=Y or K***;

X16=A, S, Aib or K***;

X17=A, K or Aib;

X18=Y, S or R;

X21=E or K***;

X24=I, Aib or K***;

X31=S or K***;

X33=G, K or K***; and

X34=A or S;

where K*** is lysine, and the ε-amino group of the lysine sidechain is covalently joined to a lipophilic substituent, optionally via a spacer, both as defined herein.

In some embodiments, X1=Y. In some embodiments, X1=W.

In some embodiments, X3=H. In some embodiments, X3=Q.

In some embodiments, X10=Y. In some embodiments, X10=K. In some embodiments, X10=K***.

In some embodiments, X16=A. In some embodiments, X16=S. In some embodiments, X16=Aib. In some embodiments, X16=K. In some embodiments, X16=K***.

In some embodiments, X17=A. In some embodiments, X17=K. In some embodiments, X17=Aib. In some embodiments, X17=K***.

In some embodiments, X18=Y. In some embodiments, X18=S. In some embodiments, X18=R.

In some embodiments, X21=E. In some embodiments, X21=K. In some embodiments, X21=K***.

In some embodiments, X24=I. In some embodiments, X24=Aib. In some embodiments, X24=K. In some embodiments, X24=K***.

In some embodiments, X31=S. In some embodiments, X31=K. In some embodiments, X31=K***.

In some embodiments, X33=G. In some embodiments, X33=K. In some embodiments, X33=K***.

In some embodiments, X16 is K***; in some embodiments, X21 is K***; in some embodiments, X24 is K***; in some embodiments, X33 is K***, where K*** is lysine, and the ε-amino group of the lysine is covalently joined to a lipophilic substituent and a spacer of Formula II:

embedded image

In some embodiments, one or more of X10, X16, X17, X21, X24, X31 or X33 is K***, where K*** is lysine, and the ε-amino group of the lysine is covalently joined to a lipophilic substituent and a spacer of Formula II.

In some embodiments, one of X10, X16, X17, X21, X24, X31 or X33 is K***, where K*** is lysine, and the ε-amino group of the lysine is covalently joined to a lipophilic substituent and a spacer of Formula II.

In some embodiments, provided is a pharmaceutical composition comprising any of the disclosed peptides formulated as a trifluoroacetate salt, acetate salt or hydrochloride salt. In some embodiments, provided is a pharmaceutical composition comprising any of the disclosed peptides formulated as a trifluoroacetate salt. In some embodiments, provided is a pharmaceutical composition comprising any of the disclosed peptides formulated as an acetate salt. In some embodiments, provided is a pharmaceutical composition comprising any of the disclosed peptides formulated as a hydrochloride salt.

In some embodiments, provided is a pharmaceutical composition comprising any of the disclosed peptides formulated as a trifluoroacetate salt, acetate salt or hydrochloride salt, and the peptide comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by the consensus sequence of SEQ ID NO: 151. In some embodiments, provided is a pharmaceutical composition comprising any of the disclosed peptides formulated as a trifluoroacetate salt and the peptide comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by the consensus sequence of SEQ ID NO: 151. In some embodiments, provided is a pharmaceutical composition comprising any of the disclosed peptides formulated as an acetate salt and the peptide comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by the consensus sequence of SEQ ID NO: 151. In some embodiments, provided is a pharmaceutical composition comprising any of the disclosed peptides formulated as a hydrochloride salt and the peptide comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by the consensus sequence of SEQ ID NO: 151. In some embodiments, in the peptide of SEQ ID NO: 151, one of X10, X16, X17, X21, X24, X31 or X33 is K***, where K*** is lysine, and the ε-amino group of the lysine is covalently joined to a lipophilic substituent and a spacer of Formula II. In some embodiments, in the peptide of SEQ ID NO: 151, one of X10, X16, X17, X21, X24, X31 or X33 is K***, where K*** is lysine, and the ε-amino group of the lysine is covalently joined to a lipophilic substituent and a spacer of Formula III.

In some embodiments, provided is any of the isolated polypeptides of the disclosure, wherein the polypeptide comprises one or more amino acid residues, each of which is covalently joined to a lipophilic substituent, optionally via a spacer, both as defined herein. In some embodiments, each of the one or more amino acid residues is covalently joined to a lipophilic substituent and a spacer of Formula II or III.

In some embodiments, any of the isolated polypeptides of the disclosure, wherein the polypeptide comprises at least one lysine residue, where the ε-amino group of the lysine sidechain is covalently joined to a lipophilic substituent, optionally via a spacer, both as defined herein. In some embodiments, each of the at least one lysine residues has an ε-amino group that is covalently joined to a lipophilic substituent and a spacer of Formula II or III.

In some embodiments, the isolated polypeptide is selected from the group consisting of Compounds B1-B48.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSK****APPPS-OH (SEQ ID NO: 152) which is also referred to herein as Compound B1.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFVK****WLEDEPSSGAPPPS-OH (SEQ ID NO: 153) which is also referred to herein as Compound B2.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDK****KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 154) which is also referred to herein as Compound B3.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDK****SKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 155) which is also referred to herein as Compound B4.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDEPSSK*****APPPS-OH (SEQ ID NO: 156) which is also referred to herein as Compound B5.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KYAQEFVK*****WLEDEPSSGAPPPS-OH (SEQ ID NO: 157) which is also referred to herein as Compound B6.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDK*****KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 158) which is also referred to herein as Compound B7.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDK*****SKYLD(Aib)KYAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 159) which is also referred to herein as Compound B8.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSK****APPPS-OH (SEQ ID NO: 160) which is also referred to herein as Compound B9.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFVK****WLEDEPSSGAPPPS-OH (SEQ ID NO: 161) which is also referred to herein as Compound B10.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDK****KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 162) which is also referred to herein as Compound B11.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDK****SKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 163) which is also referred to herein as Compound B12.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDEPSSK*****APPPS-OH (SEQ ID NO: 164) which is also referred to herein as Compound B13.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KSAQEFVK*****WLEDEPSSGAPPPS-OH (SEQ ID NO: 165) which is also referred to herein as Compound B14.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDK*****KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 166) which is also referred to herein as Compound B15.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDK*****SKYLD(Aib)KSAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 167) which is also referred to herein as Compound B16.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSK****APPPS-OH (SEQ ID NO: 168) which is also referred to herein as Compound B17.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFVK****WLEDEPSSGAPPPS-OH (SEQ ID NO: 169) which is also referred to herein as Compound B18.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence WSQGTFTSDYSKYLDK****KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 170) which is also referred to herein as Compound B19.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence WSQGTFTSDK****SKYLD(Aib)KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 171) which is also referred to herein as Compound B20.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDEPSSK*****APPPS-OH (SEQ ID NO: 172) which is also referred to herein as Compound B21.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence WSQGTFTSDYSKYLD(Aib)KRAQEFVK*****WLEDEPSSGAPPPS-OH (SEQ ID NO: 173) which is also referred to herein as Compound B22.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence WSQGTFTSDYSKYLDK*****KRAQEFV(Aib)WLEDEPSSGAPPPS-OH (SEQ ID NO: 174) which is also referred to herein as Compound B23.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence WSQGTFTSDK*****SKYLD(Aib)KRAQEFV(Aib)WLEDEPSS GAPPPS-OH (SEQ ID NO: 175) which is also referred to herein as Compound B24.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSK****APPPS-OH (SEQ ID NO: 176) which is also referred to herein as Compound B25.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDK****KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 177) which is also referred to herein as Compound B26.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDK****SKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 178) which is also referred to herein as Compound B27.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQK****FVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 179) which is also referred to herein as Compound B28.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSSK*****APPPS-OH (SEQ ID NO: 180) which is also referred to herein as Compound B29.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLD(Aib)KRAQK*****FVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 181) which is also referred to herein as Compound B30.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDK*****KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 182) which is also referred to herein as Compound B31.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDK*****SKYLD(Aib)KRAQEFVIWLEDEPSSGAPPPS-OH (SEQ ID NO: 183) which is also referred to herein as Compound B32.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSK****SPPPS-NH₂(SEQ ID NO: 184) which is also referred to herein as Compound B33.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVK****WLEDEPKSKSPPPS-NH₂(SEQ ID NO: 185) which is also referred to herein as Compound B34.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSQGTFTSDYSKYLDK****ARAQEFVKWLEDEPKSKSPPPS-NH₂(SEQ ID NO: 186) which is also referred to herein as Compound B35.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSQGTFTSDK****SKYLDAARAQEFVKWLEDEPKSKSPPPS-NH₂(SEQ ID NO: 187) which is also referred to herein as Compound B36.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSK*****SPPPS-NH₂(SEQ ID NO: 188) which is also referred to herein as Compound B37.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSQGTFTSDYSKYLDAARAQEFVK*****WLEDEPKSKSPPPS-NH₂(SEQ ID NO: 189) which is also referred to herein as Compound B38.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSQGTFTSDYSKYLDK*****ARAQEFVKWLEDEPKSKSPPPS-NH₂(SEQ ID NO: 190) which is also referred to herein as Compound B39.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSQGTFTSDK*****SKYLDAARAQEFVKWLEDEPKSKSPPPS-NH₂(SEQ ID NO: 191) which is also referred to herein as Compound B40.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSK****APPPS-NH₂(SEQ ID NO: 192) which is also referred to herein as Compound B41.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVK****WLEDEPSSGAPPPS-NH₂(SEQ ID NO: 193) which is also referred to herein as Compound B42.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDK****ARAQEFVKWLEDEPSSGAPPPS-NH₂(SEQ ID NO: 194) which is also referred to herein as Compound B43.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDK****SKYLDSARAQEFVKWLEDEPSSGAPPPS-NH₂(SEQ ID NO: 195) which is also referred to herein as Compound B44.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSK*****APPPS-NH₂(SEQ ID NO: 196) which is also referred to herein as Compound B45.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDSARAQEFVK*****WLEDEPSSGAPPPS-NH₂(SEQ ID NO: 197) which is also referred to herein as Compound B46.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDYSKYLDK*****ARAQEFVKWLEDEPSSGAPPPS-NH₂(SEQ ID NO: 198) which is also referred to herein as Compound B47.

In some embodiments, an isolated polypeptide of the disclosure comprises the amino acid sequence YSHGTFTSDK*****SKYLDSARAQEFVKWLEDEPSSGAPPPS-NH₂(SEQ ID NO: 199) which is also referred to herein as Compound B48.

In the above embodiments, each ε-amino group of the lysine residue located at the indicated peptide residue (K****) in the disclosed compounds is covalently bound to the indicated carbonyl of Formula II to form an amide:

embedded image

In the above embodiments, each ε-amino group of the lysine residue located at the indicated peptide residue (K*****) in the disclosed compounds is covalently bound to the indicated carbonyl of Formula III to form an amide:

embedded image

In some embodiments, an isolated polypeptide of the disclosure is selected from the group consisting of Compound B1 through Compound B8.

In some embodiments, an isolated polypeptide of the disclosure is selected from the group consisting of Compound B9 through Compound B16.

In some embodiments, an isolated polypeptide of the disclosure is selected from the group consisting of Compound B17 through Compound B24.

In some embodiments, an isolated polypeptide of the disclosure is selected from the group consisting of Compound B25 through Compound B32.

In some embodiments, an isolated polypeptide of the disclosure is selected from the group consisting of Compound B33 through Compound B40.

In some embodiments, an isolated polypeptide of the disclosure is selected from the group consisting of Compound B41 through Compound B48.

In some embodiments, an isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 154, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190 and SEQ ID NO: 192.

In some embodiments, an isolated polypeptide of the disclosure is selected from the group consisting of Compound B1 through Compound B48.

In some embodiments, an isolated polypeptide of the disclosure is selected from the group consisting of Compound B3, Compound B6, Compound B7, Compound B9, Compound B10, Compound B11, Compound B14, Compound B15, Compound B17, Compound B21, Compound B22, Compound B34, Compound B37, Compound B38, Compound B39 and Compound B41.

In some embodiments, an isolated polypeptide of the disclosure is selected from the group consisting of Compound B9, Compound B14 and Compound B15.

In some embodiments, an isolated polypeptide of the disclosure is Compound B9. In some embodiments, an isolated polypeptide of the disclosure is Compound B14. In some embodiments, an isolated polypeptide of the disclosure is Compound B15.

In some embodiments, an isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by Compound B1 through Compound B48.

2.0.2 Peptide Combinations

In some embodiments, an isolated glucagon analog polypeptide of the disclosure, which is a selective glucagon receptor agonist, is co-formulated in combination with a second agent selected from the group consisting of oxyntomodulin, exenatide, a derivative of exenatide, an analogue of exenatide, glucagon-like peptide-1 (GLP-1), a derivative of GLP-1, and an analogue of GLP-1. In some embodiments, any of the glucagon analogs of the disclosure is co-formulated in combination with exenatide.

In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is not covalently joined to a lipophilic substituent and a spacer, as described herein.

In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is selected from the group consisting of Compounds A1-A105. In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is selected from the group consisting of Compounds A1, A2, A3, A4, A5 and A6. In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A1. In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A2. In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A3. In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A4. In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A5. In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound A6.

In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide, wherein the glucagon analog has at least one amino acid that is covalently joined to a lipophilic substituent and a spacer, as described herein. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide, wherein the glucagon analog has at least one lysine residue that has an ε-amino group that is covalently joined to a lipophilic substituent and a spacer of Formula II or III.

In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is selected from the group consisting of Compound B3, Compound B6, Compound B7, Compound B9, Compound B10, Compound B11, Compound B14, Compound B15, Compound B17, Compound B21, Compound B22, Compound B34, Compound B37, Compound B38, Compound B39 and Compound B41.

In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound B9. In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound B14. In some embodiments, a glucagon analog of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is Compound B15.

In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 1000:1 to 1:1000. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 500:1 to 1:500. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 100:1 to 1:100. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 50:1 to 1:50. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 25:1 to 1:25. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 10:1 to 1:10. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 1:1 to 1:10. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 10:1 to 1:1. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 5:1 to 1:5. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 1:1 to 1:5. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide in a fixed ratio of glucagon analog:exenatide of 5:1 to 1:1.

The invention also provides a method of treating type 2 diabetes mellitus in a subject in need of treatment. The method comprises providing one or more of the isolated glucagon-receptor selective agonist polypeptides of the disclosure. In some embodiments, the method comprises providing continuous delivery of an isolated glucagon-receptor selective agonist polypeptide from an osmotic delivery device, wherein substantial steady-state delivery of the isolated glucagon-specific agonist polypeptide at a therapeutic concentration is achieved within a time period of about 7 days or less after implantation of the osmotic delivery device in the subject. The substantial steady-state delivery of the isolated glucagon-specific agonist polypeptide from the osmotic delivery device is continuous over an administration period. Humans are preferred subjects for the practice of the present invention.

In some embodiments of the present invention, the administration period is, for example, at least about 3 months, at least about 3 months to about a year, at least about 4 months to about a year, at least about 5 months to about a year, at least about 6 months to about a year, at least about 8 months to about a year, at least about 9 months to about a year, at least about 10 months to about a year, at least about one year to about two years, at least about two years to about three years.

In some embodiments of the present invention, the substantial steady-state delivery of an isolated glucagon-specific agonist polypeptide at therapeutic concentrations is achieved within about 5 days or less after implantation of the osmotic delivery device in the subject, within about 4 days or less after implantation of the osmotic delivery device in the subject, within about 3 days or less after implantation of the osmotic delivery device in the subject, within about 2 days or less after implantation of the osmotic delivery device in the subject, or within about 1 day or less after implantation of the osmotic delivery device in the subject. In preferred embodiments of the present invention, the substantial steady-state delivery of the isolated glucagon-specific agonist polypeptide at therapeutic concentrations is achieved within about 2 days or less, more preferably within about 1 day or less after implantation of the osmotic delivery device in the subject.

In further embodiments, the treatment methods of the present invention provide significant decrease in the subject's fasting plasma glucose concentration after implantation of the osmotic delivery device in the subject (relative to the subject's fasting plasma glucose concentration before implantation of the osmotic delivery device) that is achieved within about 7 days or less after implantation of the osmotic delivery device in the subject, within about 6 days or less after implantation of the osmotic delivery device in the subject, within about 5 days or less after implantation of the osmotic delivery device in the subject, within about 4 days or less after implantation of the osmotic delivery device in the subject, within about 3 days or less after implantation of the osmotic delivery device in the subject, within about 2 days or less after implantation of the osmotic delivery device in the subject, or within about 1 day or less after implantation of the osmotic delivery device in the subject. In preferred embodiments of the present invention, the significant decrease in the subject's fasting plasma glucose concentration after implantation of the osmotic delivery device, relative to the subject's fasting plasma glucose concentration before implantation, is achieved within about 2 days or less, preferably within about 1 day or less after implantation of the osmotic delivery device in the subject, or more preferably within about 1 day after implantation of the osmotic delivery device in the subject. The significant decrease in fasting plasma glucose is typically statistically significant as demonstrated by application of an appropriate statistical test or is considered significant for the subject by a medical practitioner. A significant decrease in fasting plasma glucose relative to the baseline before implantation is typically maintained over the administration period.

In yet further embodiments of the first aspect of the present invention, the treatment methods further comprise the capability to terminate the continuous delivery of the glucagon-specific agonist polypeptide such that the concentration of the glucagon-specific agonist polypeptide is substantially undetectable in a blood sample from the subject within about 6 half-lives or less of the glucagon-specific agonist polypeptide after termination of continuous delivery, within about 5 half-lives or less of the glucagon-specific agonist polypeptide after termination of continuous delivery, within about 4 half-lives or less of the glucagon-specific agonist polypeptide after termination of continuous delivery, or within about 3 half-lives or less of the glucagon-specific agonist polypeptide after termination of continuous delivery. The glucagon-specific agonist polypeptide may be detected, for example, by an RIA, a chromatographic method, an ECL assay, an ELISA, or an IEMA. Termination of the continuous delivery can be accomplished, for example, by removal of the osmotic delivery device from the subject.

In related embodiments of the present invention, the treatment methods further comprise the capability to terminate the continuous delivery of the glucagon-receptor selective agonist polypeptide such that the concentration of the polypeptide is substantially undetectable in a blood sample from the subject in less than about 72 hours after termination of continuous delivery, in less than about 48 hours after termination of continuous delivery, in less than about 24 hours after termination of continuous delivery, in less than about 18 hours after termination of continuous delivery, in less than about 14 hours after termination of continuous delivery, in less than about 12 hours after termination of continuous delivery, in less than about 6 hours after termination of continuous delivery, or in less than about 4 hours after termination of continuous delivery. In preferred embodiments, the glucagon-specific agonist polypeptide is substantially undetectable in a blood sample from the subject in less than about 24 hours after termination of continuous delivery, in less than about 18 hours after termination of continuous delivery, or more preferably in less than about 14 hours after termination of continuous delivery.

In some embodiments, the glucagon-receptor selective agonist polypeptide is formulated in a suspension formulation. In some embodiments, the suspension formulation comprises a particle formulation comprising the glucagon-receptor selective agonist polypeptide, and a vehicle formulation. In some embodiments, the glucagon-receptor selective agonist polypeptide comprises an isolated polypeptide of the disclosure, a peptide analog thereof, or a peptide derivative thereof. In some embodiments, the glucagon-receptor selective agonist polypeptide comprises an amino acid sequence encompassed by the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 137. In some embodiments, the glucagon-receptor selective agonist polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-139.

In embodiments of all aspects of the present invention relating to methods of treating type 2 diabetes mellitus, suspension formulations for use in the methods can, for example, comprise a particle formulation comprising an isolated glucagon-receptor selective agonist polypeptide, and a vehicle formulation.

The reservoir of the osmotic delivery devices may, for example, comprise titanium or a titanium alloy.

In a fifth aspect, the present invention relates to a method of treating a disease or condition in a subject in need of treatment. The method comprises providing continuous delivery of a drug from an osmotic delivery device, wherein substantial steady-state delivery of the drug at therapeutic concentrations is achieved within a time period of about 7 days or less after implantation of the osmotic delivery device in the subject. The substantial steady-state delivery of the drug from the osmotic delivery device is continuous over an administration period of at least about 3 months. The drug has a known or determined half-life in a typical subject. Humans are preferred subjects for the practice of the present invention. The present invention includes a drug effective for treatment of the disease or condition, as well as an osmotic delivery device comprising the drug for use in the present methods of treating the disease or condition in a subject in need of treatment. Advantages of the present invention include mitigation of peak-associated drug toxicities and attenuation of sub-optimal drug therapy associated with troughs.

In some embodiments of this aspect of the present invention, the substantial steady-state delivery of a drug at therapeutic concentrations is achieved within a period of about 7 days or less after implantation of the osmotic delivery device in the subject, about 5 days or less after implantation of the osmotic delivery device in the subject, about 4 days or less after implantation of the osmotic delivery device in the subject, about 3 days or less after implantation of the osmotic delivery device in the subject, about 2 days or less after implantation of the osmotic delivery device in the subject, or about 1 day or less after implantation of the osmotic delivery device in the subject.

In some embodiments of this aspect of the present invention, establishment of the substantial steady-state delivery of the drug at therapeutic concentrations, after implantation of the osmotic delivery device in the subject, may take a longer period of time, for example, a period of about 2 weeks or less, or within less than about 6 half-lives of the drug within the subject after implantation of the device.

The invention also provides a method for promoting weight loss in a subject in need thereof, a method for treating excess weight or obesity in a subject in need thereof, and/or a method for suppressing appetite in a subject in need thereof. The method comprises providing delivery of an isolated glucagon-receptor selective agonist polypeptide. In some embodiments, the isolated glucagon-receptor selective agonist polypeptide is continuously delivered from an osmotic delivery device, wherein substantial steady-state delivery of the glucagon-receptor selective agonist polypeptide at a therapeutic concentration is achieved within a time period of about 7 days or less after implantation of the osmotic delivery device in the subject. The substantial steady-state delivery of the glucagon-receptor selective agonist polypeptide from the osmotic delivery device is continuous over an administration period. Humans are preferred subjects for the practice of the present invention. The present invention includes an isolated glucagon-receptor selective agonist polypeptide, as well as an osmotic delivery device comprising an isolated glucagon-receptor selective agonist polypeptide for use in the present methods in a subject in need of treatment. They subject may have type 2 diabetes. The subject in need thereof may have a baseline HbA1c % of greater than 10.0%, i.e., a high baseline (HBL) subject. The subject may not have previously received a drug for treating type 2 diabetes mellitus.

In some embodiments of the present invention, the administration period is, for example, at least about 3 months, at least about 3 months to about a year, at least about 4 months to about a year, at least about 5 months to about a year, at least about 6 months to about a year, at least about 8 months to about a year, at least about 9 months to about a year, or at least about 10 months to about a year, at least about one year to about two years, or at least about two years to about three years.

In embodiments of all aspects of the present invention relating to methods of treating a disease or condition in a subject, an exemplary osmotic delivery device comprises the following: an impermeable reservoir comprising interior and exterior surfaces and first and second open ends; a semi-permeable membrane in sealing relationship with the first open end of the reservoir; an osmotic engine within the reservoir and adjacent the semi-permeable membrane; a piston adjacent the osmotic engine, wherein the piston forms a movable seal with the interior surface of the reservoir, the piston divides the reservoir into a first chamber and a second chamber, the first chamber comprising the osmotic engine; a drug formulation or suspension formulation comprising the drug, wherein the second chamber comprises the drug formulation or suspension formulation and the drug formulation or suspension formulation is flowable; and a diffusion moderator inserted in the second open end of the reservoir, the diffusion moderator adjacent the suspension formulation. In preferred embodiments, the reservoir comprises titanium or a titanium alloy.

In embodiments of all aspects of the present invention relating to methods of treating a disease or condition in a subject, the drug formulation can comprise the drug and a vehicle formulation. Alternatively, suspension formulations are used in the methods and can, for example, comprise a particle formulation comprising the drug and a vehicle formulation. Vehicle formulations for use in forming the suspension formulations of the present invention can, for example, comprise a solvent and a polymer.

The reservoir of the osmotic delivery devices may, for example, comprise titanium or a titanium alloy.

In embodiments of all aspects of the present invention the implanted osmotic delivery device can be used to provide subcutaneous delivery.

In embodiments of all aspects of the present invention the continuous delivery can, for example, be zero-order, controlled continuous delivery.

Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed herein.

3.0.0 Formulations and Compositions

Drugs for use in the practice of the present invention are typically uniformly suspended, dissolved or dispersed in a suspension vehicle to form a suspension formulation.

The isolated polypeptides of the disclosure (also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the fusion protein and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subdermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, rectal, or combinations thereof. In some embodiments, a pharmaceutical composition or an isolated polypeptide of the disclosure is formulated for administration by topical administration. In some embodiments, a pharmaceutical composition or an isolated polypeptide of the disclosure is formulated for administration by inhalation administration. In some embodiments, the pharmaceutical composition is formulated for administration by a device or other suitable delivery mechanism that is suitable for subdermal or subcutaneous implantation and delivers the pharmaceutical composition subcutaneously. In some embodiments, the pharmaceutical composition is formulated for administration by an implant device that is suitable for subdermal or subcutaneous implantation and delivers the pharmaceutical composition subcutaneously. In some embodiments, the pharmaceutical composition is formulated for administration by an osmotic delivery device, e.g., an implantable osmotic delivery device, that is suitable for subdermal or subcutaneous placement or other implantation and delivers the pharmaceutical composition subcutaneously. Solutions or suspensions used for parenteral application, intradermal application, subdermal application, subcutaneous application, or combinations thereof can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

3.1.0 Drug Particle Formulations

In one aspect, the present invention provides drug particle formulations for pharmaceutical use. The particle formulation typically comprises a drug and includes one or more stabilizing component (also referred to herein as “excipients”). Examples of stabilizing components include, but are not limited to, carbohydrates, antioxidants, amino acids, buffers, inorganic compounds, and surfactants.

In any of the embodiments, the particle formulation may comprise about 50 wt % to about 90 wt % drug, about 50 wt % to about 85 wt % drug, about 55 wt % to about 90 wt % drug, about 60 wt % to about 90 wt % drug, about 65 wt % to about 85 wt % drug, about 65 wt % to about 90 wt % drug, about 70 wt % to about 90 wt % drug, about 70 wt % to about 85 wt % drug, about 70 wt % to about 80 wt % drug, or about 70 wt % to about 75 wt % drug.

In any of the embodiments, a particle formulation comprises a drug, as described above, and one or more stabilizer. The stabilizers may be, for example, carbohydrate, antioxidant, amino acid, buffer, inorganic compound, or surfactant. The amounts of stabilizers in the particle formulation can be determined experimentally based on the activities of the stabilizers and the desired characteristics of the formulation, in view of the teachings of the present specification.

Typically, the amount of carbohydrate in the formulation is determined by aggregation concerns. In general, the carbohydrate amount should not be too high so as to avoid promoting crystal growth in the presence of water due to excess carbohydrate unbound to drug.

Typically, the amount of antioxidant in the formulation is determined by oxidation concerns, while the amount of amino acid in the formulation is determined by oxidation concerns and/or formability of particles during spray drying.

Typically, the amount of buffer in the formulation is determined by pre-processing concerns, stability concerns, and formability of particles during spray drying. Buffer may be required to stabilize drug during processing, e.g., solution preparation and spray drying, when all stabilizers are solubilized.

Examples of carbohydrates that may be included in the particle formulation include, but are not limited to, monosaccharides (e.g., fructose, maltose, galactose, glucose, D-mannose, and sorbose), disaccharides (e.g., lactose, sucrose, trehalose, and cellobiose), polysaccharides (e.g., raffinose, melezitose, maltodextrins, dextrans, and starches), and alditols (acyclic polyols; e.g., mannitol, xylitol, maltitol, lactitol, xylitol sorbitol, pyranosyl sorbitol, and myoinsitol). Suitable carbohydrates include disaccharides and/or non-reducing sugars, such as sucrose, trehalose, and raffinose.

Examples of antioxidants that may be included in the particle formulation include, but are not limited to, methionine, ascorbic acid, sodium thiosulfate, catalase, platinum, ethylenediaminetetraacetic acid (EDTA), citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxyltoluene, and propyl gallate. Further, amino acids that readily oxidize can be used as antioxidants, for example, cysteine, methionine, and tryptophan.

Examples of amino acids that may be included in the particle formulation include, but are not limited to, arginine, methionine, glycine, histidine, alanine, L-leucine, glutamic acid, iso-leucine, L-threonine, 2-phenylamine, valine, norvaline, proline, phenylalanine, tryptophan, serine, asparagines, cysteine, tyrosine, lysine, and norleucine. Suitable amino acids include those that readily oxidize, e.g., cysteine, methionine, and tryptophan.

Examples of buffers that may be included in the particle formulation include, but are not limited to, citrate, histidine, succinate, phosphate, maleate, tris, acetate, carbohydrate, and gly-gly. Suitable buffers include citrate, histidine, succinate, and tris.

Examples of inorganic compounds that may be included in the particle formulation include, but are not limited to, NaCl, Na₂SO₄, NaHCO₃, KCl, KH₂PO₄, CaCl₂, and MgCl₂.

In addition, the particle formulation may include other stabilizers/excipients, such as surfactants and salts. Examples of surfactants include, but are not limited to, Polysorbate 20, Polysorbate 80, PLURONIC® (BASF Corporation, Mount Olive, N.J.) F68, and sodium dodecyl sulfate (SDS). Examples of salts include, but are not limited to, sodium chloride, calcium chloride, and magnesium chloride.

3.1.1 Exemplary Drugs

The drug particle formulations comprise a drug. The drug may be any physiologically or pharmacologically active substance, particularly those known to be delivered to the body of a human or an animal.

Suitable drugs include, but are not limited to: peptides, proteins, polypeptides or synthetic analogs of these species, as well as mixtures thereof.

In one embodiment, preferred drugs include macromolecules. Such macromolecules include, but are not limited to, pharmacologically active peptides, proteins, or polypeptides. Numerous peptides, proteins, or polypeptides that are useful in the practice of the present invention are described herein. In addition to the peptides, proteins, or polypeptides described, modifications of these peptides, proteins, or polypeptides are also known to one of skill in the art and can be used in the practice of the present invention following the guidance presented herein. Such modifications include, but are not limited to, amino acid analogs, amino acid mimetics, analog polypeptides, or derivative polypeptides. Further, the drugs disclosed herein may be formulated or administered singly or in combination (e.g., using mixtures of drugs or multiple devices; U.S. Patent Publication No. 2009/0202608).

Some embodiments of the present invention comprise use of polypeptides of SEQ ID NOs: 4-136, 138, 139 and 143-149.

Some embodiments of the present invention comprise use of a glucagon-receptor selective agonist polypeptide in combination with a second polypeptide, such as, by way of, non-limiting example, insulinotropic peptides, peptide hormones, for example, glucagon and incretin mimetics (e.g., GLP-1 and exenatide), as well as peptide analogs and peptide derivatives thereof; PYY (also known as peptide YY, peptide tyrosine tyrosine), as well as peptide analogs and peptide derivatives thereof, for example, PYY(3-36); oxyntomodulin, as well as peptide analogs and peptide derivatives thereof); and gastric inhibitory peptide (GIP), as well as peptide analogs and peptide derivatives thereof.

GLP-1, including three forms of the peptide, GLP-1(1-37), GLP-1(7-37) and GLP-1(7-36) amide, as well as peptide analogs of GLP-1 have been shown to stimulate insulin secretion (i.e., is insulinotropic), which induces glucose uptake by cells and results in decreases in serum glucose concentrations (see, e g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)).

Numerous GLP-1 peptide derivatives and peptide analogs demonstrating insulinotropic action are known in the art (see, e.g., U.S. Pat. Nos. 5,118,666; 5,120,712; 5,512,549; 5,545,618; 5,574,008; 5,574,008; 5,614,492; 5,958,909; 6,191,102; 6,268,343; 6,329,336; 6,451,974; 6,458,924; 6,514,500; 6,593,295; 6,703,359; 6,706,689; 6,720,407; 6,821,949; 6,849,708; 6,849,714; 6,887,470; 6,887,849; 6,903,186; 7,022,674; 7,041,646; 7,084,243; 7,101,843; 7,138,486; 7,141,547; 7,144,863; and 7,199,217), as well as in clinical trials (e.g., taspoglutide and albiglutide). One example of a GLP-1 peptide derivative useful in the practice of the present invention is Victoza® (Novo Nordisk A/S, Bagsvaerd D K) (liraglutide; U.S. Pat. Nos. 6,268,343, 6,458,924, and 7,235,627). Once-daily injectable Victoza® (liraglutide) is commercially available in the United States, Europe, and Japan. For ease of reference herein, the family of GLP-1 peptides, GLP-1 peptide derivatives and GLP-1 peptide analogs having insulinotropic activity is referred to collectively as “GLP-1.”

The molecule exenatide has the amino acid sequence of exendin-4 (Kolterman O. G., et al., J. Clin. Endocrinol. Metab. 88(7):3082-9 (2003)) and is produced by chemical synthesis or recombinant expression. Twice-daily injectable exenatide is commercially available in the United States and Europe, and sold under the trade name of Byetta® (Amylin Pharmaceuticals, Inc., San Diego, Calif.). Exendin-3 and exendin-4 are known in the art and were originally isolated from Heloderma spp. (Eng, J., et al., J. Biol. Chem., 265:20259-62 (1990); Eng., J., et al., J. Biol. Chem., 267:7402-05 (1992)). Use of exendin-3 and exendin-4 for the treatment of type 2 diabetes mellitus and the prevention of hyperglycemia has been proposed (see, e.g., U.S. Pat. No. 5,424,286). Numerous exenatide peptide derivatives and peptide analogs (including, e.g., exendin-4 agonists) are known in the art (see, e.g., U.S. Pat. Nos. 5,424,286; 6,268,343; 6,329,336; 6,506,724; 6,514,500; 6,528,486; 6,593,295; 6,703,359; 6,706,689; 6,767,887; 6,821,949; 6,849,714; 6,858,576; 6,872,700; 6,887,470; 6,887,849; 6,924,264; 6,956,026; 6,989,366; 7,022,674; 7,041,646; 7,115,569; 7,138,375; 7,141,547; 7,153,825; and 7,157,555). One example of an exenatide derivative useful in the practice of the present invention is lixisenatide (also known as ZP10, AVE0010) (see, e.g., U.S. Pat. No. 6,528,486), which is in clinical trials. For ease of reference herein, the family of exenatide peptides (e.g., including exendin-3, exendin-4, and exendin-4-amide), exenatide peptide derivatives, and exenatide peptide analogs is referred to collectively as “exenatide.”

Peptide YY (PYY) is a 36 amino acid residue peptide amide. PYY inhibits gut motility and blood flow (Laburthe, M., Trends Endocrinol Metab. 1(3): 168-74 (1990), mediates intestinal secretion (Cox, H. M., et al., Br J Pharmacol 101(2):247-52 (1990); Playford, R. J., et al., Lancet 335(8705):1555-7 (1990)), and stimulate net absorption (MacFayden, R. J., et al., Neuropeptides 7(3):219-27 (1986)). Two major in vivo variants, PYY(1-36) and PYY(3-36), have been identified (e.g., Eberlein, G. A., et al., Peptides 10(4), 797-803 (1989)). The sequence of PYY, as well as peptide analogs and peptide derivatives thereof, are known in the art (e.g., U.S. Pat. Nos. 5,574,010 and 5,552,520).

Oxyntomodulin is a naturally occurring 37 amino acid peptide hormone found in the colon that has been found to suppress appetite and facilitate weight loss (Wynne K, et al., Int J Obes (Lond) 30(12):1729-36(2006)). The sequence of oxyntomodulin, as well as peptide analogs and peptide derivatives thereof, are known in the art (e.g., Bataille D, et al., Peptides 2(Suppl 2):41-44 (1981); and U.S. Patent Publication Nos. 2005/0070469 and 2006/0094652).

Gastric Inhibitory Peptide (GIP) is an insulinotropic peptide hormone (Efendic, S., et al., Horm Metab Res. 36:742-6 (2004)) and is secreted by the mucosa of the duodenum and jejunum in response to absorbed fat and carbohydrate that stimulate the pancreas to secrete insulin. GIP circulates as a biologically active 42-amino acid peptide. GIP is also known as glucose-dependent insulinotropic protein. GIP is a 42-amino acid gastrointestinal regulatory peptide that stimulates insulin secretion from pancreatic beta cells in the presence of glucose (Tseng, C., et al., PNAS 90:1992-1996 (1993)). The sequence of GIP, as well as peptide analogs and peptide derivatives thereof, are known in the art (e.g., Meier J. J., Diabetes Metab Res Rev. 21(2):91-117 (2005) and Efendic S., Horm Metab Res. 36(11-12):742-6 (2004)).

Glucagon is a peptide hormone, produced by alpha cells of the pancreas, which raises the concentration of glucose in the bloodstream. Its effect is opposite that of insulin, which lowers the glucose concentration. The pancreas releases glucagon when the concentration of glucose in the bloodstream falls too low. Glucagon causes the liver to convert stored glycogen into glucose, which is released into the bloodstream. High blood glucose levels stimulate the release of insulin. Insulin allows glucose to be taken up and used by insulin-dependent tissues. Thus, glucagon and insulin are part of a feedback system that keeps blood glucose levels at a stable level.

Glucagon-like peptide-2 (GLP-2) is a 33 amino acid peptide with the sequence HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO: 200) in humans. GLP-2 is created by specific post-translational proteolytic cleavage of proglucagon in a process that also liberates the related glucagon-like peptide-1 (GLP-1). GLP-2 is produced by the intestinal endocrine L cell and by various neurons in the central nervous system. Intestinal GLP-2 is co-secreted along with GLP-1 upon nutrient ingestion. When externally administered, GLP-2 produces a number of effects in humans and rodents, including intestinal growth, enhancement of intestinal function, reduction in bone breakdown and neuroprotection. GLP-2 may act in an endocrine fashion to link intestinal growth and metabolism with nutrient intake.

Examples of half-lives of some of the peptides are as follows: exenatide, approximately 2.5 hours; GLP-1, approximately 2 minutes; GIP, approximately 5 minutes; PYY, approximately 8 minutes; glucagon, approximately 6 minutes; oxyntomodulin, approximately 6 minutes; and GLP-2, approximately 6 minutes.

The drugs can also be in various forms including, but not limited to, the following: uncharged molecules; components of molecular complexes; and pharmacologically acceptable salts such as hydrochloride, hydrobromide, sulfate, laurates, palmatates, phosphate, nitrate, borate, acetate, maleate, tartrate, oleates, or salicylates. For acidic drugs, salts of metals, amines or organic cations, for example, quaternary ammonium, can be employed. Furthermore, simple derivatives of the drug such as esters, ethers, amides and the like that have solubility characteristics suitable for the purpose of the invention can also be used herein.

The above drugs and other drugs known to those of skill in the art are useful in methods of treatment for a variety of conditions including but not limited to the following: chronic pain, hemophilia and other blood disorders, endocrine disorders, metabolic disorders, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), Alzheimer's disease, cardiovascular diseases (e.g., heart failure, atherosclerosis, and acute coronary syndrome), rheumatologic disorders, diabetes (including type 1, type 2 diabetes mellitus, human immunodeficiency virus treatment-induced, latent autoimmune diabetes in adults, and steroid-induced), hypoglycemia unawareness, restrictive lung disease, chronic obstructive pulmonary disease, lipoatrophy, metabolic syndrome, leukemia, hepatitis, renal failure, infectious diseases (including bacterial infection, viral infection (e.g., infection by human immunodeficiency virus, hepatitis C virus, hepatitis B virus, yellow fever virus, West Nile virus, Dengue virus, Marburg virus, and Ebola virus), and parasitic infection), hereditary diseases (such as cerbrosidase deficiency and adenosine deaminase deficiency), hypertension, septic shock, autoimmune diseases (e.g., Grave's disease, systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis), shock and wasting disorders, cystic fibrosis, lactose intolerance, Crohn's diseases, inflammatory bowel disease, gastrointestinal cancers (including colon cancer and rectal cancer), breast cancer, leukemia, lung cancer, bladder cancer, kidney cancer, non-Hodgkin lymphoma, pancreatic cancer, thyroid cancer, endometrial cancer, and other cancers. Further, some of the above agents are useful for the treatment of infectious diseases requiring chronic treatments including, but not limited to, tuberculosis, malaria, leishmaniasis, trypanosomiasis (sleeping sickness and Chagas disease), and parasitic worms.

The amount of drug in drug particle formulations is that amount necessary to deliver a therapeutically effective amount of the agent to achieve the desired therapeutic result in the subject to which the drug is being delivered. In practice, this will vary depending upon such variables, for example, as the particular agent, the severity of the condition, and the desired therapeutic effect. Beneficial agents and their dosage unit amounts are known to the prior art in Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., (2005), McGraw Hill; Remington's Pharmaceutical Sciences, 18th Ed., (1995), Mack Publishing Co.; and Martin's Physical Pharmacy and Pharmaceutical Sciences, 1.00 edition (2005), Lippincott Williams & Wilkins. Further, highly concentrated drug particles are described in U.S. Patent Publication No. 2010/0092566. Typically, for an osmotic delivery system, the volume of the chamber comprising the drug formulation is between about 100 μl to about 1000 μl, more preferably between about 140 μl and about 200 μl. In one embodiment, the volume of the chamber comprising the drug formulation is about 150 μl.

Drug particle formulations of the invention are preferably chemically and physically stable for at least 1 month, preferably at least 3 months, more preferably at least 6 months, more preferably at least 12 months at delivery temperature. The delivery temperature is typically normal human body temperature, for example, about 37° C., or slightly higher, for example, about 40° C. Further, drug particle formulations of the present invention are preferably chemically and physically stable for at least 3 months, preferably at least 6 months, more preferably at least 12 months, at storage temperature. Examples of storage temperatures include refrigeration temperature, for example, about 5° C.; or room temperature, for example, about 25° C.

A drug particle formulation may be considered chemically stable if less than about 25%; preferably less than about 20%, more preferably less than about 15%, more preferably less than about 10%, and more preferably less than about 5% breakdown products of the drug particles are formed after about 3 months, preferably after about 6 months, preferably after about 12 months at delivery temperature and after about 6 months, after about 12 months, and preferably after about 24 months at storage temperature.

A drug particle formulation may be considered physically stable if less than about 10%, preferably less than about 5%, more preferably less than about 3%, more preferably less than 1% aggregates of the drug are formed after about 3 months, preferably after about 6 months, at delivery temperature and about 6 months, preferably about 12 months, at storage temperature.

When the drug in the drug particle formulation is a protein, the protein solution is kept in a frozen condition and lyophilized or spray dried to a solid state. Tg (glass transition temperature) may be one factor to consider in achieving stable compositions of protein. While not intending to be bound by any particular theory, the theory of formation of a high Tg amorphous solid to stabilize peptides, polypeptides, or proteins has been utilized in pharmaceutical industry. Generally, if an amorphous solid has a higher Tg, such as 100° C., peptide products will not have mobility when stored at room temp or even at 40° C. because the storage temperature is below the Tg. Calculations using molecular information have shown that if a glass transition temperature is above a storage temperature of 50° C. that there is zero mobility for molecules. Zero mobility of molecules correlates with better stability. Tg is also dependent on the moisture concentration in the product formulation. Generally, the more moisture, the lower the Tg of the composition.

Accordingly, in some aspects of the present invention, excipients with higher Tg may be included in the protein formulation to improve stability, for example, sucrose (Tg=75° C.) and trehalose (Tg=110° C.). Preferably, particle formulations are formable into particles using processes such as spray drying, lyophilization, desiccation, freeze-drying, milling, granulation, ultrasonic drop creation, crystallization, precipitation, or other techniques available in the art for forming particles from a mixture of components. In one embodiment of the invention the particles are spray dried. The particles are preferably substantially uniform in shape and size.

The particles are typically sized such that they can be delivered via an implantable osmotic delivery device. Uniform shape and size of the particles typically helps to provide a consistent and uniform rate of release from such a delivery device; however, a particle preparation having a non-normal particle size distribution profile may also be used. For example, in a typical implantable osmotic delivery device having a delivery orifice, the size of the particles is less than about 30%, more preferably is less than about 20%, more preferably is less than about than 10%, of the diameter of the delivery orifice. In an embodiment of the particle formulation for use with an osmotic delivery system, wherein the delivery orifice diameter of the implant is about 0.5 mm, particle sizes may be, for example, less than about 150 microns to about 50 microns. In an embodiment of the particle formulation for use with an osmotic delivery system, wherein the delivery orifice diameter of the implant is about 0.1 mm, particle sizes may be, for example, less than about 30 microns to about 10 microns. In one embodiment, the orifice is about 0.25 mm (250 microns) and the particle size is about 2 microns to about 5 microns.

Those of ordinary skill in the art will appreciate that a population of particles follow principles of particle size distribution. Widely used, art-recognized methods of describing particle size distributions include, for example, average diameters and D values, such as the D₅₀value, which is commonly used to represent the mean diameter of the range of the particle sizes of a given sample.

Particles of a particle formulation have diameters of between about 2 microns to about 150 micron, e.g., less than 150 microns in diameter, less than 100 microns in diameter, less than 50 microns in diameter, less than 30 microns in diameter, less than 10 microns in diameter, less than 5 microns in diameter, and about 2 microns in diameter. Preferably, particles have diameters of between about 2 microns and about 50 microns.

Particles of a particle formulation comprising an isolated glucagon-specific agonist polypeptide have average diameters of between about 0.3 microns to about 150 microns. Particles of a particle formulation comprising an isolated glucagon-specific agonist polypeptide have average diameters of between about 2 microns to about 150 microns, e.g., less than 150 microns in average diameter, less than 100 microns in average diameter, less than 50 microns in average diameter, less than 30 microns in average diameter, less than 10 microns in average diameter, less than 5 microns in average diameter, and about 2 microns in average diameter. In some embodiments, particles have average diameters of between about 0.3 microns and 50 microns, for example, between about 2 microns and about 50 microns. In some embodiments, the particles have an average diameter between 0.3 microns and 50 microns, for example, between about 2 microns and about 50 microns, where each particle is less than about 50 microns in diameter.

Typically, the particles of the particle formulations, when incorporated in a suspension vehicle, do not settle in less than about 3 months, preferably do not settle in less than about 6 months, more preferably do not settle in less than about 12 months, more preferably do not settle in less than about 24 months at delivery temperature, and most preferably do not settle in less than about 36 months at delivery temperature. The suspension vehicles typically have a viscosity of between about 5,000 to about 30,000 poise, preferably between about 8,000 to about 25,000 poise, more preferably between about 10,000 to about 20,000 poise. In one embodiment, the suspension vehicle has a viscosity of about 15,000 poise, plus or minus about 3,000 poise. Generally speaking, smaller particles tend to have a lower settling rate in viscous suspension vehicles than larger particles. Accordingly, micron- to nano-sized particles are typically desirable. In viscous suspension formulation, particles of about 2 microns to about 7 microns of the present invention will not settle for at least 20 years at room temperature based on simulation modeling studies. In an embodiment of the particle formulation of the present invention, for use in an implantable osmotic delivery device, comprises particles of sizes less than about 50 microns, more preferably less than about 10 microns, more preferably in a range from about 2 microns to about 7 microns.

In one embodiment, a drug particle formulation comprises a drug, as described above, one or more stabilizers, and optionally a buffer. The stabilizers may be, for example, carbohydrate, antioxidant, amino acid, buffer, inorganic compound, or surfactant. The amounts of stabilizers and buffer in the particle formulation can be determined experimentally based on the activities of the stabilizers and buffers and the desired characteristics of the formulation, in view of the teachings of the present specification. Typically, the amount of carbohydrate in the formulation is determined by aggregation concerns. In general, the carbohydrate amount should not be too high so as to avoid promoting crystal growth in the presence of water due to excess carbohydrate unbound to drug. Typically, the amount of antioxidant in the formulation is determined by oxidation concerns, while the amount of amino acid in the formulation is determined by oxidation concerns and/or formability of particles during spray drying. Typically, the amount of buffer in the formulation is determined by pre-processing concerns, stability concerns, and formability of particles during spray drying. Buffer may be required to stabilize drug during processing, e.g., solution preparation and spray drying, when all excipients are solubilized.

Examples of carbohydrates that may be included in the particle formulation include, but are not limited to, monosaccharides (e.g., fructose, maltose, galactose, glucose, D-mannose, and sorbose), disaccharides (e.g., lactose, sucrose, trehalose, and cellobiose), polysaccharides (e.g., raffinose, melezitose, maltodextrins, dextrans, and starches), and alditols (acyclic polyols; e.g., mannitol, xylitol, maltitol, lactitol, xylitol sorbitol, pyranosyl sorbitol, and myoinsitol). Preferred carbohydrates include disaccharides and/or non-reducing sugars, such as sucrose, trehalose, and raffinose.

Examples of amino acids that may be included in the particle formulation include, but are not limited to, arginine, methionine, glycine, histidine, alanine, L-leucine, glutamic acid, iso-leucine, L-threonine, 2-phenylamine, valine, norvaline, praline, phenylalanine, tryptophan, serine, asparagines, cysteine, tyrosine, lysine, and norleucine.

Examples of buffers that may be included in the particle formulation include, but are not limited to, citrate, histidine, succinate, phosphate, maleate, tris, acetate, carbohydrate, and gly-gly.

Examples of inorganic compounds that may be included in the particle formulation include, but are not limited to, NaCl, Na₂SO₄, NaHCO₃, KCl, KH₂PO₄, CaCl₂, and MgCl₂.

In addition, the particle formulation may include other excipients, such as surfactants, and salts. Examples of surfactants include, but are not limited to, Polysorbate 20, Polysorbate 80, PLURONIC® (BASF Corporation, Mount Olive, N.J.) F68, and sodium dodecyl sulfate (SDS). Examples of salts include, but are not limited to, sodium chloride, calcium chloride, and magnesium chloride.

All components included in the particle formulation are typically acceptable for pharmaceutical use in mammals, particularly, in humans.

In summary, a selected drug or combination of drugs is formulated into dried powders in solid state, which preserve maximum chemical and biological stability of the drug. The particle formulation offers long-term storage stability at high temperature, and therefore, allows delivery to a subject of stable and biologically effective drug for extended periods of time.

3.2.0 Vehicle Formulations and Suspension Formulations

In one aspect, the suspension vehicle provides a stable environment in which the drug particle formulation is dispersed. The drug particle formulations are chemically and physically stable (as described above) in the suspension vehicle. The suspension vehicle typically comprises one or more polymer and one or more solvent that form a solution of sufficient viscosity to uniformly suspend the particles comprising the drug. The suspension vehicle may comprise further components, including, but not limited to, surfactants, antioxidants, and/or other compounds soluble in the vehicle.

The viscosity of the suspension vehicle is typically sufficient to prevent the drug particle formulation from settling during storage and use in a method of delivery, for example, in an implantable, osmotic delivery device. The suspension vehicle is biodegradable in that the suspension vehicle disintegrates or breaks down over a period of time in response to a biological environment, while the drug particle is dissolved in the biological environment and the active pharmaceutical ingredient (i.e., the drug) in the particle is absorbed.

In embodiments, the suspension vehicle is a “single-phase” suspension vehicle, which is a solid, semisolid, or liquid homogeneous system that is physically and chemically uniform throughout.

The solvent in which the polymer is dissolved may affect characteristics of the suspension formulation, such as the behavior of drug particle formulation during storage. A solvent may be selected in combination with a polymer so that the resulting suspension vehicle exhibits phase separation upon contact with the aqueous environment. In some embodiments of the invention, the solvent may be selected in combination with the polymer so that the resulting suspension vehicle exhibits phase separation upon contact with the aqueous environment having less than approximately about 10% water.

The solvent may be an acceptable solvent that is not miscible with water. The solvent may also be selected so that the polymer is soluble in the solvent at high concentrations, such as at a polymer concentration of greater than about 30%. Examples of solvents useful in the practice of the present invention include, but are not limited to, lauryl alcohol, benzyl benzoate, benzyl alcohol, lauryl lactate, decanol (also called decyl alcohol), ethyl hexyl lactate, and long chain (C₈to C₂₄) aliphatic alcohols, esters, or mixtures thereof. The solvent used in the suspension vehicle may be “dry,” in that it has a low moisture content. Preferred solvents for use in formulation of the suspension vehicle include lauryl lactate, lauryl alcohol, benzyl benzoate, and mixtures thereof.

Examples of polymers for formulation of the suspension vehicles of the present invention include, but are not limited to, a polyester (e.g., polylactic acid and polylacticpolyglycolic acid), a polymer comprising pyrrolidones (e.g., polyvinylpyrrolidone having a molecular weight ranging from approximately 2,000 to approximately 1,000,000), ester or ether of an unsaturated alcohol (e.g., vinyl acetate), polyoxyethylenepolyoxypropylene block copolymer, or mixtures thereof. Polyvinylpyrrolidone can be characterized by its K-value (e.g., K-17), which is a viscosity index. In one embodiment, the polymer is polyvinylpyrrolidone having a molecular weight of 2,000 to 1,000,000. In a preferred embodiment, the polymer is polyvinylpyrrolidone K-17 (typically having an approximate average molecular weight range of 7,900-10,800). The polymer used in the suspension vehicle may include one or more different polymers or may include different grades of a single polymer. The polymer used in the suspension vehicle may also be dry or have a low moisture content.

Generally speaking, a suspension vehicle for use in the present invention may vary in composition based on the desired performance characteristics. In one embodiment, the suspension vehicle may comprise about 40 wt % to about 80 wt % polymer(s) and about 20 wt % to about 60 wt % solvent(s). Preferred embodiments of a suspension vehicle include vehicles formed of polymer(s) and solvent(s) combined at the following ratios: about 25 wt % solvent and about 75 wt % polymer; about 50 wt % solvent and about 50 wt % polymer; about 75 wt % solvent and about 25 wt % polymer. Accordingly, in some embodiments, the suspension vehicle may comprise selected components and in other embodiments consist essentially of selected components.

The suspension vehicle may exhibit Newtonian behavior. The suspension vehicle is typically formulated to provide a viscosity that maintains a uniform dispersion of the particle formulation for a predetermined period of time. This helps facilitate making a suspension formulation tailored to provide controlled delivery of the drug contained in the drug particle formulation. The viscosity of the suspension vehicle may vary depending on the desired application, the size and type of the particle formulation, and the loading of the particle formulation in the suspension vehicle. The viscosity of the suspension vehicle may be varied by altering the type or relative amount of the solvent or polymer used.

The suspension vehicle may have a viscosity ranging from about 100 poise to about 1,000,000 poise, preferably from about 1,000 poise to about 100,000 poise. In preferred embodiments, the suspension vehicles typically have a viscosity, at 33° C., of between about 5,000 to about 30,000 poise, preferably between about 8,000 to about 25,000 poise, more preferably between about 10,000 to about 20,000 poise. In one embodiment, the suspension vehicle has a viscosity of about 15,000 poise, plus or minus about 3,000 poise, at 33° C. The viscosity may be measured at 33° C., at a shear rate of 10⁻⁴/sec, using a parallel plate rheometer.

The suspension vehicle may exhibit phase separation when contacted with the aqueous environment; however, typically the suspension vehicle exhibits substantially no phase separation as a function of temperature. For example, at a temperature ranging from approximately 0° C. to approximately 70° C. and upon temperature cycling, such as cycling from 4° C. to 37° C. to 4° C., the suspension vehicle typically exhibits no phase separation.

The suspension vehicle may be prepared by combining the polymer and the solvent under dry conditions, such as in a dry box. The polymer and solvent may be combined at an elevated temperature, such as from approximately 40° C. to approximately 70° C., and allowed to liquefy and form the single phase. The ingredients may be blended under vacuum to remove air bubbles produced from the dry ingredients. The ingredients may be combined using a conventional mixer, such as a dual helix blade or similar mixer, set at a speed of approximately 40 rpm. However, higher speeds may also be used to mix the ingredients. Once a liquid solution of the ingredients is achieved, the suspension vehicle may be cooled to room temperature. Differential scanning calorimetry (DSC) may be used to verify that the suspension vehicle is a single phase. Further, the components of the vehicle (e.g., the solvent and/or the polymer) may be treated to substantially reduce or substantially remove peroxides (e.g., by treatment with methionine; see, e.g., U.S., Patent Application Publication No. 2007-0027105).

The drug particle formulation is added to the suspension vehicle to form a suspension formulation. In some embodiments, the suspension formulation may comprise a drug particle formulation and a suspension vehicle and in other embodiments consist essentially of a drug particle formulation and a suspension vehicle.

The suspension formulation may be prepared by dispersing the particle formulation in the suspension vehicle. The suspension vehicle may be heated and the particle formulation added to the suspension vehicle under dry conditions. The ingredients may be mixed under vacuum at an elevated temperature, such as from about 40° C. to about 70° C. The ingredients may be mixed at a sufficient speed, such as from about 40 rpm to about 120 rpm, and for a sufficient amount of time, such as about 15 minutes, to achieve a uniform dispersion of the particle formulation in the suspension vehicle. The mixer may be a dual helix blade or other suitable mixer. The resulting mixture may be removed from the mixer, sealed in a dry container to prevent water from contaminating the suspension formulation, and allowed to cool to room temperature before further use, for example, loading into an implantable, drug delivery device, unit dose container, or multiple-dose container.

The suspension formulation typically has an overall moisture content of less than about 10 wt %, preferably less than about 5 wt %, and more preferably less than about 4 wt %.

In preferred embodiments, the suspension formulations of the present invention are substantially homogeneous and flowable to provide delivery of the drug particle formulation from the osmotic delivery device to the subject.

In summary, the components of the suspension vehicle provide biocompatibility. Components of the suspension vehicle offer suitable chemico-physical properties to form stable suspensions of drug particle formulations. These properties include, but are not limited to, the following: viscosity of the suspension; purity of the vehicle; residual moisture of the vehicle; density of the vehicle; compatibility with the dry powders; compatibility with implantable devices; molecular weight of the polymer; stability of the vehicle; and hydrophobicity and hydrophilicity of the vehicle. These properties can be manipulated and controlled, for example, by variation of the vehicle composition and manipulation of the ratio of components used in the suspension vehicle.

4.0.0 Delivery of Suspension Formulations

The suspension formulations described herein may be used in an implantable, osmotic delivery device to provide zero-order, continuous, controlled, and sustained delivery of a compound over an extended period of time, such as over weeks, months, or up to about one year or more. Such an implantable osmotic delivery device is typically capable of delivering the suspension formulation, comprising the drug, at a desired flow rate over a desired period of time. The suspension formulation may be loaded into the implantable, osmotic delivery device by conventional techniques.

A dose and delivery rate can be selected to achieve a desired blood concentration of a drug generally within less than about 6 half-lives of the drug within the subject after implantation of the device. The blood concentration of the drug is selected to give the optimal therapeutic effects of the drug while avoiding undesirable side effects that may be induced by excess concentration of the drug, while at the same time avoiding peaks and troughs that may induce side effects associated with peak or trough plasma concentrations of the drug.

The implantable, osmotic delivery device typically includes a reservoir having at least one orifice through which the suspension formulation is delivered. The suspension formulation may be stored within the reservoir. In a preferred embodiment, the implantable, drug delivery device is an osmotic delivery device, wherein delivery of the drug is osmotically driven. Some osmotic delivery devices and their component parts have been described, for example, the DUROS® delivery device or similar devices (see, e.g., U.S. Pat. Nos. 5,609,885; 5,728,396; 5,985,305; 5,997,527; 6,113,938; 6,132,420; 6,156,331; 6,217,906; 6,261,584; 6,270,787; 6,287,295; 6,375,978; 6,395,292; 6,508,808; 6,544,252; 6,635,268; 6,682,522; 6,923,800; 6,939,556; 6,976,981; 6,997,922; 7,014,636; 7,207,982; and 7,112,335; 7,163,688; U.S. Patent Publication Nos. 2005/0175701, 2007/0281024, 2008/0091176, and 2009/0202608).

The osmotic delivery device typically consists of a cylindrical reservoir which contains the osmotic engine, piston, and drug formulation. The reservoir is capped at one end by a controlled-rate, semi-permeable membrane and capped at the other end by a diffusion moderator through which suspension formulation, comprising the drug, is released from the drug reservoir. The piston separates the drug formulation from the osmotic engine and utilizes a seal to prevent the water in the osmotic engine compartment from entering the drug reservoir. The diffusion moderator is designed, in conjunction with the drug formulation, to prevent body fluid from entering the drug reservoir through the orifice.

The osmotic device releases a drug at a predetermined rate based on the principle of osmosis. Extracellular fluid enters the osmotic delivery device through a semi-permeable membrane directly into a salt engine that expands to drive the piston at a slow and even delivery rate. Movement of the piston forces the drug formulation to be released through the orifice or exit port at a predetermined shear rate. In one embodiment of the present invention, the reservoir of the osmotic device is loaded with a suspension formulation wherein the device is capable of delivering the suspension formulation to a subject over an extended period of time (e.g., about 1, about 3, about 6, about 9, about 10, and about 12 months) at a pre-determined, therapeutically effective delivery rate.

The release rate of the drug from the osmotic delivery device typically provides a subject with a predetermined target dose of a drug, for example, a therapeutically effective daily dose delivered over the course of a day; that is, the release rate of the drug from the device, provides substantial steady-state delivery of the drug at a therapeutic concentration to the subject.

Typically, for an osmotic delivery device, the volume of a beneficial agent chamber comprising the beneficial agent formulation is between about 100 μl to about 1000 μl, more preferably between about 120 μl and about 500 μl, more preferably between about 150 μl and about 200 μl.

Typically, the osmotic delivery device is implanted within the subject, for example, subdermally or subcutaneously to provide subcutaneous drug delivery. The device(s) can be implanted subdermally or subcutaneously into either or both arms (e.g., in the inside, outside, or back of the upper arm) or the abdomen. Preferred locations in the abdominal area are under the abdominal skin in the area extending below the ribs and above the belt line. To provide a number of locations for implantation of one or more osmotic delivery device within the abdomen, the abdominal wall can be divided into 4 quadrants as follows: the upper right quadrant extending at least 2-3 centimeters below the right ribs, e.g., at least about 5-8 centimeters below the right ribs, and at least 2-3 centimeters to the right of the midline, e.g., at least about 5-8 centimeters to the right of the midline; the lower right quadrant extending at least 2-3 centimeters above the belt line, e.g., at least about 5-8 centimeters above the belt line, and at least 2-3 centimeters to the right of the midline, e.g., at least about 5-8 centimeters to the right of the midline; the upper left quadrant extending at least 2-3 centimeters below the left ribs, e.g., at least about 5-8 centimeters below the left ribs, and at least 2-3 centimeters to the left of the midline, e.g., at least about 5-8 centimeters to the left of the midline; and the lower left quadrant extending at least 2-3 centimeters above the belt line, e.g., at least about 5-8 centimeters above the belt line, and at least 2-3 centimeters to the left of the midline, e.g., at least about 5-8 centimeters to the left of the midline. This provides multiple available locations for implantation of one or more devices on one or more occasions. Implantation and removal of osmotic delivery devices are generally carried out by medical professionals using local anesthesia (e.g., lidocaine).

Termination of treatment by removal of an osmotic delivery device from a subject is straightforward, and provides the important advantage of immediate cessation of delivery of the drug to the subject.

Preferably, the osmotic delivery device has a fail-safe mechanism to prevent an inadvertent excess or bolus delivery of drug in a theoretical situation like the plugging or clogging of the outlet (diffusion moderator) through which the drug formulation is delivered. To prevent an inadvertent excess or bolus delivery of drug the osmotic delivery device is designed and constructed such that the pressure needed to partially or wholly dislodge or expel the diffusion moderator from the reservoir exceeds the pressure needed to partially or wholly dislodge or expel the semi-permeable membrane to the extent necessary to de-pressurize the reservoir. In such a scenario, pressure would build within the device until it would push the semi-permeable membrane at the other end outward, thereby releasing the osmotic pressure. The osmotic delivery device would then become static and no longer deliver the drug formulation provided that the piston is in a sealing relationship with the reservoir.

The suspension formulations may also be used in infusion pumps, for example, the ALZET® (DURECT Corporation, Cupertino, Calif.) osmotic pumps which are miniature, infusion pumps for the continuous dosing of laboratory animals (e.g., mice and rats).

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to practice the present invention, and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, concentrations, and percent changes) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, temperature is in degrees Centigrade and pressure is at or near atmospheric.

The compositions used to practice the methods of the present invention meet the specifications for content and purity required of pharmaceutical products.

Example 1: Generation of Glucagon-Receptor Selective Agonist Polypeptides

Glucagon polypeptides of the invention, as provided in Table 3, were synthesized on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, Ariz.) by solid-phase methods using Fmoc strategy with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate (HBTU) or 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) activation (5 fold molar excess) in N,N-dimethyiformamide (DMF), and N′N-Diisopropylethylamine (DIEA) as base, 20% piperidine/DMF for Fmoc deprotection. The resin was Rink Amide MBHA LL (Novabiochem) or N-α-Fmoc protected, pre-loaded Wang LL (Novabiochem), with loadings of 0.29-0.35 mmol/g on a 20-400 μmol scale. Final deprotection and cleavage of the peptide from the solid support was performed by treatment of the resin with (92.5% TFA, 2.5% phenol, 2.5% water and 2.5% triisopropylsilane) for 2-3 hours. The cleaved peptide was precipitated using cold diethyl ether. The diethyl ether was decanted and the solids triturated again with cold diethyl ether, pelleted by centrifugation and lyophilized. The lyophilized solid was re-dissolved in a 1:1 solution of acetonitrile/water, with 0.1% TFA (10-15 mL), purified via reverse phase HPLC on a Waters XBridge™ BEH 130, CIS, 10 pm, 130 Å, 30×250 mm ID column, using a 30 gradient within the ranges of 5-75% acetonitrile/water with 0.1% TFA over 30-45 minutes at a flow rate of 30 mL/min, λ—215 nm. The purified product was lyophilized and analyzed by ESI-LC/MS and analytical HPLC; and were demonstrated to be pure (>98%). Mass results all agreed with calculated values.

Characterizations of peptide analogs were performed via C18 HPLC and LC/MS analysis (Acquity SQD Waters Corp, Milford, Mass.) and UV detection provided by dual absorbance signals at 215 nm and 280 nm, using one of the three methods A or Method B or Method C.

LC/MS Conditions: Method A: Performed using a Phenomenex UPLC Aeris™ Peptide XB C18 35 column, 1.7 pm, 2.1×100 mm or ACQUiTY BEH300 or BEH130 CT8 column, 1.77 pm. 2.1×100 mm using 5-65% acetonitrile/water with 0.05% TFA over 30 minutes with a flow rate 0.5 mL/min, λ—215 nm, 280 nm.

C18 HPLC Conditions: Method A: UPLC analysis was conducted on an Acquity BEH130, C18 column, 1.7 μm, 100×2.10 mm column at 25° C., 5-65% acetonitrile/water with 0.05% TFA over 30 minutes, flow rate 0.5 mL/min, λ 215 nm, λ 280 nm.

Method B: UPLC analysis was conducted on an Acquity BEH130, C18 column, 1.7 μm, 100×2.10 mm column at 25° C., 5-65% acetonitrile/water with 0.05% TFA over 20 minutes, flow rate 0.5 mL/min, λ 215 nm, λ 280 nm.

Method C: UPLC analysis was conducted on an Acquity BEH130, C18 column, 1.7 μm, 100×2.10 mm column at 25° C., 5-65% acetonitrile/water with 0.05% TFA over 10 minutes, flow rate 0.5 mL/min, λ215 nm, λ 280 nm. 5.0 uL of sample was injected using a PLNO (partial loop w/ needle overfill) injection mode.

Table 3 provides the amino acid sequence and experimental data for the selective glucagon receptor analogs of the disclosure, including the selective glucagon receptor analogs referred to herein as Compound A4 (SEQ ID NO: 44), Compound A5 (SEQ ID NO: 45), Compound A6 (SEQ ID NO: 46), Compound A2 (SEQ ID NO: 42), Compound A1 (SEQ ID NO: 41), Compound A3 (SEQ ID NO: 43), Compound A97 (SEQ ID NO: 143), Compound A98 (SEQ ID NO: 144), Compound A99 (SEQ ID NO: 145), Compound A100 (SEQ ID NO: 146), Compound A101 (SEQ ID NO: 147), Compound A102 (SEQ ID NO: 148), and Compound A97 (SEQ ID NO: 149).

Example 2: Generation of Lactam-Bridged Glucagon-Receptor Selective Agonist Polypeptides

The synthesis, purification and analytical methods were performed as described in Example 1 with following modifications. Glucagon polypeptides of the invention, as provided in Table 3, were synthesized on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, Ariz.)) by solid-phase methods using Fmoc strategy and using Rink Amide MBHA LL (Novabiochem) or N-α-Fmoc protected, pre-loaded Wang LL (Novabiochem), with loadings of 0.29-0.35 mmol/g on a 20-400 μmol scale. Fmoc amino acid (4.0 eq, 1.0 mmol) residues were activated using 4.0 eq HBTU, 4.0 eq of HOBT, 8.0 eq DIEA and coupled to the resin for 1 hour. The Fmoc group was removed by treatment with 20% (v/v) piperidine in dimethylformamide. The side chain protection groups used were Trt for Asn, Gln, Cys and His; t-Bu for Ser, Thr, and Tyr; Boc for Lys and Trp; Ot-Bu for Asp and Glu; and Pbf for Arg.

The Lactam Bridges between positions 17-21 and between positions 24-27 were introduced using an orthogonally protected Lysine (Alloc)17 and Glu (Allyl)21; and Glu(Allyl)24 and Lysine (Alloc)27 is represented by E in parenthesis in the sequence Lysine (Alloc) is represented by K in parenthesis in the sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20.

Synthesis was carried out from C-terminal to N-terminal on the solid support. To incorporate the first lactam bridge (residue 17-residue 21) the synthesis was paused at Phe21. The resin was washed with DCM (6×10 ml) that had been flushed previously with nitrogen for 30 minutes. Next, Palladium Tetrakis, Pd(PPh3)3 (3 equiv) was added to a solution of CHCl3/AcOH/NMM (37:2:1). Nitrogen was bubbled through the solution until all of the solid had dissolved leaving a dark amber solution. This solution was transferred to an amino acid bottle wherein it was degassed for an additional 5 minutes with nitrogen. It was then placed on to the prelude synthesizer. 20 ml of the Palladium solution was added to each RV and the reaction mixture was allowed to agitate for 3 hours. Synthesis was continued using standard protocol. To incorporate the second lactam bridge (residue 24-residue 28) the synthesis was paused at Leu14 and the above procedure for lactam bride formation was repeated.

Before cleaving peptide from resin the allyl protecting groups were removed using Pd(PPh3)₃in CHCl₃/AcOH/NMM (37:2:1). Cyclization to the lactam was effected using Pybop (6 equivs), HOBT (6 equivs), and DIEA (12 equivs). The resin was then washed with DMF and dried under vacuum for 15 minutes under nitrogen.

Final deprotection and cleavage of the peptide from the solid support was performed by treatment of the resin with reagent B (92.5% TFA, 2.5% phenol, 2.5% water and 2.5% triisopropylsilane) for 2-3 hours. The cleaved peptide was precipitated using cold diethyl ether, the diethyl ether was decanted and the solids triturated again with cold diethyl ether, pelleted by centrifugation and lyophilized. The pellet was re-dissolved in water (10-15 mL), filtered purified by reverse phase HPLC on a Waters XBridge™ BEH 130, CIS, 10 pm, 130 Å, 30×250 mm ID column, using 30 a gradient within the ranges of 5-75% acetonitrile/water with 0.1% TFA over 30-45 minutes at a flow rate of 30 mL/min, λ—215 nm. Alternatively, purification was afforded using a Gilson Preparative HPLC System via reverse phase chromatography using a Waters XBridge BEH 130, C18, 10 μm, 130 Å, 30×250 mm ID column, with a gradient range of 5-45% acetonitrile/water with 0.1% TFA over 30-45 minutes at a flow rate of 30 mL/min, λ 215 nm. The purified product was lyophilized and analyzed by ESI-LC/MS and analytical HPLC; and are demonstrated to be pure (>98%). Mass results all agreed with calculated values.

The chemical structure of Compound A104 is shown in FIG. 4A, and the chemical structure of Compound A105 is shown in FIG. 4B.

Example 3: Stability and Solubility of Glucagon-Receptor Selective Agonist Polypeptides

Glucagon and the analogs described herein, e.g. Compound A98, Compound A99, Compound A100, Compound A101, Compound A102, Compound A2, Compound A1, Compound A5, Compound A6, Compound A3, Compound A44, and Compound A97, were tested for stability in saline 5% NaCl in water (i.e., bioassay buffer) or in aqueous (i.e., in water). Glucagon and these analogs were incubated at 37° C. and at room temperature. Samples were withdrawn at a regular interval for 30 days and analyzed by LC/MS and HPLC for determination of purity and mass of the degradation product. The results of this analysis are shown in Table 3.

Glucagon and the analogs described herein, e.g. Compound A98, Compound A99, Compound A100, Compound A101, Compound A102, Compound A2, Compound A1, Compound A5, Compound A6, Compound A3, Compound A44, and Compound A97 were tested for solubility in saline 5% NaCl in water (i.e., bioassay buffer) or in aqueous at room temperature. Samples were visually inspected for clarity of the sample, any appearance of turbidity or haziness. The results of this analysis are shown in Table 3.

Example 4: In Vitro Screening of Analogs, Peptide-Receptor Mediated cAMP Accumulation

CMV promoter-driven plasmids encoding human GCGR (NM_000160) and human GLP-1R (NM_002062) were transfected into CHO-K1 cells for 36 hours using Lipofectamine 2000. Following transfection, cells were removed from the flask using cell disruption media and dispensed in white 384-well plates at 1000 cells per well in 5 uL of stimulation buffer. Peptide-receptor activity was determined using the LANCE Ultra cAMP detection kit according to the manufacturer's protocol (Perkin Elmer). Peptides were serially diluted in stimulation buffer and 5 uL of each peptide dilution was added to cells and incubated for thirty minutes at room temperature. For results shown in Table 2A, tested concentrations ranged from 1 nM to 15 fM in the GCGR and GLP-1R assays. For results shown in Table 2B, tested concentrations ranged from 1 uM to 0.1 fM for GCGR, and 500 uM to 10 fM for GLP-1R assays. Following peptide incubation, 5 uL Europium labelled cAMP and 5 uL Ulight™ anti-cAMP antibody were added to wells and incubated for an additional 60 minutes. Plates were read on an Envision® fluorescent plate reader and data was analyzed using GraphPad Prism®. Potency was determined from baseline-corrected fit curves using the formula: Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)) with the Hill Slope constrained to −1.0.

TABLE 2A

hGCGR pEC50
hGLP1 pEC50

(cAMP) mean
(cAMP) mean

(n = 9): 11
(n = 9): 11

point curve
point curve

Compound
SEQ ID NO:
starting at 1 uM
starting at 500 uM

Glucagon
141
10
9.9

A4
44
11.8
<5

A5
45
11.8
5.8

A6
46
11.7
5.3

A2
42
11.7
5.4

A1
41
12.3
5.2

A3
43
12.1
6.7

TABLE 2B

hGCGR pEC50
hGLP1 pEC50

(cAMP) mean
(cAMP) mean

(n = 3): 11
(n = 3): 11

Compound

point curve
point curve

ID#
SEQ ID NO:
starting at 1 nM
starting at 1 nM

A97
143
12.3
<9

A98
144
12.2
<9

A99
145
12.5
<9

A100
146
12.6
<9

A101
147
11.3
<9

A102
148
12.4
<9

A103
149
11.9
<9

In some embodiments, provided is an isolated polypeptide of the disclosure corresponding to Compound A97, A98, A99, A100, A101, A102 or A103.

Peptide-receptor activation profiles at the human glucagon receptor (GCGR) and the human GLP-1 receptor (GLP-1R) by human glucagon, GLP-1 (7-37), and two glucagon receptor selective peptide agonists. FIG. 1A demonstrates that peptides Compound A2, Compound A1 and glucagon are nearly equipotent on GCGR, while GLP-1 (7-37) activates GCGR at a much lower potency. FIG. 1B demonstrates that, in contrast to the results shown FIG. 1A, peptides Compound A2 and Compound A1 are inactive on GLP-1R demonstrating that peptides Compound A2 and Compound A1 profile as GCGR selective agonists. Data of all the analogs are recorded in Table 3.

Sequences of synthesized glucagon receptor agonist peptides and their pEC50 values determined in cAMP assays are shown below in Table 3.

TABLE 3

hGCGR
hGLP1

pEC50
pEC50

(cAMP)
(cAMP)

mean
mean

(n =
(n =

3): 11
3): 11

Sta-

point
point

Calc.

bility

curve
curve

Mass
Ob-

Sol-
1 mg/

start-
start-

Calc.
(Parent
served

ubility
ml in

ing
ing
CL

SEQ
Mass
MW,
Mass

in
water at

Com-
at
at
(ml/

ID
(Parent
M +
(M +

water
37 deg

pound
1 nM
1 nM
min/kg)
Sequence
NO:
MW)
3/3))
3/3)
Purity
mg/ml
C.

Glu-
10
9.9
can't
HSQGTFTSDYSKYLDSRRAQDFVQWL
140
3482.76
1161.92
1162.2
>95%
0.03-10
Aggre-

cagon

measure
MNT-OH

ug/ml
gates

due to

insol-

ubility

A1
12.3
5.2
11.5
YSHGTFTSDYSKYLD(Aib)KYAQEF
41
4270.59
1424.5
1425.5
>90%
>200
>71%

V(Aib)WLEDEPSSGAPPPS-OH

A2
11.7
5.4
10.8
YSHGTFTSDYSKYLD(Aib)KSAQEF
42
4194.49
1399.16
1400.1
>90%
>200
>84%

V(Aib)WLEDEPSSGAPPPS-OH

A3
12.1
6.7
10.7
YSQGTFTSDYSKYLDAARAQEFVKWL
43
4353.78
1452.258
1453.4
>95%
>200
>64%

EDEPKSKSPPPS-NH₂

A4
11.8
<5
12.6
YSHGTFTSDYSKYLD(Aib)KRAQEF
44
4291.66
1431.55
1432.4
>98%
>200
>81%

VIWLEDEPSSGAPPPS-OH

A5
11.8
5.8
11.7
YSHGTFTSDYSKYLDSARAQEFVKWL
45
4250.57
1417.83
1418.9
>98%
>200
>94%

EDEPSSGAPPPS-NH₂

A6
11.7
5.3
12.4
WSQGTFTSDYSKYLD(Aib)KRAQEF
46
4277.63
1426.88
1427.5
>98%
>200
>80%

V(Aib)WLEDEPSSGAPPPS-OH

A7
11.9
<9
28.5
YSHGTFTSDYSKYLD(Aib)ARAQEF
47
4206.5
1403.17
1403.8
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A8
12.3
<9
17
YSHGTFTSDYSKYLD(Aib)KRAQEF
48
4262.6
1421.87
1423
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-NH₂

A9
13.0
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
49
4847.6
1426.87
1427
>98%
—
—

V(Aib)WLEEEPSSGAPPPS-OH

A10
11.3
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
50
4962.7
1465.23
1465.7
>98%
—
—

V(Aib)WLEDEEPSSGAPPPS-OH

A11
11.2
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
51
5091.8
1508.27
1509.6
>98%
—
—

V(Aib)WLEDEEEPSSGAPPPS-OH

A12
13.2
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
52
4562.3
1331.77
1332.5
>98%
—
—

V(Aib)WLEDEGAPPPS-OH

A13
12.3
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
53
4649.4
1360.8
1361.6
>98%
—
—

V(Aib)WLEDESGAPPPS-OH

A14
12.0
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
54
4736.5
1389.83
1390.6
>98%
—
—

V(Aib)WLEDESSGAPPPS-OH

A15
11.4
<9
—
YSHGTFTSDYSKYLD(Aib)SRAQEF
55
4222.5
1408.5
1409.4
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A16
11.9
<9
—
YSHGTFTSDYSKYLD(Aib)TRAQEF
56
4236.5
1413.17
1413.5
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A17
11.1
<9
—
YSHGTFTSDYSKYLD(Aib)ERAQEF
57
4264.5
1422.5
1422.9
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A18
12.4
<9
—
YSHGTFTSDYSKWLD(Aib)ARAQEF
58
4229.5
1410.83
1411.7
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A19
11.0
<9
—
YSHGTFTSDYSKWLD(Aib)SRAQEF
59
4245.5
1409.17
1417
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A20
13.3
<9
15
YSHGTFTSDYSKYLD(Aib)KRAQEF
60
4320.7
1441.23
1442.2
>98%
—
—

V(Aib)WLEDEPSSGKPPPS-OH

A21
11.1
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
61
4321.6
1441.53
1442.9
>98%
—
—

V(Aib)WLEDEPSSGEPPPS-OH

A22
11.0
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
62
4279.6
1427.53
1428.4
>98%
—
—

V(Aib)WLEDEPSSGSPPPS-OH

A23
11.2
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
63
4334.7
1445.9
1447.1
>98%
—
—

V(Aib)WLEDEPSSKAPPPS-OH

A24
12.2
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
64
4391.7
1464.9
1465.2
>98%
—
—

V(Aib)WLEDEPSSKGAPPPS-OH

A25
11.3
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
65
4350.7
1451.2
1451.8
>98%
—
—

V(Aib)WLEDEPSSGAPPPSS-OH

A26
11.1
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
66
4392.72
1465.23
1465.9
>98%
—
—

V(Aib)WLEDEPSSGAPPPSE-OH

A27
12.1
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
67
4377.71
1460.23
1461.4
>98%
—
—

V(Aib)WLEDEGGPSSGAPPPS-OH

A28
12.2
<9
34.7
YSHGTFTSDYSKYLD(Aib)KRAQEF
68
4320.66
1441.23
1441.6
>98%
—
—

V(Aib)WLEDEGPSSGAPPPS-OH

A29
11.1
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
69
4304.70
1435.9
1436.4
>98%
—
—

V(Aib)WLEDEPKSGAPPPS-OH

A30
10.9
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
70
4304.70
1435.9
1436.6
>98%
—
—

V(Aib)WLEDEPSKGAPPPS-OH

A31
11.7
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
71
4305.64
1436.2
1436.4
>98%
—
—

V(Aib)WLEDEPESGAPPPS-OH

A32
12.5
<9
—
YTHGTFTSDYSKWLD(Aib)KRAQEF
72
4300.67
1434.56
1435.3
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A33
10.9
<9
—
YSHGTFTSDHSKWLD(Aib)KRAQEF
73
4260.61
1421.2
1422.8
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A34
10.3
<9
—
YTHGTFTSDHSKWLD(Aib)KRAQEF
74
4274.64
1425.88
1426.9
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A35
13.2
<9
30.1
YTHGTFTSDYSKWLD(Aib)ARAQEF
75
4243.57
1415.52
1416.3
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A36
10.2
<9
—
YSHGTFTSDHSKWLD(Aib)ARAQEF
76
4203.51
1402.17
1402.9
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A37
13.0
<9
—
YSHGTFTSDYSKYLDSARAQEFVKWL
77
4251.55
1418.17
1419.3
>98%
—
—

EDEPSSGAPPPS-OH

A38
13.7
<9
—
YSHGTFTSDYSKWLDSARAQEFVKWL
78
4274.59
1425.83
1426.9
>98%
—
—

EDEPSSGAPPPS-OH

A39
9.4
<9
—
YTHGTFTSDYSKWLDSARAQEFVKWL
79
4288.62
1430.53
1431.9
>98%
—
—

EDEPSSGAPPPS-OH

A40
10.1
<9
—
YSHGTFTSDHSKWLDSARAQEFVKWL
80
4248.55
1417.17
1418.1
>98%
—
—

EDEPSSGAPPPS-OH

A41
9.2
<9
—
YTHGTFTSDHSKWLDEARAQEFVKWL
81
4304.62
1435.87
1436.6
>98%
—
—

EDEPSSGAPPPS-OH

A42
9.4
<9
—
YTHGTFTSDYSKWLDSKRAQEFVKWL
82
4345.71
1449.57
1450.8
>98%
—
—

EDEPSSGAPPPS-OH

A43
11.8
<9
47
YSHGTFTSDYSKYLDKARAQEFVKWL
83
4292.65
1431.88
1433
>98%
—
—

EDEPSSGAPPPS-OH

A44
11.9
<9
23.9
YSHGTFTSDYSKYLDQARAQEFVKWL
84
4292.60
1431.87
1433
>98%
—
—

EDEPSSGAPPPS-OH

A45
11.7
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
85
4374.85
1459.27
1460.9
>90%
—
—

V(Aib)WLEDEKRNKPPPA-OH

A46
12.5
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
86
4390.89
1464.63
1465.4
>90%
—
—

V(Aib)WLEDEKRNKPPIA-OH

A47
11.5
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
87
4407.83
1470.28
1471.5
>98%
—
—

V(Aib)WLEDEKRNKNPPS-OH

A48
12.9
<9
37
YSHGTFTSDYSKYLD(Aib)KRAQEF
88
4504.95
1502.63
1503.1
>98%
—
—

V(Aib)WLEDEKRNKNPPPS-OH

A49
12.4
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
89
4490.88
1497.96
1498.8
>98%
—
—

V(Aib)WLEDEPRNKNNPPS-OH

A50
11.5
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
90
4390.84
1464.6
1464.8
>98%
—
—

V(Aib)WLEDEKRNKPPPS-OH

A51
12.3
<9
—
YSHGTFTSDYSKYLDLKRAQEFV
91
4291.66
1431.55
1432.55
>98%
—
—

(Aib)WLEDEPSSGAPPPS-OH

A52
12.4
<9
29
YSHGTFTSDYSKYLDIKRAQEFV
92
4291.66
1431.55
1432.4
>98%
—
—

(Aib)WLEDEPSSGAPPPS-OH

A53
11.6
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
93
4291.66
1431.55
1432.4
>98%
—
—

VLWLEDEPSSGAPPPS-OH

A54
12.4
<9
24
YSHGTFTSDYSKYLD(Aib)KRAQEF
94
4392.8
1465.27
1465.5
>95%
—
—

V(Aib)WLEDEPSSKEPPPS-OH

A55
12.2
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
95
4350.7
1451.23
1465.7
>95%
—
—

V(Aib)WLEDEPSSKSPPPS-OH

A56
12.3
<9
39.9
YSHGTFTSDYSKYLD(Aib)KRAQEF
96
4375.8
1459.6
1460.6
>95%
—
—

V(Aib)WLEDEPKSKAPPPS-OH

A57
12.0
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
97
4432.9
1478.63
1479.7
>95%
—
—

V(Aib)WLEDEPKSKQPPPS-OH

A58
12.2
<9
37.7
YSHGTFTSDYSKYLD(Aib)KRAQEF
98
4391.8
1464.94
1465.7
>95%
—
—

V(Aib)WLEDEPKSKSPPPS-OH

A59
12.2
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
99
4391.8
1464.93
1465.5
>95%
—
—

V(Aib)WLEDEPSSKQPPPS-OH

A60
11.3
<9
—
YTHGTFTSDYSKYLD(Aib)KRAQEF
100
4334.73
1445.9
1447.1
>98%
—
—

V(Aib)WLEDEPSSGKPPPS-OH

A61
11.4
<9
29.4
YSHGTFTSDYSKYLDSARAQEFVKWL
101
4308.65
1437.23
1437.9
>98%
—
—

EDEPSSGKPPPS-OH

A62
11.5
<9
—
YTHGTFTSDYSKYLD(Aib)KRAQEF
102
4277.63
1426.87
1427.4
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A63
10.4
<9
—
YTHGTFTSDYSKYLDSARAQEFVKWL
103
4265.58
1422.87
1424.1
>98%
—
—

EDEPSSGAPPPS-OH

A64
10.7
<9
—
YTHGTFTSDYSKYLD(Aib)ARAQEF
104
4220.54
1407.83
1408.6
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A65
12.5
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
105
4432.88
1478.6
1479.8
>98%
—
—

V(Aib)WLEDEPKSKEPPPS-NH₂

A66
12.5
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
106
4390.84
1464.6
1465
>98%
—
—

V(Aib)WLEDEPKSKSPPPS-NH₂

A67
10.9
<9
—
YSHGTFTSDYSKYLD(Aib)ARAQEF
107
4333.74
1445.57
1446.4
>98%
—
—

V(Aib)WLEDEPKSKSPPPS-NH₂

A68
11.3
<9
—
YSHGTFTSDYSKYLD(Aib)ARAQEF
108
4375.78
1459.57
1460.6
>98%
—
—

V(Aib)WLEDEPKSKEPPPS-NH₂

A69
11.8
<9
—
YSHGTFTSDYSKYLDSARAQEFVKWL
109
4421.81
1474.94
1476.2
>98%
—
—

EDEPKSKEPPPS-OH

A70
12.0
<9
20.9
YSHGTFTSDYSKYLDSARAQEFVKWL
110
4379.77
1460.92
1461.4
>98%
—
—

EDEPKSKSPPPS-OH

A71
11.7
<9
—
YSHGTFTSDYSKYLDSARAQEFVKWL
111
4363.77
1455.59
1456.6
>98%
—
—

EDEPKSKAPPPS-OH

A72
12.1
<9
31.3
YSHGTFTSDYSKYLDSARAQEFVKWL
112
4420.82
1474.61
1475.1
>98%
—
—

EDEPKSKEPPPS-NH₂

A73
11.7
<9
23
YSHGTFTSDYSKYLDSARAQEFVKWL
113
4378.79
1460.6
1461.2
>98%
—
—

EDEPKSKSPPPS-NH₂

A74
11.6
<9
—
YSHGTFTSDYSKYLDSARAQEFVKWL
114
4362.79
1455.26
1456.3
>98%
—
—

EDEPKSKAPPPS-NH₂

A75
11.3
<9
—
WSHGTFTSDYSKYLD(Aib)KRAQEF
115
4286.6
1429.88
1430.8
>90%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A76
12.7
<9
15.1
YSHGTFTSDYSKYLD(Aib)KAAQEF
116
4178.50
1393.83
1394.8
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A77
11.5
<9
—
YSHGTFTSDYSKYLD(Aib)KTAQEF
117
4208.52
1403.83
1404.8
>90%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A78
10.8
<9
—
YSHGTFTSDYSKYLD(Aib)KLAQEF
118
4220.58
1407.83
1408.6
>90%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A79
10.2
<9
—
YSHGTFTSDYSKYLD(Aib)KEAQEF
119
4236.53
1413.17
1416.3
>90%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A80
12.5
<9
—
YSHGTFTSDYSKYLDAARAQEFVKWL
120
4362.79
1455.262
1456.2
>95%
—
—

EDEPKSKSPPPS-NH₂

A81
11.0
<9
—
YSQGTFTSDYSKYLDSARAQEFVKWL
121
4370.76
1457.9
1458.4
>95%
—
—

EDEPKSKSPPPS-OH

A82
11.0
<9
—
YSQGTFTSDYSKYLDSARAQEFVKWL
122
4354.76
1452.57
1453.7
>95%
—
—

EDEPKSKAPPPS-OH

A83
10.2
<9
—
YSHGTFTSDYSKYLDSARAQEFTKWL
123
4381.74
1461.57
1462.4
>95%
—
—

EDEPKSKSPPPS-OH

A84
9.4
<9
—
YSHGTFTSDYSKYLDSARAQEFVKHL
124
4330.70
1444.57
1445.3
>95%
—
—

EDEPKSKSPPPS-OH

A85
13.6
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
125
4319.72
1441.23
1441.3
>98%
—
—

V(Aib)WLEDEPSSGKPPPS-NH₂

A86
13.3
<9
14.7
YSQGTFTSDYSKYLD(Aib)KRAQEF
126
4311.69
1438.2
1439.1
>98%
—
—

V(Aib)WLEDEPSSGKPPPS-OH

A87
13.6
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
127
4351.76
1451.57
1452.2
>95%
—
—

V(Aib)WLEDEKSSGKPPPS-OH

A88
13.8
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
128
4351.76
1451.57
1451.7
>95%
—
—

V(Aib)WLEDEPSSGKKPPS-OH

A89
12.7
<9
—
YSHTFTSDYSKYLD(Aib)KRAQEF
129
4271.63
1424.87
1426.1
>98%
—
—

V(Aib)HLEDEPSSGKPPPS-OH

A90
13.1
<9
—
YSHGTFTSDYSKYLD(Aib)KAAQEF
130
4235.59
1412.83
1413.8
>98%
—
—

V(Aib)WLEDEPSSGKPPPS-OH

A91
12.8
<9
13.7
WSQGTFTSDYSKYLD(Aib)KRAQEF
131
4334.73
1445.9
1446.7
>98%
—
—

V(Aib)WLEDEPSSGKPPPS-OH

A92
10.7
<9
—
WSQGTFTSDYSKYLD(Aib)KAAQEF
132
4192.52
1398.5
1399.3
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A93
10.9
<9
—
WSQGTFTSDYSKYLD(Aib)KAAQEF
133
4249.62
1417.53
1418.7
>98%
—
—

V(Aib)WLEDEPSSGKPPPS-OH

A94
14.1
<9
16.5
YSQGTFTSDYSKYLD(Aib)KRAQEF
134
4254.59
1419.17
1420.6
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A95
13.3
<9
15.4
YSQGTFTSDYSKYLD(Aib)KAAQEF
135
4169.48
1390.8
1391.5
>98%
—
—

V(Aib)WLEDEPSSGAPPPS-OH

A96
13.0
<9
—
YSQGTFTSDYSKYLD(Aib)KAAQEF
136
4226.58
1409.83
1410.4
>98%
—
—

V(Aib)WLEDEPSSGKPPPS-OH

A97
12.3
<9
18.9
YSHGTFTSDYSKYLDAARAQEFVKWL
143
4235.55
1412.87
1413.7
>98%
>200
>59

EDEPSSGAPPPS-OH

A98
12.2
<9
—
YSHGTFTSDYTRLLESKRAQEFVKWL
144
4314.7
1439.23
1439.6
91%
>200
>75

EDEPSSGAPPPS-OH

A99
12.5
<9
23
YSHGTFTSDYSKYLD(Aib)KRAQEF
145
4263.6
1422.2
1423
>98%
>200
>64

V(Aib)WLEDEPSSGAPPPS-OH

A100
12.6
<9
—
YSHGTFTSDYSKYLD(Aib)KRAQEF
146
3485.8
1162.92
1163.9
>98%
>200
>69

V(Aib)WLEDE-OH

A101
11.3
<9
—
YGHGTFTSDHSKYLD(Aib)KRAQEF
147
3472.8
1158.59
1159.8
>98%
>200
>50

VKWLEDE-OH

A102
12.4
<9
—
YSHGTFTSDYSKWLD(Aib)KRAQEF
148
3551.9
1184.96
1185.4
>98%
>200
>51

VKWLEDE-OH

A103
11.9
<9
28.5
YSHGTFTSDYSKYLD(Aib)ARAQEF
149
4206.5
1403.17
1403.8
>98%
>200
—

V(Aib)WLEDEPSSGAPPPS-OH

Example 5: Intravenous Infusion: Pharmacokinetic Studies to Assess Peptide Clearance from Kidney (CL)

Peptides were formulated in sterile saline and administered as a 3-hour intravenous infusion to non-fasted male Wistar Han or Sprague-Dawley rats (n=3 per group) via jugular vein cannula at a final dose of 0.3 or 0.1 mg/kg. Formulations were administered at a rate of 1.67 mL/kg/h. Blood samples (approximately 250 uL) were collected for pharmacokinetic analysis via a femoral vein cannula at 1, 2, 3, 3.17, 3.33, 3.5, 4, 4.5, 5 and 6 h post-start of infusion into microtainer tubes containing K2EDTA as anticoagulant and 25 uL of a protease inhibitor cocktail. Plasma was prepared by centrifugation and stored at −80° C. until analysis.

Example 6: Subcutaneous Infusion: Pharmacokinetic Studies to Assess Peptide Clearance from Kidney (CL)

Peptides were formulated in sterile saline and administered as a 3-hour subcutaneous infusion to non-fasted male Wistar Han or Sprague-Dawley rats (n=3 per group) at a final dose of 0.3 or 0.1 mg/kg via a cannula placed into the subcutaneous space between the scapulae. Formulations were administered at a rate of 0.145 mL/kg/h. Blood samples (approximately 250 uL) were collected for pharmacokinetic analysis via a jugular vein cannula at 1, 2, 3, 3.17, 3.33, 3.5, 4, 4.5, 5 and 6 h post-start of infusion into microtainer tubes containing K2EDTA as anticoagulant and 25 uL of a protease inhibitor cocktail. Plasma was prepared by centrifugation and stored at −80° C. until analysis. The results of this analysis are shown below in Table 3 and in FIGS. 2A and 2B.

Example 7: Intravenous Bolus Injection: Pharmacokinetic Studies to Assess Peptide Clearance from Kidney (CL) Cyclic Peptide Glucagon Analogs

Peptides were administered as a single intravenous bolus dose to non-fasted male Wistar Han rats (n=3 per group) via jugular vein cannula. Compounds were formulated as solutions in either sterile saline, acidified saline (pH 2.0 or 4.5), or 5% DMSO in water and administered at a volume of 1.5 mL/kg and a final dose of 0.1 mg/kg. Blood samples (approximately 250 uL) were collected for pharmacokinetic analysis via a femoral vein cannula at 0.083, 0.167, 0.25, 0.33, 0.5, 1, 2, 4, 8, 12 and 24 h post-dose into microtainer tubes containing K2EDTA as anticoagulant and 25 uL of a protease inhibitor cocktail. Plasma was prepared by centrifugation and stored at −80° C. until analysis. The results of this analysis are shown below in Table 4.

TABLE 4

IV Bolus Clearance from Kidney (CL)

Compound
IV Bolus Clearance Kidney (CL)

Glucagon
94.1 ± 16.1

Compound A104
80.7 ± 15.4

Compound A105
67.0 ± 7.3

In some embodiments, provided is an isolated polypeptide of the disclosure corresponding to Compound A104 or A105.

Example 8: Generic Method of Plasma Sample Preparation for Pharmacokinetic Studies

Protein Precipitation: All 96-well plates were coated with a blocking agent to mitigate non-specific binding of peptides. Plasma samples (75 uL) were added to a 96-well plate containing 200 uL of 2:1 ethanol:acetonitrile containing 0.1% TFA and mixed well via aspiration. Plates were capped, vortex-mixed and centrifuged. Supernatants (215 uL) were transferred to a clean 96-well plate, evaporated to dryness under nitrogen flow and then reconstituted in 75 μL of 20% acetonitrile in water containing 0.1% formic acid.

Solid-Phase Extraction: Samples were diluted 3-fold with 5% NH₄OH (aq) and loaded onto an Oasis MAX microElution plate (Waters Corp, Milford, Mass.) that had been pre-conditioned with 200 uL each of methanol and 5% NH₄OH (aq). The plate was washed with 200 uL 5% NH₄OH (aq), followed by 200 uL 20% acetonitrile in water. Peptides were eluted using 200 uL 5% formic acid in methanol and evaporated under nitrogen flow. Samples were reconstituted in 80 uL 0.1% formic acid in water.

Example 9: LC/MS Quantification of Peptides in Plasma

All calibration standards were prepared in control rat plasma containing K2EDTA and protease inhibitor cocktail. Samples and standards were analyzed by TurbolonSpray™ UPLC-MS/MS using a system consisting of a CTC HTS PAL auto-injector (Leap, Carrboro, N.C.), an Agilent Infinity 1290 system with column oven (Palo Alto, Calif.), a Valco switching valve (Houston, Tex.), and either an AB Sciex API 5600 TripleTOF™ or Sciex API 4000QTrap mass spectrometer (Framingham, Mass.). Samples were injected onto a 2.1×50 mm reverse phase C18 analytical column, typically a Waters ACQUITY UPLC™ HSS T3, 1.8 μm (Waters Corporation, Milford, Mass.) or similar. Chromatographic separation was achieved with a gradient method using water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) as mobile phase. Initial conditions consisted of 95% A and 5% B. The organic component was increased linearly to 95% B over a period of 3-4 minutes, depending on the peptide. Typical flow rates were 600 μL/min. The column temperature was held constant at 40 or 45° C. Peptides were quantified my monitoring one or more product ions produced from a multiply charged parent ion.

Example 10: In Vivo Efficacy of Glucagon Analogs with Reduction in Body Weight

Chronic (13 days) in vivo efficacy studies were conducted in a rodent model for obesity (diet-induced obese (DIO) Long Evans rat) to investigate the efficacy and durability of examples 11 and 12 singly and in combination with exendin-4 as anti-obesity agents.

Male Diet-Induced Obese (DIO) Long Evans (LE) rats were used (Harlan Laboratories, Inc., Indianapolis, Ind.) and beginning at weaning (about 3 weeks of age), the rats were fed a high fat chow (Teklad TD 95217, 40% kcal from fat, Harlan Laboratories, Madison, Wis.). Rats were 15-17 weeks old at the start of the study. The rats were housed 1 per cage and given ad libitum access to high fat diet (Harlan TD.95217, 4.3 kcal/g) and water, maintained on a 12 hr light/dark cycle from 5:00 AM to 5:00 PM at 21° C. and 50% relative humidity and allowed to acclimate for at least 10 days prior to the surgeries. Baseline fat mass and non-fat mass measurements were taken 3 days before the start of peptide infusion using a QMR instrument (Echo Medical Systems, Houston, Tex.). Body weight measurements were taken 2 times/week starting three days before the surgery. Rats were randomized according to their percent body fat mass and/or body weight into the various treatment groups (n=4-6 rats/group). Alzet mini-osmotic pumps (2 week; Model 2002, Durect Corporation, Cupertino, Calif.) were filled under sterile condition with either vehicle or peptide one day prior to the surgery. On the day of surgery, rats were anesthetized under isoflurane and the dorsal skin surface was shaved and cleansed. Rats were injected SC with Flunexin (2.5 mg/kg). A 1-2 cm surgical incision was made between the scapulae. Using blunt dissection, a 2-3 cm subcutaneous tunnel was created into which the sterile, filled, mini-osmotic pump was introduced. The skin opening was closed with a skin staple. Each rat was implanted with either one or two osmotic pumps containing vehicle or peptide according to their treatment group. All the data are presented as mean±SEM. The data were analyzed in Excel and/or Prism (GraphPad Software, Inc., La Jolla, Calif.) using one-way ANOVA to compare each group to the appropriate control group. P-values <0.05 were considered to indicate a significant difference between treatment groups.

Animals were housed and maintained in an AAALAC, international accredited care and use programs. All procedures were performed in compliance with the Animal Welfare Act, USDA regulations and approved by either the GlaxoSmithKline or the Mispro Institutional Animal Care and Use Committees.

Example 11: Efficacy: Body Weight Change in DIO Rats after 13 Days

In DIO LE rats, continuous dosing of Compound A2 and Compound A1 led to dose-dependent decreases in body weight after 13 days. Significant efficacy of 3.7, 3.9, 8.6 and 23.7% weight loss was achieved at the 0.01, 0.03, 0.1 and 0.3 mg/kg/day doses of Compound A2 when compared to vehicle control (p<0.05), respectively (FIG. 3B). Whereas, Compound A1 achieved weight loss of 5.6, 3.5, 10 and 22.6% at the 0.01, 0.03, 0.1 and 0.3 mg/kg/day doses when compared to vehicle control (p<0.05), respectively (FIG. 3C).

Example 12. Efficacy of Compound A104 Singly or in Combination with Exendin-4 in DIO LE Rats

Continuous dosing of Compound A104 led to dose-dependent decreases in body weight after 13 days of dosing in DIO LE rats. Significant efficacy of 4.2%, 16.5% and 25.2% weight loss was achieved at the 0.1, 0.3 and 1.0 mg/kg/day doses of Compound A104 when compared to vehicle control (p<0.05), respectively (FIG. 3A, left panel). The cyclic peptide glucagon analogs were then used in combination with Exendin-4, a polypeptide comprising the amino acid sequence H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂(SEQ ID NO: 142). In combination with Exendin-4 at 0.01 mg/kg/d (2.5% weight loss singly), doses of 0.03, 0.1, 0.3 and 1 mg/kg/d Compound A104 achieved 6.7, 11.7, 24.3 and 29.7% weight loss vs. vehicle control (p<0.05) after 13 days in DIO LE rats (FIG. 3A, right panel).

Example 13. Steady State Plasma Concentrations and Intravenous Infusion Rates for Exenatide and GLP-1

Methods: With reference to the graph of FIG. 5, male Sprague-Dawley rats (Harlan, Indianapolis, Ind.) weighing 350-370 g, had ad libitum access to water and ad libitum access to food (Diet LM-485, Teklad, Madison, Wis.) until 18 hours prior to experimentation. Animals were anesthetized with halothane during catheterization of right saphenous vein (for peptide infusions) and right femoral artery (for blood sampling and monitoring of arterial pressure). Colonic temperature was measured and controlled. Exenatide or GLP-1-(7-36)amide was continuously infused for 3 hours at one of 3 infusion rates, 0.5, 5, or 50 nmol/h (n=4-6 for each group). Arterial samples were collected every 30 min into heparinized Natelson capillaries, the plasma separated on a bench-top centrifuge and then frozen at −20° C. until assay. Protease inhibitors were added to blood samples collected for GLP-1 measurement, which was assayed using Linco, Kit No. EGLP-35K. Exenatide was assayed using a two-site sandwich assay.

Results: Plasma concentrations of exendin-4 and GLP-1 during infusions of 0.05, 0.5, 5, and 50 nmol/h approached steady state within −30 min. Steady-state plasma concentrations of both peptides were each infusion rate-dependent. The relationship between infusion rate and steady state plasma concentration (mean of values in final 2 hours) is shown by the three data points that are farthest below GFR, and the relationship for exenatide is shown by the three data points nearest GFR (note that both X-axis and Y-axis are in logarithmic units). These relationships enabled calculation of clearance of each agent. Plasma clearance rates for exenatide ranged from 3.7+0.5 to 8.3+0.7 mL/min. Clearance of GLP-1 ranged from 34+4 to 38+3 mL/min, and was ˜12-fold higher than that of exenatide. The clearance of exenatide approximated the reported clearance of inulin (a marker of glomerular filtration rate). The relationship between steady-state concentration and infusion rate expected for insulin (cleared only by glomerular filtration, GFR) is shown as a black dashed line.

Example 14. Steady State Plasma Concentrations and Intravenous Infusion Rates for Selected Glucagon Analogs

With reference to the graph of FIG. 6, these data include those shown in the graph of FIG. 5, and additionally contain steady state concentrations (inverted triangles) of native glucagon when it was infused intravenously at 2 different infusion rates into anesthetized rats (using procedures described in Example 13). The square data points, closest to GFR, denote measured plasma concentrations of 2 glucagon analogs (Compounds A1 and A2) when they were administered continuously into the subcutaneous space via osmotic minipump. The proximity of the square data points to the GFR dashed line indicates (a) that subcutaneous bioavailability was high, and that (b) clearance from the vascular compartment was consistent with being via glomerular filtration.

Example 15. Clearance Values for Selected Glucagon Analogs

With reference to the illustration of FIG. 7, clearance was determined in numerous experiments performed as described above (for Example 14), except that intravenous infusion rate was uniformly 10 mcg/kg/hr. Selected values for clearance, expressed in mL/min per kg body weight, obtained such experiments, are provided in Table 5 below.

TABLE 5

Row
Selected Compound #
CL mL/min/kg

1
A1
10.6

2
A2
10.8

3
A2
11.3

4
A1
11.5

5
A3
10.7

26
Glucagon
94

The three dark points at the top of the distribution, closest to GFR, are from rows 1, 2 and 5 in Table 5, corresponding to Compounds A1, A2 and A3. Duplicate experiments in rows 3 and 4 returned values very similar to those in rows 1 and 2. Note that the vertical axis is logarithmic and reversed, so that ligands with lowest clearance are at the top of the scatter plot. The solid line labelled GFR represents published values in rats. The values from rows 1, 2 and 5 are close to this bound, consistent with these analogs being cleared from plasma via glomerular filtration. The other dark point, on the dotted line labelled “glucagon,” farthest from GFR, represents the value obtained for native (human/rat) glucagon in this study. The other dotted line, closest to GFR, labelled “exenatide” is from published values for exenatide obtained in this model system. It appears that clearance of exenatide is close to glomerular filtration, with a small fraction cleared by means other than glomerular filtration. Several analogs exceeded exenatide in approaching glomerular filtration as their limiting mode of clearance. Without being bound by theory, these analogs may be even more resistant to peptidase digestion than was exenatide. Several analogs had a higher clearance than did native glucagon, illustrating that all changes in sequence were not necessarily beneficial.

Example 16. Comparison of Dose Dependency of Weight Loss in Rats Upon Continuous Administration of Exenatide or a Selected Glucagon Analog

With reference to the graph of FIG. 8, the black circles plot mean change in body weight over 13 days, expressed as a percent of initial body weight, in diet-induced obese rats into which had been implanted mini pumps delivering Compound A1 (as described for example 10). Symbols are means±SEM (n=4/dose group). Delivery rates into the subcutaneous space were 10, 30, 100, 200, 300 and 1000 mcg/kg/day. Percent change in body weight is relative to the vehicle-infused group (at “zero” on the x-axis). The curve based on data point circles is the best-fitting 4-parameter sigmoid function, constraining vehicle response to 0, and maximal response to −28%. The derived ED50 was 65.7 mcg/kg/day. The Hill slope was −1.26.

Data for exenatide tested in the same model are shown as the inverted triangles. Exenatide infusion rates were 3, 10, 40, 100, 150 and 500 mcg/kg/day. The resulting curve, based on data point triangles is the best-fitting 4-parameter sigmoid function, constraining vehicle response to 0. The derived maximal response was −12.2% weight loss. ED50 was 10.4 mcg/kg/day and Hill slope was −1.85.

As illustrated by the data in Table 6 below, Compound A1 administered alone was almost as potent in vivo as exenatide, and invoked 2.3-fold greater maximal weight loss than did exenatide.

TABLE 6

Rat in vivo

Potency & Efficacy

Human in vitro Potency

%
%

(pEC50)
Physiochemical
Rat PK

Weight
Weight

hGLP-

Properties
Cl/
ED₅₀/
loss
loss

hGCGR
1R
hGIPR
Solubility
Stability
% F
EC₅₀
Single
Combo

Native
11.3
9.3
<5
<5
No
94;
nd
nd
—

Glucagon

~10-

20%

% F

Exenatide
9.2
11.7
<5
>100
>95%
17;
10.4
12%
—

~100
0.45

% F

Desired
>10.0
<7
<6
>40
>90%
5-15;
<100
>10%
>20%

≥40

% F

Compound
12.3
5.2
<5
>200
>95%
11.5;
65.7
26%
—

A1

74% F
4.4

Example 17. Comparison of Dose Dependency of Weight Loss in Rats Upon Continuous Administration of Selected Glucagon Analogs

With reference to the graph of FIG. 9, symbols denote mean±SEM of percent change in body weight over 13 days' infusion of Compound A1 (circles), Compound A2 (squares) or Compound A3 (triangles) into diet-induced obese rats (as in FIG. 8). Infusion rates for Compound A1 were 0, 10, 30, 100, 200, 300 and 1000 mcg/kg/day. For Compound A2, they were 0, 3, 10, 30, 100, 200 and 300 mcg/kg/day, and for Compound A3, 0, 3, 10, 30, 100 and 300 mcg/kg/day.

The three curves denote the best-fitting 4-parameter sigmoid curves for each data set. The fits were constrained to share the same percent change in weight for vehicle groups. Because all were working via a common (glucagon signaling) pathway, they were constrained to share a common maximal weight loss response, derived as 27.2% over 13 days. For Compounds A1, A2 and A3, ED50's were 51, 271 and 535 mcg/kg/day, respectively. Hill slopes were −1.43, −1.25 and −1.12, respectively. The experiment demonstrates that even though each analog had been optimized for pharmacokinetic properties, in vivo potencies were different, and that it required experimentation to select the analog that had highest in vivo potency.

Example 18. Comparison of Dose Dependency of Weight Loss in Rats Upon Continuous Administration of a Selected Glucagon Analog, Alone or in Combination with Exenatide

Data on the graph of FIG. 10 illustrate mean (±SEM) percentage change in body weight of diet-induced obese rats over 27 days treatment. Mean initial body weight was 564+4 g. Animals (n=8/dose group) were implanted with osmotic mini pumps delivering Compound A1 with or without concomitant delivery of exenatide at 10 mcg/kg/day (as described for example 10). Compound A1 was delivered at rates of 0, 7.5, 24 or 50 mcg/kg/day. There were, therefore, 2 dose-response experiments for Compound A1, one without, and one with exenatide. There was an additional group, “pair-fed”, that was offered each day, the same quantity of food that the group treated with Compound A1 50 mcg/kg/day+exenatide 10 mcg/kg/day had consumed the previous day. The objective of the pair-fed group was to estimate the amount of weight loss in the latter group that was attributable to reduction in caloric intake versus via other mechanisms.

The black circles plot the 27-day change in body weight in animals treated with Compound A1 alone (or vehicle). Animals infused with vehicle or with Compound A1 at 7.5 and 24 mcg/kg/day showed a small increase in body weight of ˜4%, while the highest infusion rate showed a 16.8% decrease.

The triangles denote the 27-day percent change in body weight in similarly-treated groups, but with exenatide at 10 mcg/kg/day added. This infusion on its own resulted in a 4.2% decrease in body weight, a relative difference from the vehicle-only group of 7.9%. The squares represent the 7.9% loss expected of exenatide-only, added to the observed weight changes in the Compound A1-only groups, representing the arithmetic sum of responses of each component.

The dotted line is the fitted sigmoid curve through the arithmetically summed responses obtained by this method. In contrast to the expected response (being the arithmetic sum of the responses of single agents), the observed responses of the combination of the 2 highest infusion rates of Compound A1 (24 and 50 mcg/kg/day) with exenatide gave weight loss that was greater than predicted. That is, there was a supra-additive (synergistic) effect. The maximal weight loss response, instead of being ˜27%, was instead ˜47%.

Example 19. Effects of a Glucagon Analog on Weight Loss in DIO LE Rats after 27 Days

The above procedures were used to monitor weight loss in DIO LE rats upon administration of exenatide, a glucagon (“GCG”) analog at derived ED10, ED30 and ED50 values, or a combination of exenatide and the GCG analog at derived ED10, ED30 and ED50 values. Weight loss percentages were calculated 27 days after treatment (FIG. 11). FIG. 11 is a bar graph that alternatively depicts that data shown in FIG. 10.

Example 20. Effects of a Glucagon Analog on Serum Triglycerides, Liver Fat Content and Liver Weight in Zucker Diabetic Fatty (ZDF) Rats after 14 Days

Male Zucker Diabetic Fatty (ZDF) rats were obtained 6 weeks of age (Charles River, Raleigh, N.C.) and used on study at 8 weeks old. Upon receipt, the rats were housed 1 per cage on alpha dri bedding (Shepherd Specialty Papers, Inc., Kalamazoo, Mich.) with free access to Purina 5008 chow (Lab Diet, St. Louis, Mo.) and water, maintained on a 12 hr light/dark cycle from 5:00 AM to 5:00 PM at 21° C. and 50% relative humidity and allowed to acclimate for 9 days before the start of the study.

The rats were dosed with exenatide (0.01 mg/kg/day in 10% DMSO/water) and/or a glucagon analog, compound A104, (0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg/day in 10% DMSO/water) via subcutaneously (s.c.) placed Alzet osmotic pumps, model 2002 (DURECT Corporation, Cupertino, Calif.). Alzet pumps were filled under sterile condition with either vehicle (10% DMSO/sterile water) or exenatide on the day of surgery. Rats were anesthetized under isoflurane and the dorsal skin surface was shaved and cleansed with chlorhexidine and sterile water. Rats were injected ID with Lidocaine (analgesic, 0.1 ml of 0.125% Lidocaine). A 1-2 cm surgical incision was made between the scapulae. Using blunt dissection, a 2-3 cm subcutaneous tunnel was created into which the sterile, filled, mini-osmotic pump was introduced. The skin opening was closed with a skin staple. Rats were monitored after surgery for their recovery from the isoflurane anesthesia.

Blood samples were taken for baseline bleeds (day −3) via tail vein to measure triglyceride levels. Final blood samples were collected by cardiac puncture under isoflurane anesthesia (day 14). Serums were prepared according to manufacturer's protocol using EDTA and T-MG tubes, respectively [Terumo Medical Corporation, Elkton, Md.]. After the terminal bleeds, livers were collected, weighed and homogenized for biochemical analysis (FIG. 13). Clinical chemistry analysis of serum samples and liver homogenate was performed by using an Olympus AU640 Clinical Chemistry analyzer (Olympus America Inc., Melville, N.Y.) with the protocol and method parameters as described by the manufacturer for the triglyceride assay.

Animals were housed and maintained in an AAALAC, internationally accredited care and use programs. All procedures were performed in compliance with the Animal Welfare Act, USDA regulations and approved by either the GlaxoSmithKline or the Mispro Institutional Animal Care and Use Committees.

In ZDF rats, continuous dosing of compound A104, singly and in combination with exenatide (Ex4), led to dose-dependent decreases in serum triglycerides after 14 days (FIG. 12). Compound A104 also dose-dependently reduced liver fat content and liver weights after 14 days (FIG. 13).

Example 21. General Synthetic Method for Installation of Lipophilic Substituent and Spacer of Formula II

The lipophilic substituent and spacer of Formula II was incorporated into numerous disclosed peptides:

embedded image

where the indicated carbonyl on the structure of Formula II was covalently joined to an ε-amino group of a lysine residue of the disclosed peptide to form an amide linkage.

The linear peptide sequence was synthesized on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, Ariz.)) by solid-phase methods using Fmoc strategy with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate (HBTU) or 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) activation (5 fold molar excess) in N,N-dimethyiformamide (DMF), and N′N-Diisopropylethylamine (DIEA) as base, 20% piperidine/DMF for Fmoc deprotection. The resin was Rink Amide MBHA LL (Novabiochem) or N-α-Fmoc protected, pre-loaded Wang LL (Novabiochem), with loadings of 0.29-0.35 mmol/g on a 20-400 μmol scale. At the desired position for acyl-chain substitution, FMOC-Lys(Alloc)-OH was preferentially substituted into the peptide chain. Next, Boc-Tyr(tbu)-OH or Boc-Trp(Boc)-OH was used as the N-terminal amino acid residue. Upon completion of synthesis the resin was washed with Dichloromethane (DCM) and dried under vacuum for 30 minutes. Next, the Alloc protecting group was removed using a solution of Palladium Tetrakis in (CHCl₃/Acetic Acid/N-methylmorpholine, 37:2:1 ratio). The resin was subsequently washed with a 0.5% solution of Sodium Diethyldithiocarbamate trihydrate in DMF, followed by a 0.5% solution of DIEA in DMF, and finally DMF. Next, FMOC-Glu(Otbu)-OH was coupled to the free Lysine side-chain using normal solid-phase conditions. The C-16 acyl side-chain was added using Palmitic Acid under normal solid-phase methods. Final deprotection and cleavage of the peptide from the solid support was performed by treatment of the resin with (92.5% TFA, 2.5% phenol, 2.5% water and 2.5% triisopropylsilane) for 2-3 hours. The cleaved peptide was precipitated using cold diethyl ether. The diethyl ether was decanted and the solids triturated again with cold diethyl ether, pelleted by centrifugation and lyophilized. The lyophilized solid was re-dissolved in a 1:1 solution of acetonitrile/water, with 0.1% TFA (10-15 mL), purified via reverse phase HPLC on a Waters XBridge™ BEH 130, C18, 10 um, 130 Å, 30×250 mm ID column, using a gradient within the ranges of 5-75% acetonitrile/water with 0.1% TFA over 30 minutes at a flow rate of 30 mL/min, λ—215 nm. Column heater set at 60° C. The purified product was lyophilized and analyzed by ESI-LC/MS and analytical HPLC; and were demonstrated to be pure (>98%). Mass results all agreed with calculated values.

HPLC analysis conditions: 4.6×250 mm XBridge BEH130, 5 um, C18 column using an analytical Agilent 1100 with the following gradient: 20% to 100% over 15 minutes and holding at 100% to 20 minutes. The column temperature was set at 40° C. The flow rate was set at 1.0 mL/min. The solvents consisted of A=H2O+0.1% TFA and B=Acetonitrile+0.1% TFA. The crude and final LCMS were observed and product mass was identified using the following conditions: UV detection set at 215 and 280 nm.

LCMS analysis conditions: 4.6×250 mm XBridge BEH130, 5 um, C18 column using an analytical Agilent 1100 in combination with an API-4000 Sciex LC/MS/MS system with the following gradient: 20% B to 95% B over 10 minutes, holding at 95% to wash the column over 1.2 min. Equilibrate column at 5% B, 95% A to 12.5 min. The column temperature was set at 40° C. The flow rate was set at 1.5 mL/min. The solvents consisted of A=H2O+0.1% TFA and B=Acetonitrile+0.1% TFA. UV detection set at 215 and 280 nm. Method=Q1 MS. Syringe size 100 uL, UV range 190-400 nm, Slit width=4 mm, Sampling rate=>20 Hz., Ion source: Turbo spray, Polarity=Positive.

Example 22. General Synthetic Method for Installation of Lipophilic Substituent and Spacer of Formula III

The lipophilic substituent and spacer of Formula III was incorporated into numerous disclosed peptides:

embedded image

where the indicated carbonyl on the structure of Formula III was covalently joined to an ε-amino group of a lysine residue of the disclosed peptide to form an amide linkage.

The linear peptide sequence was synthesized on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, Ariz.)) by solid-phase methods using Fmoc strategy with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate (HBTU) or 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) activation (5 fold molar excess) in N,N-dimethyiformamide (DMF), and N′N-Diisopropylethylamine (DIEA) as base, 20% piperidine/DMF for Fmoc deprotection. The resin was Rink Amide MBHA LL (Novabiochem) or N-α-Fmoc protected, pre-loaded Wang LL (Novabiochem), with loadings of 0.29-0.35 mmol/g on a 20-400 μmol scale. At the desired location for acyl-chain substitution, FMOC-Lys(Alloc)-OH was preferentially substituted into the peptide chain. Next, Boc-Tyr(tbu)-OH or Boc-Trp(Boc)-OH was used as the N-terminal amino acid residue. Upon completion of synthesis the resin was washed with Dichloromethane (DCM) and dried under vacuum for 30 minutes. Next, the Alloc protecting group was removed using a solution of Palladium Tetrakis in (CHCl₃/Acetic Acid/N-methylmorpholine, 37:2:1 ratio). The resin was subsequently washed with a 0.5% solution of Sodium Diethyldithiocarbamate trihydrate in DMF, followed by a 0.5% solution of DIEA in DMF, and finally DMF. Next, elongation of the spacer region proceeded with coupling of {2-[2-(Fmoc-amino)ethoxy}acetic acid, followed by FMOC-Glu(Otbu)-OH using normal solid-phase conditions. The C-18, acid terminating side-chain, was added using Octadecanedioic Acid under normal solid-phase methods. Final deprotection and cleavage of the peptide from the solid support was performed by treatment of the resin with (92.5% TFA, 2.5% phenol, 2.5% water and 2.5% triisopropylsilane) for 2-3 hours. The cleaved peptide was precipitated using cold diethyl ether. The diethyl ether was decanted and the solids triturated again with cold diethyl ether, pelleted by centrifugation and lyophilized. The lyophilized solid was re-dissolved in a 1:1 solution of acetonitrile/water, with 0.1% TFA (10-15 mL), purified via reverse phase HPLC on a Waters XBridge™ BEH 130, C18, 10 um, 130 Å, 30×250 mm ID column, using a gradient within the ranges of 5-75% acetonitrile/water with 0.1% TFA over 30 minutes at a flow rate of 30 mL/min, λ—215 nm. Column heater set at 60° C. The purified product was lyophilized and analyzed by ESI-LC/MS and analytical HPLC; and were demonstrated to be pure (>98%). Mass results all agreed with calculated values.

HPLC analysis conditions: 4.6×250 mm XBridge BEH130, 5 um, C18 column using an analytical Agilent 1100 with the following gradient: 5% to 70% over 15 minutes and holding at 70% to 20 minutes. The column temperature was set at 40° C. The flow rate was set at 1.5 mL/min. The solvents consisted of A=H2O+0.1% TFA and B=Acetonitrile+0.1% TFA. The crude and final LCMS were observed and product mass was identified using the following conditions: UV detection set at 215 and 280 nm.

LCMS analysis conditions: 4.6×250 mm XBridge BEH130, 5 um, C18 column using an analytical Agilent 1100 in combination with an API-4000 Sciex LC/MS/MS system with the following gradient: 5% to 65% over 10 minutes, ramping up to 95% to wash the column over 1 minute and equilibrating back to 5% organic to 12.5 minutes. The column temperature was set at 40° C. The flow rate was set at 1.5 mL/min. The solvents consisted of A=H2O+0.1% TFA and B=Acetonitrile+0.1% TFA. UV detection set at 215 and 280 nm. Method=Q1 MS. Syringe size 100 uL, UV range 190-400 nm, Slit width=4 mm, Sampling rate=>20 Hz., Ion source: Turbo spray, Polarity=Positive.

TABLE 7

Modification:
Site of

Lipophilic
Mod-

GLP-

Com-
Substituent
ification:

GluR
1R

pound
and
Peptide

pEC
pEC

ID
Spacer
Residue #
Sequence
50
50

Compound
none
n/a
YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDE
10.6
<6

A1

PSSGAPPPS-OH (SEQ ID NO: 41)

Compounds B1-B8: Substitutions & Modifications to Compound A1

Compound
Formula II
33
YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDE

B1

PSSK****APPPS-OH (SEQ ID NO: 152)

Compound
Formula II
24
YSHGTFTSDYSKYLD(Aib)KYAQEFVK****WLED
11.1
<6

B2

EPSSGAPPPS-OH (SEQ ID NO: 153)

Compound
Formula II
16
YSHGTFTSDYSKYLDK****KYAQEFV(Aib)WLED
11.4
<6

B3

EPSSGAPPPS-OH (SEQ ID NO: 154)

Compound
Formula II
10
YSHGTFTSDK****SKYLD(Aib)KYAQEFV(Aib)WL
9.1
<6.0

B4

EDEPSSGAPPPS-OH (SEQ ID NO: 155)

Compound
Formula
33
YSHGTFTSDYSKYLD(Aib)KYAQEFV(Aib)WLEDE
9.6
<6

B5
III

PSSK*****APPPS-OH (SEQ ID NO: 156)

Compound
Formula
24
YSHGTFTSDYSKYLD(Aib)KYAQEFVK*****WLE
10.1
<6

B6
III

DEPSSGAPPPS-OH (SEQ ID NO: 157)

Compound
Formula
16
YSHGTFTSDYSKYLDK*****KYAQEFV(Aib)WLE
8.6
<6

B7
III

DEPSSGAPPPS-OH (SEQ ID NO: 158)

Compound
Formula
10
YSHGTFTSDK*****SKYLD(Aib)KYAQEFV(Aib)W
10.6
<6

B8
III

LEDEPSSGAPPPS-OH (SEQ ID NO: 159)

Compound
none
n/a
YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDE

A2

PSSGAPPPS-OH (SEQ ID NO: 42)

Compounds B9-B16: Substitutions & Modifications to Compound A2

Compound
Formula
33
YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDE
11.1
<6

B9
II

PSSK****APPPS-OH (SEQ ID NO: 160)

Compound
Formula
24
YSHGTFTSDYSKYLD(Aib)KSAQEFVK****WLED
9.9
<6

B10
II

EPSSGAPPPS-OH (SEQ ID NO: 161)

Compound
Formula
16
YSHGTFTSDYSKYLDK****KSAQEFV(Aib)WLED
10.3
6.3

B11
II

EPSSGAPPPS-OH (SEQ ID NO: 162)

Compound
Formula
10
YSHGTFTSDK****SKYLD(Aib)KSAQEFV(Aib)WL
11.5
7.2

B12
II

EDEPSSGAPPPS-OH (SEQ ID NO: 163)

Compound
Formula
33
YSHGTFTSDYSKYLD(Aib)KSAQEFV(Aib)WLEDE
9.2
<6

B13
III

PSSK*****APPPS-OH (SEQ ID NO: 164)

Compound
Formula
24
YSHGTFTSDYSKYLD(Aib)KSAQEFVK*****WLED
9.3
<6

B14
III

EPSSGAPPPS-OH (SEQ ID NO: 165)

Compound
Formula
16
YSHGTFTSDYSKYLDK*****KSAQEFV(Aib)WLED
9.9
<6.0

B15
III

EPSSGAPPPS-OH (SEQ ID NO: 166)

Compound
Formula
10
YSHGTFTSDK*****SKYLD(Aib)KSAQEFV(Aib)W
8.5
8.3

B16
III

LEDEPSSGAPPPS-OH (SEQ ID NO: 167)

Compound
none
n/a
WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDE

A6

PSSGAPPPS-OH (SEQ ID NO: 42)

Compounds B17-B24: Substitutions & Modifications to Compound A6

Compound
Formula
33
WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDE
10.5
<6.0

B17
II

PSSK****APPPS-OH (SEQ ID NO: 168)

Compound
Formula
24
WSQGTFTSDYSKYLD(Aib)KRAQEFVK****WLED
10.2
<6.0

B18
II

EPSSGAPPPS-OH (SEQ ID NO: 169)

Compound
Formula
16
WSQGTFTSDYSKYLDK****KRAQEFV(Aib)WLED
10.6
6.3

B19
II

EPSSGAPPPS-OH (SEQ ID NO: 170)

Compound
Formula
10
WSQGTFTSDK****SKYLD(Aib)KRAQEFV(Aib)WL
11.1
6.5

B20
II

EDEPSSGAPPPS-OH (SEQ ID NO: 171)

Compound
Formula
33
WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLEDE

B21
III

PSSK*****APPPS-OH (SEQ ID NO: 172)

Compound
Formula
24
WSQGTFTSDYSKYLD(Aib)KRAQEFV(Aib)WLE

B22
III

DEPSSGAPPPS-OH (SEQ ID NO: 173)

Compound
Formula
16
WSQGTFTSDYSKYLDK*****KRAQEFV(Aib)WLE
9.5
6.5

B23
III

DEPSSGAPPPS-OH (SEQ ID NO: 174)

Compound
Formula
10
WSQGTFTSDK*****SKYLD(Aib)KRAQEFV(Aib)W
8.2
6.8

B24
III

LEDEPSSGAPPPS-OH (SEQ ID NO: 175)

Compound
none
n/a
YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSS

A4

GAPPPS (SEQ ID NO: 44)

Compounds B25-B32: Substitutions & Modifications to Compound A4

Compound
Formula
33
YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSS
10.5
<6

B25
II

K****APPPS-OH (SEQ ID NO: 176)

Compound
Formula
16
YSHGTFTSDYSKYLDK****KRAQEFVIWLEDEPSS
11
<6

B26
II

GAPPPS-OH (SEQ ID NO: 177)

Compound
Formula
10
YSHGTFTSDK****SKYLD(Aib)KRAQEFVIWLEDE
11.5
6.6

B27
II

PSSGAPPPS-OH (SEQ ID NO: 178)

Compound
Formula
21
YSHGTFTSDYSKYLD(Aib)KRAQK****FVIWLEDE

B28
II

PSSGAPPPS-OH (SEQ ID NO: 179)

Compound
Formula
33
YSHGTFTSDYSKYLD(Aib)KRAQEFVIWLEDEPSS
9.8
7.0

B29
III

K*****APPPS-OH (SEQ ID NO: 180)

Compound
Formula
21
YSHGTFTSDYSKYLD(Aib)KRAQK*****FVIWLED
9.7
7.0

B30
III

EPSSGAPPPS-OH (SEQ ID NO: 181)

Compound
Formula
16
YSHGTFTSDYSKYLDK*****KRAQEFVIWLEDEPS
10.6
7.4

B31
III

SGAPPPS-OH (SEQ ID NO: 182)

Compound
Formula
10
YSHGTFTSDK*****SKYLD(Aib)KRAQEFVIWLED
9.4
7.8

B32
III

EPSSGAPPPS-OH (SEQ ID NO: 183)

Compound
none
n/a
YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSKS

A3

PPPS-NH₂ (SEQ ID NO: 43)

Compounds B33-B40: Substitutions & Modifications to Compound A3

Compound
Formula
33
YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSK*
10.6
<6

B33
II

***SPPPS-NH₂ (SEQ ID NO: 184)

Compound
Formula
24
YSQGTFTSDYSKYLDAARAQEFVK****WLEDEPK
9.8
<6

B34
II

SKSPPPS-NH₂(SEQ ID NO: 185)

Compound
Formula
16
YSQGTFTSDYSKYLDK****ARAQEFVKWLEDEPK
11.1
6.6

B35
II

SKSPPPS-NH₂ (SEQ ID NO: 186)

Compound
Formula
10
YSQGTFTSDK****SKYLDAARAQEFVKWLEDEPK
11.5
8.1

B36
II

SKSPPPS-NH₂ (SEQ ID NO: 187)

Compound
Formula
33
YSQGTFTSDYSKYLDAARAQEFVKWLEDEPKSK*
10.1
6.2

B37
III

****SPPS-NH₂ (SEQ ID NO: 188)

Compound
Formula
24
YSQGTFTSDYSKYLDAARAQEFVK*****WLEDEP
9.6
6

B38
III

KSKSPPPS-NH₂ (SEQ ID NO: 189)

Compound
Formula
16
YSQGTFTSDYSKYLDK*****ARAQEFVKWLEDEP
10.9
6

B39
III

KSKSPPPS-NH₂ (SEQ ID NO: 190)

Compound
Formula
10
YSQGTFTSDK*****SKYLDAARAQEFVKWLEDEP
11.9
7.2

B40
III

KSKSPPPS-NH₂ (SEQ ID NO: 191)

Compound
none
n/a
YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSGA

A5

PPPS-NH₂ (SEQ ID NO: 45)

Compounds B41-B48: Substitutions & Modifications to Compound A5

Compound
Formula
33
YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSK**
10.6
<6

B41
II

**APPPS-NH₂ (SEQ ID NO: 192)

Compound
Formula
24
YSHGTFTSDYSKYLDSARAQEFVK****WLEDEPS
9.7
<6

B42
II

SGAPPPS-NH₂ (SEQ ID NO: 193)

Compound
Formula
16
YSHGTFTSDYSKYLDK****ARAQEFVKWLEDEPS
11.1
<6

B43
II

SGAPPPS-NH₂ (SEQ ID NO: 194)

Compound
Formula
10
YSHGTFTSDK****SKYLDSARAQEFVKWLEDEPS
11.9
7.2

B44
II

SGAPPPS-NH₂ (SEQ ID NO: 195)

Compound
Formula
33
YSHGTFTSDYSKYLDSARAQEFVKWLEDEPSSK**
9.8
<6

B45
III

***APPPS-NH₂ (SEQ ID NO: 196)

Compound
Formula
24
YSHGTFTSDYSKYLDSARAQEFVK*****WLEDEP
9.5
<6

B46
III

SSGAPPPS-NH₂ (SEQ ID NO: 197)

Compound
Formula
16
YSHGTFTSDYSKYLDK*****ARAQEFVKWLEDEP
10.8
<6

B47
III

SSGAPPPS-NH₂ (SEQ ID NO: 198)

Compound
Formula
10
YSHGTFTSDK*****SKYLDSARAQEFVKWLEDEP
9.2
<6

B48
III

SSGAPPPS-NH₂ (SEQ ID NO: 199)

Each ε-amino group of the lysine residue located at the indicated peptide residue (K****) in the disclosed compounds is covalently bound to the indicated carbonyl of the structure of Formula II to form an amide:

embedded image

Each ε-amino group of the lysine residue located at the indicated peptide residue (K*****) in the disclosed compounds is covalently bound to the indicated carbonyl of the structure of Formula III to form an amide:

embedded image

Example 23. Clearance of Peptide from Kidney (CL) Following Intravenous Infusion

Acylated peptides were formulated in sterile saline and administered as a 1-hour intravenous infusion to non-fasted male Sprague-Dawley rats (n=3 per group) via jugular vein cannula at a final dose of 0.033 mg/kg. Formulations were administered at a rate of 1.67 mL/kg/h. Blood samples (approximately 250 uL) were collected for pharmacokinetic analysis via a femoral vein cannula at 0, 0.25, 0.5, 1, 1.17, 1.33, 1.5, 2, 4, 6, 8, 24, 30 and 48 h post-start of infusion into microtainer tubes containing K2EDTA as anticoagulant and 25 uL of a protease inhibitor cocktail. Plasma was prepared by centrifugation and stored at −80° C. until analysis. Representative results are provided in Table 8 below.

TABLE 8

Modification:
Site of

Lipophilic
Modification:

Substituent
Peptide
CL

Compound ID
and Spacer
Residue #
(IV/SubQ)
T ½

Compound A1
none
none
11.5
0.83

Compound B3
Formula II
16
18.8
1.79

Compound B9
Formula II
33
0.594
4.98

Compound B14
Formula III
24
2.65
13.9

Compound B15
Formula III
16
0.457
9.34

Compound B33
Formula II
33
6.96
0.429

Compound B41
Formula II
33
4.856
1.09

Example 24. Weight Loss in Rats Upon Continuous Administration of Exenatide or a Glucagon Analog

Diet-induced-obese LE rats with initial body weights of 586+86 (mean±SD) were each implanted with 2 osmotic minipumps (Alzet) delivering, respectively, exenatide or a glucagon analog i.e., selective glucagon receptor agonist (as described for example 10). The treatment groups comprised 30 different combinations of exenatide and glucagon analog, with 10-20 animals in each group.

Body weight was measured every 7 days. Results are illustrated in FIG. 14 as a 3-D plot of weight loss on the vertical (Z) axis, represented as a percentage of initial body weight after 21 days of continuous dosing. A positive weight loss, shown as a circle atop a riser emerging from the base plane, thus represents a decrease in body weight relative to pre-treatment body weight. To maintain clarity, error bars are not shown.

The horizontal axes in FIG. 14 relate to the logarithm of the daily dose in units of micrograms per kg body weight per day for each of the exenatide and glucagon analog singletons. Log exenatide dose of −1 denotes treatments where exenatide dose was zero (glucagon analog-only treatments), and −1 on the glucagon log dose axis denotes treatments where glucagon was not dosed (exenatide-only treatments). The series of points in the left foreground and in the right foreground therefore depict dose-responses for exenatide alone, and for the glucagon analog alone, respectively.

The weight loss response to exenatide alone after 21 days treatment could be described by a 4-parameter sigmoid curve with a maximal response of 12.5% weight loss, an ED₅₀of 9.6 μg/kg/day, with a Hill slope of 0.88. For the glucagon analog alone, the parameters for 21 day weight loss were a maximum of 28.5%, an ED₅₀of 45 μg/kg/day, and a Hill slope of 2.7.

Example 25. Evaluation of Exenatide/Glucagon Analog Combinations

The raw data in FIG. 14 were fitted to a continuous function to enable characterization of certain key features of the response to exenatide/glucagon analog combinations. The general form of the continuous response surface was

Response to combination=(a*Resp_EX)+(b*Resp_GGN)+(c*Resp_EX*Resp_GGN)

where Resp_EXand Resp_GGNrepresent the dose-dependent responses observed with each singleton.

The combination response was further constrained by a hyperbolic relationship such that it could not exceed 100% weight loss. The best-fitting continuous response surface (R=0.9) was obtained by least-squares iterative approximation of the above-mentioned user-defined equation using GraphPad Prism v7.0 (GraphPad software, San Diego, Calif.). Where C>0, a multiplicative (supra-additive) component was present.

The symbols representing each treatment group are shown as spheres embedded within the mesh of the best-fitting surface in FIG. 15. The 4 broken curves joining the exenatide-only and the glucagon analog-only planes represent lines of equal effect (isoboles) that would be obtained with an additive interaction as one agent was substituted for equi-effective doses of the other (expected 2%, 4%, 6% and 8% weight loss isoboles shown). The 4 heavy solid lines depict what was instead observed, being the response surface corresponding to such dose-substitution. The differences between the solid line (observed) and broken line (expected) define a synergistic interaction. For each level of predicted effect, there was a dose ratio where the effect peaked, and the synergy was maximal.

Example 26. Evaluation of Fixed-Ratio Combinations of Exenatide/Glucagon Analog

Because a combination product may comprise agents administered in a fixed ratio, the response surface was analyzed from the viewpoint of increasing doses of fixed-ratio mixtures.

Fixed dose ratios are depicted in FIG. 16 as parallel diagonal planes above the Log Exenatide (“EX”) vs Log Glucagon Analog (“GGN”) base plane. The dose-response of each mixture is shown as the series of black curves where the response surface intersects each plane. The 4 gray curves on the surface (shown as thick black lines in FIG. 15) are the observed responses when one agent is substituted for an equi-effective dose of the other, a so-called “dose-ratio scan”.

The heavy black line, where the gray plane of a fixed dose ratio intersects with the response surface, appears to transect the 4 gray lines near their dose-ratio optima. The gray plane defines a 3:1 GGN:EX dose ratio. Mixtures of 10:1 and 1:1 GGN:EX also performed well.

Example 27. Evaluation of In Vivo Potencies

Evaluation of in vivo potencies, defined as effect per unit total mass of drug, was undertaken for different fixed-ratio combinations of exenatide/glucagon analog.

For some applications, such as in a mini osmotic pump where the volume of the drug reservoir is limited, the mixture with the highest apparent in vivo potency is most beneficial. The apparent in vivo potency of different mixtures may be defined as effect per unit total mass of drug (or more strictly, per volume of formulation).

The responses to mixtures shown as surfaces in FIG. 15 and FIG. 16 may thereby be reduced to the 2-D dose responses shown in FIG. 17. The X-axis in FIG. 17 is not the dose of a single agent, but is the mass of both agents in a fixed-ratio mixture.

The family of dose responses for different mixtures includes one where the least combined dose evokes a given effect (exemplified by 20% weight loss, as indicated by the horizontal arrow). A promising mixture thus identified was an approximately 3:1 mixture of GGN:EX. The response to this mixture, exhibiting highest apparent potency, is shown as a broken line.

Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method of making an isolated polypeptide comprising: (i) the amino acid sequence:
2. The method of claim 1, wherein each of the amino acids of steps (a), (b), and (c) comprises an Fmoc-protected amino group, and wherein at the end of each of steps (a), (b), and (c), the Fmoc group is deprotected with piperidine.
3. The method of claim 1, wherein each of the peptide coupling agents of steps (b) and (c) is selected from the group consisting of HBTU, HCTU, and HOBT.
4. The method of claim 1, wherein steps (a), (b), and (c) further comprise the addition of N,N-Diisopropylethylamine (DIEA).
5. The method of claim 1, wherein the isolated polypeptide comprises the amino acid sequence:
6. The method of claim 1, wherein the isolated polypeptide comprises the amino acid sequence:
7. The method of claim 6, wherein the isolated polypeptide comprises an amino acid sequence represented by SEQ ID NO: 41 (Compound A1).
8. The method of claim 6, wherein the isolated polypeptide comprises an amino acid sequence represented by SEQ ID NO: 42 (Compound A2).
9. The method of claim 1, wherein the isolated polypeptide comprises an amino acid sequence represented by SEQ ID NO: 137.
10. The method of claim 9, wherein the lysine and glutamate residues that form the lactam bridge comprise alloc and allyl protecting groups, respectively, on their side chains at the time they are coupled to the elongated resin-bound peptide.
11. The method of claim 10, wherein the alloc and allyl protecting groups are removed before step (e).
12. The method of claim 9, wherein the lactam bridge is formed before step (e).
13. The method of claim 9, wherein the isolated polypeptide comprises an amino acid sequence of SEQ ID NO: 138 (Compound A104) or SEQ ID NO: 139 (Compound A105).
14. The method of claim 1, wherein the isolated polypeptide comprises an amino acid sequence represented by SEQ ID NO: 151.
15. The method of claim 14, wherein the lysine that is covalently joined to a lipophilic substituent, optionally via a spacer, comprises an alloc-protected amino group on its side chain at the time it is coupled to the elongated resin-bound peptide.
16. The method of claim 15, wherein covalent attachment of the lipophilic substituent, optionally via a spacer, occurs between step (d) and step (e) and comprises the following steps: (d.1) removal of the alloc protecting group to provide a free lysine side chain;(d.2) coupling the free lysine side chain with Fmoc-Glu(Otbu)-OH; or coupling the free lysine side chain with the spacer elongation group {2-[2-(Fmoc-amino)ethoxy}acetic acid and subsequently coupling the spacer elongation group with Fmoc-Glu(Otbu)-OH;(d.3) coupling the glutamate residue of step (d.2) with palmitic acid or octadecanedioic acid.
17. The method of claim 14, wherein each lipophilic substituent and spacer are of Formula II:
18. A method of treating or alleviating a symptom of a disease or disorder in which agonism at the glucagon receptor is desired in a subject in need thereof, the method comprising administering to the subject an isolated polypeptide selected from the group consisting of SEQ ID NOs: 41-136, 138, 139, 143-149, and 152-199 (Compounds A1-A105 and B1-48).
19. The method of claim 18, wherein the disease or disorder is type 2 diabetes mellitus, obesity, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).
20. The method of claim 18, wherein the subject is a human.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/595,809, filed May 15, 2017, which application claims the benefit of U.S. Provisional Application No. 62/337,005, filed May 16, 2016; U.S. Provisional Application No. 62/414,146, filed Oct. 28, 2016; and U.S. Provisional Application No. 62/420,937, filed Nov. 11, 2016, the contents of each of which are incorporated herein by reference in their entirety.

US Referenced Citations (770)

Number	Name	Date	Kind
2110208	Eggert	Mar 1938	A
2168437	Buercklin	Aug 1939	A
2531724	Cevasco	Nov 1950	A
D179537	Floyd et al.	Jan 1957	S
3025991	Gillon	Mar 1962	A
3122162	Sands	Feb 1964	A
3523906	Vrancken et al.	Aug 1970	A
3625214	Higuchi	Dec 1971	A
3632768	Bergy et al.	Jan 1972	A
3691090	Kitajima et al.	Sep 1972	A
D226915	Huggins	May 1973	S
3732865	Higuchi et al.	May 1973	A
3737337	Schnoring et al.	Jun 1973	A
3773919	Boswell et al.	Nov 1973	A
3797492	Place	Mar 1974	A
3869549	Geller	Mar 1975	A
3891570	Fukushima et al.	Jun 1975	A
D236035	Ciencewicki	Jul 1975	S
3960757	Morishita et al.	Jun 1976	A
3987790	Eckenhoff et al.	Oct 1976	A
3995631	Higuchi et al.	Dec 1976	A
3995632	Nakano et al.	Dec 1976	A
4008719	Theeuwes et al.	Feb 1977	A
4034756	Higuchi et al.	Jul 1977	A
4078060	Benson et al.	Mar 1978	A
4111201	Theeuwes	Sep 1978	A
4111202	Theeuwes	Sep 1978	A
4111203	Theeuwes	Sep 1978	A
4203439	Theeuwes	May 1980	A
4211771	Witkowski et al.	Jul 1980	A
4221862	Naito et al.	Sep 1980	A
4243030	Lynch et al.	Jan 1981	A
D258837	Spranger et al.	Apr 1981	S
D259458	Fuller et al.	Jun 1981	S
4305927	Theeuwes et al.	Dec 1981	A
4310516	Chang et al.	Jan 1982	A
4340054	Michaels	Jul 1982	A
4350271	Eckenhoff	Sep 1982	A
4373527	Fischell	Feb 1983	A
4376118	Daher et al.	Mar 1983	A
4384975	Fong	May 1983	A
4389330	Tice et al.	Jun 1983	A
4439196	Higuchi	Mar 1984	A
4444498	Heinemann	Apr 1984	A
4455143	Theeuwes et al.	Jun 1984	A
4455145	Theeuwes	Jun 1984	A
4530840	Tice et al.	Jul 1985	A
4552561	Eckenhoff et al.	Nov 1985	A
4588614	Lauchenauer	May 1986	A
4594108	Greminger, Jr. et al.	Jun 1986	A
4609374	Ayer	Sep 1986	A
4639244	Rizk et al.	Jan 1987	A
4655462	Balsells	Apr 1987	A
4673405	Guittard et al.	Jun 1987	A
4675184	Hasegawa et al.	Jun 1987	A
4695623	Stabinsky	Sep 1987	A
4727138	Goeddel et al.	Feb 1988	A
4734284	Terada et al.	Mar 1988	A
4737437	Gutsell, Jr. et al.	Apr 1988	A
4743449	Yoshida et al.	May 1988	A
4753651	Eckenhoff	Jun 1988	A
4762791	Goeddel et al.	Aug 1988	A
4765989	Wong et al.	Aug 1988	A
4783337	Wong et al.	Nov 1988	A
4818517	Kwee et al.	Apr 1989	A
4820638	Swetly et al.	Apr 1989	A
4826144	Balsells	May 1989	A
4830344	Balsells	May 1989	A
4840396	Reddy et al.	Jun 1989	A
4845196	Cowling	Jul 1989	A
4847079	Kwan	Jul 1989	A
4851228	Zentner et al.	Jul 1989	A
4865845	Eckenhoff et al.	Sep 1989	A
4873080	Brickl et al.	Oct 1989	A
4874388	Wong et al.	Oct 1989	A
4876781	Balsells	Oct 1989	A
4885166	Meyer et al.	Dec 1989	A
4886668	Haslam et al.	Dec 1989	A
4892778	Theeuwes et al.	Jan 1990	A
4893795	Balsells	Jan 1990	A
4897471	Stabinsky	Jan 1990	A
4907788	Balsells	Mar 1990	A
4915366	Balsells	Apr 1990	A
4915949	Wong et al.	Apr 1990	A
4915954	Ayer et al.	Apr 1990	A
4917887	Hauptmann et al.	Apr 1990	A
4917895	Lee et al.	Apr 1990	A
4923805	Reddy et al.	May 1990	A
4927687	Nuwayser	May 1990	A
4929554	Goeddel et al.	May 1990	A
4931285	Edgren et al.	Jun 1990	A
4934666	Balsells	Jun 1990	A
4940465	Theeuwes et al.	Jul 1990	A
4940588	Sparks et al.	Jul 1990	A
4952402	Sparks et al.	Aug 1990	A
4957119	de Nijs	Sep 1990	A
4961253	Balsells	Oct 1990	A
4964204	Balsells	Oct 1990	A
4969884	Yum	Nov 1990	A
4974821	Balsells	Dec 1990	A
4976966	Theeuwes et al.	Dec 1990	A
5004689	Fiers et al.	Apr 1991	A
5006346	Theeuwes et al.	Apr 1991	A
5019382	Cummins, Jr.	May 1991	A
5019400	Gombotz et al.	May 1991	A
5023088	Wong et al.	Jun 1991	A
5024842	Edgren et al.	Jun 1991	A
5030216	Theeuwes et al.	Jul 1991	A
5034229	Magruder et al.	Jul 1991	A
5057318	Magruder et al.	Oct 1991	A
5059423	Magruder et al.	Oct 1991	A
5066436	Komen et al.	Nov 1991	A
5071642	Lahr et al.	Dec 1991	A
5072070	Balsells	Dec 1991	A
5079388	Balsells	Jan 1992	A
5091188	Haynes	Feb 1992	A
5108078	Balsells	Apr 1992	A
5110596	Magruder et al.	May 1992	A
5112614	Magruder et al.	May 1992	A
5113938	Clayton	May 1992	A
5117066	Balsells	May 1992	A
D326718	Maxwell	Jun 1992	S
5118666	Habener	Jun 1992	A
5120306	Gosselin	Jun 1992	A
5120712	Habener	Jun 1992	A
5120832	Goeddel et al.	Jun 1992	A
5122128	Cardinal et al.	Jun 1992	A
5122377	Miller	Jun 1992	A
5126142	Ayer et al.	Jun 1992	A
5126147	Silvestri et al.	Jun 1992	A
5134244	Balsells	Jul 1992	A
5137727	Eckenhoff	Aug 1992	A
D329278	Gallup	Sep 1992	S
5151093	Theeuwes et al.	Sep 1992	A
5160122	Balsells	Nov 1992	A
5160743	Edgren et al.	Nov 1992	A
5161806	Balsells	Nov 1992	A
5180591	Margruder et al.	Jan 1993	A
5190765	Jao et al.	Mar 1993	A
5203849	Balsells	Apr 1993	A
5204108	Ilium	Apr 1993	A
5207752	Sorensen et al.	May 1993	A
5209746	Balaban et al.	May 1993	A
5213310	Steber	May 1993	A
5213809	Wright et al.	May 1993	A
5219572	Sivaramakrishnan	Jun 1993	A
5221278	Linkwitz et al.	Jun 1993	A
5223265	Wong	Jun 1993	A
5225205	Orsolini	Jul 1993	A
5231176	Goeddel et al.	Jul 1993	A
5234424	Yum et al.	Aug 1993	A
5234692	Magruder et al.	Aug 1993	A
5234693	Magruder et al.	Aug 1993	A
5234695	Hobbs et al.	Aug 1993	A
5252338	Jao et al.	Oct 1993	A
5260069	Chen	Nov 1993	A
D342855	Butler, II	Jan 1994	S
5278151	Korb et al.	Jan 1994	A
5279608	Cherif Cheikh	Jan 1994	A
5284655	Bogdansky et al.	Feb 1994	A
5288501	Nürnberg et al.	Feb 1994	A
5288502	Mcginity et al.	Feb 1994	A
5290271	Jernberg	Mar 1994	A
5300079	Niezink et al.	Apr 1994	A
5300302	Tachon et al.	Apr 1994	A
5308348	Balaban et al.	May 1994	A
5312335	McKinnon et al.	May 1994	A
5312389	Theeuwes et al.	May 1994	A
5312390	Wong	May 1994	A
5318558	Linkwitz et al.	Jun 1994	A
5318780	Viegas et al.	Jun 1994	A
5320616	Magndu et al.	Jun 1994	A
5324280	Wong et al.	Jun 1994	A
5336057	Fukuda et al.	Aug 1994	A
5336505	Ng et al.	Aug 1994	A
5352662	Brooks et al.	Oct 1994	A
5368588	Bettinger	Nov 1994	A
5368863	Eckenhoff et al.	Nov 1994	A
5371089	Rattan	Dec 1994	A
5374620	Clark et al.	Dec 1994	A
5385738	Yamahira et al.	Jan 1995	A
5385887	Yim et al.	Jan 1995	A
5407609	Tice et al.	Apr 1995	A
D358644	Park	May 1995	S
5411951	Mitchell	May 1995	A
5413572	Wong et al.	May 1995	A
5413672	Hirotsuji et al.	May 1995	A
5424286	Eng	Jun 1995	A
5428024	Chu et al.	Jun 1995	A
5429602	Hauser	Jul 1995	A
443459	Wong et al.	Aug 1995	A
5439688	Orsolini et al.	Aug 1995	A
5445829	Paradissis et al.	Aug 1995	A
5456679	Balaban et al.	Oct 1995	A
5458888	Chen	Oct 1995	A
5464929	Bezwada et al.	Nov 1995	A
5472708	Chen	Dec 1995	A
5478564	Wantier et al.	Dec 1995	A
5486365	Takado et al.	Jan 1996	A
5498255	Wong et al.	Mar 1996	A
5511355	Dingler	Apr 1996	A
5512293	Landrau et al.	Apr 1996	A
5512549	Chen et al.	Apr 1996	A
5514110	Teh	May 1996	A
5529914	Hubbell et al.	Jun 1996	A
5531736	Wong et al.	Jul 1996	A
5540665	Mercado et al.	Jul 1996	A
5540912	Roorda et al.	Jul 1996	A
5541172	Labrie et al.	Jul 1996	A
5542682	Goldstein et al.	Aug 1996	A
5543156	Roorda et al.	Aug 1996	A
5545618	Buckley et al.	Aug 1996	A
5556642	Kobayashi et al.	Sep 1996	A
5557318	Gabriel	Sep 1996	A
5571525	Roorda et al.	Nov 1996	A
5574008	Johnson et al.	Nov 1996	A
5574137	Gray et al.	Nov 1996	A
5580578	Oshiack et al.	Dec 1996	A
5589187	Cleland et al.	Dec 1996	A
5595751	Bezwada	Jan 1997	A
5595759	Wright et al.	Jan 1997	A
5597579	Bezwada et al.	Jan 1997	A
5602010	Hauptmann et al.	Feb 1997	A
5605688	Himmler et al.	Feb 1997	A
5607687	Bezwada et al.	Mar 1997	A
5609885	Rivera et al.	Mar 1997	A
5614221	Fjellstrom	Mar 1997	A
5614492	Habener	Mar 1997	A
5618552	Bezwada et al.	Apr 1997	A
5620698	Bezwada et al.	Apr 1997	A
5620705	Dong et al.	Apr 1997	A
5630796	Bellhouse et al.	May 1997	A
5633011	Dong et al.	May 1997	A
5635213	Nystrom et al.	Jun 1997	A
5639477	Maruyama et al.	Jun 1997	A
5639640	Reddy et al.	Jun 1997	A
5645850	Bezwada et al.	Jul 1997	A
5648088	Bezwada et al.	Jul 1997	A
5650173	Ramstack et al.	Jul 1997	A
5654008	Herbert et al.	Aug 1997	A
5654010	Johnson et al.	Aug 1997	A
5656207	Bernstein et al.	Aug 1997	A
5656299	Kino et al.	Aug 1997	A
5658593	Orly et al.	Aug 1997	A
5660847	Magruder et al.	Aug 1997	A
5660858	Parikh et al.	Aug 1997	A
5660861	Jao et al.	Aug 1997	A
5667808	Johnson et al.	Sep 1997	A
5668170	Gyory	Sep 1997	A
5676942	Testa et al.	Oct 1997	A
5686097	Taskovich et al.	Nov 1997	A
5688801	Mesens et al.	Nov 1997	A
5690925	Gray et al.	Nov 1997	A
5690952	Magruder et al.	Nov 1997	A
5697113	Shatz et al.	Dec 1997	A
5698213	Jamiolkowski et al.	Dec 1997	A
5700486	Canal et al.	Dec 1997	A
5700583	Jamiolkowski et al.	Dec 1997	A
5703200	Bezwada et al.	Dec 1997	A
5707644	Ilium	Jan 1998	A
5711967	Juch	Jan 1998	A
5713847	Howard, III et al.	Feb 1998	A
5718022	Herrero-Vanrell	Feb 1998	A
5728088	Margruder et al.	Mar 1998	A
5728396	Peery et al.	Mar 1998	A
5733572	Unger et al.	Mar 1998	A
5736159	Chen et al.	Apr 1998	A
5738845	Imakawa	Apr 1998	A
5747058	Tipton et al.	May 1998	A
5756450	Hahn et al.	May 1998	A
5767251	Reddy et al.	Jun 1998	A
5770231	Mesens et al.	Jun 1998	A
5782396	Mastri et al.	Jul 1998	A
5792477	Rickey et al.	Aug 1998	A
5795591	Lee et al.	Aug 1998	A
5795779	McCormick et al.	Aug 1998	A
5807876	Armistead et al.	Sep 1998	A
5814323	Lyle	Sep 1998	A
D399821	Tyneski et al.	Oct 1998	S
5817129	Labrecque et al.	Oct 1998	A
5830501	Dong et al.	Nov 1998	A
5836935	Ashton et al.	Nov 1998	A
5843891	Sherman	Dec 1998	A
5844017	Jamiolkowski et al.	Dec 1998	A
5851451	Takechi et al.	Dec 1998	A
5858746	Hubbell et al.	Jan 1999	A
5859150	Jamiolkowski et al.	Jan 1999	A
5861166	Eckenhoff	Jan 1999	A
5871770	Margruder et al.	Feb 1999	A
5871778	Kino et al.	Feb 1999	A
5874388	Hsu	Feb 1999	A
5876746	Jona et al.	Mar 1999	A
5882676	Lee et al.	Mar 1999	A
D408917	Hacker	Apr 1999	S
5904935	Eckenhoff et al.	May 1999	A
5906816	Soos et al.	May 1999	A
5906830	Farinas et al.	May 1999	A
5908621	Glue et al.	Jun 1999	A
5916598	Rickey et al.	Jun 1999	A
5922253	Herbert et al.	Jul 1999	A
5928666	Farinas et al.	Jul 1999	A
5932547	Stevenson et al.	Aug 1999	A
5938654	Wong et al.	Aug 1999	A
5939286	Johnson et al.	Aug 1999	A
5942223	Bazer et al.	Aug 1999	A
5942253	Gombotz et al.	Aug 1999	A
5945126	Thanoo et al.	Aug 1999	A
5948430	Zerbe et al.	Sep 1999	A
5958909	Habener	Sep 1999	A
D415073	Meehan et al.	Oct 1999	S
5962023	Jamiolkowski et al.	Oct 1999	A
5965168	Mesens et al.	Oct 1999	A
5972370	Eckenhoff et al.	Oct 1999	A
5972373	Yajima et al.	Oct 1999	A
5976109	Heruth	Nov 1999	A
5980945	Ruiz	Nov 1999	A
5981719	Woiszwillo et al.	Nov 1999	A
5984890	Gast et al.	Nov 1999	A
5985305	Peery et al.	Nov 1999	A
5989463	Tracy et al.	Nov 1999	A
5997527	Gumucio et al.	Dec 1999	A
5997902	Maruyama et al.	Dec 1999	A
6007805	Foster et al.	Dec 1999	A
6017545	Modi	Jan 2000	A
6022561	Carlsson et al.	Feb 2000	A
6023802	King	Feb 2000	A
6029361	Newman	Feb 2000	A
6056718	Funderburk et al.	May 2000	A
6060450	Soos et al.	May 2000	A
6069133	Carlo et al.	May 2000	A
6074660	Jamiolkowski et al.	Jun 2000	A
6074673	Guillen	Jun 2000	A
6100346	Jamiolkowski et al.	Aug 2000	A
6110503	Rickey et al.	Aug 2000	A
6113938	Chen et al.	Sep 2000	A
6113947	Cleland et al.	Sep 2000	A
6120787	Gustafsson et al.	Sep 2000	A
6124261	Stevenson et al.	Sep 2000	A
6124281	Lewis et al.	Sep 2000	A
6127520	Ueda et al.	Oct 2000	A
6129761	Hubbell	Oct 2000	A
6130200	Brodbeck et al.	Oct 2000	A
6132420	Dionne et al.	Oct 2000	A
6133249	Hills	Oct 2000	A
6133429	Davis et al.	Oct 2000	A
6147168	Jamiolkowski et al.	Nov 2000	A
6156331	Peery et al.	Dec 2000	A
6172046	Albrecht	Jan 2001	B1
6174547	Dong et al.	Jan 2001	B1
6177096	Zerbe et al.	Jan 2001	B1
6183461	Matsuura et al.	Feb 2001	B1
6187095	Labrecque et al.	Feb 2001	B1
6190350	Davis et al.	Feb 2001	B1
6190700	Okada et al.	Feb 2001	B1
6190702	Takada et al.	Feb 2001	B1
6191102	DiMarchi et al.	Feb 2001	B1
6204022	Johnson et al.	Mar 2001	B1
6217893	Pellet et al.	Apr 2001	B1
6217906	Gumucio et al.	Apr 2001	B1
6217908	Mathiowitz et al.	Apr 2001	B1
6218431	Schoen et al.	Apr 2001	B1
6224894	Jamiolkowski et al.	May 2001	B1
6235712	Stevenson et al.	May 2001	B1
6245349	Yiv et al.	Jun 2001	B1
6245357	Edgren et al.	Jun 2001	B1
6248112	Gambale et al.	Jun 2001	B1
6251435	Jamiolkowski et al.	Jun 2001	B1
D445975	Stratford	Jul 2001	S
6258377	New et al.	Jul 2001	B1
6261584	Peery et al.	Jul 2001	B1
6268343	Knudsen et al.	Jul 2001	B1
6270700	Ignatious	Aug 2001	B1
6270787	Ayer	Aug 2001	B1
6277413	Sankaram	Aug 2001	B1
6283949	Roorda	Sep 2001	B1
6284264	Zerbe et al.	Sep 2001	B1
6284725	Coolidge et al.	Sep 2001	B1
6284727	Kim et al.	Sep 2001	B1
6287295	Chen et al.	Sep 2001	B1
6329336	Bridon et al.	Dec 2001	B1
6331311	Brodbeck et al.	Dec 2001	B1
6372218	Cummins, Jr.	Apr 2002	B1
6372256	Jamiolkowski et al.	Apr 2002	B2
6375978	Kleiner et al.	Apr 2002	B1
6395292	Peery et al.	May 2002	B2
6403655	Bezwada et al.	Jun 2002	B1
6419952	Wong et al.	Jul 2002	B2
6433144	Morris et al.	Aug 2002	B1
6436091	Harper et al.	Aug 2002	B1
6447522	Gambale et al.	Sep 2002	B2
6451974	Hansen	Sep 2002	B1
6458385	Jamiolkowski et al.	Oct 2002	B2
6458387	Scott et al.	Oct 2002	B1
6458924	Knudsen et al.	Oct 2002	B2
6461605	Cutler et al.	Oct 2002	B1
6464688	Harper et al.	Oct 2002	B1
6468961	Brodbeck et al.	Oct 2002	B1
6471688	Harper et al.	Oct 2002	B1
6472512	LaFleur et al.	Oct 2002	B1
6485706	McCoy et al.	Nov 2002	B1
6495164	Ramstack et al.	Dec 2002	B1
6506724	Hiles et al.	Jan 2003	B1
6508808	Carr et al.	Jan 2003	B1
6514500	Bridon et al.	Feb 2003	B1
6514517	Jamilolkowski et al.	Feb 2003	B2
6524305	Peterson et al.	Feb 2003	B1
6528093	Kamei et al.	Mar 2003	B1
6528486	Larsen et al.	Mar 2003	B1
D472896	Peiker	Apr 2003	S
6541021	Johnson et al.	Apr 2003	B1
6544252	Theeuwes et al.	Apr 2003	B1
6547250	Noble	Apr 2003	B1
6551613	Dong et al.	Apr 2003	B1
6569420	Chen et al.	May 2003	B2
6572890	Faour et al.	Jun 2003	B2
6579851	Goeke et al.	Jun 2003	B2
6592887	Zerbe et al.	Jul 2003	B2
6593295	Bridon et al.	Jul 2003	B2
6635268	Peery et al.	Oct 2003	B2
6667061	Ramstack et al.	Dec 2003	B2
6670368	Breault et al.	Dec 2003	B1
6673767	Brodbeck et al.	Jan 2004	B1
6682522	Carr et al.	Jan 2004	B2
6703225	Kojima et al.	Mar 2004	B1
6703359	Young et al.	Mar 2004	B1
6706689	Coolidge et al.	Mar 2004	B2
6709671	Zerbe et al.	Mar 2004	B2
6720407	Hughes et al.	Apr 2004	B1
6730328	Maskiwicz et al.	May 2004	B2
6767887	Hoffmann et al.	Jul 2004	B1
6821949	Bridon et al.	Nov 2004	B2
6833256	Pontzer et al.	Dec 2004	B1
6835194	Johnson et al.	Dec 2004	B2
6840931	Peterson et al.	Jan 2005	B2
6849708	Habener	Feb 2005	B1
6849714	Bridon et al.	Feb 2005	B1
6858576	Young et al.	Feb 2005	B1
6872700	Young et al.	Mar 2005	B1
6875748	Manthorpe et al.	Apr 2005	B2
6887470	Bridon et al.	May 2005	B1
6887849	Bridon et al.	May 2005	B2
6899887	Ayer	May 2005	B2
6899898	Albayrak	May 2005	B2
6902744	Kolterman et al.	Jun 2005	B1
6903186	Dong	Jun 2005	B1
6913767	Cleland et al.	Jul 2005	B1
6923800	Chen et al.	Aug 2005	B2
6924264	Prickett et al.	Aug 2005	B1
6939556	Lautenbach	Sep 2005	B2
6956026	Beeley et al.	Oct 2005	B2
6969702	Bertilsson et al.	Nov 2005	B2
6976981	Ayer	Dec 2005	B2
6989366	Beeley et al.	Jan 2006	B2
6992065	Okumu	Jan 2006	B2
6997922	Theeuwes et al.	Feb 2006	B2
7014636	Gilbert	Mar 2006	B2
7022674	DeFelippis et al.	Apr 2006	B2
7041646	Pan	May 2006	B2
7074423	Fereira et al.	Jul 2006	B2
7084243	Glaesner et al.	Aug 2006	B2
7101567	Sano et al.	Sep 2006	B1
7101843	Glaesner et al.	Sep 2006	B2
7112335	Lautenbach	Sep 2006	B2
7115569	Beeley et al.	Oct 2006	B2
7138375	Beeley et al.	Nov 2006	B2
7138486	Habener et al.	Nov 2006	B2
7141547	Rosen et al.	Nov 2006	B2
7144863	DeFelippis et al.	Dec 2006	B2
7153825	Young et al.	Dec 2006	B2
7157555	Beeley et al.	Jan 2007	B1
7163688	Peery et al.	Jan 2007	B2
7163697	Hanes et al.	Jan 2007	B2
7199217	DiMarchi et al.	Apr 2007	B2
7205409	Pei et al.	Apr 2007	B2
7207982	Dionne et al.	Apr 2007	B2
7241457	Chen et al.	Jul 2007	B2
7258869	Berry et al.	Aug 2007	B1
D555589	Hussaini et al.	Nov 2007	S
7297761	Beeley et al.	Nov 2007	B2
7316680	Gilbert	Jan 2008	B2
7393827	Nadler	Jul 2008	B2
7407499	Trautman	Aug 2008	B2
7442682	Kitaura et al.	Oct 2008	B2
7456254	Wright et al.	Nov 2008	B2
7459432	Cowley et al.	Dec 2008	B2
7521423	Young et al.	Apr 2009	B2
7563871	Wright et al.	Jul 2009	B2
7589169	Bolotin	Sep 2009	B2
7612176	Wright et al.	Nov 2009	B2
7635463	Bolotin et al.	Dec 2009	B2
D608447	Meyer et al.	Jan 2010	S
7655254	Dennis et al.	Feb 2010	B2
7655257	Peery et al.	Feb 2010	B2
7666835	Bloom et al.	Feb 2010	B2
7682356	Alessi et al.	Mar 2010	B2
7727519	Moran	Jun 2010	B2
7731947	Eliaz et al.	Jun 2010	B2
7736665	Patel et al.	Jun 2010	B2
7741269	Young et al.	Jun 2010	B2
7790140	Bolotin	Sep 2010	B2
7825091	Bloom et al.	Nov 2010	B2
7829109	Chen et al.	Nov 2010	B2
7833543	Gibson et al.	Nov 2010	B2
7879028	Alessi et al.	Feb 2011	B2
7879794	Weyer et al.	Feb 2011	B2
7919109	Berry et al.	Apr 2011	B2
7928065	Young et al.	Apr 2011	B2
D638478	Block	May 2011	S
7959938	Rohloff et al.	Jun 2011	B2
7964183	Eliaz et al.	Jun 2011	B2
8039432	Bridon et al.	Oct 2011	B2
8048438	Berry et al.	Nov 2011	B2
8052996	Lautenbach et al.	Nov 2011	B2
8058233	Cowley et al.	Nov 2011	B2
8101576	Bloom	Jan 2012	B2
8114430	Rohloff et al.	Feb 2012	B2
8114437	Rohloff et al.	Feb 2012	B2
8158150	Lautenbach et al.	Apr 2012	B2
8173150	Berry et al.	May 2012	B2
8202836	Moore et al.	Jun 2012	B2
8206745	Rohloff et al.	Jun 2012	B2
8211467	Rohloff et al.	Jul 2012	B2
8217001	Cowley et al.	Jul 2012	B2
8231859	Bolotin et al.	Jul 2012	B2
8257682	Bolotin et al.	Sep 2012	B2
8257691	Eliaz et al.	Sep 2012	B2
8262667	Silver et al.	Sep 2012	B1
8263545	Levy et al.	Sep 2012	B2
8263736	Berry	Sep 2012	B2
8268341	Berry	Sep 2012	B2
8273365	Lautenbach et al.	Sep 2012	B2
8273713	Pittner et al.	Sep 2012	B2
D669589	Delaey	Oct 2012	S
8277776	Bolotin et al.	Oct 2012	B2
8278267	Weyer et al.	Oct 2012	B2
8288338	Young et al.	Oct 2012	B2
8298561	Alessi et al.	Oct 2012	B2
8299025	Alessi et al.	Oct 2012	B2
8343140	Alessi et al.	Jan 2013	B2
8367095	Lautenbach et al.	Feb 2013	B2
8372424	Berry et al.	Feb 2013	B2
D678889	Chiu	Mar 2013	S
8398967	Eliaz et al.	Mar 2013	B2
8440226	Rohloff et al.	May 2013	B2
8460694	Rohloff et al.	Jun 2013	B2
8470353	Lautenbach et al.	Jun 2013	B2
8801700	Alessi et al.	Aug 2014	B2
8815802	Kalthoff et al.	Aug 2014	B2
8858621	Oba et al.	Oct 2014	B2
8865202	Zerbe et al.	Oct 2014	B2
8888745	Van Der Graaf et al.	Nov 2014	B2
8926595	Alessi et al.	Jan 2015	B2
8940316	Alessi et al.	Jan 2015	B2
8992961	Berry et al.	Mar 2015	B2
8992962	Lautenbach et al.	Mar 2015	B2
9044209	Dayton et al.	Jun 2015	B2
9078900	Kuzma et al.	Jul 2015	B2
9095553	Rohloff et al.	Aug 2015	B2
9241722	Yu	Jan 2016	B2
D750764	DeSocio	Mar 2016	S
9332995	Russo	May 2016	B2
9526763	Rohloff et al.	Dec 2016	B2
9539200	Lautenbach	Jan 2017	B2
9572889	Alessi et al.	Feb 2017	B2
D789539	Kleiner et al.	Jun 2017	S
D789540	Gyorgy	Jun 2017	S
9682127	Alessi et al.	Jun 2017	B2
RE46577	Collins et al.	Oct 2017	E
9889085	Alessi et al.	Feb 2018	B1
10501517	Blackwell	Dec 2019	B2
20010012511	Bezwada et al.	Aug 2001	A1
20010021377	Jamiolkowski et al.	Sep 2001	A1
20010021822	Ayer	Sep 2001	A1
20010022974	Ayer	Sep 2001	A1
20010026793	Jamiolkowski et al.	Oct 2001	A1
20010027311	Chen et al.	Oct 2001	A1
20010031790	Beisswenger	Nov 2001	A1
20010036472	Wong et al.	Nov 2001	A1
20010040326	Balczun	Nov 2001	A1
20020001631	Okumu	Jan 2002	A1
20020004481	Cleland et al.	Jan 2002	A1
20020012818	Ruppi et al.	Jan 2002	A1
20020034532	Brodbeck et al.	Mar 2002	A1
20020037309	Jaworowicz et al.	Mar 2002	A1
20020048600	Bhatt et al.	Apr 2002	A1
20020098180	Lei	Jul 2002	A1
20020136848	Yoshii et al.	Sep 2002	A1
20020137666	Beeley et al.	Sep 2002	A1
20020141985	Pittner et al.	Oct 2002	A1
20020197185	Jamiolkowski et al.	Dec 2002	A1
20020197235	Moran	Dec 2002	A1
20030032947	Harper et al.	Feb 2003	A1
20030044467	Brodbeck et al.	Mar 2003	A1
20030045454	Okumu et al.	Mar 2003	A1
20030059376	Libbey et al.	Mar 2003	A1
20030060425	Ahlem et al.	Mar 2003	A1
20030097121	Jolly et al.	May 2003	A1
20030104063	Babcock et al.	Jun 2003	A1
20030108608	Laridon et al.	Jun 2003	A1
20030108609	Berry et al.	Jun 2003	A1
20030113380	Ramstack et al.	Jun 2003	A1
20030114837	Peterson et al.	Jun 2003	A1
20030118660	Rickey et al.	Jun 2003	A1
20030138403	Drustrup	Jul 2003	A1
20030138491	Tracy et al.	Jul 2003	A1
20030157178	Chen et al.	Aug 2003	A1
20030170289	Chen et al.	Sep 2003	A1
20030180364	Chen et al.	Sep 2003	A1
20030186858	Arentsen	Oct 2003	A1
20030191099	Bohlmann et al.	Oct 2003	A1
20030211974	Brodbeck et al.	Nov 2003	A1
20030215515	Truong-Le et al.	Nov 2003	A1
20040001689	Goldsmith et al.	Jan 2004	A1
20040001889	Chen et al.	Jan 2004	A1
20040002442	Pan et al.	Jan 2004	A1
20040022859	Chen et al.	Feb 2004	A1
20040024068	Levy et al.	Feb 2004	A1
20040024069	Chen et al.	Feb 2004	A1
20040029784	Hathaway	Feb 2004	A1
20040039376	Peery et al.	Feb 2004	A1
20040097906	Fereira et al.	May 2004	A1
20040101557	Gibson et al.	May 2004	A1
20040102762	Gilbert	May 2004	A1
20040115236	Chan et al.	Jun 2004	A1
20040142867	Oi et al.	Jul 2004	A1
20040142902	Ker-Boudier	Jul 2004	A1
20040151753	Chen et al.	Aug 2004	A1
20040157951	Wolf	Aug 2004	A1
20040198654	Glaesner et al.	Oct 2004	A1
20040209801	Brand et al.	Oct 2004	A1
20040224903	Berry et al.	Nov 2004	A1
20040225113	LaFleur et al.	Nov 2004	A1
20040243106	Ayer	Dec 2004	A1
20040265273	Li et al.	Dec 2004	A1
20040266683	Hathaway et al.	Dec 2004	A1
20040266692	Young et al.	Dec 2004	A1
20050004557	Russell	Jan 2005	A1
20050008661	Fereira et al.	Jan 2005	A1
20050009742	Bertilsson et al.	Jan 2005	A1
20050010196	Fereira et al.	Jan 2005	A1
20050010942	Kim et al.	Jan 2005	A1
20050070883	Brown et al.	Mar 2005	A1
20050070927	Feinberg	Mar 2005	A1
20050079200	Rathenow et al.	Apr 2005	A1
20050079202	Chen et al.	Apr 2005	A1
20050095284	Trautman	May 2005	A1
20050101943	Ayer et al.	May 2005	A1
20050106214	Chen	May 2005	A1
20050112188	Eliaz et al.	May 2005	A1
20050118206	Luk et al.	Jun 2005	A1
20050118221	Blakely et al.	Jun 2005	A1
20050131386	Freeman et al.	Jun 2005	A1
20050131389	Peterson et al.	Jun 2005	A1
20050175701	Pan et al.	Aug 2005	A1
20050201980	Moran	Sep 2005	A1
20050215475	Ong et al.	Sep 2005	A1
20050216087	Zucherman, Jr. et al.	Sep 2005	A1
20050266087	Junnarkar et al.	Dec 2005	A1
20050271702	Wright et al.	Dec 2005	A1
20050276856	Fereira et al.	Dec 2005	A1
20050281879	Chen et al.	Dec 2005	A1
20060013879	Brodbeck et al.	Jan 2006	A9
20060014678	Cowley et al.	Jan 2006	A1
20060030526	Liu et al.	Feb 2006	A1
20060069029	Kolterman et al.	Mar 2006	A1
20060073182	Wong et al.	Apr 2006	A1
20060084604	Kitaura et al.	Apr 2006	A1
20060084922	Botha	Apr 2006	A1
20060094652	Levy et al.	May 2006	A1
20060094693	Aziz et al.	May 2006	A1
20060106399	Taras et al.	May 2006	A1
20060141040	Chen et al.	Jun 2006	A1
20060142234	Chen et al.	Jun 2006	A1
20060160736	Nadler	Jul 2006	A1
20060178304	Juul-Mortensen et al.	Aug 2006	A1
20060193918	Rohloff et al.	Aug 2006	A1
20060216242	Rohloff et al.	Sep 2006	A1
20060224145	Gills	Oct 2006	A1
20060233841	Brodbeck et al.	Oct 2006	A1
20060246138	Rohloff et al.	Nov 2006	A1
20060251618	Dennis et al.	Nov 2006	A1
20060263433	Ayer et al.	Nov 2006	A1
20060264890	Moberg et al.	Nov 2006	A1
20060280795	Penhasi et al.	Dec 2006	A1
20060293232	Levy et al.	Dec 2006	A1
20070027105	Junnarkar et al.	Feb 2007	A1
20070149011	Kent et al.	Jun 2007	A1
20070166352	Wright et al.	Jul 2007	A1
20070207958	Bridon et al.	Sep 2007	A1
20070248572	Moran et al.	Oct 2007	A1
20070281024	Lautenbach et al.	Dec 2007	A1
20080020016	Li et al.	Jan 2008	A1
20080038316	Wong et al.	Feb 2008	A1
20080064636	Bloom et al.	Mar 2008	A1
20080065090	Scribner et al.	Mar 2008	A1
20080091176	Alessi et al.	Apr 2008	A1
20080110515	Angelosanto et al.	May 2008	A1
20080112994	Junnarkar et al.	May 2008	A1
20080200383	Jennings et al.	Aug 2008	A1
20080207512	Roth et al.	Aug 2008	A1
20080208194	Bickenbach	Aug 2008	A1
20080226625	Berry et al.	Sep 2008	A1
20080226689	Berry et al.	Sep 2008	A1
20080260838	Hokenson et al.	Oct 2008	A1
20080260840	Alessi et al.	Oct 2008	A1
20080269725	Deem et al.	Oct 2008	A1
20080312157	Levy et al.	Dec 2008	A1
20090022727	Houston et al.	Jan 2009	A1
20090036364	Levy et al.	Feb 2009	A1
20090042781	Petersen et al.	Feb 2009	A1
20090074734	Rottiers	Mar 2009	A1
20090087408	Berry et al.	Apr 2009	A1
20090156474	Roth et al.	Jun 2009	A1
20090163447	Maggio	Jun 2009	A1
20090186817	Ghosh et al.	Jul 2009	A1
20090202481	Li et al.	Aug 2009	A1
20090202608	Alessi et al.	Aug 2009	A1
20090209460	Young et al.	Aug 2009	A1
20090210019	Kim et al.	Aug 2009	A1
20090215694	Kolterman et al.	Aug 2009	A1
20090234392	Dziedzic	Sep 2009	A1
20090247463	Wright et al.	Oct 2009	A1
20090254143	Tweden et al.	Oct 2009	A1
20090286723	Levy et al.	Nov 2009	A1
20090312246	Baron et al.	Dec 2009	A1
20100092566	Alessi et al.	Apr 2010	A1
20100105627	Salama et al.	Apr 2010	A1
20100144621	Kim et al.	Jun 2010	A1
20100185184	Alessi et al.	Jul 2010	A1
20100297209	Rohloff et al.	Nov 2010	A1
20100298840	Schwartz	Nov 2010	A1
20110076317	Alessi et al.	Mar 2011	A1
20110091527	Moonen et al.	Apr 2011	A1
20110104111	Rohloff et al.	May 2011	A1
20110152181	Alsina-Fernandez et al.	Jun 2011	A1
20110152182	Alsina-Fernandez et al.	Jun 2011	A1
20110160708	Berry et al.	Jun 2011	A1
20110166554	Alessi et al.	Jul 2011	A1
20110264077	Rohloff et al.	Oct 2011	A1
20110306549	Tatarkiewicz et al.	Dec 2011	A1
20120208755	Leung	Aug 2012	A1
20130030417	Alessi	Jan 2013	A1
20130034210	Rohloff et al.	Feb 2013	A1
20130052237	Eliaz et al.	Feb 2013	A1
20140058425	Porat	Feb 2014	A1
20140121741	Bennett et al.	May 2014	A1
20140257272	Clark, III et al.	Sep 2014	A1
20140378900	Alessi et al.	Dec 2014	A1
20150001118	Selepack et al.	Jan 2015	A1
20150057227	Leung	Feb 2015	A1
20150133791	Sato et al.	May 2015	A1
20150231062	Lautenbach et al.	Aug 2015	A1
20150231256	Berry et al.	Aug 2015	A1
20150297509	Schwarz	Oct 2015	A1
20160022582	Alessi et al.	Jan 2016	A1
20160030337	Kuzma et al.	Feb 2016	A1
20160354115	Smith et al.	Dec 2016	A1
20160354305	Alessi et al.	Dec 2016	A1
20170056476	Rohloff et al.	Mar 2017	A1
20170079906	Alessi et al.	Mar 2017	A1
20170119854	Alessi et al.	May 2017	A1
20170119855	Berry et al.	May 2017	A1
20170181964	Lautenbach et al.	Jun 2017	A1
20170252409	Leung	Sep 2017	A1
20170273706	Mirza et al.	Sep 2017	A1
20170319470	Eliaz et al.	Nov 2017	A1
20170319662	Berry et al.	Nov 2017	A1
20170348392	Rohloff et al.	Dec 2017	A1
20170368145	Alessi et al.	Dec 2017	A1
20180185451	Young et al.	Jul 2018	A1

Foreign Referenced Citations (179)

Number	Date	Country
0052510	May 1982	EP
0079405	May 1983	EP
0254394	Jan 1988	EP
0295411	Dec 1988	EP
0302582	Feb 1989	EP
0368339	May 1990	EP
0373867	Jun 1990	EP
0431942	Jun 1991	EP
0486959	May 1992	EP
0521586	Jan 1993	EP
0596161	May 1994	EP
0379147	Sep 1994	EP
0627231	Dec 1994	EP
0729747	May 1997	EP
0771817	May 1997	EP
0841359	May 1998	EP
0767689	Jun 1999	EP
1046399	Oct 2000	EP
1084703	Mar 2001	EP
1300129	Apr 2003	EP
1300173	Apr 2003	EP
1600187	Jan 2009	EP
2133073	Dec 2009	EP
2020990	Sep 2010	EP
640907	Jul 1928	FR
1049104	Nov 1966	GB
1518683	Jul 1978	GB
2501400	Oct 2013	GB
H02124814	May 1990	JP
H07196479	Aug 1995	JP
9241153	Sep 1997	JP
11-100353	Apr 1999	JP
2006213727	Aug 2006	JP
9100160	Apr 1994	NL
592113	Aug 2012	NZ
200634060	Oct 2006	TW
WO1989003678	May 1989	WO
WO1990013285	Nov 1990	WO
WO1990013361	Nov 1990	WO
WO1990013780	Nov 1990	WO
WO 9107160	May 1991	WO
WO1992019241	Nov 1992	WO
WO 9306819	Apr 1993	WO
WO 9306821	Apr 1993	WO
WO 93008832	May 1993	WO
WO 9309763	May 1993	WO
WO 9323083	Nov 1993	WO
WO 9409743	May 1994	WO
WO1994010982	May 1994	WO
WO 9421262	Sep 1994	WO
WO 9501167	Jan 1995	WO
WO 9509006	Apr 1995	WO
WO 9509007	Apr 1995	WO
WO1995013799	May 1995	WO
WO 9534285	Dec 1995	WO
WO 96001134	Jan 1996	WO
WO 96003116	Feb 1996	WO
WO1996036317	Nov 1996	WO
WO 9639142	Dec 1996	WO
WO 9640049	Dec 1996	WO
WO 9640139	Dec 1996	WO
WO 9640355	Dec 1996	WO
WO1996040049	Dec 1996	WO
WO 9715289	May 1997	WO
WO 9715296	May 1997	WO
WO 9728181	Aug 1997	WO
WO1997031943	Sep 1997	WO
WO1997044039	Nov 1997	WO
WO 9746204	Dec 1997	WO
WO 9747339	Dec 1997	WO
WO 9800152	Jan 1998	WO
WO 9800157	Jan 1998	WO
WO 9800158	Jan 1998	WO
WO 9802169	Jan 1998	WO
WO1997041837	Feb 1998	WO
WO1998007412	Feb 1998	WO
WO 9816250	Apr 1998	WO
WO 9817315	Apr 1998	WO
WO 9820930	May 1998	WO
WO 9827960	Jul 1998	WO
WO 98027962	Jul 1998	WO
WO 9827963	Jul 1998	WO
WO 98030231	Jul 1998	WO
WO 9832463	Jul 1998	WO
WO1998030231	Jul 1998	WO
WO 9842317	Oct 1998	WO
WO 9847487	Oct 1998	WO
WO 9851282	Nov 1998	WO
WO 9903453	Jan 1999	WO
WO 9904767	Feb 1999	WO
WO 99004768	Feb 1999	WO
WO1999012549	Mar 1999	WO
WO 9916419	Apr 1999	WO
WO 99025728	May 1999	WO
WO 9929306	Jun 1999	WO
WO 99033446	Jul 1999	WO
WO 9933449	Jul 1999	WO
WO 9939700	Aug 1999	WO
WO 99040788	Aug 1999	WO
9944659	Sep 1999	WO
WO 99044659	Sep 1999	WO
WO 99062501	Dec 1999	WO
WO 99064061	Dec 1999	WO
WO 00013663	Mar 2000	WO
WO 00029206	May 2000	WO
WO 00038652	Jul 2000	WO
WO 00039280	Jul 2000	WO
WO 00040273	Jul 2000	WO
WO 00041548	Jul 2000	WO
WO 00045790	Aug 2000	WO
WO 00054745	Sep 2000	WO
WO2000059476	Oct 2000	WO
WO 00066138	Nov 2000	WO
WO 00067728	Nov 2000	WO
WO2000066087	Nov 2000	WO
WO2001019345	Mar 2001	WO
WO2001028525	Apr 2001	WO
WO 01043528	Jun 2001	WO
WO 01051041	Jul 2001	WO
WO 0178683	Oct 2001	WO
WO 02028366	Apr 2002	WO
WO 02036072	May 2002	WO
WO 02043800	Jun 2002	WO
WO 02045752	Jun 2002	WO
WO 0247716	Jun 2002	WO
WO 02067895	Sep 2002	WO
WO 02069983	Sep 2002	WO
WO 0276344	Oct 2002	WO
WO 02085428	Oct 2002	WO
WO 03000230	Jan 2003	WO
WO 03007981	Jan 2003	WO
WO 03011892	Feb 2003	WO
WO 03024357	Mar 2003	WO
WO 03024503	Mar 2003	WO
WO2003020245	Mar 2003	WO
WO 03030923	Apr 2003	WO
WO 03041684	May 2003	WO
WO 03041757	May 2003	WO
WO 03053400	Jul 2003	WO
WO2003066585	Aug 2003	WO
WO 03072113	Sep 2003	WO
WO 03072133	Sep 2003	WO
WO 04002565	Jan 2004	WO
WO2004034975	Apr 2004	WO
WO2004035754	Apr 2004	WO
WO2004035762	Apr 2004	WO
WO2004036186	Apr 2004	WO
WO 04052336	Jun 2004	WO
WO 04056338	Jul 2004	WO
WO 04089335	Oct 2004	WO
WO2004103342	Dec 2004	WO
WO 05048930	Jun 2005	WO
WO 05048952	Jun 2005	WO
WO 05102293	Nov 2005	WO
WO20051 10425	Nov 2005	WO
WO2005102293	Nov 2005	WO
WO 06017772	Feb 2006	WO
WO 06023526	Mar 2006	WO
WO 06081279	Aug 2006	WO
WO 06083761	Aug 2006	WO
WO 06084139	Aug 2006	WO
WO 06086727	Aug 2006	WO
WO 06101815	Sep 2006	WO
WO 06111169	Oct 2006	WO
WO2006131730	Dec 2006	WO
WO 07024700	Mar 2007	WO
WO 07056681	May 2007	WO
WO 07075534	Jul 2007	WO
WO 07084460	Jul 2007	WO
WO 07133778	Nov 2007	WO
WO 07140416	Dec 2007	WO
WO 08021133	Feb 2008	WO
WO2008041245	Apr 2008	WO
WO 08061355	May 2008	WO
WO 08133908	Nov 2008	WO
WO 08134425	Nov 2008	WO
WO 09109927	Sep 2009	WO
WO2009143285	Nov 2009	WO
WO 2013004983	Jan 2013	WO

Non-Patent Literature Citations (259)

Entry
US 5,672,540 A, 09/1997, Minami et al. (withdrawn)
Akers, et al., “Formulation Design and Development of Parenteral Suspensions,” Journal of Parenteral Science & Technology, 41(3): 88-96 (1987).
Alonso, et al., “Determinants of Release Rate of Tetanus Vaccine from Polyester Microspheres,” Pharmaceutical Research, 10(7):945-953 (1993).
Beck, et al., “Poly(dl-lactide-co-glycolide)/norethisterone microcapsules: An injectable biodegradable contraceptive,” Biology of Reproduction, 28(1): 186-195 (1983).
Bodmeier and McGinity, “Solvent selection in the preparation of poly(dl-lactide) microspheres prepared by the solvent evaporation method,” International Journal of Pharmaceutics, 43(1-2): 179-186 (Apr. 1988).
Cha and Pitt, “A one-week subdermal delivery system for I-methadone based on biodegradable microcapsules,” Journal of Controlled Release, 7: 69-78 (1988).
Cha and Pitt, “The acceleration of degradation-controlled drug delivery from polyester microspheres,” Journal of Controlled Release, 8: 259-265 (1989).
Cohen, et al., “Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres,” Pharmaceutical Research, 8(6): 713-720 (1991).
Conti, et al., “Use of polylactic acid for the preparation of microparticulate drug delivery systems,” Journal of Microencapsulation, 9(2): 153-166 (1992).
Hodgman, et al., Eds., Handbook of Chemistry and Physics, 35th Edition, 1024-1025 (1953).
Jalil and Nixon, “Biodegradable poly(lactic acid) and poly(lactide-co-glycolide) microcapsules: Problems associated with preparative techniques and release properties,” Journal of Microencapsulation, 7(3): 297-325 (Jul.-Sep. 1990).
Lee and Timasheff, “The stabilization of proteins by sucrose,” J. Biological Chern., 256(14): 7193-7201 (Jul. 1981).
Li, et al., “Prediction of solvent removal profile and effect on properties for peptide-loaded PLGA microspheres prepared by solvent extraction/evaporation method,” Journal of Controlled Release, 37: 199-214 (1995).
Maa and Hsu, “Liquid-liquid emulsification by static mixers for use in microencapsulation,” Journal of Microencapsulation, 13(4): 419-433 (Jul.-Aug. 1996).
Maulding, et al., “Biodegradable microcapsules: Acceleration of polymeric excipient hydrolytic rate by incorporation of a basic medicament,” Journal of Controlled Release, 3: 103-117 (1986).
Mehta, et al.,“Peptide containing microspheres from low molecular weight and hydrophilic poly(d,l-lactide-co-glycolide),” Journal of Controlled Release, 41: 249-257 (1996).
Sah, et al., “A novel method of preparing PLGA microcapsules utilizing methylethyl ketone,” Pharmaceutical Research, 13(3): 360-367 (1996).
Sato, et al., “Porous biodegradable microspheres for controlled drug delivery. I. Assessment of processing conditions and solvent removal techniques,” Pharmaceutical Research, 5(1): 21-30 (1988).
Szayna, et al., “Exendin-4 decelerates food intake, weight gain, and fat deposition in Zucker rats,” Endocrinology, 141(6): 1936-1941 (2000).
Thomasin, et al., “A contribution to overcoming the problem of residual solvents in biodegradable microspheres prepared by coacervation,” Eur. J. Pharm. Biopharm., 42(1): 16-24 (1996).
van Santbrink and Fauser, “Urinary follicle-stimulating hormone for normogonadotropic colomiphene-resistant anovulatory infertility: Prospective, randomized comparison between low dose step-up and step-down dose regimens,” J. Clin. Endocrin. Metab., 82(11): 3597-3602 (1997).
Tracy et al., “Factors affecting the degradation rate of poly(lactide-co-glycolide) microspheresin vivo and in vitro.” Biomaterials. 20(11:): 1057-1062 (1999).
Ertl et al., “Poly (DL-lactide-co-glycolide) microspheres as carriers for peptide vaccines,” Vaccine 14(9):879-885.(1996).
Thompson et al., “Biodegradable microspheres as a delivery system for rismorelin porcine, a porcine-growth-hormone-releasing hormone,” Journal of Controlled Release 43(1):9-22 (1997).
Glumetza Brochure 2009, 13 Pages.
EROWID,“Introduction to the Federal Controlled Substance Analog Act” 2001, 4 pages.
Li et al. (“Glucagon-Like Peptide-I Receptor Agonists Versus Insulin Glargine for Type 2 Diabetes Mellitus: A Systematic Review and Meta-Analysis of Randomized Controlled Trials” in Current Therapeutic Research, vol. 71, No. 4, Aug. 2010.
Adolf, “Human interferon omega-a review,” Mult. Sclr. 1:S44-47 (1995).
Costaniino et al., “Protein Spray Freeze Drying. 2. Effect of Formulation Variables on particle Size and Stability,” J. Pharm. Sci. 91:388-395 (2002).
Henry et al., “Comparing ITCA 650, continuous subcutaneous delivery of exenatide via DUROS® device, vs. twice daily exenatide injections in metformin-treated type 2 diabetes,” oral presentation at the 46th Annual Meeting of the European Association for the Study of Diabetes in Stockholm, Sweden , 21 pages (Sep. 20-24, 2010).
Huggins et al., “Synergistic antiviral effects of ribavirin and the C-nucleoside analogs tiazofurin and selenazofurin against togaviruses, bunyaviruses, and arenaviruses,” Antimicrobial Agents & Chemotherapy, 26(4):476-480 (1984).
Ishiwata et al., “Clinical effects of the recombinant feline interferon-omega on experimental parvovirus infection in beagle dogs,” J. Vet. Med. Sci. 60(8):911-917 (1998).
Johnson et al., “How interferons fight disease,” Sci. Am. 270(5):68-75 (May 1994).
Lublin et al., “Defining the clinical course of multiple sclerosis: results of an international survey,” Neurology. 46:907-911 (1996).
Madsbad, “Exenatide and liraglutide: different approaches to develop GLP-1 receptor agonists (incretin mimetics)—preclinical and clinical results,” Best Practice & Research Clinical Endocrinology & Metabolism 23:463-77 (2009).
Nielsen, “Incretin mimetics and DPP-IV inhibitors for the treatment of type 2 diabetes,” Drug Discovery Today 10(10):703-710 (May 15, 2005).
Palmeri et al., “5-Fluorouracil and recombinant α-interferon-2a in the treatment of advanced colorectal carcinoma: a dose optimization study,” J. Chemotherapy 2(5):327-330 (Oct. 1990).
Patti et al., “Natural interferon-b treatment of relapsing-remitting and secondary-progressive multiple sclerosis patients: two-year study,” Acta. Neurol. Scand. 100:283-289 (1999).
Paty et al., “Interferon beta-1 b is effective in relapsing-remitting multiple sclerosis,” Neurology 43:662-667 (1993).
PCT International Search Report for PCT/US2009/000916, 4 pages (dated Aug. 12, 2009).
“Intarcia Therapeutics Announces Final Results from a Phase 2 Study of Injectable Omega Interferon plus Ribavirin for the Treatment of Hepatitis C Genotype-1,” NLV Partners Press Coverage Portfolio News (Apr. 12, 2007) (Press Release).
Quianzon et al., “Lixisenatide-Once-daily Glucagon-like Peptide-1 Diabetes,” US Endocrinology 7(2):104-109 (2011).
Ratner et al., “Dose-dependent effects of the one-daily GLP-1 receptor agonist lixisenatide in patients with Type 2 diabetes inadequately controlled with metfinmin: a randomized, double-blind, placebo-controlled trial,” Diabetic Medicine 27(9): 1024-1032 (Sep. 2010).
Roberts et al., “The Evolution of the Type I Interferons1,” J. Interferon Cytokine Res. 18(10):805-816(0ct. 1998).
Rohloff et al., “DUROS Technology Delivers Peptides and Proteins at Consistent Rate Continuously for 3 to 12 Months,” J. Diabetes Sci. & Tech., 2(3):461-467 (May 1, 2008).
“Sequence Listings for International Patent Application Publication No. W02009109927, WIPO Patentscope”, http://patentscope.wipo.int/search/docservicepdf_pct/id00000008776887, 1 page (last visited Nov. 14, 2012).
Shire et al., “Challenges in the Development of High Protein Concentration Formulations,” J. Pharm. Sci. 93:1390-1402 (2004).
Smith, “Peripheral Neuro-hormones as a Strategy to Treat Obesity,” oral presentation at the 2007 Cardiometabolic Health Congress in Boston, MA, pp. 1-35 (Sep. 26-29, 2007).
Written Opinion for International Patent Application No. PCT/US2009/005629 (corresponding to U.S. Appl. No. 12/587,946), 5 pages (dated Apr. 15, 2011).
Zhang et al., “Efficacy observations of different dosages of interferon to treat 150 Hepatitis B carriers,” Current Physician 2(12):45-46 (1997).
Pratley et al., “Targeting Incretins in Type 2 Diabetes: Role of GLP-1 Receptor Agonists and DPP-4 Inhibitors,” Rev. Diabet. Stud., 5(2):73-94 (2008).
Gonzalez, et al., “Hemoglobin Alc: A Reliable and Accurate Test for Diabetes Care? A Prospective Study in Mexico,” Salud Publica Mex 55:462-468 (2013).
Ahn et al., “A New Approach to Search for the Bioactive Confirmation of Glucagon: Positional Cyclization Scanning” Journal of Medicinal Chemistry, vol. 44, No. 19, (2001): 3109-3116.
Glue et al., “A dose-ranging study of Peg-intron and ribavirin in chronic hepatitis C- safety, efficacy, and virological Yationale,” Prog. Abstr. 50th Annu. Mtg. Postgrad. Courses Am. Assn. Study Liver Dis., Dallas, TX(Nov.5-9, 1999) (Abstract 571).
McHutchison et al., “Open-label phase 1B study of hepatitis C viral dynamics with omega interferon treatment,” Hepatology 34(4):A333 (Oct. 1, 2001) (XP004716177)(Abstract Only).
Georgios, et al., “Pharmacokinetics and Tolerability of Exenatide Delivered by 7-Day Continuous Subcutaneous Infusion in Healthy Volunteers”, Advances in Therapy, Health Communications, Metuchen, NJ, US, vol. 32, No. 7, Jul. 10, 2015, pp. 650-661.
Yu et al., “Glucagon-like peptide 1 based therapy for type 2 diabetes”, World Journal of Pediatrics vol. 4, No. 1, Feb. 1, 2008, pp. 8-13.
Taylor et al., “Day-long subcutaneous infusion of exenatide lowers glycemia in patients with type 2 diabetes”, Horm Metab Res 37: 627-632 (2005).
Gao et al., “Target-Mediated Pharmacokinetics and Pharmacodynamics Model of Exendin-4 in Rats, Monkeys, and Humans,” Drug Metabolism and Disposition, vol. 40, No. 5, pp. 990-997 (2012).
Fioravante et al., “Weight loss and resting energy expenditure in patients with chronic hepatitis C before and during standard treatment”, Nutrition, 2012, pp. 630-634 (Year: 2012).
Kosmiski, “Energy expenditure in HIV infection”, Am J Clin Nutr, 2011, p. 1677S-82S (Year: 2011).
“Abstracts 2007,” Diabetologia Clinical & Experimental Diabetes & Metabolism, Springer, Berlin, Germany, vol. 50 S243 (Aug. 21, 2007) (paragraph [0586]) (XP002538652).
Jetschmann et al., “Open-label rising-dose study of omega interferon in IFN-naive patients with chronic hepatitis C,” Gastroenterology 122:A278-A347 (Apr. 1, 2002) (Abstract M1454).
Bray, “Gut Signals and Energy Balance: Ghrelin, Peptide YY, Leptin, and Amylin,” (Dec. 19, 2007) (slides and transcript for presentation at Medscape CME).
“Implantable infusion pumps: technology poised for takeoff,” BBI Newsletter 17(12):209-211 (Dec. 1994).
Adamson et al., “Phase I trial and pharmacokinetic study of all-trans-retinoic acid administered on an intermittent schedule in combination with interferon-alpha2a in pediatric patients with refractory cancer,” J. Clin. Oncol. 15(11):3330-3337 (Nov. 1997).
Adolf et al., “Monoclonal antibodies and enzyme immunoassays specific for human interferon (IFN) ω1: evidence that IFN-ω1 is a component of human leukocyte IFN,” Virology 175(2): 410-471 (Apr. 1990).
Adolf et al., “Antigenic structure of human interferon ω1 (Interferon αl11): comparison with other human interferons,” J. Gen. Virol. 68(6): 1669-1676 (Jun. 1987).
Adolf et al., “Purification and characterization of natural human interferon col,” J. Bio. Chem. 265(16):9290-9295 (Jun. 1990).
Adolf et al., “Human interferon col. isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein,” Biochim. Biophys. Acta 108(9): 167-174 (Jun. 1991).
Andrx Pharmaceu'Iicals, Llc, Anda for Concerta® Extended-Release Tablets, 6 pages (correspondence dated Sep. 6, 2005).
ASTM International, Annual Book of ASTM Standards, 8.02:208-211, 584-587 (1984).
Ansel et al., “Dosage Form Design: Pharmaceutical and Formulation Considerations,” Pharmaceutical Dosage Forms and Drug Delivery Systems, Ch. 3 at 87-92 (7th ed. Lippincott Williams & Wilkins 1999).
Ansel et al., “Modified-Release Dosage Forms and Drug Delivery Systems,” Pharmaceutical Dosage Forms and Drug Delivery Systems, Ch. 8 at 229-243 (7th ed. Lippincott Williams & Wilkins 1999).
Aulitzky, “ Successful Treatment of Metastatic Renal Cell Carcinoma With a Biologically Active Dose of Recombinant Interferon-Gama,” Journal of Clinical Oncology 7(12):1875-1884 (1989).
Hauck, “Engineer's Guide to Plastics,” Materials Engineering 5(72):3 8-45 (Jul. 17, 1972).
Bailon et al., “Rational Design of a Potent, Long-lasting Form of Interferon: A 40 kDa Branched Polyethylene Glycol-conjugated Interferon Alpha-2a for the Treatment of Hepatitis C,” Bioconjugate Chemistry 12(2): 195-202 (2001).
Bakan et al., “Physicochemical Characterization of a Synthetic Lipid Emulsion for Hepatocyte-Selective Delivery of Lipophilic Compounds: Application to Polyiodinated triglycerides as Contrast Agents for Computed Tomography,” J. Pharm. Sci., 85(9):908-914 (1996).
Bakhtiar et al., “Taking Delivery,” Soap Perfumery & Cosmetics 76(3):59-65 (2003) (liposomes in cosmetic delivery systems).
BALKWILL,F., “Interferons,” Lancet 1(8646): 1060-1063 (May 1989).
Bauer et al., “Non-aqueous emulsions as vehicles for capsule fillings,” Drug Dev. & Industrial Pharmacy 10(5):699-712 (1984).
Bekkering et al., “Estimation of early hepatitis C viral clearance in patients receiving daily interferon and ribavirin therapy using a mathematical model,” Hepatology 33(2):419-423 (Feb. 2001).
Bell et al., “Hamster preproglucagon contains the sequence of glucagon and two related peptides,” Nature 302:716-718 (1983).
Bell et al., “Impact of moisture on thermally induced denaturation and decomposition of lyophilized bovine somatotropin,” Drug Delivery Research & Dev. Biopolymers, (35):201-209 (1995).
Bertoncello et al., “Haematopoietic radioprotection by Cremophor EL: a polyethoxylated castor oil,” Int. J. Radiat. Biol. 67(l):57-64 (1995).
Bohlinder et al., “Use and characteristics of a novel lipid particle-forming matrix as a drug-carrier system,” Euro. J. Pharm. Sci. 2(4):271-279 (1994).
Bolinger et al., “Recombinant interferon γ for treatment of chronic granulomatous disease and other disorders,” Clin. Pharm. 11(10):834-850 (Oct. 1992).
Bonkovsky et al., “Outcomes research in chronic viral hepatitis C: effects of interferon therapy,” Can. J. Gastroenterol. 14(Supp. B):21B-29B (Jul.-Aug. 2000).
Borden et al., “Second-generation interferons for cancer: clinical targets,” Semin. Cancer Biol. 10(2): 125-144 (Apr. 2000).
Bouéet al., “Antiviral and antiluteolytic activity of recombinant bovine IFN-ol obtained from Pichia pastoris,” J. Interferon & Cytokine Res. 20:677-683 (2000).
Buckwold et al. “Antiviral activity of CHO-SS cell-derived human omega interferon and other human interferons against HCV RNA replicons and related viruses,” Antiviral Res. 73(2): 118-25 (Feb. 2007) (Epub Sep. 11, 2006).
Cantor, “Theory of lipid monolayers comprised of mixtures of flexible and stiff amphiphiles in anthermal solvents: fluid phase coexistence,” J. Chem. Physics 104(20):8082-8095 (1996).
CAS No. 56-81-5 (Nov. 16, 1984).
Chang et al., “Biodegradable polyester implants and suspension injection for sustained release of a cognitive enhancer,” Pharm. Tech. 20(1): 80-84 (1996).
Chapman et al., “Physical Studies of Phospholipids. VI. Thermotropic and Lyotropic Mesomorphism of Some 1,2-Diacylphosphatidylcholines (lecithins),” Chem. & Physics of Lipids 1(5):445-475 (1967).
Chaumeil, “Micronization: a method of improving the bioavailability of poorly soluble drugs,” Methods & Findings in Experimental & Clinical Pharmacology 20(3):211-215 (1998).
Clark et al., “The diabetic Zucker fatty rat,” Proc. Soc. Exp. Biol. 173(1):68-75 (1983).
Condino-Neto, “Interferon-Y improves splicing efficiency of CYBB gene transcripts in an interferon responsive variant of chronic granulomatous disease due to a splice site consensus region mutation,” Blood 95(11):3548-3554 (Jun. 2000).
Darney, “Subdermal progestin implant contraception,” Current Opinion in Obstetrics & Gynecology 3:470-476 (1991).
Das et al., “Reviewing Antisense Oligonucleotide Therapy: Part 2, Delivery Issues,” BioPharm, 2(11):44-51 (1999).
Dash et al., “Therapeutic applications of implantable drug delivery systems,” Journal of Pharmacological and Toxicological Methods, 40(1): 1-12 (1998).
Davis et al., “Durability of viral response to interferon alone or in combination with oral ribavirin in patients with chronic hepatitis C,” Prog. Abstr. 50th Annu. Mtg. Postgrad. Courses Am. Assn. Study Liver Dis., Dallas, TX (Nov. 5-9, 1999)(Abstract 570 ).
Deacon et al., “GLP-1-(9-36) amide reduces blood glucose in anesthetized pigs by a mechanism that does not involve insulin secretion,” Am. J. Physiol. Endocrinol. Metab., 282:E873-E879 (2002).
Desai et al., “Protein structure in the lyophilized state: a hydrogen isotope exchange/NMR study with bovine pancreatic trypsin inhibitor,” J. Am. Chem. Soc. 116(21):9420-9422 (1994).
Di Marco et al., “Combined treatment of relapse of chronic hepatitis C with high-dose a-2B interferon plus ribavirin for 6 or 12 months,” Prog. Abstr. 50th Annu. Mtg. Postgrad. Courses Am. Assn. Study Liver Dis., Dallas, TX (Nov. 5-9, 1999)(Abstract 569).
Dorr et al., “Phase I-II trial of interferon-alpha 2b by continuous subcutaneous infusion over 28 days,” J. Interferon Res. 8:717-725 (1988).
Uhlig et al., “The electro-osmotic actuation of implantable insulin micropumps,” J. Biomed. Materials Res. 17:931-943 (1983).
Efendic et al., “Overview of incretin hormones,” Horm. Metab. Res., 36(11-12):742-746 (2004).
Eissele et al., “Rat gastric somatostatin and gastrin release: interactions of exendin-4 and truncated glucagon-like peptide-1 (GLP-1) amide,” Life Sci., 55(8):629-634 (1994).
Elias et al., “Infusional Interleukin-2 and 5-fluorouracil with subcutaneous interferon-a for the treatment of patients with advanced renal cell carcinoma: a southwest oncology group Phase II study,” Cancer 89(3):597-603 (Aug. 2000).
Eng et al., “Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas,” J. Biol. Chem., 267(11):7402-7405 (1992).
Eng et al., “Purification and structure of exendin-3, a new pancreatic secretagogue isolated from Heloderma horridum venom,” J. Biol. Chem., 265(33):20259-20262 (1990).
Eppstein et al., “Biological activity of liposome-encapsulated murine interferon γ is mediated by a cell membrane receptor,” PNAS USA 82:3688-3692 (1985).
Eros et al., “Multiple phase emulsions as controlled drug delivery therapeutic systems,” Proc.-Conf. Colloid Chem. 193-196 (1993).
Fang et al., “The impact of baseline liver histology on virologic response to interferon a-2b±p ribavirin therapy in patients with chronic hepatitis C,” Prog. Abstr. 50th Annu. Mtg. Postgrad. Courses Am. Assn. Study Liver Dis., Dallas, TX (Nov. 5-9, 1999)(Abstract 572).
Felker et al., “The Rate of Transfer of Unesterified Cholesterol from Rat Erythrocytes to Emulsions Modeling Nascent Triglyceride-Rich Lipoproteins and Chylomicrons Depends on the Degree of Fluidity of the Surface,” J. Nutritional Biochem. 4(1):630-634 (1993).
Ferenci et al., “Combination of interferon (IFN) induction therapy and ribavirin in chronic hepatitis C,” Prog. Abstr. Dig. Dis. Week 2000, San Diego, CA (May 21-24, 2000) (Abstract 977).
Fontaine et al., “Recovery from chronic hepatitis C in long-term responders to ribarivin plus interferon α,” Lancet 356(9223):41 (Jul. 2000).
Franchetti et al., “Furanfurin and Thiophenfurin: Two Novel Tiazofurin Analogues. Synthesis, Structure, Antitumor Activity, and Interactions with Inosine Monophosphate Dehydrogenase,” J. Medicinal Chem. 38(19):3829-3837 (1995).
Fujii et al., “Effect of phosphatidylcholine on Skin Permeation of Indomethacin from gel prepared with Liquid Paraffin and Hydrogenated Phospholipid,” Int'1 J. Pharmaceutics 222(1):57-64 (2001).
Fujii et al., “Enhancement of skin permeation of miconazole by phospholipid and dodecyl 2-(N, N-dimethylamino) propionate (DDAIP),” Int'1 J. Pharmaceutics 234(1-2): 121-128 (2002).
Luft et al., “Electro-osmotic valve for the controlled administration of drugs,” Med. & Biological Engineering & Computing 45-50 (Jan. 1978) (non-English with English abstract).
Gan To Kagaku Ryoho, “Phase II study of recombinant leukocyte A interferon (Ro22-8181) in malignant brain tumors,” Cancer & Chemotherapy 12(4):913-920 (Apr. 1985) (non-English with English abstract).
Gappa et al., “Juvenile laryngeal papillomatosis—a case report,” Pneumologie 45(11):936-938 (Nov. 1991) (XP009079028) (non-English with English abstract).
Gause et al., “Phase I study of subcutaneously administered interleukin-2 in combination with interferon alfa-2a in patients with advanced cancer,” J. Clin. Oncol. 14(8):2234-2241 (Aug. 1996).
Ghiglione et al., “How glucagon-like is glucagon-like peptide-1?” Diabetologia 27:599-600 (1984).
Glue et al., “A dose-ranging study of Peg-intron and ribavirin in chronic hepatitis C—safety, efficacy, and virological rationale,” Prog. Abstr. 50th Annu. Mtg. Postgrad. Courses Am. Assn. Study Liver Dis., Dallas, TX(Nov. 5-9, 1999)(Abstract 571).
Goke et al., “Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like peptide 1-(7-36)-amide receptor of insulin-secreting betacells,” J. Biol. Chem., 268(26): 19650-19655 (1993).
Gonzales et al., “Randomized controlled trial including an initial 4-week ‘induction’ period during one year of high-dose interferon α-2B treatment for chronic hepatitis C,” Prog. Abstr. Dig. Dis. Week 2000, San Diego, CA (May 21-24, 2000) (Abstract 975).
Gosland et al., “A phase I trial of 5-day continuous infusion cisplatin and interferon alpha,” Cancer Chemother. Pharmacol. 37(1-2):39-46 (1995).
Grant et al., “Combination therapy with interferon-α plus N-acetyl cysteine for chronic hepatitis C: a placebo controlled double-blind multicentre study,” J. Med. Virol. 61(4):439-442 (Aug. 2000).
Gutniak et al., “Antidiabetogenic effect of glucagon-like peptide-1 (7-36)amide in normal subjects and patients with diabetes mellitus,” N. Engl. J. Med., 326(20): 1316-1322 (1992).
Hageman, “The Role of Moisture in Protein Stability, ” Drug Dev. & Ind. Pharm. 14(14):2047-2070 (1988).
Heathcote et al., “Peginterferon alfa-2a in Patients With Chronic Hepatitis C and Cirrhosis,” New England J. Med. 343(23): 1673-1680 (2000).
Heim et al., “Intracellular signaling and antiviral effects of interferons,” Dig. Liver Dis. 32(3):257-263 (Apr. 2000).
Heinrich et al., “Pre-proglucagon messenger ribonucleic acid: nucleotide and encoded amino acid sequences of the rat pancreatic complementary deoxyribonucleic acid,” Endocrinol., 115:2176-2181 (1984).
Hellstrand et al., “Histamine and cytokine therapy,” Acta Oncol. 37(4):347-353 (1998).
Hellstrand et al., “Histamine and the response to IFN-α in chronic hepatitis C,” Interferon Cytokine Res. 18(1):21-22 (Jan. 1998).
Hellstrand et al., “Histamine in immunotherapy of advanced melanoma: a pilot study,” Cancer Immunol Immunother. 39(6):416-419 (Dec. 1994).
Hisatomi et al., “Toxicity of polyoxyethylene hydrogenated castor oil 60 (HCO-60) in experimental animals,” J. Toxicol. Sci., 18(3): 1-9 (1993).
Hodoshima et al., “Lipid nanoparticles for delivering antitumor drugs,” International Joumal of Pharmaceutics, 146(1):81-92 (1997).
Hoffmann-La Roche Inc., Pegasys® (peginterferon alfa-2a), 15 pages (2002).
Horton et al., “Antitumor effects of interferon-omega: in vivo therapy of human tumor xenografts in nude mice” Cancer Res 59(16):4064-4068 (Aug. 1999).
Hubel et al., “A phase I/II study of idarubicin, dexamethasone and interferon-alpha (1-Dexa) in patients with relapsed or refractory multiple myeloma” Leukemia 11 Suppl 5:S47-S51 (Dec. 1997).
Iacobelli et al., “A phase I study of recombinant interferon-alpha administered as a seven-day continuous venous infusion at circadian-rhythm modulated rate in patients with cancer,” Am. J. Clin. Oncol. 18(1):27-31 (1995).
IFNB Multiple Sclerosis Study Group, “Interferonβ-lb is effective in relapsingremitting multiple sclerosis,” Neurology 43(4):655-667 (Apr. 1993).
INTERMUNE® INC., Tnfergen® (Interferon alfacon-1), 5 pages (2002).
“Introduction to Antibodies”, http://www.chemicon.com/resource/ANT101/a1.asp, 8 pages (retrieved May 2, 2007).
Isaacs et al., “Virus interference. I. The interferon,” Pro. R. Soc. Lond. B. Biol. Sci. 147:258-267 (1957).
Jain et al., “Controlled delivery of drugs from a novel injectable in situ formed biodegradable PLGA microsphere system,” J. Microencapsulation 17(3):343-362 (2000).
Jordan et al., “Guidelines for Antiemetic Treatment of Chemotherapy-Induced Nausea and Vomiting: Past, Present and Future Recommendations,” The Oncologist 12(9): 1143-1150 (2007).
Kabalnov et al., “Macroemulsion type and stability of alkane-water-phospholipid systems,” Abstracts of Papers, Part 1, 210th ACS National Meeting, 0-8412-3222-9, American Chemical Society, Chicago, IL (Aug. 20-24, 1995) (Abstract only).
Kabalnov et al., “Phospholipids as Emulsion Stabilizers.2. Phase Behavior Versus Emulsion Stability,” Journal of Colloid and Interface Science 184(l):227-235 (1996).
Khalili et al., “Interferon and ribavirin versus interferon and amantadine in interferon nonresponders with chronic hepatitis C,” Am. J. Gastroenterol. 95(5): 1284-1289 (May 2000).
Kildsig et al., “Theoretical Justification of Reciprocal Rate Plots in Studies of Water Vapor Transmission through Films,” J. Pharma. Sci. 29(11): 1634-01637 (Nov. 17, 1970).
Kirkwood et al., “Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: The Eastern Cooperative Oncology Group Trial EST 1684,” J. Clin. Oncol. 14(1):7-17 (1996).
Kita et al., “Characterization of a polyethylene glycol conjugate of recombinant human interferon-y,” Drug Des. Deliv. 6(3): 157-0167 (Sep. 1990).
Knepp et al., “Identification of antioxidants for prevention of peroxide-mediated oxidation of recombinant human ciliary neurotrophic factor and recombinant human nerve growth factor,” J. Pharm. Sci. Tech. 50(3): 163-171 (1996).
Knepp et al., “Stability of nonaqueous suspension formulations of plasma derived factor IX and recombinant human alpha interferon at elevated temperatures,” Pharma. Res. 15(7): 1090-1095 (1998).
Knobler et al., “Systemic α-interferon therapy of multiple sclerosis,” Neurology 34(10): 1273-1279 (Oct. 1984).
Kovacevic et al., “Treatment of chronic viral hepatitis B in secondary membranoproliferative glomerulonephritis using recombinant α-2 interferon,” Maksic Dj Vojnosanit. Pregl. 57(2):235-240 (Mar.-Apr. 2000) (non-English with English abstract).
Kracke et al., “Mx proteins in blood leukocytes for monitoring interferon β-1b therapy in patients with MS,” Neurology 54(1): 193-199 (Jan. 2000).
Kronenberger et al., “Influence of interferon-α on CD82-expression in HCV-positive patients,” Prog. Abstr. Dig. Dis. Week 2000, San Diego, CA (May 21-24, 2000) (Abstract 976).
Krown et al., “Interferons and interferon inducers in cancer treatment,” Semin. Oncol. 13(2):207-217 (1986).
Kubes et al., “Cross-species antiviral and antiproliferative activity of human interferon-co,” J. Interferon Res. 14:57-59 (1994).
Kunzi et al., “Role of interferon-stimulated gene ISG-15 in the interferon-co-mediated inhibition of human immunodeficiency virus replication,” J. Interferon Cytokine Res. 16(11):919-927 (Nov. 1996).
Larsson, “Stability of emulsions formed by polar lipids,” Progress in the Chemistry of Fats and Other Lipids 16:163-0169 (1978).
Lee et al., “Dynamics of hepatitis C virus quasispecies turnover during interferon-A treatment,” Prog. Abstr. Dig. Dis. Week 2000, San Diego, CA (May 21-24, 2000) (Abstract 974).
Lee, “Therapy of hepatitis C: interferon alfa-2A trials,” Hepatology 26: 89S-95S (Sep. 1997) (XP000981288).
Lopez et al., “Mammalian pancreatic preproglucagon contains three glucagon-related peptides,” Proc. Natl. Acad. Sci. USA, 80(18):5485-5489 (1983).
Lukaszewski et al., “Pegylated α interferon is an effective treatment for virulent Venezuelan equine encephalitis virus and has profound effects on host immune response to infection,” J. Virol. 74(11):5006-5015 (Jun. 2000).
Lund et al., “Pancreatic preproglucagon cDNA contains two glucagon-related coding sequences arranged in tandem,” Proc. Natl. Acad. Sci. USA, 79(2):345-349 (1982).
Lundberg, “A submicron lipid emulsion coated with amphipathic polyethylene glycol for parenteral administration of paclitaxel (Taxol),” J. Pharm. & Pharmacol. 49(1): 16-21 (1997).
Magnuson et al. “Enhanced recovery of a secreted mammalian protein from suspension culture of genetically modified tobacco cells,” Protein Expression & Purification 7:220-228 (1996).
Malley et al., “Chronic Toxicity And Oncogenicity Of N-Methylpyrrolidone (Nmp) In Rats And Mice By Dietary Administration,” Drug Chem Toxicol. 24(4):315-38 (Nov. 2001).
Manning et al., “Stability of protein pharmaceuticals,” Pharm. Res. 6(11): 903-918 (1989).
Marincola et al., “Combination therapy with interferon alfa-2a and interleukin-2 for the treatment of metastatic cancer,” J. Clinical Oncol. 13(5): 1110-1122 (1995) (XP009078965).
Massey, “Interaction of vitamin E with saturated phospholipid bilayers,” Biochem. & Biophys. Res. Comms. 106(3):842-847 (1982).
Mchutchison et al., “Interferon a-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C,” N. Engl. J. Med. 339(21): 1485-1492 (Nov. 1998).
Meier et al., “The glucagon-like peptide-1 metabolite GLP-1-(9-36) amide reduces postprandial glycemia independently of gastric emptying and insulin secretion in humans,” Am. J. Physiol. Endocrinol. Metab., 290(6):E1118-E1123 (2006).
Merad et al., “Generation of monocyte-derived dendritic cells from patients with renal cell cancer: modulation of their functional properties after therapy with biological response modifiers (IFN-α plus IL-2 and IL-12),” J. Immunother. 23(3):369-378 (May-Jun. 2000).
Milella et al., “Neutralizing antibodies to recombinant α-interferon and response to therapy in chronic hepatitis C virus infection,” Liver 13(3): 146-150 (Jun. 1993).
Mohler, “Primer on electrodeposited coatings,” Materials Engineering 5:38-45 (1972).
Mojsov, “Structural requirements for biological activity of glucagon-like peptide-I,” Int. J. Peptide Protein Research, 40:333-343 (1992).
Morgan, “Structure and Moisture Permeability of Film-Forming Poloyers,” Ind. Eng. Chem. 45(10):2296-2306 (1953).
Motzer et al., “Phase I trial of 40-kd branched pegylated interferon alfa-2a for patients with advanced renal cell carcinoma,” J. Clinical Oncol. 19(5): 1312-1319 (2001).
Nauck et al., “Normalization of fasting glycaemia by intravenous GLP-1 ([7-36 amide] or [7-37]) in type 2 diabetic patients,” Diabet. Med., 15(11):937-945(1998).
Neumann et al., “Hepatitis C Viral Dynamics In Vivo and the Antiviral Efficacy of Interferon-alpha Therapy,” Science 282:103-107 (Dec. 1998).
Nieforth et al., “Use of an indirect pharmacodynamic stimulation model of MX protein induction to compare in vivo activity of interferon-a-2a and a polyethylene glycol-modified derivative in healthy subjects,” Clin. Pharmacol. Ther. 59(6):636-646 (Jun. 1996).
Norden et al., “Physicochemical characterization of a drug-containing phospholipid-stabilized o / w emulsion for intravenous administration,” Eur. J. Pharm. Sci. 13(4):393-401 (2001).
Olaso et al., “Early prediction of lack of response to treatment with interferon and interferon plus ribavirin using biochemical and virological criteria in patients with chronic hepatitis C,” Esp. Quimioter. 12(3):220-228 (Sep. 1999) (non-English with English abstract).
Ortiz et al., “A differential scanning calorimetry study of the interaction of α-tocopherol with mixtures of phospholipids,” Biochim et Biophys Acta 898(2):214-222 (1987).
Panitch, “Interferons in multiple sclerosis,” Drugs 44(6):946-962 (Dec. 1992).
Patzelt et al., “Identification and processing of proglucagon in pancreatic islets,” Nature, 282:260-266 (1979).
Peterson et al., “Zucker Diabetic Fatty Rat as a Model for Non-insulin-dependent Diabetes Mellitus,” ILAR Journal, 32(3): 16-19 (1990).
Peterson et al., “Neuropathic complications in the Zucker diabetic fatty rat (ZDF/Drt-fa),” Frontiers in diabetes research. Lessons from Animal Diabetes III, Shafrir, E. (ed.), pp. 456-458, Smith-Gordon, London (1990).
Pimstone et al., “High dose (780 MIU/52 weeks) interferon monotherapy is highly effective treatment for hepatitis C,” Prog. Abstr. Dig. Dis. Week 2000, San Diego, CA (May 21-24-2000) (Abstract 973).
Plauth et al., “Open-label phase II study of omega interferon in previously untreated HCV infected patients,” Hepatology 34(4):A331 (Oct. 1, 2001) (XP004716169) (Abstract Only).
Plauth et al., “Open-label study of omega interferon in previously untreated HCV-infected patients,” J. Hepatology 36(Supp. 1): 125 (Apr. 2002) (XP002511882) (Abstract Only).
Pohl et al., “Molecular cloning of the helodermin and exendin-4 cDNAs in the lizard. Relationship to vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide and glucagon-like peptide 1 and evidence against the existence of mammalian homologues,” J. Biol. Chem., 273(16):9778-9784 (1998).
Poynard et al., “Is an ‘a la carte’ combined interferon a 2b plus ribavirin possible for the first line treatment in patients with chronic hepatitis C,” Hepatology 31(1):211-218 (Jan. 2000).
Poynard et al., “Randomized trial of interferon a 2b plus ribavirin for 48 weeks or for 24 weeks versus interferon a 2b plus placebo for 48 weeks for the treatment of chronic infection with hepatitis C virus,” Lancet 352(9138): 1426-1432 (Oct. 1998).
“Intarcia Presents Positive ITCA 650 Phase 2 Study Results for Type 2 Diabetes at EASD, ”Intarcia Therapeutics, Inc. (Sep. 22, 2010) (Press Release).
Quesada et al., “Interferons in Hematological Malignancies”, eds. Baron et al., U. Tex. 487-495 (1987).
Quintanar-Guerrero et al., “Applications of the ion-pair concept to hydrophilic substances with special emphasis on peptides,” Pharm. Res. 14(2): 119-127 (1997).
Rajkumar et al., “Phase I evaluation of radiation combined with recombinant interferon alpha-2a and BCNU for patients with high-grade glioma,” Int'1 J. Radiat. Oncol. Biol. Phys. 40(2):297-302 (Jan. 15, 1998).
Roche Pharmaceuticals, Roferon®-A (Interferon alfa-2a, recombinant), 22 pages (2003).
Roff et al., “Handbook of Common Polymers”, Cleveland Rubber Co. 72 pages (1971).
Rogers et al., “Permeability Valves,” Ind. & Eng. Chem. 49(11): 1933-1936 (Nov. 17, 1957).
Roman et al., “Cholestasis in the rat by means of intravenous administration of cyclosporine vehicle, Cremophor EL,” Transplantation 48(4):554-558 (1989).
Roth et al., “High Dose Etretinate and Interferon-alpha—A Phase I Study in Squamous Cell Carcinomas and Transitional Cell Carcinomas,” Acta Oncol. 38(5):613-617 (1999).
Roth et al., “Combination therapy with amylin and peptide YY[3-36] in obese rodents: anorexigenic synergy and weight loss additivity,” Endocrinol. 148( 12):6054-61 (Dec. 2007).
Schepp et al., “Exendin-4 and exendin-(9-39)NH2: agonist and antagonist, respectively, at the rat parietal cell receptor for glucagon-like peptide-1-(7-36)NH2,” Eur. J. Pharmacol., 269(2): 183-191 (1994).
Schering Corp., Intron® A for Injection, 6 pages (2001).
Schering Corp., PEG-Intron™(Peginterferon alfa-2b) Powder for Injection, 29 pages (2003).
Schmalfub et al., “Modification of drug penetration into human skin using microemulsions,” J. Controlled Release 46(3):279-285 (1997).
Sen et al., “The interferon system: a bird's eye view of its biochemistry,” J. Biol. Chem. 267(8):5017-5020 (Mar. 1992).
Shiftman et al., “A decline in HCV-RNA level during interferon or interferon/ribavirin therapy in patients with virologic nonresponse is associated with an improvement in hepatic histology,” Prog. Abstr. 50th Annu. Mtg. Postgrad. Courses Am. Assn. Study Liver Dis., Dallas, TX (Nov. 5-9, 1999) (Abstract 567).
Shima et al., “Serum total bile acid level as a sensitive indicator of hepatic histological improvement in chronic hepatitis C patients responding to interferon treatment,” J. Gastroenterol. Hepatol. 15(3):294-299 (Mar. 2000).
Shiratori et al., “Histologic improvement of fibrosis in patients with hepatitis C who have sustained response to interferon therapy,” Ann. Int. Med. 132(7):517-524 (Apr. 2000).
Simon et al., “A longitudinal study of T1 hypointense lesions in relapsing Ms: MSCRG trial of interferon β1a,” Neurology 55(2): 185-192 (Jul. 2000).
Sparks et al., “Lipoprotein alterations in 10- and 20-week-old Zucker diabetic fatty rats: hyperinsulinemic versus insulinopenic hyperglycemia,” Metabolism, 47(11): 1315-1324 (1998).
Sulkowski et al., “Pegylated Interferon Alfa-2A (Pegasys™) and Ribavirin Combination Therapy for Chronic Hepatitis C: A Phase II Open-Label Study,” Gastroenterology 118(4, Supp. 2) (2000) (Abstract 236).
Sulkowski et al., “Peginterferon-a-2a (40kD) and ribavirin in patients with chronic hepatitis C: a phase II open label study,” Biodrugs 16(2): 105-109 (2002).
Talpaz et al., “Phase I study of polyethylene glycol formulation of interferon alpha-2B (Schering 54031) in Philadelphia chromosome-positive chronic myelogenous leukemia,” Blood 98(6): 1708-1713 (2001).
Talsania et al., “Peripheral exendin-4 and peptide YY(3-36) synergistically reduce food intake through different mechanisms in mice,” Endocrinology 146(9):3748-56 ( Sep. 2005).
Tanaka et al., “Effect of interferon therapy on the incidence of hepatocellular carcinoma and mortality of patients with chronic hepatitis C: a retrospective cohort study of 738 patients,” Int. J. Cancer 87(5)741-749 (Sep. 2000).
Tong et al., “Prediction of response during interferon α 2b therapy in chronic hepatitis C patients using viral and biochemical characteristics: a comparison,” Hepatology 26(6): 1640-01645 (Dec. 1997).
Touza Rey et al., “The clinical response to interferon-γ in a patient with chronic granulomatous disease and brain abscesses due to Aspergillus fumigatus,” Ann. Med. Int. 17(2):86-87 (Feb. 2000).
Trudeau et al., “A phase I study of recombinant human interferon alpha-2b combined with 5-fluorouracil and cisplatin in patients with advanced cancer,” Cancer Chemother. Pharmacol. 35(6):496-500 (1995).
Tseng et al., “Glucose-dependent insulinotropic peptide: structure of the precursor and tissue-specific expression in rat,” PNAS USA, 90(5): 1992-1996 (1993).
Tsung et al., “Preparation and Stabilization of Heparin/Gelatin Complex Coacervate Microcapsules,” J. Pharm. Sci. 86(5):603-7 (May 1997).
Unniappan et al., “Effects of dipeptidyl peptidase IV on the satiety actions of peptide YY,” Diabetologia; Clinical and Experimental Diabetes and Metabolism 49(8): 1915-1923 (Jun. 27, 2006).
Vokes et al., “A phase I trial of concomitant chemoradiotherapy with cisplatin dose intensification and granulocyte-colony stimulating factor support for advanced malignancies of the chest,” Cancer Chemother. Pharmacol. 35(4):304-312 (1995).
Vrabec, “Tympanic membrane perforations in the diabetic rat: a model of impaired wound healing,” Otolaryngol. Head Neck Surg., 118(3 Pt. 1):304-308 (1998).
Wang et al., “Preferential interaction of α-tocopherol with phosphatidylcholines in mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine,” Eur. J. Biochem. 267(21):6362-6368 (2000).
Wang et al., “Ripple phases induced by α-tocopherol in saturated diacylphosphatidylcholines,” Archives of Biochem. & Biophys. 377(2):304-314 (2000).
Wang et al., “The distribution of α-tocopherol in mixed aqueous dispersions of phosphatidylcholine and phosphattidylethanolamine,” Biochimica et Biophysica Acta-Biomembranes 1509(1-2):361-372 (2000).
Wang et al., “Parenteral formulations of proteins and peptides: stability and stabilizers,” J. Parenter. Sci. Technol. 42(2S):S4-S26 (1988).
Weinstock-Guttman et al., “What is new in the treatment of multiple sclerosis?” Drugs 59(3): 401-410 (Mar. 2000).
Weissmann et al., “The interferon genes,” Prog. Nucleic Acid Res. Mol. Biol. 33:251-300 (1986).
Wright et al., “Preliminary experience with α-2b-interferon therapy of viral hepatitis in liver allograft recipients,” Transplantation 53(1): 121-123 (Jan. 1992).
Young et al., “Glucose-lowering and insulin-sensitizing actions of exendin-4: studies in obese diabetic (ob/ob, db/db) mice, diabetic fatty Zucker rats, and diabetic rhesus monkeys (Macaca mulatta),” Diabetes, 48(5): 1026-1034 (1999).
Younossi et al., “The role of amantadine, rimantadine, ursodeoxycholic acid, and NSAIDs, alone or in combination with a interferons, in the treatment of chronic hepatitis C,” Semin. Uiver Dis. 19(Supp. 1):95-102 (1999).
Yu et al., “Preparation, characterization, and in vivo evaluation of an oil suspension of a bovine growth hormone releasing factor analog,” J. Pharm. Sci. 85(4):396-401 (1996).
Zeidner et al., “Treatment of FeLV-induced immunodeficiency syndrome (feLV-FAIDS) with controlled release capsular implantation of 2',3'-dideoxycytidine,” Antivir. Res. 11(3): 147-0160 (Apr. 1989).
Zein, “Interferons in the management of viral hepatitis,” Cytokines Cell Mol. Ther. 4(4):229-241 (Dec. 1998).
Zeuzem et al., “Peginterferon Alfa-2a in Patients with Chronic Hepatitis C,” New Engl. J. Med. 343(23): 1666-1672 (2000).
Zeuzem et al., “Hepatitis C virus dynamics in vivo: effect of ribavirin and interferon a on viral turnover,” Hepatology 28(1):245-252 (Jul. 1998).
Zhang et al., “Report on Large Dosage Interferon to Treat 30 Cases of Viral Encephalitis,” J. Clinical Pediatrics 14(2):83-84 (1996).
Zhang et al., “A new strategy for enhancing the stability of lyophilized protein: the effect of the reconstitution medium on keratinocyte growth factor,” Pharm. Res. 12(10): 1447-1452 (1995).
Zheng et al. “Therapeutic Effect of Interferon Varied Dose in Treating Virus Encephalitis,” Beijing Med. J. 13(2):80-81 (1998).
Ziesche et al., “A preliminary study of long-term treatment with interferon γ-1b and low-dose prednisolone in patients with idiopathic pulmonary fibrosis,” New Engl. J. Med. 341(17): 1264-1269 (Oct. 1999).
Sanofi-Aventis U.S. LLC, Prescribing Information for ADLYXINR® (Lixisenatide) Injection, for Subcutaneous Use, rev. Jul. 2016, 31 pages.
Amylin Pharmaceuticals, Inc., Prescribing Information for BYETTA® (Exenatide) Injection, rev. Oct. 2009, 34 pages.
Astrazeneca Pharmaceuticals LP, Prescribing Information for BYDUREON® (Exenatide Extended-Release for Injectable Suspension), rev. Mar. 2015, 60 pages.
Novo Nordisk A/S, Prescribing Information for Victoza® (Liraglutide [rDNA Origin] Injection), Solution for Subcutaneous Use, v. 1, Jan. 2010, 23 pages.
Glaxosmithkline LLC, Prescribing Information for TANZEUM® (Albiglutide) for Injection, for Subcutaneous Use, rev. Jun. 2014, 55 pages.
Eli Lilly & Company, Prescribing Information for TRULICITY® (Dulaglutide) Injection, for Subcutaneous Use, rev. Mar. 2015, 19 pages.

Related Publications (1)

	Number	Date	Country
	20200299350 A1	Sep 2020	US

Provisional Applications (3)

Number	Date	Country
62337005	May 2016	US
62414146	Oct 2016	US
62420937	Nov 2016	US

Continuations (1)

	Number	Date	Country
Parent	15595809	May 2017	US
Child	16667502		US

Glucagon-receptor selective polypeptides and methods of use thereof

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

Field of Search

CPC

International Classifications

Abstract

Description

Claims

RELATED APPLICATIONS

US Referenced Citations (770)

Foreign Referenced Citations (179)

Non-Patent Literature Citations (259)

Related Publications (1)

Provisional Applications (3)

Continuations (1)