Patent Grant
11840559
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.
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.
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.
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, or at least 16 amino acid substitutions, wherein the amino acid substitution(s) is selected from the group consisting of:
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:
wherein:
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: 2:
wherein:
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: 3:
wherein:
In some embodiments, an isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of
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
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
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: 137:
wherein:
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 insulinotrophic peptide. For example, the insulinotrophic 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 insulinotrophic 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.
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.
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-C4 alkyl). 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 “insulinotrophic” 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 insulinotrophic hormone). Such compounds typically stimulate or otherwise affect the secretion or biosynthesis of insulin in a subject. Thus, an “insulinotrophic peptide” is an amino acid-containing molecule capable of stimulating or otherwise affecting secretion or biosynthesis of insulin.
The term “insulinotrophic 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; 1, 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: 1, 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: 1), exenatide-LAR, lixisenatide, GLP-1 (7-36), liraglutide, semaglutide, dulaglutide, albiglutide, and taspoglutide. Incretin mimetics are also referred to herein as “insulinotrophic 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: 1, 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 β 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:
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)
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).
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).
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.
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, or at least 16 amino acid substitutions, wherein the amino acid substitution(s) is selected from the group consisting of:
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:
wherein:
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: 2:
wherein:
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: 3:
wherein:
In some embodiments, an isolated polypeptide of the disclosure comprises an amino acid sequence selected from the group consisting of
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
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.
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: 137:
wherein:
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.
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 (t1/2) in humans of at least about 5 hours following subcutaneous administration. In some embodiments, the disclosed peptide has an elimination half-life (t1/2) 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)
In some embodiments, each R1 is hydrogen. In some embodiments, each R3 is hydrogen. In some embodiments, each R1 and each R3 are hydrogen.
In some embodiments, at least one R2 is CO2H. In some embodiments, one R2 is 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(CH2)2O)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:
In some embodiments, the lipophilic substituent and spacer combine to form the structure of Formula III:
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 A1, A2, A3, A4, A5 or A6, via a spacer, and the lipophilic substituent and spacer are of Formula III.
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, 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 III.
In some embodiments, isolated polypeptide comprises a lysine, and the β-amino group of the lysine is covalently bound to the lipophilic substituent, optionally via a spacer.
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: 151:
wherein:
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.
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: 151 wherein:
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.
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:
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 III:
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 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 III.
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, 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 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 β-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 β-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 β-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)WLEDEPSSGAPPP S-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:
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:
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: 152 through SEQ ID NO: 199.
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 comprises an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 160, SEQ ID NO: 165, and SEQ ID NO: 166.
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.
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 Compounds B1-B48. In some embodiments, any of the glucagon analogs of the disclosure is formulated in combination with exenatide, wherein the glucagon analog is selected from the group consisting of amino acid sequences represented by SEQ ID NO: 152-SEQ ID NO: 199.
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 selected from the group consisting of Compound B9, Compound B14 and Compound B15.
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 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.
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 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 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.
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.
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 myoinisitol). 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 hydroxyanisol, butylated hydroxytoluene, 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.
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, insulinotrophic 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, palmitates, 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 D50 value, 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 myoinisitol). Preferred 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 hydroxyanisol, butylated hydroxytoluene, 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, 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.
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 (C8 to 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.
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).
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.
Glucagon polypeptides of the invention, as provided in Table 3, were synthesized on a Prelude peptide synthesizer (Protein Technologies Inc., Tucson, AZ) 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-dimethylformamide (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 L L (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, MA) 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, k 215 nm, k 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).
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, AZ)) by solid-phase methods using Fmoc strategy and using Rink Amide MBHA LL (Novabiochem) or N-α-Fmoc protected, pre-loaded Wang L L (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)3 in 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 μm, 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.
Characterizations of peptide analogs were performed via C18 HPLC and LC/MS analysis (Acquity SQD Waters Corp, Milford, MA) 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.
The chemical structure of Compound A104 is shown in
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.
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.
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.
Sequences of synthesized glucagon receptor agonist peptides and their pEC50 values determined in cAMP assays are shown below in Table 3.
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.
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
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.
In some embodiments, provided is an isolated polypeptide of the disclosure corresponding to Compound A104 or A105.
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, MA) 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.
All calibration standards were prepared in control rat plasma containing K2EDTA and protease inhibitor cocktail. Samples and standards were analyzed by TurboIonSpray™ UPLC-MS/MS using a system consisting of a CTC HTS PAL auto-injector (Leap, Carrboro, NC), an Agilent Infinity 1290 system with column oven (Palo Alto, CA), a Valco switching valve (Houston, TX), and either an AB Sciex API 5600 TripleTOF™ or Sciex API 4000QTrap mass spectrometer (Framingham, MA). 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, MA) 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.
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, IN) 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, WI). 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, TX). 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, CA) 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, CA) using one-way ANOVA to compare each group to the appropriate control group. β-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.
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 (
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 (
Methods: With reference to the graph of
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.
With reference to the graph of
With reference to the illustration of
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.
With reference to the graph of
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.
With reference to the graph of
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.
Data on the graph of
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 ˜7%, was instead ˜47%.
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 (
Male Zucker Diabetic Fatty (ZDF) rats were obtained 6 weeks of age (Charles River, Raleigh, NC) 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, MI) 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, CA). 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 (
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 (
The lipophilic substituent and spacer of Formula II was incorporated into numerous disclosed peptides:
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, AZ)) 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-dimethylformamide (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 L L (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, CI8, 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.
The lipophilic substituent and spacer of Formula III was incorporated into numerous disclosed peptides:
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, AZ)) 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-dimethylformamide (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 L L (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, CI8, 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.
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:
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:
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.
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.
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
The horizontal axes in
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 ED50 of 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 ED50 of 45 μg/kg/day, and a Hill slope of 2.7.
The raw data in
Response to combination=(a*RespEX)+(b*RespGGN)+(c*RespEX*RespGGN)
where RespEX and RespGGN represent 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, CA). 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
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
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.
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
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.
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.
This 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.
| 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 |
| 3713919 | Tomic | Jan 1973 | 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 |
| 4522811 | Eppstein et al. | Jun 1985 | 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 |
| 4840896 | 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 | Edgren 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 et al. | 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 et al. | Jun 1992 | A |
| 5126141 | Henry | 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 | Illum | Apr 1993 | A |
| 5207752 | Sorensen et al. | May 1993 | A |
| 5209746 | Balaban et al. | May 1993 | A |
| 5213809 | Wright et al. | May 1993 | A |
| 5213810 | Steber | May 1993 | A |
| 5219572 | Sivaramakrishnan et al. | 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 | Nurnberg 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 | Magruder 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 |
| 5439688 | Orsolini et al. | Aug 1995 | A |
| 5443459 | Wong 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 |
| 5552520 | Kim et al. | Sep 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 |
| 5574010 | McFadden | Nov 1996 | A |
| 5574137 | Gray et al. | Nov 1996 | A |
| 5580578 | Oshlack et al. | Dec 1996 | A |
| 5589161 | Fuller | Dec 1996 | A |
| 5589167 | Cleland et al. | Dec 1996 | A |
| 5595751 | Bezwada et al. | 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 |
| 5656297 | 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 |
| 5672549 | Minami et al. | 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 | Illum | Jan 1998 | A |
| 5711967 | Juch | Jan 1998 | A |
| 5713847 | Howard, III et al. | Feb 1998 | A |
| 5718922 | 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 | Magruder 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 | Chiou 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 |
| 6120781 | Le Bras 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 | 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 et al. | 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 | Maskiewicz 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 et al. | 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 |
| 7235627 | Knudson et al. | Jun 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 |
| 2133073 | Wright et al. | Dec 2009 | A1 |
| 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 | Bloom | Sep 2012 | B2 |
| 8268341 | Berry et al. | 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 et al. | May 2016 | B2 |
| D769540 | Meier | Oct 2016 | S |
| 9509001 | Usami | Nov 2016 | B2 |
| 9526763 | Rohloff et al. | Dec 2016 | B2 |
| 9539200 | Lautenbach et al. | Jan 2017 | B2 |
| 9572889 | Alessi et al. | Feb 2017 | B2 |
| D789539 | Kleiner et al. | Jun 2017 | S |
| 9682127 | Alessi et al. | Jun 2017 | B2 |
| RE46577 | Collins et al. | Oct 2017 | E |
| 9800157 | Kawano et al. | Oct 2017 | B2 |
| 9889085 | Alessi et al. | Feb 2018 | B1 |
| 10501517 | Blackwell | Dec 2019 | B2 |
| 11214607 | Blackwell | Jan 2022 | 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 et al. | Oct 2001 | A1 |
| 20010036472 | Wong et al. | Nov 2001 | A1 |
| 20010040326 | Balczun et al. | 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 et al. | 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 | 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 | Struijker-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 |
| 20050070469 | Bloom et al. | Mar 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 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 et al. | 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 et al. | 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 | Schwartz | 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 |
| 20180009871 | Blackwell et al. | Jan 2018 | A1 |
| 20180185451 | Young et al. | Jul 2018 | A1 |
| 20200299350 | Blackwell et al. | Sep 2020 | A1 |
| Number | Date | Country |
|---|---|---|
| 102459325 | May 2012 | CN |
| 104447980 | Mar 2015 | CN |
| 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 |
| 1536767 | Jun 2005 | EP |
| 1600187 | Jan 2009 | EP |
| 2133073 | Dec 2009 | EP |
| 2020990 | Sep 2010 | EP |
| 3458084 | Mar 2019 | EP |
| 3733694 | Nov 2020 | 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 |
| H09241153 | Sep 1997 | JP |
| H11100353 | Apr 1999 | JP |
| 2006213727 | Aug 2006 | JP |
| 2014520789 | Aug 2014 | JP |
| 9100160 | Aug 1992 | NL |
| 592113 | Apr 2012 | NZ |
| 200634060 | Oct 2006 | TW |
| WO 1989003678 | May 1989 | WO |
| WO 1990013285 | Nov 1990 | WO |
| WO 1990013361 | Nov 1990 | WO |
| WO 1990013780 | Nov 1990 | WO |
| WO 1991007160 | May 1991 | WO |
| WO 1992019241 | Nov 1992 | WO |
| WO 1993006819 | Apr 1993 | WO |
| WO 1993006821 | Apr 1993 | WO |
| WO 1993008832 | May 1993 | WO |
| WO 1993009763 | May 1993 | WO |
| WO 1993023083 | Nov 1993 | WO |
| WO 1994009743 | May 1994 | WO |
| WO 1994010982 | May 1994 | WO |
| WO 1994021262 | Sep 1994 | WO |
| WO 1995001161 | Jan 1995 | WO |
| WO 1995009006 | Apr 1995 | WO |
| WO 1995009007 | Apr 1995 | WO |
| WO 1995013799 | May 1995 | WO |
| WO 1995034285 | Dec 1995 | WO |
| WO 1996001134 | Jan 1996 | WO |
| WO 1996003116 | Feb 1996 | WO |
| WO 1996036317 | Nov 1996 | WO |
| WO 1996039142 | Dec 1996 | WO |
| WO 1996040049 | Dec 1996 | WO |
| WO 1996040139 | Dec 1996 | WO |
| WO 1996040355 | Dec 1996 | WO |
| WO 1997015289 | May 1997 | WO |
| WO 1997015296 | May 1997 | WO |
| WO 1997028181 | Aug 1997 | WO |
| WO 1997031943 | Sep 1997 | WO |
| WO 1997044039 | Nov 1997 | WO |
| WO 1997046204 | Dec 1997 | WO |
| WO 1997047339 | Dec 1997 | WO |
| WO 1998000152 | Jan 1998 | WO |
| WO 1998000157 | Jan 1998 | WO |
| WO 1998000158 | Jan 1998 | WO |
| WO 1998002169 | Jan 1998 | WO |
| WO 1997041837 | Feb 1998 | WO |
| WO 1998007412 | Feb 1998 | WO |
| WO 1998016250 | Apr 1998 | WO |
| WO 1998017315 | Apr 1998 | WO |
| WO 1998020930 | May 1998 | WO |
| WO 1998027960 | Jul 1998 | WO |
| WO 1998027962 | Jul 1998 | WO |
| WO 1998027963 | Jul 1998 | WO |
| WO 1998030231 | Jul 1998 | WO |
| WO 1998032463 | Jul 1998 | WO |
| WO 1998042317 | Oct 1998 | WO |
| WO 1998047487 | Oct 1998 | WO |
| WO 1998051282 | Nov 1998 | WO |
| WO 1999003453 | Jan 1999 | WO |
| WO 1999004767 | Feb 1999 | WO |
| WO 1999004768 | Feb 1999 | WO |
| WO 1999012549 | Mar 1999 | WO |
| WO 1999016419 | Apr 1999 | WO |
| WO 1999025728 | May 1999 | WO |
| WO 1999029306 | Jun 1999 | WO |
| WO 1999033446 | Jul 1999 | WO |
| WO 1999033449 | Jul 1999 | WO |
| WO 1999039700 | Aug 1999 | WO |
| WO 1999040788 | Aug 1999 | WO |
| WO 1999044659 | Sep 1999 | WO |
| WO 1999062501 | Dec 1999 | WO |
| WO 1999064061 | Dec 1999 | WO |
| WO 2000013663 | Mar 2000 | WO |
| WO 2000029206 | May 2000 | WO |
| WO 2000038652 | Jul 2000 | WO |
| WO 2000039280 | Jul 2000 | WO |
| WO 2000040273 | Jul 2000 | WO |
| WO 2000041548 | Jul 2000 | WO |
| WO 2000045790 | Aug 2000 | WO |
| WO 2000054745 | Sep 2000 | WO |
| WO 2000059476 | Oct 2000 | WO |
| WO 2000066087 | Nov 2000 | WO |
| WO 2000066138 | Nov 2000 | WO |
| WO 2000067728 | Nov 2000 | WO |
| WO 2001019345 | Mar 2001 | WO |
| WO 2001028525 | Apr 2001 | WO |
| WO 2001029206 | Apr 2001 | WO |
| WO 2001043528 | Jun 2001 | WO |
| WO 2001051041 | Jul 2001 | WO |
| WO 2001078683 | Oct 2001 | WO |
| WO 2002028366 | Apr 2002 | WO |
| WO 2002036072 | May 2002 | WO |
| WO 2002043800 | Jun 2002 | WO |
| WO 2002045752 | Jun 2002 | WO |
| WO 2002047716 | Jun 2002 | WO |
| WO 2002067895 | Sep 2002 | WO |
| WO 2002069983 | Sep 2002 | WO |
| WO 2002076344 | Oct 2002 | WO |
| WO 2002085428 | Oct 2002 | WO |
| WO 2003000230 | Jan 2003 | WO |
| WO 2003007981 | Jan 2003 | WO |
| WO 2003011892 | Feb 2003 | WO |
| WO 2003020245 | Mar 2003 | WO |
| WO 2003024357 | Mar 2003 | WO |
| WO 2003024503 | Mar 2003 | WO |
| WO 2003030923 | Apr 2003 | WO |
| WO 2003041684 | May 2003 | WO |
| WO 2003041757 | May 2003 | WO |
| WO 2003053400 | Jul 2003 | WO |
| WO 2003066585 | Aug 2003 | WO |
| WO 2003072113 | Sep 2003 | WO |
| WO 2003072133 | Sep 2003 | WO |
| WO 2004002565 | Jan 2004 | WO |
| WO 2004034975 | Jan 2004 | WO |
| WO 2004035754 | Apr 2004 | WO |
| WO 2004035762 | Apr 2004 | WO |
| WO 2004036186 | Apr 2004 | WO |
| WO 2004052336 | Jun 2004 | WO |
| WO 2004056338 | Jul 2004 | WO |
| WO 2004089335 | Oct 2004 | WO |
| WO 2004103342 | Dec 2004 | WO |
| WO 2005048930 | Jun 2005 | WO |
| WO 2005048952 | Jun 2005 | WO |
| WO 2005102293 | Nov 2005 | WO |
| WO 2005110425 | Nov 2005 | WO |
| WO 2006017772 | Feb 2006 | WO |
| WO 2006023526 | Mar 2006 | WO |
| WO 2006081279 | Aug 2006 | WO |
| WO 2006083761 | Aug 2006 | WO |
| WO 2006084139 | Aug 2006 | WO |
| WO 2006086727 | Aug 2006 | WO |
| WO 2006101815 | Sep 2006 | WO |
| WO 2006111169 | Oct 2006 | WO |
| WO 2006131730 | Dec 2006 | WO |
| WO 2007024700 | Mar 2007 | WO |
| WO 2007056681 | May 2007 | WO |
| WO 2007075534 | Jul 2007 | WO |
| WO 2007084460 | Jul 2007 | WO |
| WO 2007133778 | Nov 2007 | WO |
| WO 2007140416 | Dec 2007 | WO |
| WO 2008021133 | Feb 2008 | WO |
| WO 2008041245 | Apr 2008 | WO |
| WO 2008061355 | May 2008 | WO |
| WO 2008133908 | Nov 2008 | WO |
| WO 2008134425 | Nov 2008 | WO |
| WO 2009109921 | Sep 2009 | WO |
| WO 2009143285 | Nov 2009 | WO |
| WO 2013004983 | Jan 2013 | WO |
| WO 2013074910 | May 2013 | WO |
| WO 2014152460 | Sep 2014 | WO |
| Entry |
|---|
| Du et al. “Drug Carriers for the Delivery of Therapeutic Peptides”, Biomacromolecules, 2014, 1097-1114 (Year: 2014). |
| U.S. Appl. No. 15/595,809 / 2018/0009871 A1 / U.S. Pat. No. 10,501,517, filed May 15, 2017 / Jan. 11, 2018 / Dec. 10, 2019, William Blackwell. |
| U.S. Appl. No. 16/667,502 / 2020/0299350 A1 / U.S. Pat. No. 11,214,607. filed Oct. 29, 2019/ Sep. 24, 2020 / Jan. 4, 2022, William Blackwell. |
| “Abstracts 2007,” Diabetologia Clinical & Experimental Diabetes & Metabolism, Springer, Berlin, Germany, vol. 50 S243 (Aug. 21, 2007) (paragraph [0586]) (XP002538652). |
| 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. |
| CAS No. 56-81-5 (Nov. 16, 1984). |
| Eli Lilly & Company, Prescribing Information for Trulicity® (Dulaglutide) Injection, for Subcutaneous Use, rev. Mar. 2015, 19 pages. |
| Extended European Search Report Received for EP Patent Application No. 20166273.1, dated Oct. 7, 2020, 6 pages. |
| Glaxosmithkline LLC, Prescribing Information for Tanzeum® (Albiglutide) for Injection, for Subcutaneous Use, rev. Jun. 2014, 55 pages. |
| Glumetza Brochure 2009, 13 Pages. |
| “Implantable infusion pumps: technology poised for takeoff,” BBI Newsletter 17(12):209211 (Dec. 1994). |
| International Search Report and Written Opinion received for PCT Patent Application No. PCT/US2009/000916, dated Aug. 12, 2009, 12 pages. |
| International Search Report and Written Opinion Received for PCT Application No. PCT/US2017/032714, dated Jul. 28, 2017, 9 pages. |
| “Introduction to Antibodies”, http://www.chemicon.com/resource/ANT101/a 1 .asp, 8 pages (retrieved May 2, 2007). |
| “Intarcia Presents Positive ITCA 650 Phase 2 Study Results for Type 2 Diabetes at EASD,” Intarcia Therapeutics, Inc. (Sep. 22, 2010) (Press Release). |
| Novo Nordisk A/S, Prescribing Information for Victoza® (Liraglutide [rDNA Origin] Injection), Solution for Subcutaneous Use, v. 1, Jan. 2010, 23 pages. |
| Sanofi-Aventis U.S. LLC, Prescribing Information for ADLYXINO (Lixisenatide) Injection, for Subcutaneous Use, rev. Jul. 2016, 31 pages. |
| “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). |
| 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). |
| 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”, Journal of Clinical Oncology 15(11):3330- 3337 (Nov. 1997). |
| Adolf et al., “Antigenic Structure of Human Interferon ω1 (Interferon αll1): Comparison with Other Human Interferons”, The Journal of General Virology 68(6):1669-1676 (Jun. 1987). |
| Adolf, “Human Interferon Omega—A review”, Multiple Sclerosis 1:S44-47 (1995). |
| Adolf et al., “Human Interferon ω1: Isolation of the Gene, Expression in Chinese Hamster Ovary Cells and Characterization of the Recombinant Protein”, Biochimca et Biophysica Acta 108(9):167-174 (Jun. 1991). |
| Adolf et al., “Monoclonal Antibodies and Enzyme Immunoassays Specific for Human Interferon (IFN) Gw1: Evidence That IFN-ω1 Is a Component of Human Leukocyte IFN”, Virology 175(2):410-471 (Apr. 1990). |
| Adolf et al., “Purification and Characterization of Natural Human Interferon ω1”, Journal of Biological Chemistry 265(16):9290-9295 (Jun. 1990). |
| Ahn et al., “A New Approach to Search for the Bioactive Confirmation of Glucagon: Positional Cyclization Scanning”, Journal of Medicinal Chemistry 44(19) 3109-3116 (2001). |
| Akers et al., “Formulation Design and Development of Parenteral Suspensions”, Journal of Parenteral Science and 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). |
| Andrx Pharmaceuticals, LLC, ANDA for Concerta® Extended-Release Tablets, 6 pages (correspondence dated Sep. 6, 2005). |
| 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). |
| ASTM International, Annual Book of ASTM Standards, 8.02:208-211, 584-587 (1984). |
| Aulitzky et al., “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). |
| Baggio et al., “Biology of Incretins: GLP-1 and GIP”, Gastroenterology 132(6):2131-2157 (2007). |
| 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”, Journal of Pharmaceutical Sciences 85(9):908-914 (1996). |
| Bakhtiar et al., “Taking Delivery”, Soap Perfumery & Cosmetics 76(3):59-65 (2003) (liposomes in cosmetic delivery systems). |
| Balkwill, “Interferons”, Lancet 1(8646):1060-1063 (May 1989). |
| Bataille et al., “Bioactive Enteroglucagon (Oxyntomodulin): Present Knowledge on Its Chemical Structure and Its Biological Activities”, Peptides 2(2):41-44 (1981). |
| Bauer et al., “Non-Aqueous Emulsions as Vehicles for Capsule Fillings”, Drug Development and Industrial Pharmacy 10(5):699-712 (1984). |
| Beck et al., “Poly(dl-Lactide-Co-Glycolide)/norethisterone Microcapsules: An Injectable Biodegradable Contraceptive”, Biology of Reproduction 28(1):186-195 (1983). |
| Bekkering, “Estimation of Early Hepatitis C Viral Clearance in Patients Receiving Daily Interferon and Ribavirin Therapy Using a Mathematical Model”, Hepatology 33(2):419 (Feb. 2001). |
| Bell et al., “Hamster Preproglucagon Contains the Sequence of Glucagon and Two Related Peptides”, Nature 302(5910):716-718 (1983). |
| Bell et al., “Impact of Moisture on Thermally Induced Denaturation and Decomposition of Lyophilized Bovine Somatotropin”, Drug Delivery Research and Development Biopolymers 35(2):201-209 (1995). |
| Bertoncello et al., “Haematopoietic Radioprotection by Cremophor EL: A Polyethoxylated Castor Oil”, International Journal of Radiation Biology 67(1):57-64 (1995). |
| Bodmeier et al., “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). |
| Bohlinder et al., “Use and Characteristics of a Novel Lipid Particle-Forming Matrix as a Drug-Carrier System”, European Journal of Pharmaceutical Sciences 2(4):271-279 (1994). |
| Bolinger et al., “Recombinant Interferon γ for Treatment of Chronic Granulomatous Disease and Other Disorders”, Clinical Pharmacology 11(10):834-850 (Oct. 1992). |
| Bonkovsky et al., “Outcomes Research in Chronic Viral Hepatitis C: Effects of Interferon Therapy”, Canadian Journal of Gastroenterology and Hepatology 14(Supp. B):21B-29B (Jul.-Aug. 2000). |
| Borden et al., “Second-Generation Interferons for Cancer: Clinical Targets”, Seminars in Cancer Biology 10(2):125-144 (Apr. 2000). |
| Boue et al., “Antiviral and Antiluteolytic Activity of Recombinant Bovine IFN-ω1 Obtained from Pichia Pastoris”, Journal of Interferon & Cytokine Research 20:677-683 (2000). |
| Bray et al., “Gut Signals and Energy Balance: Ghrelin, Peptide YY, Leptin, and Amylin”, (Slides and transcript for presentation at Medscape CME) (Dec. 19, 2007). |
| 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 Research 73(2):118-25 (Feb. 2007) (Epub Sep. 11, 2006). |
| Cantor et al., “Theory of Lipid Monolayers Comprised of Mixtures of Flexible and Stiff Amphiphiles in Athermal Solvents: Fluid Phase Coexistence”, The Journal of Chemical Physics 104(20):8082-8095 (1996). |
| Cha et al., “A One-Week Subdermal Delivery System for I-Methadone Based on Biodegradable Microcapsules”, Journal of Controlled Release 7:69-78 (1988). |
| Cha et al., “The Acceleration of Degradation-Controlled Drug Delivery from Polyester Microspheres”, Journal of Controlled Release 8(3):259-265 (1989). |
| Chang et al., “Biodegradable Polyester Implants and Suspension Injection for Sustained Release of a Cognitive Enhancer”, Pharmaceutical Technology 20(1):80-84 (1996). |
| Chapman et al., “Physical Studies of Phospholipids. VI. Thermotropic and Lyotropic Mesomorphism of Some 1,2-Diacylphosphatidylcholines (Lecithins)”, Chemistry and 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”, Proceedings of the Society for Experimental Biology and Medicine 173(1):68-75 (1983). |
| Cohen et al. “Controlled Delivery Systems for Proteins Based on Poly(lactic/glycolic Acid) Microspheres”, Pharmaceutical Research 8(6):713-720 (1991). |
| Condino-Neto et al., “Interferon-Gamma 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). |
| Conti et al., “Use of Polylactic Acid for the Preparation of Microparticulate Drug Delivery Systems”, Journal of Microencapsulation 9(2): 153-166 (1992). |
| Costantino et al., “Protein Spray Freeze Drying. 2. Effect of Formulation Variables on particle Size and Stability”, Journal of Pharmaceutical Sciences 91:388-395 (2002). |
| Cox et al., “The Effects of Neuropeptide Y and Its Fragments Upon Basal and Electrically Stimulated lon Secretion in Rat Jejunum Mucosa”, British Journal of Pharmacology 101(2):246-252 (Oct. 1990). |
| Darney et al., “Subdermal Progestin Implant Contraception”, Current Opinion in Obstetrics & Gynecology 3(4):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”, Progress Abstract 50th Annual. Meeting Postgraduate Courses American Association Study Liver Disease, Dallas, TX (Nov. 5-9, 1999) (Abstract 570). |
| Deacon, “GLP-1-(9-36) Amide Reduces Blood Glucose in Anesthetized Pigs by a Mechanism That Does not Involve Insulin Secretion”, American Journal of Physiology—Endocrinology and Metabolism 282(4):E873-E879 (2002). |
| Desai et al., “Protein Structure in the Lyophilized State: A Hydrogen Isotope Exchange/NMR Study with Bovine Pancreatic Trypsin Inhibitor”, Journal of the American Chemical Society 116(21):9420-9422 (1994). |
| Di Marco et al., “Combined Treatment of Relapse of Chronic Hepatitis C with High-Dose α2b Interferon plus Ribavirin for 6 or 12 Months”, Progress Abstract 50th Annual Meeting Postgraduate Courses American Association Study Liver Disease, Dallas, TX (Nov. 5-9, 1999) (Abstract 569). |
| Dorr et al., “Phase I-II Trial of Interferon-α 2b by Continuous Subcutaneous Infusion over 28 Days”, Journal of Interferon & Cytokine Research 8(6):717-725 (1988). |
| Eberlein et al., “A New Molecular form of PYY: Structural Characterization of Human PYY(3-36) and PYY(1-36)”, Peptides 10(4):797-803 (1989). |
| Efendic et al., “Overview of Incretin Hormones,” Hormone and Metabolic Research 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 Sciences 55(8):629-634 (1994). |
| Elias et al., “Infusional Interleukin-2 and 5-Fluorouracil with Subcutaneous Interferon-α 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”, Journal of Biological Chemistry 267(11):7402-7405 (1992). |
| Eng et al., Purification and Structure of Exendin-3, a New Pancreatic Secretagogue Isolated from Heloderma Horridum Venom, Journal of Biological Chemistry 265(33):20259-20262 (1990). |
| Eppstein et al., Biological Activity of Liposome-Encapsulated Murine Interferon Gamma is Mediated by a Cell Membrane Receptor, PNAS USA 82(11):3688-3692 (1985). |
| Eros et al., “Multiple Phase Emulsions as Controlled Drug Delivery Therapeutic Systems”, Proceeding of Conference Colloid Chemistry 193-196 (1993). |
| Erowid, “Introduction to the Federal Controlled Substance Analog Act”, 4 pages (2001). |
| Ertl et al., “Poly (DL-lactide-co-glycolide) Microspheres as Carriers for Peptide Vaccines”, Vaccine 14(9):879-885.(1996). |
| Fang et al., “The Impact of Baseline Liver Histology on Virologic Response to Interferon α-2b±ρ Ribavirin Therapy in Patients with Chronic Hepatitis C”, Progress Abstract 50th Annual Meeting Postgraduate Courses American Association Study Liver Disease, 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”, The Journal of Nutritional Biochemistry 4(11):630-634 (1993). |
| Ferenci et al., “Combination of Interferon (IFN) Induction Therapy and Ribavirin in Chronic Hepatitis C”, Progress Abstract Digestive Disease Week 2000, San Diego, CA (May 21-24, 2000) (Abstract 977). |
| Fioravante et al., “Weight Loss and Resting Energy Expenditure in Patients With Chronic Hepatitis C Before and During Standard Treatment Kosmiski”, Nutrition 28(6):630-634 (Jun. 2012). |
| Fontaine et al., “Recovery from Chronic Hepatitis C in Long-Term Responders to Ribavirin 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”, Journal of Medicinal Chemistry 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”, International Journal of 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)”, International Journal of Pharmaceutics 234(1-2):121-128 (2002). |
| Gan To Kagaku Ryoho, “Phase II Study of Recombinant Leukocyte a Interferon (Ro 22-8181) in Malignant Brain Tumors”, Cancer & Chemotherapy 12(4):913-920 (Apr. 1985). |
| Gao et al., “Target-Mediated Pharmacokinetic and Pharmacodynamic Model of Exendin-4 in Rats, Monkeys, and Humans”, Drug Metabolism and Disposition 40(5):990-997 (May 2012). |
| Gappa et al., “Juvenile Laryngeal Papillomatosis—A Case Report”, Pneumologie 45(11):936-938 (Nov. 1991) (XP009079028). |
| Gause et al., “Phase I Study of Subcutaneously Administered Interleukin-2 in Combination with Interferon Alfa-2a in Patients with Advanced Cancer”, Journal of Clinical Oncology 14(8):2234-2241 (Aug. 1996). |
| Ghiglione et al., “How Glucagon-Like is Glucagon-Like Peptide-1?”, Diabetologia 27(6):599-600 (1984). |
| Glue et al., “A Dose-Ranging Study of Peg-Intron and Ribavirin in Chronic Hepatitis C-Safety, Efficacy, and Virological Rationale”, Progress Abstract 50th Annual Meeting Postgraduate Courses American Association Study Liver Disease, 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 Beta-Cells”, Journal of Biological Chemistry 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”, Progress Abstract Digestive Disease Week 2000, San Diego, CA (May 21-24, 2000) (Abstract 975). |
| Gonzalez, et al., “Hemoglobin A1c: A Reliable and Accurate Test for Diabetes Care? Prospective Study in Mexico”, Salud Pública de México 55:462-468 (2013). |
| Gosland et al., “A Phase I Trial of 5-Day Continuous Infusion Cisplatin and Interferon α”, Cancer Chemotherapy and Pharmacology 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”, Journal of Medical Virology 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”, The New England Journal of Medicine 326(20):1316-1322 (1992). |
| Hageman, “The Role of Moisture in Protein Stability,” Drug Development and Industrial Pharmacy 14(14):2047-2070 (1988). |
| Hauck, “Engineer's Guide to Plastics”, Materials Engineering 5(72):38-45 (Jul. 17, 1972). |
| Heathcote et al., “Peginterferon alfa-2a in Patients With Chronic Hepatitis C and Cirrhosis”, The New England Journal of Medicine 343(23):1673-1680 (2000). |
| Heim et al., “Intracellular Signaling and Antiviral Effects of Interferons,” Digestive and Liver Disease 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”, Endocrinology 115(6):2176-2181 (1984). |
| Hellstrand et al., “Histamine and Cytokine Therapy”, Acta Oncologica 37(4):347-353 (1998). |
| Hellstrand et al., “Histamine and the Response to IFN-α in Chronic Hepatitis C”, Journal of Interferon & Cytokine Research 18(1):21-22 (Jan. 1998). |
| Hellstrand et al., “Histamine in Immunotherapy of Advanced Melanoma: A Pilot Study”, Cancer Immunology, Immunotherapy 39(6):416-419 (Dec. 1994). |
| 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). |
| Hisatomi et al., “Toxicity of Polyoxyethylene Hydrogenated Castor oil 60 (HCO-60) in Experimental Animals”, The Journal of Toxicological Sciences 18(3):1-9 (1993). |
| Hodgman et al., Handbook of Chemistry and Physics, 35th Edition, 1024-1025 (1953). |
| Hodoshima et al., “Lipid Nanoparticles for Delivering Antitumor Drugs”, International Journal of Pharmaceutics 146(1):81-92 (1997). |
| Hoffmann-La Roche Inc., Pegasys® (peginterferon alfa-2a), 15 pages (2002). |
| Holst, “Incretin Hormones and the Satiation Signal”, International Journal of Obesity 37(9):1161-1168 (Jan. 2013). |
| Holst et al., “The Incretin System and its Role in Type 2 Diabetes Mellitus”, Molecular and Cellular Endocrinology 297(1-2):127-136 (2008). |
| Horton et al., “Antitumor Effects of Interferon-ω: In Vivo Therapy of Human Tumor Xenografts in Nude Mice”, Cancer Research 59(16):4064-4068 (Aug. 1999). |
| Hubel et al., “A Phase I/II Study of Idarubicin, Dexamethasone and Interferon-α (I-Dexa) in Patients with Relapsed or Refractory Multiple Myeloma” Leukemia 11 Suppl 5:S47-S51 (Dec. 1997). |
| 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). |
| Iacobelli et al., “A Phase | Study of Recombinant Interferon-α Administered as a Seven-Day Continuous Venous Infusion at Circadian-Rhythm Modulated Rate in Patients with Cancer”, American Journal of Clinical Oncology 18(1):27-31 (1995). |
| IFNB Multiple Sclerosis Study Group, “Interferon β-1b Is Effective in Relapsing-Remitting Multiple Sclerosis”, Neurology 43(4):655-667 (Apr. 1993). |
| Intarcia Therapeutics “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 XP002504917 (Apr. 12, 2007) (Press Release). |
| Intermuner Inc., Infergen® (Interferon alfacon-1), 5 pages (2002). |
| Isaacs et al., “Virus interference. I. The interferon”, Proceedings of the Royal Society B: Biological Sciences 147(927):258-267 (1957). |
| Ishiwata et al., “Clinical Effects of the Recombinant Feline Interferon-Omega on Experimental Parvovirus Infection in Beagle Dogs”, The Journal of Veterinary Medical Science 60(8):911-917 (1998). |
| Jain et al., “Controlled Delivery of Drugs from a Novel Injectable in Situ Formed Biodegradable PLGA Microsphere System”, Journal of Microencapsulation 17(3):343-362 (2000). |
| Jalil et al., “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). |
| Jetschmann et al., “Open-Label Rising-Dose Study of ω Interferon in IFN-Naive Patients with Chronic Hepatitis C”, Gastroenterology 122:A278-A347 (Apr. 1, 2002) (Abstract M1454). |
| Johnson et al., “How Interferons Fight Disease”, Scientific American 270(5):68-75 (May 1994). |
| 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). |
| Kabalnov et al., “Phospholipids as Emulsion Stabilizers.2. Phase Behavior Versus Emulsion Stability”, Journal of Colloid and Interface Science 184(1):227-235 (1996). |
| Khalili et al., “Interferon and Ribavirin Versus Interferon and Amantadine in Interferon Nonresponders with Chronic Hepatitis C”, The American Journal of Gastroenterology 95(5):1284-1289 (May 2000). |
| Kildsig et al., “Theoretical Justification of Reciprocal Rate Plots in Studies of Water Vapor Transmission through Films”, Journal of Pharmaceutical Sciences 29(11):1634-1637 (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”, Journal of Clinical Oncology 14(1):7-17 (1996). |
| Kita et al., “Characterization of a Polyethylene Glycol Conjugate of Recombinant Human Interferon-Yrmulations of Plasma Derived Factor IX and Recombinant Human Alpha Interferon at Elevated Temperatures”, Drug Design Development and Delivery Journal 6(3):157-167 (Sep. 1990). |
| Kjems et al., “The Influence of GLP-1 on Glucose-Stimulated Insulin Secretion: Effects on Beta-cell Sensitivity in Type 2 and Nondiabetic Subjects”, Diabetes 52(2):380-386 (Feb. 2003). |
| Knepp et al., “Identification of Antioxidants for Prevention of Peroxide-Mediated Oxidation of Recombinant Human Ciliary Neurotrophic Factor and Recombinant Human Nerve Growth Factor”, Journal of Pharmaceutical Science and Technology 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”, Pharmaceutical Research 15(7):1090-1095 (1998). |
| Knobler et al., “Systemic α-interferon Therapy of Multiple Sclerosis”, Neurology 34(10):1273-1279 (Oct. 1984). |
| Kolterman et al., “Synthetic Exendin-4 (Exenatide) Significantly Reduces Postprandial and Fasting Plasma Glucose in Subjects with Type 2 Diabetes”, The Journal of Clinical Endocrinology & Metabolism 88(7):3082-3089 (2003). |
| Kosmiski, “Energy Expenditure in HIV Infection”, The American Journal of Clinical Nutrition 94(6):1677S-1682S (Dec. 2011). |
| Kovacevic et al., “Treatment of Chronic Viral Hepatitis B in Secondary Membranoproliferative Glomerulonephritis Using Recombinant Alfa-2 Interferon”, Maksic Dj Vojnosanit. Pregl. 57(2):235-240 (Mar.-Apr. 2000). |
| Kracke et al., “Mx Proteins in Blood Leukocytes for Monitoring Interferon 13-1 B Therapy in Patients with MS”, Neurology 54(1):193-199 (Jan. 2000). |
| Kronenberger et al., “Influence of Interferon-a 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”, Seminars in Oncology 13(2):207-217 (1986). |
| Kubes et al., “Cross-Species Antiviral and Antiproliferative Activity of Human Interferon-Omega”, Journal of Interferon & Cytokine Research 14:57-59 (1994). |
| Kunzi et al., “Role of Interferon-Stimulated Gene ISG-15 in the Interferon-W-Mediated Inhibition of Human Immunodeficiency Virus Replication”, Journal of Interferon & Cytokine Research 16(11):919-927 (Nov. 1996). |
| Laburthe, “Peptide YY and Neuropeptide Y in the Gut: Availability, Biological Actions, and Receptors”, Trends in Endocrinology & Metabolism 1(3):168-174 (1990). |
| Larsson, et al., “Stability of Emulsions Formed by Polar Lipids”, Progress in the Chemistry of Fats and Other Lipids 16:163-169 (1978). |
| Lee et al., “The Stabilization of Proteins by Sucrose”, Journal of Biological Chemistry 256(14):7193-7201 (Jul. 1981). |
| 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 et al., “Therapy of Hepatitis C: Interferon alfa-2A Trials”, Hepatology 26:89S-95S (XP000981288) (Sep. 1997). |
| Li et al., “Prediction of Solvent Removal Profile and Effect on Properties for Peptideloaded PLGA Microspheres Prepared by Solvent Extraction/evaporation Method”, Journal of Controlled Release, 37:199-214 (1995). |
| 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”, Current Therapeutic Research 71(4):211-238 (Aug. 2010). |
| Lopez et al., “Mammalian Pancreatic Preproglucagon contains Three Glucagon-related Peptides”, PNAS USA, 80(18):5485-5489 (1983). |
| Lublin et al., “Defining the Clinical Course of Multiple Sclerosis: Results of an International Survey”, Neurology 46:907-911 (1996). |
| Luft et al., “Electro-Osmotic Valve for the Controlled Administration of Drugs”, Medical & Biological Engineering & Computing 16(1):45-50 (Jan. 1978). |
| 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”, Journal of Virology 74(11):5006-5015 (Jun. 2000). |
| Lund et al., “Pancreatic Preproglucagon cDNA Contains Two Glucagon-Related Coding Sequences Arranged in Tandem”, PNAS USA 79(2):345-349 (1982). |
| Lundberg, “A Submicron Lipid Emulsion Coated with Amphipathic Polyethylene Glycol for Parenteral Administration of Paclitaxel (Taxol)”, Journal of Pharmacy and Pharmacology 49(1):16-21 (1997). |
| Maa et al., “Liquid-Liquid Emulsification by Static Mixers for Use in Microencapsulation”, Journal of Microencapsulation 13(4):419-433 (Jul.-Aug. 1996). |
| MacFayden et al., “NPY Stimulates Nett Absorption Across Rat Intestinal Mucosa in Vivo”, Neuropeptides 7:219-227 (1986). |
| Madsbad et al., “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). |
| Magnuson et al. “Enhanced Recovery of a Secreted Mammalian Protein from Suspension Culture of Genetically Modified Tobacco Cells”, Protein Expression and Purification 7(2):220-228 (1996). |
| Malley et al., “Chronic Toxicity and Oncogenicity of N-Methylpyrrolidone (Nmp) in Rats and Mice by Dietary Administration”, Drug and Chemical Toxicology 24(4):315-38 (Nov. 2001). |
| Manning et al., “Stability of Protein Pharmaceuticals”, Pharmaceutical Research 6(11):903-918 (1989). |
| Marincola et al., “Combination Therapy with Interferon alfa-2a and Interleukin-2 for the Treatment of Metastatic Cancer”, Journal of Clinical Oncology 13(5):1110-1122 (XP009078965) (1995). |
| Massey et al., “Interaction of Vitamin E with Saturated Phospholipid Bilayers”, Biochemical and Biophysical Research Communications 106(3):842-847 (1982). |
| Maulding et al., “Biodegradable Microcapsules: Acceleration of Polymeric Excipient Hydrolytic Rate by Incorporation of a Basic Medicament”, Journal of Controlled Release 3(1-4):103-117 (1986). |
| McHutchison et al., “Interferon α-2b Alone or in Combination with Ribavirin as Initial Treatment for Chronic Hepatitis C”, The New England Journal of Medicine 339(21):1485-1492 (Nov. 1998). |
| 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). |
| Mehta et al., “Peptide Containing Microspheres from Low Molecular Weight and Hydrophilic Poly(d, 1-Lactide-Co-Glycolide)”, Journal of Controlled Release 41:249-257 (1996). |
| Meier et al., “Glucagon-like Peptide 1(GLP-1) in Biology and Pathology”, Diabetes/Metabolism Research and Reviews 21(2):91-117 (Mar. 9, 2005). |
| 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”, American Journal of Physiology-Endocrinology and Metabolism 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)”, Journal of Immunotherapy 23(3):369-378 (May-Jun. 2000). |
| Milella et al., “Neutralizing Antibodies to Recombinant a-Interferon and Response to Therapy in Chronic Hepatitis C Virus Infection”, Liver 13(3):146-150 (Jun. 1993). |
| Mohler et al., “Primer on Electrodeposited Coatings”, Materials Engineering 5:38-45 (1972). |
| Mojsov, “Structural Requirements for Biological Activity of Glucagon-Like Peptide-I”, International Journal of Peptide and Protein Research 40(3-4):333-343 (1992). |
| Morgan et al., “Structure and Moisture Permeability of Film-Forming Poloyers”, Industrial & Engineering Chemistry Research 45(10):2296-2306 (1953). |
| Motzer et al., “Phase I Trial of 40-Kd Branched Pegylated Interferon α-2a for Patients with Advanced Renal Cell Carcinoma”, Journal of Clinical Oncology 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”, Diabetic Medicine 15(11):937-945 (1998). |
| NCBI “Homo sapiens Glucagon Receptor (GCGR), mRNA”, NCBI Reference Sequence: NM_000160.1, Retrieved from: <<https://www.ncbi.nlm.nih.gov/nuccore/NM_000160.1>>, 4 pages (2006). |
| NCBI “Homo sapiens Glucagon-like Peptide 1 Receptor (GLP1R), mRN”, NCBI Reference Sequence: NM_002062.1, Retrieved from: <<https://www.ncbi.nlm.nih.gov/nuccore/NM_002062.1>>, 3 pages (2000). |
| Neumann et al., “Hepatitis C Viral Dynamics In Vivo and the Antiviral Efficacy of Interferon-α 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 α-2a and a Polyethylene Glycol-Modified Derivative in Healthy Subjects”, Clinical Pharmacology & Therapeutics 59(6):636-646 (Jun. 1996). |
| Nielsen et al. “Incretin Mimetics and DPP-IV Inhibitors for the Treatment of Type 2 Diabetes”, Drug Discovery Today 20(10):703-710 (May 15, 2005). |
| Norden et al., “Physicochemical Characterisation of a Drug-Containing Phospholipid-Stabilised O/w Emulsion for Intravenous Administration”, European Journal of Pharmaceutical Sciences 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). |
| Ortiz et al., “A Differential Scanning Calorimetry Study of the Interaction of Alpha-Tocopherol with Mixtures of Phospholipids”, Biochimica et Biophysica Acta 898(2):214-222 (1987). |
| Palmeri et al., “5-Fluorouracil and Recombinant Alpha Interferon-2a in the Treatment of Advanced Colorectal Carcinoma: A Dose Optimization Study”, Journal of Chemotherapy 2(5):327-330 (Oct. 1990). |
| Panitch, “Interferons in Multiple Sclerosis”, Drugs 44(6):946-962 (Dec. 1992). |
| Patti et al., “Natural Interferon-B Treatment of Relapsing-Remitting and Secondary-progressive Multiple Sclerosis Patients: Two-Year Study”, Acta Neurologica Scandinavica 100:283-289 (1999). |
| Paty et al., “Interferon beta-1 b is effective in Relapsing-Remitting Multiple Sclerosis”, Neurology 43:662-667 (1993). |
| Patzelt et al., “Identification and Processing of Proglucagon in Pancreatic Islets”, Nature 282(5736):260-266 (1979). |
| Peterson et al., “Neuropathic complications in the Zucker Diabetic Fatty Rat”, Frontiers in Diabetic Research: Lessons from Animal Diabetes III, Edited by E. Shafrir, London: Smith-Gordon 456-458 (1990). |
| Peterson et al., “Zucker Diabetic Fatty Rat as a Model for Non-Insulin-Dependent Diabetes Mellitus”, Institute for Laboratory Animal Research (ILAR) Journal 32(3):16-19 (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 Study of Omega Interferon in Previously Untreated HCV-Infected Patients”, Hepatology 34(4):A331 (XP004716169) (Oct. 1, 2001) (Abstract Only). |
| Plauth et al., “Open-Label Study of Omega Interferon in Previously Untreated HCV-Infected Patients”, Journal of Hepatology 36(Supp.1):125, XP002511882 (Apr. 2002) (Abstract Only). |
| Playford et al., “Preliminary Report: Role of Peptide YY in Defence Against Diarrhoea”, The Lancet 335(8705):1555-1557 (Jun. 30, 1990). |
| Pohl et al., “Molecular cloning of the helodermin and exendin-4 cDNAs in the Lizard”, Journal of Biological Chemistry 273(16):9778-9784 (1998). |
| Poynard et al., “Is an “A La Carte” Combination Interferon α-2b plus Ribavirin Regimen Possible for the First Line Treatment in Patients with Chronic Hepatitis C? the ALGOVIRC Project Group”, Hepatology 31(1):211-218 (Jan. 2000). |
| Poynard et al., “Randomised Trial of Interferon α2b plus Ribavirin for 48 Weeks or for 24 Weeks Versus Interferon α2b plus Placebo for 48 Weeks for Treatment of Chronic Infection with Hepatitis C Virus. International Hepatitis Interventional Therapy Group (IHIT)”, Lancet 352(9138):1426-1432 (Oct. 1998). |
| Pratley et al., “Targeting Incretins in Type 2 Diabetes: Role of GLP-1 Receptor Agonists and DPP-4 Inhibitors”, Review of Diabetic Studies 5(2):73-94 (2008). |
| Quesada et al., “Interferons in Hematological Malignancies”, eds. Baron et al., U. Tex. 487-495 (1987). |
| Quianzon et al., “Lixisenatide-Once daily Glucagon-Like Peptide-1 Receptor Agonist in the Management of Type 2 Diabetes”, US Endocrinology 7(2):104-109 (Dec. 2011). |
| Quintanar-Guerrero et al., “Applications of the Ion-Pair Concept to Hydrophilic Substances with Special Emphasis on Peptides”, Pharmaceutical Research 14(2):119-127 (1997). |
| Rajkumar et al., “Phase I Evaluation of Radiation Combined with Recombinant Interferon α-2a and BCNU for Patients with High-Grade Glioma”, International Journal of Radiation Oncology 40(2):297-302 (Jan. 15, 1998). |
| Ratner et al., “Dose-Dependent Effects of the Once-Daily GLP-1 Receptor Agonist Lixisenatide In Patients with Type 2 Diabetes Inadequately Controlled with Metformin: A Randomized Double-Blind, Placebo-Controlled Trial”, Diabetic Medicine 27(9):1024-1032 (Aug. 2010). |
| Roberts et al., “The Evolution of the Type I Interferons”, Journal of Interferon & Cytokine Research 18(10):805-816 (Oct. 1998). |
| Roche Pharmaceuticals, Roferon®-a (Interferon α-2a, Recombinant), 22 pages (2003). |
| Roff et al., “Handbook of Common Polymers”, Cleveland Rubber Co. 72 pages (1971). |
| Rogers et al., “Permeability Valves”, Industrial & Engineering Chemistry Research 49(11):1933-1936 (Nov. 17, 1957). |
| Rohloff et al., “DUROS Technology Delivers Peptides and Proteins at Consistent Rate Continuously for 3 to 12 Months”, Journal of Diabetes Science and Technology 2(3):461-467 (May 2008). |
| 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., “Combination Therapy with Amylin and Peptide YY[3-36] in Obese Rodents: Anorexigenic Synergy and Weight Loss Additivity”, Endocrinology 148(12):6054-6061 (Dec. 2007). |
| Roth et al., “High Dose Etretinate and Interferon-alpha-A Phase I Study in Squamous Cell Carcinomas and Transitional Cell Carcinomas”, Acta Oncologica 38(5):613-617 (1999). |
| 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). |
| 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”, Journal of Pharmacology: Molecular Pharmacology 269(2):183-191 (1994). |
| Schering Corporation, Intron® A for Injection, 6 pages (2001). |
| Schering Corporation, PEG-Intron™ (Peginterferon α-2b) Powder for Injection, 29 pages (2003). |
| Schmalfub et al., “Modification of Drug Penetration into Human Skin Using Microemulsions”, Journal of Controlled Release 46(3):279-285 (1997). |
| Sen et al., “The Interferon System: A Bird's Eye View of Its Biochemistry”, Journal of Biological Chemistry 267(8):5017-5020 (Mar. 1992). |
| Seufert et al., “The Extra-pancreatic Effects of GLP-1 Receptor Agonists: a Focus on the Cardiovascular, Gastrointestinal and Central Nervous Systems”, Diabetes, Obesity and Metabolism 16(8):673-688 (Aug. 2014). |
| Shiffman et al., “A Decline in HCV-RNA Level During Interferon or Ihterferon/ribavirin Therapy in Patients with Virologic Nonresponse Is Associated with an Improvement in Hepatic Histology”, Progress Abstract 50th Annual Meeting Postgraduate Courses American Association Study Liver Disease, 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”, Journal of Gastroenterology & Hepatology 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”, Annals of Internal Medicine 132(7):517-524 (Apr. 2000). |
| Shire et al., “Challenges in the Development of High Protein Concentration Formulations,” Journal of Pharmaceutical Sciences 93:1390-1402 (2004). |
| Simon et al., “A Longitudinal Study of T1 Hypointense Lesions in Relapsing MS: MSCRG Trial of Interferon β-1a”, Neurology 55(2):185-192 (Jul. 2000). |
| Smith, “Peripheral Neuro-Horrnones as a Strategy to Treat Obesity”, Oral Presentation at the 2007 Cardiometabolic Health Congress in Boston, MA, pp. 1-35 (Sep. 26-29, 2007). |
| 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., “Peginterferon-a-2a (40kD) and ribavirin in patients with chronic hepatitis C: a phase II open label study,” Gastroenterology 118(4, Supp. 2) (2000) (Abstract 236). |
| Sulkowski et al., “Peginterferon-α-2a (40kD) and Ribavirin in Patients with Chronic Hepatitis C: A Phase II Open-Label Study”, Biodrugs 16(2):105-109 (2002). |
| Szayna et al., “Exendin-4 Decelerates Food Intake, Weight Gain, and Fat Deposition in Zucker Rats”, Endocrinology 141(6):1936-1941 (2000). |
| Talpaz et al., “Phase I Study of Polyethylene Glycol Formulation of Interferon α-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”, International Journal of Cancer 87(5):741-749 (Sep. 2000). |
| Taylor et al., “Day-long Subcutaneous Infusion of Exenatide Lowers Glycemia in Patients with Type 2 Diabetes”, Hormone and Metabolic Research 37(10):627-632 (Aug. 2010). |
| Thomasin et al., “VA Contribution to Overcoming the Problem of Residual Solvents in Biodegradable Microspheres Prepared by Coacervation”, European Journal of Pharmaceutics and Biopharmaceutics 42(1):16-24 (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). |
| Tong et al., “Prediction of Response During Interferon a 2b Therapy in Chronic Hepatitis C Patients Using Viral and Biochemical Characteristics: A Comparison”, Hepatology 26(6):1640-1645 (Dec. 1997). |
| Touza Rey et al., “The Clinical Response to Interferon-Gamma in a Patient with Chronic Granulomatous Disease and Brain Abscesses Due to Aspergillus Fumigatus”, Anales de Medicina Interna 17(2):86-87 (Feb. 2000). |
| Tracy et al., “Factors Affecting the Degradation Rate of Poly(lactide-Co-Glycolide) Microspheresin Vivo and in Vitro”, Biomaterials 20(11:):1057-1062 (1999). |
| Trudeau et al., “A Phase I Study of Recombinant Human Interferon α-2b Combined with 5-Fluorouracil and Cisplatin in Patients with Advanced Cancer”, Cancer Chemotherapy and Pharmacology 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 (Mar. 1993). |
| Tsung et al., “Preparation and Stabilization of Heparin/Gelatin Complex Coacervate Microcapsules,” Journal of Pharmaceutical Sciences 86(5):603-607 (May 1997). |
| Uhlig et al., “The Electro-Osmotic Actuation of Implantable Insulin Micropumps”, Journal of Biomedical Materials Research 17(6):931-943 (1983). |
| 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). |
| Van Santbrink et al., “Urinary Follicle-Stimulating Hormone for Normogonadotropic Clomiphene-Resistant Anovulatory Infertility: Prospective, Randomized Comparison Between Low Dose Step-up and Step-down Dose Regimens”, The Journal of Clinical Endocrinology and Metabolism 82(11):3597-3602 (1997). |
| Vlasakakis et al., “Pharmacokinetics and Tolerability of Exenatide Delivered by 7-Day Continuous Subcutaneous Infusion in Healthy Volunteers”, Advances in Therapy 32:650-661 (Jul. 10, 2015). |
| 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 Chemotherapy and Pharmacology 35(4):304-312 (1995). |
| Vrabec, “Tympanic Membrane Perforations in the Diabetic Rat: A Model of Impaired Wound Healing”, Otolaryngology—Head and Neck Surgery 118(3):304-308 (Mar. 1998). |
| Wang et al., “Parenteral Formulations of Proteins and Peptides: Stability and Stabilizersple Sclerosis?”, Journal of Parenteral Science and Technology 42(2S):S4-S26 (1988). |
| Wang et al., “Preferential Interaction of α-Tocopherol with Phosphatidylcholines in Mixed Aqueous Dispersions of Phosphatidylcholine and Phosphatidylethanolamine”, European Journal of Biochemistry 267(21):6362-6368 (2000). |
| Wang et al., “Ripple Phases Induced by Alpha-Tocopherol in Saturated Diacylphosphatidylcholines”, Archives of Biochemistry and Biophysics 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). |
| 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”, Progress in Nucleic Acid Research and Molecular Biology 33:251-300 (1986). |
| Wright et al., “Preliminary Experience with α-2b-Interferon Therapy of Viral Hepatitis in Liver Allograft Recipients”, Transplantation 53(1):121-124 (Jan. 1992). |
| Wynne et al., “Oxyntomodulin Increases Energy Expenditure in Addition to Decreasing Energy Intake in Overweight and Obese Humans: a Randomised Controlled Trial”, International Journal of Obesity 30:1729-1736 (2006). |
| Young et al., “Glucose-Lowering and Insulin-Sensitizing Actions of Exendin-4”, Diabetes 48(5):1026-1034 (May 1999). |
| Younossi et al., “The Roles of Amantadine, Rimantadine, Ursodeoxycholic Acid, and NSAIDs, Alone or in Combination with Alpha Interferons, in the Treatment of Chronic Hepatitis C”, Seminars in Liver Disease 19(Supp. 1):95-102 (1999). |
| Yu et al., “Glucagon-Like Peptide-1 Prevented Abdominal Aortic Aneurysm Development in Rats”, Surgery Today 46(9):1099-1107 (Sep. 2016). |
| Yu et al., “Preparation, Characterization, and in Vivo Evaluation of an Oil Suspension of a Bovine Growth Hormone Releasing Factor Analog”, Journal of Pharmaceutical Sciences 85(4):396-401 (1996). |
| Zeidner et al., “Treatment of FeLV-Induced Immunodeficiency Syndrome (feLVFAIDS) with Controlled Release Capsular Implantation of 2′,3′-Dideoxycytidine”, Antiviral Research 11(3):147-160 (Apr. 1989). |
| Zein et al., “Interferons in the Management of Viral Hepatitis,” Cytokines, Cellular & Molecular Therapy 4(4):229-241 (Dec. 1998). |
| 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). |
| Zeuzem et al., “Peginterferon α-2a in Patients with Chronic Hepatitis C”, The New England Journal of Medicine 343(23):1666-1672 (2000). |
| Zhang et al., “A New Strategy for Enhancing the Stability of Lyophilized Protein: The Effect of the Reconstitution Medium on Keratinocyte Growth Factor”, Pharmaceutical Research 12(10):1447-1452 (1995). |
| Zhang et al., “Report on Large Dosage Interferon to Treat 30 Cases of Viral Encephalitis,” Journal of Clinical Pediatrics 14(2):83-84 (1996). |
| Zhang et al., “Efficacy Observations of Different Dosages of Interferon to Treat 150 Hepatitis B Carrier”, Current Physician 2(12):45-46 (1997). |
| Zheng et al. “Therapeutic Effect of Interferon Varied Dose in Treating Virus Encephalitis,” Beijing Medical Journal 13(2):80-81 (1998). |
| Ziesche et al., “Preliminary Study of Long-Term Treatment with Interferon Y-1 Band Low-Dose Prednisolone in Patients with Idiopathic Pulmonary Fibrosis”, The New England Journal of Medicine 341(17):1264-1269 (Oct. 1999). |
| Number | Date | Country | |
|---|---|---|---|
| 20220332781 A1 | Oct 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62420937 | Nov 2016 | US | |
| 62414146 | Oct 2016 | US | |
| 62337005 | May 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16667502 | Oct 2019 | US |
| Child | 17538507 | US | |
| Parent | 15595809 | May 2017 | US |
| Child | 16667502 | US |
