NZ785999A - Glucagon and glp-1 co-agonists for the treatment of obesity - Google Patents

Abstract

Provided herein are methods of preventing and treating obesity and diabetes in patients comprising administering GLP-1/glucagon agonist peptides.

Description

GLUCAGON AND GLP-1 CO-AGONISTS FOR THE TREATMENT OF OBESITY This application is a divisional application from New Zealand Patent Application No. , the entire disclosure of which is incorporated herein by reference.

REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62/306,121, filed March 10, 2016, which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The content of the electronically submitted sequence listing in ASCII text file (Name: Sequencelisting_ST25.txt; Size: 14,333 bytes; and Date of Creation: March 6, 2017) filed with the application is incorporated herein by reference in its entirety.

BACKGROUND Obesity is a major and growing health problem worldwide. It is ated with many life-threatening diseases such as cardiovascular e, renal disease, hypertension, stroke, infertility, respiratory dysfunction, and type 2 diabetes.

Glucagon and on-like peptide-1 (GLP-1) derive from pre-proglucagon, a 158 amino acid precursor polypeptide that is processed in different tissues to form a number of different cagon-derived es, including glucagon, glucagon-like peptide-1 (GLP- 1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM), that are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal , as well as the regulation of food intake. Glucagon is a 29- amino acid peptide that corresponds to amino acids 33 through 61 of cagon (53 to 81 of preproglucagon), while GLP-1 is produced as a no acid peptide that corresponds to amino acids 72 through 108 of proglucagon (92 to 128 of preproglucagon). GLP-1(7-36) amide or GLP-1(7-37) acid are biologically active forms of GLP-1, that demonstrate essentially equivalent activity at the GLP-1 receptor.

Glucagon is produced by the pancreas and interacts with the glucagon receptor ("glucR"). Glucagon acts in the liver to raise blood glucose via gluconeogenesis and glycogenolysis. When blood e begins to fall, glucagon signals the liver to break down glycogen and release e, causing blood glucose levels to rise toward a normal level.

GLP-l has different biological activities compared to glucagon. It is secreted from gut L cells and binds to the GLP-1 receptor. Its activities include stimulation of insulin synthesis and secretion, inhibition of glucagon secretion, and inhibition of food intake.

Both on and GLP-1, acting as agonists at their respective receptors, have been shown to be effective in weight loss. Certain GLP-l analogs are being sold or are in development for treatment of obesity including, e.g., Liraglutide (VICTOZA® from Novo Nordisk) and Exenatide (Byetta® from Eli Lilly/Amylin). Glucagon/GLP-l agonist peptides have also been disclosed in While some therapies are ble for the control of blood glucose, none currently achieve substantial weight loss, which remains a significant unmet need for patients. Fifty percent of patients progress from oral monotherapy for glucose control (usually with metformin) to tion of insulin within 10 years, often taking multiple oral combination therapies before ting insulin. The use of insulin exacerbates weight gain, which can be as great as 6 kg in the first year after starting insulin therapy. This weight gain can lead to increased insulin resistance, which is associated with hypertension, dyslipidemia, and an increased risk of major adverse cardiovascular events.

With respect to reducing insulin resistance, significant weight loss (> 5%) is the l intervention to reduce insulin resistance, gh this can only be ed reliably at present through intensive dietary and lifestyle interventions and/or ric surgery.

There remains a need for methods of administering GLP-l/Glucagon t peptides to humans using dosage regimens that are therapeutically effective in ng diabetes and obesity but avoid adverse effects.

BRIEF SUMMARY Provided herein are methods of preventing and treating obesity and diabetes and related conditions in patients comprising stering glucagon agonist peptides.

In one instance, a method of reducing body weight comprises stering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide comprising the amino acid sequence of HXZQGTFTSDX103X12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30; wherein X2 is G or S, X10 is Y or K, X12 is K, E, R, or S, X13 is K or Y, X15 is D or E, X16 is S or G, X17 is E, R, Q, or K, X13 is R, S, or A, X20 is R, K, or Q, X21 is D or E, X23 is V or I, X24 is A or Q, X27 is E or V, X28 is A or K, and X30 is G or R (SEQ ID NO:4).

In one instance, a method of reducing body fat comprises administering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

In one instance, a method of treating y comprises administering to a human subject in need thereof 50-600 ug or 0 ug of a e sing the amino acid sequence of SEQ ID NO:4.

In one instance, a method of treating or preventing a disease or ion caused or characterized by excess body weight comprises administering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

In one instance, a method of managing weight comprises administering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide sing the amino acid sequence of SEQ ID NO:4.

In one ce, a method of treating Nonalcoholic Steatohepatitis (NASH) comprises administering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

In one instance, a method of increasing lipid oxidation comprises administering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

In one instance, a method of reducing food intake comprises administering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

In one instance, a method of lowering plasma glucose comprises stering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

In one instance, a method of ing glycemic control comprises administering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

WO 53575 In one instance, a method of achieving glycemic l comprises administering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

In one instance, a method of decreasing weight and controlling glucose comprises administering to a human subject in need thereof 50-600 ug or 100-600 ug of a peptide sing the amino acid sequence of SEQ ID NO:4.

In one instance, the subject has diabetes. In one instance, the diabetes is type 2 diabetes mellitus.

In one ce, a method of treating type 2 es mellitus comprises administering to a human a peptide comprising the amino acid sequence of SEQ ID NO:4.

In one instance, a method of improving glycemic control in a human subject with type 2 diabetes mellitus comprises administering to the subject 50-600 ug or 100—600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

In one ce, the administering reduces body weight. In one instance, the administering treats obesity. In one instance, the administration reduces body fat.

In one instance, X2 is G, X10 is K, X12 is E, R, or S, X13 is K, X17 is E or K, X18 is S, X20 is R, X27 is E, or X28 is A.

In one instance, X2 is G, X10 is K, X12 is E, R, or S, X13 is K, X17 is E or K, X18 is S, X20 is R, X27 is E, and X28 is A.

In one ce, the peptide ses, consists essentially of, or consists of the amino acid sequence of SEQ ID N0219.

In one instance, the administration comprises administering an initial dose for 3 to days and a second higher dose thereafter.

In one ce, the initial dose is administered for 3 to 7 days. In one instance, the initial dose is 100 ug of the peptide. In one instance, the second dose is administered for at least four days. In one ce, the initial dose is administered for four consecutive days and the second dose is administered for at least four consecutive days. In one instance, the second dose is 150-200 ug of the peptide. In one instance, the second dose is 150 ug of the peptide or 200 ug of the peptide.

In one instance, the administration further comprises administering a third dose after the second dose, wherein the third dose is higher than the second dose. In one instance, the initial dose is administered for 3 to 10 days and the second dose is administered for 3 to 10 days. In one instance, the initial dose is administered for 3 to 7 days and the second dose is administered for 3 to 7 days. In one instance, the l dose is administered for four utive days, the second dose is administered for four consecutive days, and the third dose is administered for at least four consecutive days. In one instance, the initial dose is administered for four consecutive days, the second dose is administered for seven consecutive days, and the third dose is administered for at least four consecutive days. In one instance, the third dose is 200-400 ug of the e. In one instance, the third dose is 200 ug of the peptide, 300 ug of the peptide, or 400 ug of the peptide.

In one instance, an initial dose of 100 ug is administered for four days, a second dose of 150 ug is administered for four days, and a third dose of 200 ug is uently administered daily. In one instance, an initial dose of 100 ug is administered for five days, a second dose of 150 ug is administered for five days, and a third dose of 200 ug is administered for five days, and a fourth dose of 300 ug is subsequently administered daily. In one instance, an initial dose of 100 ug is administered for five days, a second dose of 200 ug is administered for five days, and a third dose of 300 ug is subsequently administered daily.

In one instance, 50 ug of the e is stered. In one instance, 100 ug of the peptide is administered. In one instance, 150 ug of the e is administered. In one instance, 200 ug of the peptide is administered. In one instance, 250 ug of the peptide is administered. In one instance, 300 ug of the peptide is administered. In one instance, 400 ug of the peptide is administered.

In one instance, the peptide is administered daily. In one ce, the peptide is administered once daily.

In one instance, the peptide is administered for at least one week, for at least two weeks, for at least three weeks, or for at least four weeks.

In one instance, the e is administered by injection. In one instance, the administration is subcutaneous.

In one instance, the administration results in at least 20% reduction in glucose area under the concentration-time curve after a mixed-meal test.

In one ce, glucose is reduced. In one instance, the e is fasting plasma e. In one instance, the glucose is postprandial glucose from a mixed-meal test.

In one instance, the administration results in weight loss of at least 1.0 kg, at least 1.3 kg, or about 1.3 to about 2.0 kg. In one ce, the subject’s weight is reduced by at least 3.5 kg or at least 5 kg. In one instance, the subject’s weight is reduced by at least 2%, at least 4%, at least 5%, or at least 10%. In one instance, the subject’s weight is reduced by about 2% to about 20%, about 2% to about 25%, or about 2% to about 30%.

In one instance, the fat is liver fat. In one instance, liver fat in the subject is reduced by at least 20%. In one instance, liver fat in the subject is reduced by about 20% to about 40%. In one instance, the stration results in about a one third reduction in liver fat. In one instance, liver volume is reduced in the subject.

In one instance, the administration s hemoglobin Alc (HbAlc) levels. In one instance, the HbAlc level in the subject is reduced by at least 0.6 %. In one instance, the HbAlc level in the subject is reduced by at least 0.9%. In one instance, the HbAlc level in the subject is reduced by about 0.5% to about 1.5%, about 0.5% to about 2%, or about 0.5% to about 3%. In one instance, the HbAlc level in the subject is reduced to 6.3% or lower.

In one instance, the administration reduces fructosamine levels.

In one instance, the subject’s appetite is reduced.

In one instance, the subject’s energy expenditure is sed.

In one instance, disease progression is stopped. In one instance, e ssion is reversed.

In one instance, the peptide is manufactured tically by solid-phase synthesis. In one instance, the solid-phase synthesis uses fluorenylmethyloxycarbonyl chloride chemistry.

In one instance, the carboxyl group of X30 in the peptide is unmodified G or R. In one instance, the carbonyl group of X30 in the peptide is amidated.

In one instance, the peptide comprises a palmitoyl moiety on the N(epsilon) group of a lysine residue. In one instance, the palmitoyl group is linked to the lysine via a linker.

In one instance, the linker is gamma glutamate. In one instance, the peptide comprises a WO 53575 stearoyl or sterate moiety on the N(epsilon) group of a lysine residue. In one instance, the lysine residue is X10.

In one instance, the peptide binds to a human glucagon receptor with an EC50 in the CAMP assay 1 of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.

In one instance, the peptide binds to a human GLP-1 receptor with an EC50 in the CAMP assay 1 of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.

In one instance, the peptide is an agonist of GLP-1 activity, an t of glucagon activity, or an agonist of both GLP-1 and glucagon activity.

In one instance, the peptide binds to both a glucagon receptor and a GLP-1 receptor, wherein the peptide exhibits at least about 2-fold, 5-fold, or 10-fold greater activity relative to the natural ligand at the GLP-1 receptor than at the glucagon receptor.

In one instance, the peptide further comprises a heterologous moiety. In one instance, the heterologous moiety is a protein, a peptide, a n domain, a linker, an c polymer, an inorganic polymer, a polyethylene glycol (PEG), biotin, an albumin, a human serum albumin (HSA), a HSA FcRn binding portion, an dy, 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 III ld, an epitope tag, a recombinant polypeptide polymer, a ne, a lipid, or a ation of two or more of the recited moieties.

In one instance, the subject has a body mass index (BMI) of 27 to 40 kg/mz. In one instance, the subject has a BMI of 30-399 kg/mz. In one instance, the subject has a BMI of at least 40 kg/mz.

In one instance, the subject is overweight. In one instance, the subject is obese. In one instance, the subject is (i) ight and (ii) has hypertension, Type 2 diabetes mellitus, dyslipidemia, a history of cardiovascular disease or a combination of thereof. In one instance, the subject is (i) overweight and (ii) has dysglycaemia, ension, dyslipidemia, obstructive sleep apnea, or a combination of thereof.

In one ce, the subject is ing insulin therapy. In one instance, the amount of insulin administered is reduced. In one instance, the insulin therapy is stopped.

In one instance, the subject is receiving insulin, metformin, sulphonylurea, a sodium-glucose cotransporter-2 (sglt-2) tor, a idyl peptidase-4 V) inhibitor, glutazone, an alpha glucosidase inhibitor, or a combination thereof.

In one instance, the half-life of the peptide is about 10 to about 12 hours.

In one instance, the administration is an adjunct to diet and se.

In one instance, the peptide comprises SEQ ID NO:l9, and the peptide is administered at an initial dose of 100 ug for four days, at a second dose of 150 ug for four days, and subsequently at a dose of 200 ug daily.

In one instance, the peptide comprises SEQ ID NO:l9, and the peptide is administered at an initial dose of 100 ug for five days, at a second dose of 200 ug for five days, and uently at a dose of 300 ug daily.

In one instance, the peptide comprises SEQ ID NO:l9, and the peptide is stered at an initial dose of 100 ug for five days, at a second dose of 150 ug for five days, at a second dose of 200 ug for five days, and subsequently at a dose of 300 ug daily.

In one ce, the subject has type 2 diabetes mellitus. In one instance, the subject is obese. In one instance, the subject is (i) overweight and (ii) has hypertension, Type 2 diabetes mellitus, dyslipidemia, a history of cardiovascular disease or a combination of f. In one instance, the subject is (i) overweight and (ii) has dysglycaemia, hypertension, dyslipidemia, obstructive sleep apnea, or a combination of thereof.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES shows the mean percent of change in body weight from day zero in DIO mice following administration of glucagon/GLP-l co-agonist peptide G730 at three different doses, compared to vehicle treatment, and ent with Liraglutide. Starting body weight in the ent groups were vehicle: 47.4i3.7g, G730 10 nmol/kg: 44.5i2.2g, G730 20 nmol/kg: 45.9i3.6g and G730 50 nmol/kg: 46.1i2.4g, respectively. shows the mean percent of change in body weight from day zero in DIO mice following administration of glucagon/GLP-l co-agonist e G797 at three different doses, compared to vehicle treatment, and treatment with Liraglutide. Starting body weight in the ent groups were vehicle: 47.4i3.7g, G797 5 nmol/kg: 47.5il.2g, G797 20 nmol/kg: 47.4i2.2g and G797 50 nmol/kg: 47.2il.8g, respectively. shows the mean percent of change in body weight from day zero in DIO mice following administration of glucagon/GLP-l co-agonist peptide G8l2 at a dose of nmol/kg, compared to vehicle treatment, and treatment with Liraglutide. Starting body weight in the different groups were vehicle: 47.4i3.7g and G812 20 nmol/kg: 49.2i3.4g, respectively. is a graph comparing the change in body weight results for the three glucagon/GLP-l co-agonist peptides presented in FIGS. 1, 2, and 3. shows the mean t of change in body weight from day zero in DIO mice following administration of glucagon/GLP-l co-agonist peptide G796 at two different doses, compared to vehicle treatment, and treatment with Liraglutide. shows the mean percent of change in body weight from day zero in DIO mice following administration of glucagon/GLP-l co-agonist peptide G865 at two different doses, compared to vehicle treatment, and treatment with Liraglutide. shows the mean percent of change in body weight from day zero in DIO mice following administration of glucagon/GLP-l co-agonist peptide G933 at two ent doses, compared to e treatment, and treatment with Liraglutide. is a graph ing the change in body weight results for the three glucagon/GLP-l co-agonist es presented in FIGs. 5, 6, and 7. provides flow ms of the planned and actual G933 single ascending dose study. provides the t disposition of the G933 single ascending dose study. shows the median glucose levels of ts in the G933 single ascending dose study. shows the median insulin levels of subjects in the G933 single ascending dose study. provides a flow diagram of Cohorts 1-4 in the G933 multiple ascending dose study. MEDIO3 82 refers to a 30 amino acid linear peptide with the ce of SEQ ID NO: 19. shows the mean mixed-meal test glucose levels in subjects treated with placebo or 100 ug G933 on Day 7 (Cohort 1), with placebo or 150 ug G933 on Day 11 (Cohort 2), and with placebo or 200 ug G933 on Day 15 (Cohort 3). shows the change in baseline fasting glucose levels in subjects treated with placebo or G933 on Day 7 (Cohort 1), with placebo or G933 on Day 9 (Cohort 3), and with placebo or G933 on Day 15 (Cohort 3). shows the change in weight from baseline weight over the course of the study in both Cohorts 1 and 3 and on Days 7 and 15 in Cohorts 1 and 3, respectively. shows the plasma concentration of MEDIO3 82 after repeat dosing. shows an improvement in glucose control as ed by glucose levels in patients treated with G933. The dashed lines represent e levels ed at baseline (Day 1), and the solid lines represent e levels observed on day 41. shows an improvement in glucose control as measured by HbAlc in patients d with G933. shows a decrease in te weight in patients treated with G933. (G933 is referred to as "MEDI" or "0382" in this figure; "Plac" refers to placebo.) shows a decrease in percent weight in patients treated with G933.

("MEDI" refers to "MEDIO3 82" in this figure.) shows an assessment of liver fat reduction in patients treated with G933.

Representative images from dual subjects are provided. shows the nausea and vomiting that occurred in patients treated with G933. provides a flow diagram of Cohorts 5 and 6 in the G933 multiple ascending dose study. shows an improvement in glucose control as measured by glucose levels in Cohort 5 ts treated with G933 as compared to placebo. The dashed lines represent glucose levels observed at baseline (Day 1), and the solid lines represent e levels observed on day 17. (G933 is referred to as "MEDI" in this figure.) shows an improvement in glucose control as measured by glucose levels in Cohort 6 patients treated with G933 as compared to placebo. The dashed lines represent glucose levels observed at baseline (Day 1), and the solid lines ent glucose levels observed on day 17. (G933 is referred to as "MEDI" in this figure.) shows the percent change from baseline glucose AUC in all cohorts. shows an improvement in fasting glucose levels in Cohorts 5 and 6.

(G933 is referred to as "MEDI" in this figure.) shows the weight change from baseline in Cohorts 5 and 6. (G933 is referred to as "MEDI" in this .) shows weight loss and change in glucose across all cohorts. shows the G933 plasma concentration in Cohort 5 on days 16 and 22 and in Cohort 6 on days 11 and 17.

DETAILED DESCRIPTION Definitions Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a polynucleotide," is understood to represent one or more polynucleotides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or ents with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is ed to encompass each of the following s: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with the language "comprising,’ I otherwise analogous aspects described in terms of "consisting of" and/or "consisting essentially of" are also ed. A e "comprising" a particular amino acid sequence refers to a peptide ning the amino acid sequence, wherein the peptide may or may not contain additional amino acids or other modifications to the amino acid sequence. A peptide "consisting of' a particular amino acid ce refers to a peptide containing only the amino acid ce and no additional amino acids or other modifications to the amino acid sequence. A peptide "comprising" an amino acid sequence sting of" a particular amino acid sequence refers to a peptide containing the amino acid sequence and no additional amino acids; however, the peptide may comprise other modifications to the amino acid sequence (e.g., an acyl moiety or a palmitoyl moiety).

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 this disclosure is d. For example, the e Dictionary of Biomedicine and Molecular Biology, Juo, ow, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the s defining the range.

Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The gs provided herein are not tions of the various aspects of the disclosure, which can be had by reference to the ication as a whole.

Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and comprises any chain or chains of two or more amino acids. Thus, as used herein, a "peptide," a "peptide subunit," a "protein," I I an "amino acid chain,’ an "amino acid ce,’ or any other term used to refer to a chain or chains of two or more amino acids, are included in the tion of a "polypeptide," even though each of these terms can have a more specific meaning. The term eptide" can be used instead of, or interchangeably with any of these terms.

The term r includes polypeptides which have undergone post-translational or post- synthesis modifications, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.

More specifically, the term "peptide" as used herein encompasses full length peptides and nts, ts or derivatives thereof, e.g., a GLP-l/glucagon agonist peptide (e.g., 29, 30, or 31 amino acids in length). A "peptide" as disclosed herein, e.g., a GLP-l/glucagon agonist peptide, can be part of a fusion polypeptide comprising additional components such as, e.g., an Fc domain or an albumin , to increase half-life. A peptide as described herein can also be derivatized in a number of different ways.

The term "isolated" refers to the state in which es or nucleic acids, will generally be in accordance with the present disclosure. Isolated peptides and isolated nucleic acids will be free or ntially free of material with which they are naturally associated such as other peptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practiced in vitro or in viva. es and nucleic acid can be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the peptides will normally be mixed with gelatin or other rs if used to coat itre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.

A "recombinant" peptide refers to a peptide produced via inant DNA technology. Recombinantly produced peptides expressed in host cells are considered isolated for the purpose of the present sure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

The terms ent," "analog," "derivative," or "variant" when referring to a GLP-l/glucagon agonist peptide include any peptide which retains at least some desirable activity, e.g., binding to glucagon and/or GLP-l receptors. Fragments of GLP-l/glucagon agonist es provided herein include proteolytic fragments, deletion fragments which exhibit desirable properties during expression, purification, and or administration to a subject.

The term "variant," as used herein, refers to a peptide that differs from the d peptide due to amino acid tutions, deletions, insertions, and/or modifications.

Variants can be ed using art-known mutagenesis techniques. Variants can also, or alternatively, contain other modifications— for example a peptide can be conjugated or coupled, e.g., fused to a heterologous amino acid ce or other moiety, e.g., for increasing half-life, solubility, or stability. Examples of moieties to be conjugated or coupled to a peptide provided herein include, but are not limited to, albumin, an immunoglobulin Fc region, hylene glycol (PEG), and the like. The peptide can also be conjugated or produced coupled to a linker or other sequence for ease of synthesis, purification or identification of the peptide (e.g., 6-His), or to enhance binding of the polypeptide to a solid support.

The term "sequence ty" as used herein refers to a relationship between two or more polynucleotide sequences or n two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid in the corresponding position of the comparator sequence, the sequences are said to be "identical" at that on. The percentage "sequence identity" is calculated by determining the number of positions at which the cal nucleic acid base or amino acid occurs in both sequences to yield the number of "identical" positions. The number of "identical" positions is then divided by the total number of positions in the comparison window and multiplied by 100 to yield the percentage of nce identity." tage of "sequence identity" is determined by comparing two optimally aligned sequences over a comparison window. In order to lly align sequences for comparison, the portion of a polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant. An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of "identical" ons between the reference and comparator sequences.

Percentage "sequence identity" between two sequences can be ined using the version of the program "BLAST 2 Sequences" which was available from the National Center for Biotechnology Information as of ber 1, 2004, which program orates the ms BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing "BLAST 2 Sequences," parameters that were default parameters as of September 1, 2004, can be used for word size (3), open gap penalty (1 1), extension gap penalty (1), gap drop- off (50), expect value (10), and any other required ter including but not limited to matrix option.

The terms "composition" or "pharmaceutical composition" refer to compositions containing a GLP-l/glucagon agonist peptide provided herein, along with e.g., pharmaceutically acceptable carriers, excipients, or diluents for administration to a t in need of treatment, e.g., a human subject being treated for obesity.

The term "pharmaceutically acceptable" refers to itions that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity or other complications commensurate with a reasonable benefit/risk ratio.

An tive amount" is that amount of a GLP-l/glucagon agonist peptide ed herein, the administration of which to a subject, either in a single dose or as part of a series, is effective for treatment, e.g., treatment of obesity. An amount is ive, for example, when its administration results in one or more of weight loss or weight maintenance (e.g., prevention of weight gain), loss of body fat, prevention or modulation hypoglycemia, prevention or modulation lycemia, promotion of insulin synthesis, or reduction in food . This amount can be a fixed dose for all subjects being treated, or can vary depending upon the weight, health, and physical condition of the subject to be treated, the extent of weight loss or weight maintenance desired, the formulation of peptide, a sional assessment of the medical situation, and other relevant s.

The term ct" is meant any subject, particularly a mammalian subject, in need of treatment with a GLP-l/glucagon t peptide provided herein. Mammalian subjects e, but are not limited to, , dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows, apes, monkeys, orangutans, and chimpanzees, and so on.

In one embodiment, the subject is a human subject.

As used herein, a "subject in need thereof' refers to an individual for whom it is desirable to treat, e.g., to an obese subject or a subject prone to obesity for whom it is desirable to facilitate weight or body fat loss, weight or body fat maintenance, or to prevent or minimize weight gain over a specified period of time.

Terms such as "treating" or "treatment" or "to treat" refer to therapeutic measures that cure and/or halt progression of a diagnosed pathologic condition or disorder. Terms such as "preventing" refer to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disease or condition. Those in need of prevention e those prone to have the disease or ion and those in whom the e or condition is to be prevented. For example, the phrase "treating a patient" having a e or condition caused or terized by excess body weight refers to reducing the severity of the disease or condition to an extent that the subject no longer suffers discomfort and/or d function due to it. The phrase "preventing" a disease or condition caused or terized by excess body weight refers to reducing the potential for the e or condition and/or reducing the occurrence of the disease or condition (for example a relative reduction in occurrence as compared to untreated patients).

Terms such as "decreasing the severity" refer to therapeutic measures that slow down or lessen the symptoms of a diagnosed pathologic condition or er. For example, the phrase "decreasing the severity" of a disease or condition caused or characterized by excess body weight refers to reducing the severity of the disease or condition (for example, a reduction in weight when compared to untreated patients or an increase in glucose control).

As used herein a "GLP-l/glucagon agonist peptide" is a chimeric peptide that exhibits activity at the glucagon receptor of at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more relative to native glucagon and also exhibits activity at the GLP-1 or of about at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more relative to native GLP-l, under the conditions of assay 1.

As used herein the term "native glucagon" refers to naturally-occurring on, e.g., human glucagon, comprising the sequence of SEQ ID NO: 1. The term "native GLP-l" refers to naturally-occurring GLP-l, e.g., human GLP-1, and is a generic term that asses, e.g., GLP-1(7-36) amide (SEQ ID NO: 2), GLP-1(7-37) acid (SEQ ID NO: 3), or a mixture of those two compounds. As used herein, a general reference to "glucagon" or "GLP-l" in the absence of any further ation is intended to mean native human glucagon or native human GLP-l, respectively. Unless otherwise indicated, "glucagon" refers to human glucagon, and "GLP-1" refers to human GLP-l.

GLP-l/glucagon agonistpeptides Provided herein are peptides which bind both to a glucagon receptor and to a GLP-1 receptor. Exemplary peptides are provided in incorporated by reference in its entirety. In certain embodiments, the peptide is MEDIO382, i.e., a 30 amino acid linear peptide with the sequence of SEQ ID NO:l9 that ns a gamma glutamate linker and palmitoyl group derivatization at residue 10. In certain embodiments, the peptides provided herein are co-agonists of glucagon and GLP- 1 activity. Such peptides are referred to herein as GLP-l/glucagon t peptides.

GLP-l/glucagon agonist peptides as ed herein possess GLP-1 and glucagon activities with favorable ratios to promote weight loss, t weight gain, or to maintain a desirable body weight, and possess optimized lity, formulatability, and stability.

In certain embodiments, GLP-l/glucagon agonist peptides as ed herein are active at the human GLPl and human glucagon receptors, in certain embodiment relative activity compared to the natural ligand at the GLP-1 receptor is at least about l-fold, 2-fold 5- fold, 8-fold, 10-fold, l5-fold, 20-fold, or d higher than at the glucagon receptor.

In certain embodiments, GLP-l/glucagon agonist peptides as disclosed have desirable potencies at the glucagon and GLP-1 receptors, and have desirable relative potencies for promoting weight loss. In certain embodiments, glucagon agonist peptides as disclosed exhibit in vitro potencies at the GLP-1 or as shown by an EC50 in the CAMP assay 1 (see Example 2) of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain embodiments, GLP-l/glucagon agonist es as disclosed exhibit in vitro potencies at the GLP-1 receptor as shown by EC50 in the CAMP assay in 4.4% human serum albumin (assay 2, see Example 2) of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain embodiments, GLP-l/glucagon agonist peptides as disclosed exhibit in vitro potencies at the on receptor as shown by an EC50 in the CAMP assay 1 (see Example 2) of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain embodiments, GLP- 1/glucagon agonist peptides as disclosed exhibit in vitro potencies at the glucagon receptor as shown by an EC50 in the CAMP assay in 4.4% human serum albumin (assay 2, see Example 2) of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In n embodiments, GLP-l/glucagon agonist peptides as disclosed have relative /glucR y ratios, when compared to the native ligands, in the range of about 0.01 to 0.50, e.g., from about 0.02 to 0.30, 6.57., about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11. 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, or 0.30 when using assay 2.

In certain embodiments, GLP-l/glucagon agonist peptides as disclosed exhibit in vitro potencies at the e-dependent insulinotropic peptide (gastric inhibitory peptide) (GIPR) as shown by an EC50 in the CAMP assay 1 (see Example 2) of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain embodiments, GLP-l/glucagon agonist peptides as disclosed exhibit in vitro potencies at the GIPR as shown by EC50 in the CAMP assay in 4.4% human serum albumin (assay 2, see Example 2) of less than 10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less than 20 pM, WO 53575 less than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less than 3 pM, or less than 2 pM.

In certain embodiments, GLP-l/glucagon agonist peptides provided herein possess one or more criteria of acceptable lity, ease in formulatability, plasma stability, and improved pharmacokinetic ties. In certain embodiments, GLP- l/glucagon agonist peptides as disclosed are soluble in standard buffers over a broad pH range.

] In n embodiments, GLP-l/glucagon agonist peptides are soluble in common buffer ons at a concentration up to 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, or more, in buffer systems and a range of ionic strengths, e.g., from 0.25 to 150 mM, including, but not limited to phosphate buffer, Tris buffer, glutamate buffer, acetate buffer, succinate buffer, or histidine buffer. Exemplary buffers include 100 mM glutamate pH 4.5 buffer, 100 mM acetate pH 5 buffer, 100 mM succinate pH 5 buffer, 100 mM ate pH 6 buffer, 100 mM histidine pH 6 buffer, 100 mM phosphate pH 6.5 buffer, 100 mM phosphate pH 7.0 buffer, 100 mM histidine pH 7.0 buffer, 100 mM phosphate pH 7.5 buffer, 100 mM Tris pH 7.5 buffer, and 100 mM Tris pH 8.0 buffer. In certain embodiments, GLP-l/glucagon agonist peptides as disclosed are soluble in standard buffers at 0.8mg/ml over a range of pH, e.g., from pH 4.0 to pH 8.0, e.g., at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5. In certain ments, GLP- l/glucagon agonist es as disclosed are e in standard buffers from pH 4.5 to 8.0, 5.0 to 8.0, 5.5 to 8.0, 6.0 to 8.0, 6.5 to 8.0, 7.0 to 8.0, 4.5 to 8.5, 5.5 to 8.5, 5.5 to 8.5, 6.0 to 8.5, 6.5 to 8.5, or 7.0 to 8.5.

In certain embodiments, GLP-l/glucagon agonist es as disclosed are formulatable in standard pharmaceutical formulations. Exemplary formulations include, but are not limited to: 0.1M Tris pH 7.5, l50mM Mannitol, final formulation pH= 7.2; 0.05M Tris, 50mM Arginine/Proline, final formulation pH= 8.0; or sodium phosphate buffer (pH8)/ 1.85 % W/V propylene glycol, final formulation pH= 7.0. In certain embodiments GLP-l/glucagon agonist peptides as disclosed are soluble is these or other formulations at a concentration up to 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, or more.

WO 53575 In certain embodiments, GLP-l/glucagon t peptides as disclosed are acceptably stable against proteases in serum or plasma. Common degradation products of glucagon or GLP-l include +1 products (acid) and the DPP IV-cleavage products.

Products with +1 mass may arise from deamidation at amide groups of ine or at the C-terminus. Cleavage products arise from the action of the protease DPP IV in plasma. In certain embodiments, GLP-l/glucagon agonist peptides as disclosed remain stable in plasma at levels up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after 24 hours in plasma at 370 C.

Provided herein is a GLP-l/glucagon agonist e sing the amino acid SCunIlCCC HX2QGTFTSDX10SX12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30; wherein X2 is G or S, X10 is Y or K, X12 is K, E, R, or S, X13 is K or Y, X15 is D or E.

X16 is S or G, X17 is E, R, Q, or K, X18 is R, S, or A, X20 is R, K, or Q, X21 is D or E, X23 is V or I, X24 is A or Q, X27 is E or V, X28 is A or K, and X30 is G or R (SEQ ID NO:4). In n embodiments, the isolated peptide shown above (SEQ ID NO:4) is provided, wherein X2 is G, X10 is K, X12 is E, R, or S, X13 is K, X17 is E or K, X18 is S, X20 is R, X27 is E, and/or X28 is A. In certain embodiments, the isolated peptide shown above (SEQ ID NO:4) is provided, wherein X10 is K, X12 is E, X17 is E, X20 is R, X27 is E, and/or X28 is A.

In n embodiments the isolated peptide shown above is provided, where X2 is S, X10 is Y or K, X12 is K, E, R, or S, X13 is K or Y, X15 is D, X16 is S, X17 is E, R, Q, or K, X18 is R, S, or A, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E or V, X28 is A, and X30 is G (SEQ ID NO:5). In certain embodiments the isolated peptide shown above is provided, where X2 is S, X10 is Y or K, X12 is K, E, R, or S, X13 is K or Y, X15 is D, X16 is S, ile7 is E and X18 is R, or ile7 is R and X18 is S, then X20 is R, X21 is D, X23 is V, X24 is A, X27 is E or V, X28 is A, and X30 is G (SEQ ID NO: 6 and SEQ ID NO. 7, respectively). In certain embodiments the isolated peptide shown above is provided, where X2 is S, X10 is Y, X12 is K, X13 is K, X15 is D, X16 is S, if X17 is E and X18 is R, or if X17 is R and X18 is S, X20 is R, X21 is D, X23 is V, X24 is A, X27 is V, X28 is A, and X30 is G (SEQ ID NO: 8 and SEQ ID NO: 9, respectively). In certain embodiments the isolated peptide shown above is provided, where X2 is S, X10 is K, X12 is K, E, R, or S, X13 is Y, X15 is D, X16 is S, if X17 is E and X18 is R, and if X17 is R and X18 is S, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 10 and SEQ ID NO: 11, respectively). In certain embodiments the isolated peptide shown above is provided, where X2 is S, X10 is K, X12 is E, X13 is Y, X15 is D, X16 is S, if X17 is E and X18 is R, or if X17 is R and X18 is S, X20 is R X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 12 and SEQ ID NO: 13, respectively). In certain embodiments the isolated peptide shown above is provided, where X2 is S, X10 is K, X12 is R, X13 is Y, X15 is D, X16 is S, ile7 is E and X18 is R, or if X17 is R and X18 is S, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 14 and SEQ ID NO: 15, respectively). glucagon agonist peptides provided herein include, but are not limited to G730 (SEQ ID NO: 16), G797 (SEQ ID NO: 17), G849 (SEQ ID NO: 18), G933 (SEQ ID NO: 19), G865 (SEQ ID NO: 20), G796 (SEQ ID NO: 21), G812 (SEQ ID NO: 22) and G380 (SEQ ID NO: 23). These glucagon agonist peptides are listed in Table Table 1: GLP-l/Glucagon Peptide Sequences 1 f SEQ ID NO: 1 1 16 1HSQGT FTSDY SKXLD SERAR DFVAW LVAGG—amide x13 H?1 1 1 1 1q: HSQGT FTSDX SEYLD SERAR DFVAW LEAGG-amlde X10 jHSQGTFTSDX SRYLD SRSAR DFVAW LEAGG‘amlde X10 - K(gE‘Palm) H \D HSQGT FTSDX SEYLD SERAR DFVAW LEAGG-ac1d X10 =mK(gE-palm) 1""HSQGT FTSDX SSYLD SRSAR DFVAW LEAGG-amlde X10 = alm)1 X10 WKEWAMWLWNWWEMMNWAWN W1HSQGT FTSDX SSYLD SRRAR DFVAW LEAGG-amide = K(gE-palm) 1 N Na G812 1 HSQGT FTSDX SKYLE GQAAK EFIAW LEKGR-amide X10 = K(gE-palm)‘ WWWWWWWWWWWWWWWWW MM WWMWW MMWM MWWWMMiWWMWWWMMIWWMWWWMMMWWM1WWWMHWWWWWWWWWMwmwmmwwmmmmwwmmg FTSDYsKE‘kBfiXRAQDFVQEWEXQEF‘afniSEX17 =K.S.9E‘Pa1m) 1 HSQGT FTSDY SKXLD SERAR DFVAW LVAGG-ac1d X13 =mK(gE-palm) ~ 1HSQGT FTSDX SKYLE GQAAK EFIAW LEKGR-acid X10 = K(gE-palm) g.,vmmmm,,,,,,,,,,«mn.nnn""mWigm 11;":1,.zg;g.m"An"",n"1«nuuuunnnn,11w,vnuuHWK1111N"«.111gnw117m11311;zflg;;lrllflmxwxanwxg; zy;gnnnyflxm"wv.mnnna.ma‘m«u,4;gzg;gllwxmwavnnnnnnv"flfl.m,tw.mmmmmmmfifium1"fififiwxflnxw;zg;gnankum11 1 G973 1HSQGT FTSDX SSYLD SRSAR DFVAW LEAGG-acid X10 = K(gE-palm) 1 1 amide) /SEQ 1 *ID NO:3 1 HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR ac1d) 2 WEN , SEQ ID "glucagonWEMHSQGT FTSDY SKYLD SRRAQ DFVQW LMNT . "$10.1 , _ WE K(gE-Palm)—— Lysine with a palmitoyl group ated to the epsilon nitrogen, through a gamma glutamic acid linker.

] In certain embodiments the isolated peptide is G933 (SEQ ID NO: 19).

The peptides G797 and G933 both have a glutamate residue at position 12, and maintain robust activity at both the glucagon and GLP-1 receptors, as shown in Example 2. The corresponding residue is lysine in exendin-4 and glucagon and is serine in GLP-l.

Although this residue is not thought to contact the receptor, changes in charge from ve to negative may modify the adjacent environment. Furthermore, G797, G849 and G933 have a glutamate residue at position 27. Residue 27 is Lysine in exendin 4 and is an ged hydrophobic residue in GLPl (valine) and glucagon (methionine). The lysine of ide makes electrostatic interactions with the GLPl receptor at es Glul27 and Glu24 (C.R.Underwood et al J Biol Chem 285 723-730 (2010); S.Runge et al J Biol Chem 283 11340-11347 (2008)). While a loss of GLPlR potency might be expected when the charge at position 27 is changed to negative, the change is compatible with GLPlR activity in G797, G849, and G933.

MED10382 is a synthetic peptide dual agonist of glucagon-like peptide-l (GLP-1) and on receptors. MED10382 is G933 (SEQ ID NO: 19), which contains a gamma glutamate linker and palmitoyl group derivatization at residue 10.

MED10382 is chemically synthesized. Peptide chain elongation on a resin is med with the aid of a solid phase peptide synthesizer using manufacturer-supplied protocols for coupling of mino acids. Glutamine residues 20 and 24 are substituted with amino acids that are not susceptible to deamidation, and arginine residue 17 is replaced with glutamate to reduce susceptibility to proteolysis.

The ce for MEDIO3 82 is shown below: L-Histidyl-L-seryl-L-glutaminylglycyl-L-threonyl-L-phenylalanyl-L-threonyl-L- seryl-L- alpha-aspartyl-(N6-[N-(l-oxohexadecyl)-L-gamma-glutamyl])L-lysyl-L-seryl-L- alpha-glutamyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-alpha-glutamyl-L-arginyl- L-alanyl-L-arginyl-L-alpha-L—aspartyl-L-phenylalanyl-L-valyl-L-alanyl-L-tryptophyl-L- leucyl-L-alpha-glutamyl-L-alanylglycylglycine \/\/\/\/\/\/\/\n/Him—l o o o H H H H2N N N , N, N '1 N 'I N . _ a H a N/\n/H H H - H N/Y o o o . \fl/O O OH r0H NH NH 03) HNmmOH QT"Yfirirrkrrkggfifi ANSI/N:N\:)OJ\NJYYNNJLNWON Methods ofmaking GLP-l/glucagon agonistpeptides This disclosure provides a method of making a GLP-l/glucagon agonist peptide.

GLP-l/glucagon agonist peptides ed herein can be made by any le method.

For example, in certain embodiments the GLP-l/glucagon agonist peptides provided herein are chemically synthesized by methods well known to those of ordinary skill in the art, e.g., by solid phase synthesis as described by ield (1963, J. Am. Chem. Soc. 852149-2154). Solid phase peptide sis can be accomplished, e.g., by using automated synthesizers, using standard ts, e.g., as explained in Example 1.

Alternatively, GLP-l/glucagon agonist peptides provided herein can be ed recombinantly using a convenient vector/host cell combination as would be well known to the person of ry skill in the art. A variety of methods are available for recombinantly producing GLP-l/glucagon t peptides. Generally, a polynucleotide sequence encoding the GLP-l/glucagon agonist peptide is inserted into an appropriate expression vehicle, e.g., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. The nucleic acid encoding the GLP-l/glucagon agonist e is inserted into the vector in proper reading frame.

The expression vector is then transfected into a suitable host cell which will express the WO 53575 2017/055679 GLP-l/glucagon t peptide. Suitable host cells e t limitation bacteria, yeast, or mammalian cells. A variety of commercially-available host-expression vector systems can be ed to express the GLP-l/glucagon agonist es described herein.

Modifications, Conjugates, Fusions, and Derivations In n embodiments, GLP-l/glucagon agonist peptides provided herein are stabilized via amino acid modifications. In certain embodiments, the carboxyl group of the C—terminal amino acid is amidated. In n embodiments, the C—terminal amino acid is amidated glycine, e.g., G730, G797, G849, G865, G796, G812, and G380. In certain embodiments, e.g., G933, the C-terminal glycine is the unmodified acid. In certain embodiments, GLP-l/glucagon agonist peptides are provided in which one or more amino acid residues are acylated. For example, in certain embodiments GLP- l/glucagon agonist peptides provided herein contain one or more lysine residues, in which a palmitoyl moiety is attached to the N(epsilon) group. In certain embodiments a linker is incorporated between lysine and the palmitoyl group. This linker can be a gamma glutamic acid group, or an alternative linker such as, but not limited to, beta alanine and aminohexanoic acid. Different acylation methods may be used such as addition of cholesterol or myristoyl groups. In certain embodiments, the palmitoyl moiety is added at position 13 (e.g., G730). In certain embodiments, the palmitoyl moiety is added at position 10 (e.g., G797, G849, G933, G865, G796, and G812). In n embodiments, the palmitoyl moiety is added at position 17 (e.g., G380).

The GLP-l/glucagon agonist peptides ed herein, e.g., G730, G797, G849 and G933 can be palmitoylated to extend their half-life by association with serum albumin, thus ng their propensity for renal clearance, as described in Example 1.

Alternatively or in addition, a glucagon agonist peptide as disclosed herein can be associated with a heterologous moiety, e.g., to extend half-life. The heterologous moiety can be a protein, a peptide, a protein domain, a , 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 inant polypeptide polymer, a cytokine, and a combination of two or more of such moieties.

For example, GLP-l/glucagon agonist peptides can be fused with a heterologous polypeptide. The peptides can be fused to proteins, either through recombinant gene fusion and expression or by chemical conjugation. Proteins that are suitable as partners for fusion include, without limitation, human serum albumin, antibodies and antibody fragments including fusion to the Fc portion of the antibodies. GLP-l has been fused to these proteins with retention of potency (L. Baggio et al, Diabetes 53 500 (2004); P. Barrington et al Diabetes, Obesity and Metabolism 13 426—433 (2011); P. Paulik et al American Diabetes Association 2012, Foster 1946). ed inant peptide sequences have also been described to give the peptide high molecular mass (V.Schellenberger et al Nature Biotechnol 27 190 (2009); PASylation (EP2173890)). In n embodiments GLP-l/glucagon agonist peptides are incorporated as the N-terminal part of a fusion protein, with the fusion partner, e.g., the albumin or Fc portion, at the C-terminal end. GLP-l/glucagon agonist es as described herein can also be fused to es or protein domains, such as ‘Albudabs‘ that have affinity for human serum albumin (M.S. Dennis et al J Biol Chem 277 35035—35043 (2002); A.

Walker et al Protein Eng Design Selection 23 271-278 (2010)). Methods for fusing a GLP-l/glucagon agonist peptides as disclosed herein with a heterologous polypeptide, e. g., albumin or an Fc region, are well known to those of ordinary skill in the art.

Other heterologous moieties can be conjugated to glucagon agonist peptides to further stabilize or increase half-life. For chemical fusion, n embodiments feature maintenance of a free N-terminus, but alternative points for derivatization can be made. A further alternative method is to derivatize the peptide with a large al moiety such as high molecular weight polyethylene glycol (PEG). A "pegylated GLP-l/glucagon agonist peptide" has a PEG chain covalently bound thereto.

Derivatization of GLP-l/glucagon agonist peptides, e. g., pegylation, can be done at the lysine that is palmitoylated, or alternatively at a e such as cysteine, that is tuted or incorporated by extension to allow derivatization. GLP-l/glucagon agonist peptide s above can be characterized in vitro and/or in viva for relative potency and the balance between GLP-1 and glucagon receptor activation.

WO 53575 The general term "polyethylene glycol chain" or "PEG , refers to mixtures of condensation rs of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH2CH2)nOH, where n is an integer of 3, 4, 5, 6, 7, 8, 9, or more. PEG chains include polymers of ethylene glycol with an average total molecular weight selected from the range of about 500 to about 40,000 Daltons. The average molecular weight of a PEG chain is indicated by a number, e.g., PEG-5,000 refers to polyethylene glycol chain having a total molecular weight average of about ,000.

PEGylation can be carried out by any of the PEGylation reactions known in the art. See, e. g., Focus on Growth Factors, 3: 4-10, 1992 and European patent applications EP 0 154 316 and EP 0 401 384. PEGylation may be carried out using an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).

Methods for preparing a ted GLP-l/glucagon agonist peptides generally include the steps of (a) reacting a GLP-l/glucagon agonist peptide or with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the molecule becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s).

Pharmaceutical Compositions ] Further provided are compositions, e. g., pharmaceutical compositions, that contain an effective amount of a glucagon agonist peptide as provided , formulated for the treatment of metabolic es, e.g., obesity.

Compositions of the disclosure can be formulated ing to known methods.

Suitable preparation s are described, for example, in Remington’s Pharmaceutical Sciences, 19th Edition, A.R. Gennaro, ed., Mack hing Co., Easton, PA (1995), which is incorporated herein by reference in its entirety. Composition can be in a variety of forms, including, but not limited to an aqueous solution, an emulsion, a gel, a suspension, lyophilized form, or any other form known in the art. In addition, the composition can contain pharmaceutically acceptable additives including, for example, diluents, s, izers, and preservatives. Once formulated, itions of the invention can be administered directly to the subject.

Carriers that can be used with compositions of the invention are well known in the art, and include, without limitation, e.g., thyroglobulin, ns such as human serum albumin, tetanus toxoid, and polyamino acids such as poly L-lysine, poly L-glutamic acid, za, hepatitis B virus core protein, and the like. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.8% , 0.3% glycine, hyaluronic acid and the like. Compositions can be sterilized by conventional, well known sterilization ques, or can be sterile ed. A resulting composition can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to stration. Compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering , tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

Methods oftreating Substantial non-surgical weight loss with effective glucose control has remained a key unmet need for patients with type 2 diabetes mellitus. The methods of treatment provided herein can meet this need.

GLP-l/glucagon agonist peptides (e.g., MEDIO382) can combine the effect of glucagon e.g., inhibition of food intake or regulation of glucose levels with the effect of GLP-1 e.g., inhibition ofgastric motility, or ion of n release. They can ore act to accelerate elimination of excessive adipose , induce sustainable weight loss, and improve glycemic control. GLP-l/glucagon agonist peptides (e.g., MEDIO382) can also act to reduce vascular risk factors such as high cholesterol, and high LDL-cholesterol or abnormal HDL/LDL . GLP-l/glucagon agonist peptides (e. g., MEDIO3 82) can also act to reduce triglycerides.

This disclosure provides a method of treating obesity or an obesity-related disease or disorder, sing administering to a subject in need of treatment 50-600 ug or 100- 600 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 ug) of a GLP-l/glucagon agonist peptide as disclosed herein (e. g., MEDIO382). In certain instances, the administration is an adjunct to diet and exercise. Further ed is 50-600 ug or 100—600 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 pg) of a GLP-l/glucagon agonist peptide (e.g., MEDIO382) for ent of obesity or an obesity-related disease or disorder. Further provided is use of 50-600 pg or 100—600 pg (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600pg) of a GLP-l/glucagon agonist peptide as provided herein (e. g., MEDIO3 82) in the manufacture of a ment for the treatment of obesity or an y-related disease or disorder. The 50-600 pg or 100-600 pg of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be administered in single or divided doses. In addition, the 50-600 pg or 100-600 pg of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be administered in increasing doses (e.g., a dose titration such as an initial dose of 100 pg, a second dose of 150 or 200 pg, and optionally a third dose of 200, 300 or 400 pg, e.g., wherein the initial dose and/or the second dose are administered for 3-10 days or 3-7 days.) In certain instances, the subject has type 2 diabetes mellitus. In certain instances, the subject has a body mass index (BMI) of 30 to 39.9 kg/mz. In certain instances, the subject has a BMI of at least 40.

This disclosure also provides a method of reducing body weight, sing stering to a t in need of treatment 50-600 pg or 100—600 pg (e.g., 50, 100, 125, 150, 175,200,225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500,525,550, 575, or 600 pg) of a GLP-l/glucagon agonist e as disclosed herein (e.g., MEDIO382). The 50-600 pg or 0 pg of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., 82) can be administered in single or divided doses. In addition, the 50-600 pg or 100-600 pg of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be administered in increasing doses (e.g., a dose titration such as an initial dose of 100 pg, a second dose of 150 or 200 pg, and optionally a third dose of 200, 300 or 400 pg, e.g., wherein the initial dose and/or the second dose are administered for 3-10 days or 3-7 days.) In certain instances, the administration is an t to diet and se. In certain instances, the subject has type 2 diabetes mellitus.

In certain instances, the subject has a BMI of 27 to 40 kg/mz. In certain instances, the subject has a BMI of 30 to 39.9 kg/mz. In certain instances, the subject has a BMI of at least 40. In certain instances, the subject is overweight. In n instances, the subject is obese.

This sure also provides a method of reducing body fat, comprising administering to a subject in need of treatment 50-600 pg or 100—600 pg (e.g., 50, 100, 125, 150, 175,200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 pg) of a GLP-l/glucagon agonist peptide as sed herein (e.g., MEDIO382). The 50-600 pg or 100-600 pg of the GLP-l/glucagon t peptide as disclosed herein (e.g., MEDIO382) can be administered in single or divided doses. In addition, the 50-600 pg or 100-600 pg of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be administered in increasing doses (e.g., a dose titration such as an l dose of 100 pg, a second dose of 150 or 200 pg, and optionally a third dose of 200, 300 or 400 pg, e.g., wherein the initial dose and/or the second dose are administered for 3-10 days or 3-7 days.) In certain instances, the administration is an adjunct to diet and exercise. In certain instances, the t has type 2 diabetes mellitus.

In certain instances, the subject has a BMI of 27 to 40 kg/mz. In certain instances, the subject has a BMI of 30 to 39.9 kg/mz. In certain instances, the subject has a BMI of at least 40. In certain instances, the t is overweight. In certain ces, the subject is obese. In n instances, the fat is liver fat. The reduction of liver fat can lead to enhanced insulin sensitivity and/or ed liver function. In certain instances, liver fat in the subject is reduced by at least 20%. In certain instances, liver fat in the subject is reduced by about 20% to about 40%. In certain instances, the administration results in about a one third reduction in liver fat. In certain instances, liver volume is reduced in the subject. In certain instances, the administration reduces obin Alc (HbAlc) levels (e.g., by at least 0.6%, by at least 0.9%, by about 0.5% to about 1.5%, by about 0.5% to about 2%, by about 0.5% to about 3%, or to 6.3% or lower.

This disclosure also provides a method of managing weight, comprising administering to a subject in need thereof 50-600 pg or 100—600 pg (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 pg) of a glucagon agonist peptide as disclosed herein (e.g., MEDIO382).

The 50—600 pg or 100-600 pg of the glucagon agonist peptide as disclosed herein (e. g., MEDIO3 82) can be administered in single or divided doses. In addition, the 50-600 pg or 100-600 pg of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be administered in increasing doses (e.g., a dose titration such as an l dose of 100 pg, a second dose of 150 or 200 pg, and optionally a third dose of 200, 300 or 400 ug, e. g., wherein the initial dose and/or the second dose are administered for 3-10 days or 3-7 days). In certain instances, the administration is an adjunct to diet and se. In certain instances, the subject has type 2 diabetes mellitus. In certain instances, the subject has a BMI of 27 to 40 kg/mz. In certain instances, the t has a BMI of 30 to 39.9 kg/mz. In certain instances, the subject has a BMI of at least 40. In certain instances, the subject is ight. In certain instances, the subject is obese.

This disclosure also provides a method of treating Nonalcoholic hepatitis (NASH), comprising administering to a subject in need of treatment 50-600 ug or 100- 600 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 ug) of a GLP-l/glucagon agonist peptide as disclosed herein (e. g., MEDIO382). The 50-600 ug or 0 ug of the glucagon agonist e as disclosed herein (e.g., MEDIO382) can be administered in single or divided doses. In addition, the 50-600 ug or 100-600 ug of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be administered in increasing doses (e.g., a dose titration such as an l dose of 100 ug, a second dose of 150 or 200 ug, and optionally a third dose of 200, 300 or 400 ug, e.g., wherein the initial dose and/or the second dose are administered for 3-10 days or 3-7 days). In certain instances, the administration is an t to diet and exercise. The administration can also reduce body weight or treat obesity. In certain instances, the subject has a BMI of 27 to 40 kg/mz. In certain instances, the t has a BMI of 30 to 39.9 kg/mz. In certain ces, the subject has a BMI of at least 40. In certain instances, the subject is overweight. In certain instances, the subject is obese.

As provided herein, 50-600 ug or 100-600 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 ug) of GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) can be administered for preventing weight gain, preventing fat gain (e.g., liver fat), promoting weight loss, promoting fat loss (e.g., liver fat), reducing excess body weight, reducing fat (e.g., liver fat), or treating obesity (e.g. by control of appetite, feeding, food intake, calorie , and/or energy expenditure), including morbid obesity. Increased energy expenditure can result, for example, from increased oxidation of fatty acids and/or glucose in the liver. This disclosure also provides a method of treating or preventing a disease or condition caused or characterized by excess body weight or excess body fat, comprising administering to a t in need of treatment 50-600 pg or 100—600 pg (e.g., 50, 100, 125, 150, 175,200,225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 pg) of a GLP-l/glucagon agonist e as disclosed herein (e.g., MEDIO382). In certain instances, the administration is an adjunct to diet and exercise. In addition, 50—600 pg or 100—600 pg (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 pg) GLP—l/glucagon agonist peptides provided herein (e.g., MEDIO382) can be used for treatment of other obesity-related metabolic disorders. The 50-600 pg or 100-600 pg of the GLP— 1/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be administered in single or divided doses. In addition, the 50-600 pg or 100-600 pg of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be stered in increasing doses (e.g., a dose titration such as an initial dose of 100 pg, a second dose of 150 or 200 pg, and optionally a third dose of 200, 300 or 400 pg, e.g., wherein the initial dose and/or the second dose are administered for 3-10 days or 3-7 days.) Examples of other obesity- related (excess body weight-related) disorders include without tion: insulin resistance, glucose intolerance, pre-diabetes, increased fasting glucose, type 2 diabetes, hypertension, dyslipidemia (or a combination of these lic risk factors), glucagonomas, cardiovascular diseases such as congestive heart e, atherosclerois, arteriosclerosis, coronary heart disease, or peripheral artery disease, stroke, respiratory dysfunction, or renal disease.

This disclosure also provides a method of treating type 2 diabetes mellitus, comprising administering to a subject in need of treatment 50-600 pg or 100—600 pg (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 pg) of a GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382). The 50-600 pg or 0 pg of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be stered in single or divided doses. In addition, the 50-600 pg or 100-600 pg of the GLP-l/glucagon agonist e as sed herein (e.g., MEDIO382) can be stered in increasing doses (e.g., a dose titration such as an l dose of 100 pg, a second dose of 150 or 200 pg, and optionally a third dose of 200, 300 or 400 pg, e.g., n the initial dose and/or the second dose are administered for 3-10 days or 3-7 days.) In certain instances, the administration is an adjunct to diet and exercise. The administration can also reduce body weight or treat obesity. In certain instances, the subject has a BMI of 27 to 40 kg/mz. In certain ces, the subject has a BMI of 30 to 39.9 kg/mz. In certain instances, the subject has a BMI of at least 40. In certain instances, the subject is ight. In certain ces, the subject is obese.

This disclosure also provides a method of improving ic control or achieving glycemic control, comprising administering to a subject in need of treatment 50—600 ug or 0 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 ug) of a GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382). The 50-600 ug or 100-600 ug of the GLP— 1/glucagon agonist peptide as disclosed herein (e. g., 82) can be administered in single or divided doses. In addition, the 50-600 ug or 100-600 ug of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be administered in increasing doses (e.g., a dose titration such as an initial dose of 100 ug, a second dose of 150 or 200 ug, and optionally a third dose of 200, 300 or 400 ug, e. g., wherein the initial dose and/or the second dose are administered for 3-10 days or 3-7 days.) In certain instances, the administration is an t to diet and exercise. The administration can also reduce body weight or treat obesity. In certain instances, the subject has type 2 diabetes mellitus. In certain instances, the subject has a BMI of 27 to 40 kg/mz. In certain instances, the subject has a BMI of 30 to 39.9 kg/mz. In certain instances, the subject has a BMI of at least 40. In n instances, the t is overweight. In certain instances, the subject is obese. An ement in glycemic control can be assessed using a mixed-meal test.

In certain instances, the improvement in glycemic l results in at least a 10% reduction in glucose area under the concentration-time-curve (AUC) after a mixed-meal test (e.g., as compared to the AUC prior to treatment). In certain instances, the improvement in glycemic control results in at least a 15% reduction in AUC after a mixed-meal test (e. g., as compared to the AUC prior to treatment). In certain instances, the improvement in glycemic control results in at least a 20% reduction in glucose AUC after a mixed-meal test (e.g., as compared to the AUC prior to treatment). In certain instances, the ement in glycemic control results in at least a 25% reduction in glucose AUC after a mixed-meal test (e.g., as compared to the AUC prior to treatment).

In certain instances, the ement in ic control results in at least a 30% reduction in glucose AUC after a mixed-meal test (e. g., as compared to the AUC prior to treatment). An improvement in glycemic control can also be assessed based on hemoglobin Alc and fructosamine.

This sure also provides a method of decreasing weight and controlling glucose, comprising administering to a subject in need of treatment 50-600 ug or 100—600 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 ug) of a GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382). The 50-600 ug or 100-600 ug of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., 82) can be administered in single or divided doses. In addition, the 50-600 ug or 100-600 ug of the GLP-l/glucagon agonist peptide as disclosed herein (e.g., MEDIO382) can be administered in increasing doses (e.g., a dose titration such as an l dose of 100 ug, a second dose of 150 or 200 ug, and optionally a third dose of 200, 300 or 400 ug, e.g., wherein the l dose and/or the second dose are stered for 3-10 days or 3-7 days.) In certain instances, the administration is an adjunct to diet and exercise. The administration can also reduce body weight or treat obesity. In n ces, the subject has type 2 diabetes mellitus. In certain instances, the subject has a BMI of 27 to 40 kg/m2. In certain instances, the subject has a BMI of 30 to 39.9 kg/mZ. In certain instances, the subject has a BMI of at least 40. In certain instances, the subject is overweight. In certain ces, the subject is obese. In certain instances, the administration results in weight loss of at least 1.0 kg, at least 1.3 kg, or about 1.3 to about 2.0 kg. In certain instances, the subject’s weight is reduced by at least 3.5 kg or at least 5 kg. In certain instances, the subject’s weight is reduced by about 2 to about 30 kg. In certain instances, the subject’s weight is reduced by at least 2%, at least 4%, at least 5%, or at least 10%. In certain instances, the subject’s weight is reduced by about 2% to about 20%, about 2% to about 25%, or about 2% to about 30%.

In certain instances, administration of the GLP-l/glucagon t peptides provided herein (e.g., MEDIO382) results in weight loss of at least 1 kg. In certain instances, administration of the glucagon agonist peptides provided herein (e.g., MEDIO382) s in weight loss of at least 1.3 kg. In certain instances, administration of the GLP-l/glucagon agonist peptides provided herein results in weight loss of at least 1.5 kg. In certain instances, administration of the GLP-l/glucagon t peptides provided herein (e.g., MEDIO382) results in weight loss of l to 3 kg following four weeks of repeat once-daily . In certain ces, administration of the GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) results in weight loss of 1.3 to 2 kg following four weeks of repeat once-daily dosing.

] The route of stration of GLP-l/glucagon agonist peptides provided herein (e. g., MEDIO3 82) can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, aneous, rectal, or vaginal administration. r example of a form for administration is a on for injection, in particular for intravenous or intraarterial injection or drip. GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) can be administered as a single dose or as multiple doses. In n embodiments, 50-600 ug or 100-600 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 ug) of a GLP— 1/glucagon agonist peptide is administered by subcutaneous injection. eral formulations can be a single bolus dose, an infusion or a loading bolus dose followed with a maintenance dose. These compositions can be administered at specific fixed or variable intervals, e.g., once a day, or on an "as needed" basis. Dosage regimens also can be adjusted to provide the m desired response (e.g., a therapeutic or prophylactic response).

In certain instances, 50-600 ug or 100-600 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 ug) of GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) can be administered once per day. In certain instances, 50-600 ug or 100-600 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 ug) of GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) can be administered once per day via injection (e.g., subcutaneous administration). In certain instances, 50—600 ug or 100—600 ug (e.g., 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 ug) of GLP—l/glucagon t peptides provided herein (e. g., MEDIO382) can be administered once per day via ion (e.g., subcutaneous administration) over a period of at least one week, over a period of at least two weeks, over a period of at least three weeks, or over a period of at least four weeks. In certain instances, 50-600 ug or 100-600 ug (e.g., 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 pg) of GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) can be administered in single or divided doses. In certain instances, 50-600 pg or 0 pg (e.g., 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 pg) of GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) can administered in increasing doses (e. g., a dose titration such as an initial dose of 100 pg, a second dose of 150 or 200 pg, and optionally a third dose of 200, 300 or 400 pg, e.g., wherein the initial dose and/or the second dose are administered for 3-10 days or 3-7 days.) In certain instances, GLP-l/glucagon agonist peptides provided herein (e. g., MEDIO3 82) can be administered as an initial dose of 100 pg for four days, a second dose of 150 pg for four days, and a third dose of 200 pg subsequently administered daily.

In certain instances, glucagon agonist peptides provided herein (e.g., MEDIO382) can be administered as an initial dose of 100 pg for five days, a second dose of 150 pg for five days, a third dose of 200 pg for five days, and a fourth dose of 300 pg subsequently administered daily. In certain instances, GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO3 82) can be administered as an l dose of 100 pg for five days, a second dose of 200 pg for five days, and a third dose of 300 pg subsequently administered daily.

In certain instances, the half-life of the GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) is about 7 to 13 hours. In certain instances, the half-life of the GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) is about 9 to 13 hours. In certain instances, the half-life of the GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) is about 7 to 12 hours. In certain ces, the half-life of the GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO382) is about 10 to 12 hours. In certain ces, the half-life of the GLP-l/glucagon agonist peptides provided herein (e.g., MEDIO3 82) is about 8 to 11 hours.

In yet other embodiments, the present disclosure provides kits sing GLP- l/glucagon agonist es, that can be used to m the methods described herein. In certain embodiments, a kit comprises a GLP-l/glucagon agonist peptide disclosed herein in one or more containers. One skilled in the art will y recognize that the sed GLP-l/glucagon t peptides can be readily incorporated into one of the established kit formats which are well known in the art.

EXAMPLES Example 1: Synthesis, modifications, and characterization of GLP-l/glucagon agonist peptides List of abbreviations: Boc: utyloxycarbonyl tert—Bu; tert-butyl DCM: dichloromethane DIC: diisopropylcarbodiimide Fmoc: 9-fluorenylmethoxycarbonyl HOBt: l-hydroxybenzotriazole HPLC: High Performance Liquid Chromatography Mtt: 4-methyltrityl NMP: N—methylpyrrolidone be: 2,2,4,6,7-pentamethyldihydrobenzofuransulf0nyl TFA: trifluoroacetic acid TIS: triisopropylsilane Trt: triphenylmethyl, trityl GLP-l/glucagon t peptides were sized as follows. Elongation of e chains on NovaSyn TGR or preloaded Fmoc-Wang resin (NovaBiochem) was performed with a PRELUDETM solid phase peptide synthesizer (Protein Technologies, Tucson, AZ, USA). Manufacturer-supplied protocols were applied for coupling of the hydroxybenzotriazole esters of amino acids in N—methylpyrolidone (NMP). The fluorenylmethoxycarbonyl (Fmoc) group was used for the semipermanent protection of alpha-amino groups of amino acids, whereas the side chains were protected with tert- butyl (tert-Bu) for serine, ine, aspartic acid, glutamic acid, tyrosine, and 2,2,4,6,7- pentamethyldihydrobenzofuransulf0nyl (be) for arginine, and trityl (Trt) for histidine. The N—terminal amino group of histidine in position 1 was protected with tert- butyloxycarbonyl group (Boc). Lys(Mtt) was incorporated into the peptide chain when a subsequent chemical modification of the side chain was ed.

Upon tion of the peptide chain elongation, the Mtt group was removed by washing the peptide-resin with DCM containing 2% TFA and 5% TIS (10 X 7 ml, each 0.5 min). ng of a lipid moiety to the side chain of Lys was performed on the PRELUDETM e synthesizer using DIC as a coupling reagent in the presence of HOBt.

Peptides were cleaved from the resin using mixture of TFA:TIS:water (95:2.5:2.5). After 2h at room temperature, the peptidyl resin was filtered, washed with TFA and combined filtrates were evaporated to dryness in vacuo. The residue was triturated with ether, and the precipitate which formed was filtered, washed with ether, and dried. The crude peptides were dissolved in 5% acetic acid in water and analyzed by reverse-phase high-pressure liquid chromatography on a Polaris 3 C8-A column attached to Varian 920-LC system. A standard gradient system of 10 to 90% buffer B over the course of 15 min was used for analysis. Buffer A was 0.1% TFA in water and buffer B was 0.1% TFA in acetonitrile. HPLC es were recorded at 210 nm. Preparative separations were med on Varian ProStar system with a semipreparative C18 RP e Waters . The above-described solvent system of water and acetonitrile, in a gradient of 30 to 70% buffer B over the course of 30 min, was used for separation. The chromatographically homogenous products (> 97% pure) were analyzed by electrospray mass spectrometry (MassLynx, Waters).

Example 2: In vitro studies Glucagon and GLP-1 receptor mediated CAMP production Biological activity of peptides in cell-based cAMP activity assay (assay 1): The biological activity of GLP-l/glucagon agonist peptides synthesized by the method of Example 1 were tested for biological activity, e.g., stimulation of one or more cellular receptor responses, by the following methods. Stable cell lines sing human, mouse, rat, or dog GLP-1 receptor (GLP-lR), glucagon receptor (GCGR) or glucose-dependent insulinotropic peptide ic inhibitory polypeptide) receptor (GIPR) were generated in HEK293s or CHO cells by standard methods. Peptide activation of these s receptors results in downstream production of cAMP second messenger which can be ed in a functional ty assay. cAMP assays were performed using "assay medium": Assay Medium: 10% FBS in DMEM (Gibco # 41966), ning 0.5mM IBMX (Sigma #17018).

Low n binding 384-well plates er # 781280) were used to perform eleven 1 in serial dilutions of test samples which were made in assay medium. All sample dilutions were made in duplicate.

A frozen cryo-vial of cells expressing the receptor of st was thawed rapidly in a water-bath, transferred to rmed assay media and spun at 240xg for 5 minutes.

Cells were re-suspended in assay media at an optimized concentration (e.g. hGCGR cells at 1x105 cells /ml, hGLP-lR and hGIPR cells at 5 cells /ml).

] From the dilution plate, a SuL replica was stamped onto a black shallow-well u- bottom 384-well plate (Corning # 3676). To this, SuL cell suspension was added and the plates incubated at room temperature for 30 minutes.

CAMP levels were measured using a commercially available CAMP dynamic 2 HTRF kit (Cisbio, Cat # 62AM4PEJ), following the two step protocol as per manufacturer’s recommendations. In brief; anti-CAMP cryptate (donor fluorophore) and CAMP-d2 (acceptor fluorophore) were made up separately by diluting each 1/20 in conjugate & lysis buffer provided in the kit. SuL anti-CAMP cryptate was added to all wells of the assay plate, and SuL CAMP-d2 added to all wells except non-specific binding (NSB) wells, to which conjugate and lysis buffer was added. Plates were ted at room temperature for one hour and then read on an Envision (Perkin Elmer) using excitation wavelength of 320nm and emission wavelengths of 620nm & 665nm.

Sequences of synthesized glucagon agonist peptides and their EC50 values determined in CAMP assays, performed in "assay medium," are shown in Table 2. All peptides in Table 2 were synthesized with a inal amide. Additional GLP- 1/glucagon agonist peptides were synthesized with a C—terminal acid and EC50 values determined in CAMP assays, performed in "assay ," are shown in Table 3. EC50 for additional GLP-l/glucagon agonist peptides, performed in "assay medium," are shown in Table 4. All peptides in Table 4 have a C-terminal amide, unless they are denoted as ‘acid’ in which case they have a C-terminal acid.

Table 2: CAMP activity of GLP-l/glucagon agonist peptides with C-terminal amide (assay 1) Assay in Assay Medium _ 39 _ Human GlucR EC50 Human GLP1R EC50 Human GlPr EC50 Peptide M M M G730 6.23E-12 1.8E-11 4.5E-O8 G797 6.14E-12 1.4E-11 3.4E-O9 G849 2.26E-12 9.0E-12 1.7E-O8 G865 1.26E-11 8.3E-12 2.2E-O8 G796 1.76E-12 1 1.4E-O8 G812 8.17E-12 1.1E-11 2.7E-O9 G380 2.17E-1O 7.7E-11 1.3E-O7 GLP1 8.1 E-11 Glucagon 3.3E-12 Table 3: CAMP activity of GLP-l/glucagon agonist peptides with C-terminal acid (assay 1) Peptide Human GlucR Human GLP1R Human GlPr EC50 EC50 EC50 M M M 1.78E—11 1.30E—10 00 .92E-12 3.20E-11 9.70E—09 6.30E—12 1.80E-11 09 8.90E—12 1.20E—11 08 Table 4: CAMP activity of additional GLP-l/glucagon agonist peptides (assay 1) Parent sequence HSQGT FTSDY SKYLD SRRAQ DFVQW LVAGG Peptides in this section all contain LVAGG at es 26 to 30 Site and nature of palmitoylation, Substitutions into hGlucR hGLPlR hGIPR parent sequence EC50 M EC50 M Glucagon 3.3E—12 1.99E—07 GLP1 1.53E—07 1.53E—07 q715 2.53} 8 10 K(q3*palm)10 q7l6 2.463709 1.293708 1.183708 K(q3palm)ll g702 1.493709 3.353709 0.003+00 <(g3pa1m)i2, 317 <(q3pa1m)i2, 317 R20 g728 1.693710 3.953707 A24 q729 11 2.093707 <(g3pa1m)i3 317 <(q3pa1m)i3 317 R20 g730 3 953711 5 663708 A24 <(q3pa1m) 3 320 A24, g875 2 983711 2 903708 337 Aib2 <(q3pa1m)i3 320 A24, g841 838 R12 acid 1m)i3, 320 A24, g802 9 643711 9.123708 337, 332 <(q3pa1m)i3, 320 A24, g820 3 393711 7 113708 337, 332 <(q3pa1m)i3, 320 A24, g842 11 08 337, R 2 acid g733 2.313711 8.173707 <(g3paim)i4, G2 33 <(q3pa1m)i4, 320 324, g803 2 963711 3 293708 838 <(q3pa1m)i4, 320 324, g843 838 R12 acid <(q3pa1m) 4, 320 A24, g732 2 323711 2 943708 337 G2 <(q3pa1m)i4, 320 A24, g777 2 743711 4 533709 337 <(q3paim)i4, 320 A24, g844 337 R12 Aib2 acid <(q3palm)i4, 320 A24, g845 337 R12 acid <(q3pa,m)i4, 320 A24, g821 5 583* 1.403708 337, 3,2 <(q3pa1m)i4, 320 A24, q846 31 4.383733 1.183708 337, 332 g731 31 4.2237 4.073708 <(g3pa1m)i4, 312 g670 32 2.0337 1.493708 <(g3pa1m)i4, S18 g335 31 7.3337 5.823707 <(g3ipa1m)17 q336 32 3.6637 1.963708 {(qwiqfiipalm)17 g384 31 2.7237 1.703708 Aib2 3 <(g3pa1m)i7, g380 3730 1.003730 6.093707 lm)i7, G2 <(q3pa,m)i7, G2, A20 (3736 8.5437 0.003+00 A24 (3381 9.0837 5.453707 33 ,, <(g3pa:,m),,7, g678 4.523709 1.0637,0 1.233707 <(g3pa1m)i7, G2 320 g599, q688 3731 1.2037,0 1.123707 <(g3pa1m)i7, 320 q679 3730 1.353730 1.173707 1m)i7, G2 324 g600, g689 6.66371 8.283708 <(g3pa:,m),,7, 324 <(q3palm)i7, G2 320 g680 1 953730 9 673708 324 1m) 7,82 33 q639 2 443* 0 8 213708 20 24 {(q3pa1m) 7,G2, 33 g681 2 833* 0 1 243707 20 24 <(q3pa1m)i7, G2 320 g720 5.343731 0.003+00 324 R12 <(q3pa1m)i7, G2 320 g660 1 063709 3.323707 324 WO 53575 <(q3pa1m)17, R20 324, g835 .913710 9.723708 312 g776 .023711 4.793708 <(g3pa1m)17, R20 324 <(q3pa1m)17, R20 324 g823 .733711 8.423708 R12 g867 4.483711 4.173708 <(g3pa1m)17, R20 A24 <(q3pa1m)17, A20 A24, g736 .543711 00 G2 <(q3pa1m)17, A20 A24, g737 7.343707 8.143711 00 G2 33 <(q3pa1m)17, 312 R20 (3675 1.513710 1.613706 A24 G2 Parent sequence HSQGT FTSDY SKYLD SRRAQ DFVQW LEAGG Peptides in this section all have the sequence LEAGG from residue 26 onwards unless otherwise stated, e.g. LERGG Site and nature of palmitoylation, Substitutions hGlucR hGLPlR hGIPR into parent sequence EC50 M EC50 M EC50 M q717 4.553713 5.773712 1.483709 <(q3pa1m)10, L3AGG q796 1.813712 1.403711 1.743708 <(q3pa1m)10, L3AGG, R20 A24 S12 <(q3pa1m)10, L3AGG, R20 A24 S18 q847 no data 312 Aib2 acid <(q3pa1m)10, L3AGG, R20 A24 317 q797 9.643712 2.2637 1 09 312 q798 5.1037 3 9.0737 2 1.513709 <(q3pa1m)10, L3AGG, R20 A24 317 <(q3pa1m)10, L3AGG, R20 A24 317 q848 9.6637 3 9 4237 2 2.773709 R12 <(q3pa1m)10, L3AGG, R20 A24 S18 q849 2.283712 9.073712 1.813708 R12 q701 3.833709 7.403709 0.003+00 <(q3pa1m)12, L3RGG, G2 317 q840 ,2 1.4537 0 1.023707 L3AGG, R20 A24, 317 q824 1.0537,2 4.7137 5.743708 <(q3pa1m)14, L3AGG, R20, 324 q780 7.923713 1.2037 08 R20 A24 1 <(q3pa1m)14, L3AGG, q601 4.9337,3 3.983711 7.413708 <(q3pa1m)14, L3AGG q816 ,2 11 2.003708 <(q3pa1m)14, L3AGG, 317 q817 1.6837,2 2.513711 08 <(q3pa1m)14, L3AGG, A18 <(q3pa1m)14, L3AGG, R20 , 324, q876 1.0437 1 8 6337 903708 312 q805 1.4437 2 2837 9 973708 <(q3pa1m)14, L3AGG, R20 324 <(q3pa1m)14, L3A, R20, A24, S18 q850 2.1937 2 2 1237 8 963708 R12 q836 1.553711 1.243710 1.003707 <(q3pa1m)14, L3AGG, R20 324, 317 q804 1.953712 7.153711 9.973708 <(q3pa1m) 4, L3A, R20, A24 q618 no data {(Aqxipalm)20, L3KGR q781 2.863712 1.043710 4.023707 <(q3pa1m)16, L3AGG, R20 A24 q782 1.5637,0 2.543711 06 <(q3pa1m)18, L3AGG, R20 A24 q744 3.923711 2.453709 0.003~00 <(g37pa1m)20, L3AGG q746 3.543711 1.153708 00 <(g37pa1m)24, L3AGG q747 9.423711 3.163709 1.043706 <(g37pa1m)31, L3AGG q512 6.063711 9.803711 4.073707 <(q3pa1m)17, L3AGG, G2, q513 7.233710 1.753710 2.983707 <(q3pa1m)17, L3AGG, 33, {(bAipalm)17, L3AGG, R20 A24, 33 q734 8.283708 6.953711 1.173706 312 WO 53575 g837 2.133710 4.673710 1.143707 {(gERpaLm)17, LEAGG, R20 A24 312 {(Aqxipalm)17, LEAGG, R20 A24 g838 12 2.373711 08 312 g783 9.113711 4.243711 8.463707 {(bAipaLm)17, LEAGG, R20 A24 312 <(bAepa;m)27, LEAGG, R20 A24, q851 no cata R12 acid <(bAepa;m)27, LEAGG, R20 A24, q852 no cata R12 Aib2 acid g819 2.343712 1.803711 1.033707 {(bAipaLm)17, LEAGG, R20 A24 g536 4.783712 7.453711 0.003+00 g600 5.473712 6.663711 1.243707 aLm)17, LVAGG, 324 g599 9.623711 8.763711 1.133707 {(gERpaLm)17, LVAGG, 320 Parent ce HSQGT5 FTSDYlO SKYLD15 SRRAQZO DFVQW25 LERGG—amide Peptides in this section all have the sequence LERGG from residue 26 onwards unless otherwise , e.g. LENT Site and nature of palmitoylation, Substitutions hGlucR hGLPlR into parent sequence EC50 M EC50 M {(Aqxipalm)17, LENT, R20 324, g825 3.67} .91} ' . , 1 g588 7.23} .10} 9. H 1 3pa1m)17, LERGG, G2, q614 3.65} .31} . H w12 1 fixipalm)17, EwRGG, fixipalm)17, LERGG, R20 A24 g684 1.64} .51} . H H" G2 1 sepalm)17, LERGG, R20 A24 g721 .23} .11} . H H" G2 1 fixipalm)17, LERGG, R20 A24 g724 .09} .90} . H H" G2 33 1 sepalm)17, LERGG, R20 A24 L3 RGG, R2 0 A24 fixipalm)17, LERGG, R20 A24 fixipalm)17, LERGG, R20 A24 q826 acid fixipalm)17, LERGG, R20 A24 q727 G2 acid fixipalm)17, LERGG, A20 A24 g683 G2 sepalm)17, LERGG, A20 g722 312 G2 fixipalm)17, LERGG, A20 A24 g725 G2 33 fixipalm)17, LERGG, A20 A24 fixipalm)17, LERGG, R20 324 g682 G2 sepalm)17, LERGG, R20 324 g723 G2 fixipalm)17, LERGG, R20 324 g726 G2 33 fixipalm)17, LERGG, R20 324 . L , 3pa1m)12, Parent sequence HSQGT5 FTSDYlO SKYLD15 SRRAQZO DFVQW25 LVAGG extension Peptides in this section have the residues noted C-terminal to residue 30 and a C—terminal amide hGlucR hGLPlR hGIPR 3xtension to sequence EC50 M EC50 M EC50 M g316 1.063711 3.1437 3.653709 SSGGSS q317 0. 3 2. 3 0.003700 SSGGSS K g318 9. 3 1. 3 0.003700 SSGGSSK(pa1m) q402 5. 3 8. 3 0.003700 SGSGSG q319 1. 3 3. 3 0.003700 PSSGA PPPSK q320 3. 3 9. 3 1.013709 PSSGA PPPSK(pa1m) g315 5. 3 2. 3 1.973708 GGGG q325 1. 3 2. 3 0.003700 GGGGK g326 2. 3 2. 3 1.263708 pa1m) g327 2. 3 1. 3 1.283708 GGGGK(q3pa1m) q321 2. 3 2. 3 6.413709 IAK q322 3. 3 1. 3 09 KNNRNNIAK(pa1m) Abbreviations: K(gE—palm) = Lysine with a palmitoyl group conjugated to the epsilon nitrogen, through a gamma glutamic acid linker; K (AhX—palm) = Lysine with a palmitoyl group conjugated to the epsilon nitrogen, h an aminohexanoic acid linker; K(bA—palm) = Lysine with a palmitoyl group conjugated to the epsilon nitrogen, through a beta alanine acid linker; Aib, aminoisobutyric acid. ) = Lysine with a palmitoyl group directly conjugated to the epsilon nitrogen.

Glucagon and GLP-1 receptor mediated CAMP production assays in presence of plasma concentrations of serum albumin_(assay 2). Agonist potency determinations for es inducing CAMP production were measured in CHO cells expressing human, rat or mouse glucagon receptors viated to GlucR or GCGR) or GLP-l receptors in the ce of human, rat or mouse serum albumin at 4.4, 3.2 and 3.2% respectively, as CHO cells with stable recombinant expression of the human, mouse or rat GlucR or GLP-1 receptor were cultured in DMEM 10% FBS and geneticin (100 .

Cryopreserved cell stocks were prepared in 1X cell freezing medium-DMSO serum free (Sigma Aldrich) at 2xlO7 /vial and stored at -80°C. Cells were rapidly thawed at 37°C and then d in to assay buffer (DMEM) containing serum albumin at 4.4, 3.2 and 3.2% for human, rat, and mouse serum albumin respectively. Peptides were serially diluted in DMSO and then d 100 fold into DMEM containing serum albumin at stated final concentration. Diluted peptides were then transferred into 384 black shallow well microtitre assay plates. Cells were added to the assay plates and incubated for 30 min at room temperature. Following incubation the assay was d and cAMP levels measured using the HTRF® dynamic d2 cAMP assay kit available from CisBio ays, as per the manufacturer‘s guidelines. Plates were read on Perkin Elmer ENVISION® cence plate readers. Human and rat serum albumin were purchased from Sigma Aldrich and mouse serum albumin from Equitech Bio Ltd.

Data was transformed to % Delta F as described in manufacturer’s guidelines and analysed by 4-parameter logistic fit to determine EC50 . Assay 2 EC50 values for selected peptides are shown the Table 5. The assay 2 EC50 values ined are dependent on both the intrinsic potency of the peptides tested at the GLPl and on receptors in the recombinant cell lines and on the ty of the peptide for serum albumin, which determines the amount of free peptide. Association with serum albumin increases the EC50 value obtained. The fraction of free peptide at plasma concentrations of n and the EC50 at 0% HSA can be calculated based on the variation in cAMP generation with the HSA concentration. For instance, G730 and G933 gave values of 0.85% and 0.29% for free peptide at 4.4% HSA and 7 pM and 6 pM for the EC50 at the GLPlR at 0% HSA respectively. G797 and G849 give values of 0.82% and 0.48% for free peptide at 4.4% HSA and 7 pM and 2 pM for the EC50 at the GLPlR at 0% HSA respectively. To compare the balance of activities at the GLPlR and GlucR between different peptides and across different conditions, these can be correlated using the calculation below, where the EC50’s are related to those of the natural ligands.

Table 5:EC50 Potencies for GLP-l/Glucagon Agonist Peptides in the Presence of Plasma Concentrations of Serum Albumin (Assay 2) Assay in 4.4% Human Assay in 3.2% Mouse Serum Assay in 3.2% Rat Serum Serum Albumin Albumin Albumin Human Human GLP1 R GlucR EC50 EC50 OM OM 0.122 1100 G797 739 1137 0.07 290 764 0.08 60 23170 0.08 G849 172 79 0.235 88 103 0.17 44 4055 0.33 G933 943 564 0.179 540 377 0.29 136 15500 G865 150 570 0.027 96 1100 0.021 18 87100 G796 140 53 34 0 36 G812 316 764 947 0 04 G380 6543 53590 576000 GLP1 25 Gluca-on 2.7 - 9700 4.97 557 60 - 1 GLP1 R ratios were determined as follows: Relative Potency GlucR = EC50 Glucagon/ EC50 Tested peptide Relative Potency GLP1 R = EC50 GLP1/ EC50 Tested peptide GlucR/GLP1 R Ratio = Relative Potency GlucR/ Relative Potency GLP1 R Stability testing of peptides in . The stability in plasma of the peptides G730, G797, G849 and G933 was determined as follows.

Stock solutions of the peptides of about 200 umol/L was prepared by weighing solid peptide into a Eppendorf Low Bind Tube and dissolved in DMSO. 10pL of stock solutions were added to 990 pL of plasma in an Eppendorf Low Bind Tube, resulting in initial concentrations of the peptides in plasma of about 2 . The frozen blank plasma from human, rat and mouse had been thawed and heated to a temperature of 37° C before addition of the stock solution. The spiked plasma samples were gently mixed and allowed to equilibrate for about 5 minutes before start of experiment. The plasma samples were incubated for 48 hours in a R C02 incubator at 37° C. Sampling (30 uL) was performed at 0, 1, 2, 6.5, 17, 24 and 48 hours. The s were stored at -70° C until analysis.

Plasma samples were assayed as follows. The 30 pL plasma samples were protein precipitated with 180 ml of cold ethanol in a 96-well low bind plate (Eppendorf n LoBind). After mixing and centrifugation, 100 pl the supernatant was transferred to a new plate and 1 pl was injected onto an analytical column.

] The analysis was performed using a uLC-system (LC Exigent pLC) coupled to a medium high resolution mass spectrometer (Perkin Elmer PenTOF) with positive electrospray ionisation. The analytical column was a 5 cm, 1 mm Agilent Poroshell m made) C18-column with a particle size of 2.7 pm. Flow: 0.1 ml/min using a slow reversed phase gradient. Mobile phases used were acetonitrile and water containing 0.1% formic acid.

] The resulting data were manually evaluated for the following degradation products: +1 product (acid) and the DPP avage product. Products with +1 mass may arise from deamidation at amide groups of glutamine or at the C—terminus. Cleavage ts arise from the action of the protease DPP IV in plasma. Both the degradation of the es and formation of peptide products were reported in percentage of the initial peptide concentration. Peaks were integrated and % remaining peptide was calculated: (peak area/peak area 0H)*100. Data for the 24h time point is shown in Table 6. Levels of deamidation and DPP IV cleavage were low for G797 and G933.

Table 6: Peptide ity in Plasma EPlasmaStabllltyInMouserWPlasma Stabllltyin Human W§W Plasma Stabllltyin Rat Wi g Plasma at 24h Plasma at24h Plasma at 24h g 4 j i %W WSW W%W WWlW %W W%W W‘W l %DPP ; %DPP W % WWgW %DPP l l ; stable § +1 i d stable +1 cleaved l i stable % cleaved % E i peptide l prod l product/+1 l peptide l prod ‘ product/+1 l peptide 3 +1 l product/+1 : l 1 g DPP [ . DPP g prod DPP g cleaved d cleaved l 1 g l l l E i l g product 3 product E ‘1 . .k . E product i {Peptidel ii i .1 ti;"1 16730 ’ 65 l 15 . 14/5 1. <1 1 24 i 58 I 2 16797 1 <1 l 60 1 , waan % .1 - 1.51 a g 6849 ‘ 38 r l l 3 l G933 W83 W...2.,W85.M......:..........§.....W<1.Wl Solubility Peptide solubility was assessed in a variety of buffer s within a pH range of 4.5 to 8.0, as follows. Dried powder forms of the GLP-l/Glucagon agonist peptides were reconstituted in various buffers at room temperature. The ance was measured at 280 nm using NanoDrop 2000 spectrophotometer and the peptide concentration was calculated using the following equation: C=(A280*Mw)/8 where: c- concentration 8- extinction coefficient Mw- molecular weight Azso— Absorbance at 280 nm 8 = (l X Trp = 5560) + (l X Tyr = 1200) The s are shown in Table 7. Each of the peptides was soluble at 0.8mg/ml over a range of pH (6.5 to 8.5). G730 was soluble in a pH range of 4.5 to 8.0, G797 was e in a pH range of 6. to 8.0, and G933 was soluble in a pH range of 6 to 8.0. The solubility of G933 was tested in a number of different buffer systems, also shown in Table 7. G933 was soluble at lmg/ml in at least the following buffer s: histidine (pH 6 and 7; ionic strength :0.25 to 100 mM), sodium phosphate (pH 6-7.5; ;ionic strength :0.25 to 100 mM), and tris/hydroxymethyl aminomethane (pH 7-9; ionic strength :0.25 to 100 mM).

TABLE 7: Peptide lity profile (Ionic Strength of all buffers: 100mM) Conc. (mg/ml) Buffer Target 1mg/ml G730 G797 G849 G933 Glutamate pH 4.5 0.83 NA 0023 0.02 Acetate pH 5 NA NA NA 0.03 Succinate pH 5 NA NA NA 1.1 Phosphate, pH :6 0.14 0.84 0.06 1.2 ine pH 6 NA Phosphate pH 6.5 0.83 Phosphate, pH 7.0 NA Histidine, pH 7.0 NA Phosphate pH 7.5 0.85 Tris pH 7.5 0.83 Tris pH 8.0 1.1 0.83 0.89 1.2 Formulations. Peptide solubility was assessed in three different isotonic formulations: 1. Default Formulation (DF)= 0.1M Tris pH 7.5, 150mM Mannitol. Final formulation pH: 7.2 2. Back up formulation 1 0.05M Tris, 50mM Arginine/Proline. Final formulation pH: 8.0 3. Back up formulation 2 (BF2)= Sodium Phosphate buffer (pH8)/ 1.85 % W/V propylene glycol. Final formulation pH: 7.0 lity was measured as ed above, and the s are shown in Table 8.

G730, G797 and G933 were soluble to at least 5mg/ml in the DF, the maximum solubility of G849 in DF was 3.7 mg/ml, G797 was e to at least 10mg/ml in BFl, and G933 was soluble to at least 10mg/ml in BF2.

Table 8: Peptide Solubility in Formulation Formulation Formulation 10 mg/ml Concentration solubility Lead (BFZ) Candidate G730 5mg/ml DF DF/BF1 G797 5mg/ml yes G849 n/a Concentration determined by A280nm The stability of the DF was evaluated by measuring purity reversed phase ultra— performance liquid chromatography (RP UPLC), within one month. The e conditions were 5°C, 25 0C, 40 0C and -80 0C. The results are shown in Tables 9 and 10.

Table 9 Peptide formulation purity after 1 month in stability conditions 59C 25 9C 40 9C minus 80C Peptide G730_DF 97.7 96.1 86.1 97.7 G797_BF1 98,72 98.84 77.54 NA G849 DF 95.5 NA NA NA G933_DF 97.8 95.9 88.9 98.9 Table 10: Peptide formulation purity loss (% compared to T0) after 1 month in stability conditions Lead Candidate _ 49 _ G730_DF 0.82 2.43 12.54 0.3 G797_BF1 0.24 0.12 21.65 0.3 G849_DF n/a n/a n/a n/a G933_DF 0.3 2.2 9.3 (-)0.8 The peptides all showed acceptable properties with respect to solubility, formulatability and stability Example 3: In vivo studies G730, G797, and G812 (study A). Selected GLP-l/glucagon agonist peptides disclosed herein were tested in a diet induced obesity (DIO) mouse model, as follows.

Female C57/Bl6JHstla (obtained from Harlan Laboratories, UK) were started on a high fat diet of Dl2492 (Research Diets, NJ, USA) and a chocolate confection, delicato ball (Delicata Bakverk, Sweden) at 9-11 weeks of age, and were maintained on the diet for 16 weeks prior to l to the animal facility, during a three week acclimation period and during drug treatment, caloric content of the two components of the diet is shown in Table 11. The mice were diVided into 9 groups (n=5-6), and ent was started at 29 weeks of age. The treatment groups and dosing are shown in Table 12.

Table 11: Content of DIO Diet Product Protein Carbohydrate Fat Kcal fat Total ("/0 ) (%) (%) (%) Kcal/gram Delicatoball (Delicata k AB , 5 53 31 54 5.05 Huddinge, Sweden) D12492 rch Diets, NJ, USA) 26.2 26.3 34.9 60 5.24 Table 12: Treatment Groups for Study A Peptide Dose # of Animals e NA 6 Liraglutide 26.6 nmol/kg 6 G730 10 g 6 G730 20 nmol/kg 5 G730 50 g 6 G797 5 nmol/kg 5 G797 20 nmol/kg 6 G797 50 nmol/kg 6 _ 50 _ G812 20 nmol/kg 5 GLP-l/glucagon agonist peptides G730, G797, and G812, as well as Liraglutide were formulated in the vehicle, 100 mM Tris/150mM mannitol, pH 7.4 The ents were administered subcutaneously twice daily for 14 days, whilst the animals were maintained on a high fat diet. The body weight of the animals was monitored daily throughout the dosing . At day 14, blood samples for the measurement of plasma glucose and insulin from conscious mice were obtained after a 4-hour fasting period.

Mice were then anaesthetized using isoflourane and terminal blood was ted from the capillary bed behind the eye. The following parameters were ed: blood try measurements of triglycerides, total cholesterol, non-esterified fatty acids (NEFA), beta-hydroxybutyrate and fibroblast growth factor 21 (FGF21) (Tables 14 and below).

The effect of treatment with liraglutide and the GLP-l/glucagon agonist peptides G730, G797 and G812 on body weight, in comparison to liraglutide and e, is shown in FIGs. 1-4. Animals treated with either G730 or G797 showed dose ent and continuous weight loss over the 14 day dosing period. At 50 nmol/kg, animals treated with G730 and G797 experienced an about 24% change in weight at day 14 as compared to the vehicle-treated animals.

] Mice treated with G730 or G797 showed a dose-dependent reduction in glucose levels at day 14 (Table 13). Reduced n levels were also observed, with these two treatments, especially at the higher doses (Table 13). The insulin sensitivity index tatic model assessment (HOMA) significantly improved at 20 nmol/kg G730 and and 50 nmol/kg G797. HOMA is a modeling method that uses the sum of plasma insulin and glucose levels to assess B-cell function and insulin resistance (Table 14). Total plasma cholesterol was lowered both by liraglutide, G730 and G797 at all doses, with less pronounced changes in plasma non-esterified fatty acids (NEFA) levels and plasma and c triglycerides (TG). Beta-hydroxybutyrate (BeHy) had tendencies towards increased levels, in line with the body weight loss. Fibroblast growth factor 21 (FGF21) generally increased with dual GLP-l/glucagon agonist peptide treatment.

Table 13: Effect of GLP-l/glucagon agonist peptide treatment on glucose, insulin, and BW day 14 (% change of dose vehicle Peptide (nmol/kg) start bw (g) ve h id 6 0 47,4 |+ 3,7 0,0 8,8 |+ O 0‘ |+ , 0,8 0,23 7,2 Liragl utide 27 47,5 |+ 1,8 -13,3 8,0 |+ O N |+ , 0,3 0,12 2,8 G730 10 44,5 |+ N N |+ O * — |+ , -7, 5 7,2 W , 0,4 0, 14 3,3 G730 20 45I9 |+ -I—I|+ C0‘ |+ 1I7 |+ G730 50 |+ C \l C W |+ 0 13I-|+ , I I I I G797 5 |+ |+ C W |+ |+ l‘l" oou'IHHo G797 20 |+ |+ o 0‘ 2 0 |+ H- l 5353 aux: 5353 ONkDU'I -I G797 50 47I2 |+ H 00 - |+ 0,5 C H |+ C CH -* I —Im H-IH I I G812 20 49,2 |+ 3I4_I-Il!_ 0I4_|+ |+ I6I0 I"? HH .flfl-Ifl—— Results evaluated by a two-tailed distribution, two-sample unequal variance ttest; * indicates p<0.05 compared to vehicle.

Table 14: Effect of GLP-l/glucagon agonist peptide treatment on additional blood chemistry measurements Hw:1; i: TE 55: TG;"15335 Results evaluated by a two-tailed bution, two-sample unequal variance t test; * indicates p<0.05 compared to vehicle.

G865, G933, and G796 (study B). A further set of GLP-l/glucagon peptides was tested in a diet induced obesity model using the same protocol above, but with the ent groups and dosing shown in Table 15: Table 15: ent Groups for Study B Peptide Dose # of Animals Vehicle NA 6 Liraglutide 26.6 nmol/kg 6 G865 5 nmol/kg 6 G865 10 g 6 G933 5 nmol/kg 6 G933 10 nmol/kg 6 _ 52 _ G796 20 nmol/kg 6 G796 50 g 6 GLP-l/glucagon agonist es G865, G933, and G796, as well as liraglutide were ated in the vehicle, 100 mM Tris/l50mM mannitol, pH 7.4 The treatments were administered subcutaneously twice daily for 14 days, whilst the animals were ined on a high fat diet. The body weight of the animals was monitored daily throughout the dosing period. At day 14, blood samples for the measurement of plasma glucose and insulin from conscious mice were obtained after a 4-hour fasting period.

Mice were then anaesthetized using isoflourane and al blood was collected from the capillary bed behind the eye. The following parameters were measured: blood chemistry measurements of triglycerides, total cholesterol, non-esterified fatty acids (NEFA), beta-hydroxybutyrate and last growth factor 21 (FGF21) (Table 16 and Table 17 below).

] The effect of ent with liraglutide and the GLP-l/glucagon agonist peptides G933, G865, G796 on body weight, in comparison to liraglutide and vehicle, is shown in FIGS. 5-8. Animals treated with either G933, G865 or G796 showed dose dependent and continuous weight loss over the 14 day dosing period.

Glucose levels, insulin levels and HOMA at day 14 post-treatment are shown in Table 16. Total plasma cholesterol levels, plasma non-esterified fatty acids (NEFA) levels, plasma and hepatic triglyceride (TG) levels, beta-hydroxy butyrate (BeHy) levels, and fibroblast growth factor 21 (FGF21) levels at day 14 post-treatment are shown in Table 17.

Table 16: Effect of GLP-l/glucagon agonist peptide treatment on glucose, insulin, and BW day 14 (% change of dose vehicle Peptide (nmol/kg) start bw (g) .- |+ |+ |+ , —-I|+ Liraglutide 2 ——I|+ |+ |+ |+ 6865 10 +|+|+ 47,0 |+ 0,36 |+ 0,06 2,43 535353 \l G933 |+ — —-I|+ |+ 1 ——II+ + |+ G796 20 50,9 |+ |+|+ O 0,38 |+ 0,05 2,24 |+ G796 50 49,7 |+ H 0,43 |+ 0, 14 2,87 |+ 1‘53 HM "HIEHI— Results evaluated by a two-tailed distribution, two-sample unequal variance t test; * indicates p<0.05 compared to vehicle.

Table 17: Effect of GLP-l/glucagon t peptide treatment on additional blood try measurements HepaticTG Plasma Plasma SEM SEM SEM SEM SEM SEM dose (g TG/100g Plasma TG NEFA Choleste Be Hy FG F21 Pe-tide (nmol/ _) tissue) (mM) (mM) ) (umol/l) (-; mL) e 0,5 1 Liraglutide 27 18,4 1 2,5 -1 1 6865 5 20,7 1 5,6 0,26 1 1 6865 10 22,3 1 5,1 1 6933 5 11,3 1 0,8 * 0,19 1 0,01 1 6933 10 14,7 1 4,1 0,16 -* 0,27 1 G796 20 9,6 H 0,9 * 0,26 0,24 H 6796 50 9,9 H 0,6 * 0,16 H Results evaluated by a two-tailed distribution, two-sample unequal variance t test; * indicates p<0.05 compared to vehicle.

Example 4: Single-ascending-dose study (A) SUBJECTS A total of 362 subjects consented to participate in the study in Germany. The subjects were screened for the following inclusion and exclusion criteria.

Inclusion ia: 0 Healthy volunteers, ages 18 through 45 years at the time of screening; 0 Body mass index 2 22 and S 30 kg/m2 and body weight 2 70 kg; and 0 Venous access le for multiple cannulations.

Exclusion criteria: 0 Any condition that would interfere with evaluation of the G933 or interpretation of t safety or study results. Specific examples included (a) past history of acute or chronic pancreatitis, or pancreatic amylase or lipase greater than the upper limit of normal (ULN) at screening, (b) past history of gastroparesis requiring treatment, (c) past history of surgery affecting the upper gastrointestinal tract likely to affect the interpretation of safety and tolerability data, (d) history of cholelithiasis leading to episodes of acute cholecystitis not treated by cholecystectomy, or known biliary disease, (e) history of or family history of multiple endocrine neoplasia type 2 (MEN-2), serum calcitonin suggestive of thyroid C—cell hyperplasia (calcitonin level > 50 ng/L), or medullary thyroid carcinoma at screening, (f) past history of clinically icant cardiac rhythm disturbance, e.g., permanent or smal atrial fibrillation/flutter, paroxysmal supraventricular tachycardia, smal ventricular tachycardia, presence of an implantable pacemaker device or verter/defibrillator, (g) history of treated or symptomatic cardiac failure, and (h) history of previous myocardial infarction or cerebrovascular accident (e.g., stroke); 0 History or presence of gastrointestinal, renal, or c disease (with the exception of Gilbert’s me) or any other condition known to interfere with absorption, bution, metabolism, or ion of drugs; 0 History of cancer, with the exception of non-melanoma skin cancer; 0 Any clinically important illness, medical/surgical procedure, or trauma within 4 weeks prior to Day 1 dosing; 0 Positive hepatitis B surface antigen or hepatitis C virus dy serology at screening; 0 Positive human immunodeficiency virus (HIV) test at screening or use of antiretroviral medications as determined by medical history or subject’s verbal 0 Serum potassium or calcium outside the normal range at screening; 0 Serum creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), or total bin greater than the ULN at screening; 0 Any other clinically important abnormalities in clinical try, hematology, or urinalysis results, as judged by the investigator, should also result in exclusion from the study; 0 Use of any of the following medicinal products:(a) concurrent or previous use of a GLP-1 receptor agonist, (b) current or previous use of systemic osteroids within the past 28 days prior to screening, or (c) use of any medicinal products or herbal preparations licensed for control of body weight or appetite is prohibited from 1 week prior to Day -1 through Day 7; 0 Abnormal vital signs after 10 minutes of supine rest, defined as any of the following: (a) Systolic blood pressure < 90 mmHg or 2 140 mmHg, (b) Diastolic blood pressure < 50 mmHg or 2 90 mmHg, or (c) Heart rate < 50 or > 90 beats per minute; 0 Any clinically important abnormalities in rhythm, conduction (e.g., Wolff- Parkinson-White syndrome, sick-sinus syndrome), or morphology of the resting 12- lead ECG, or any abnormalities in the ECG that, in the opinion of the investigator, may interfere with the interpretation of changes in the QT interval corrected for heart rate (QTc), including abnormal T-wave morphology, or left ventricular hypertrophy; 0 Prolonged QTc using the Fridericia formula (QTcF) > 450 milliseconds, or shortened QTcF < 340 milliseconds based on 12-lead ECG, or family history of long QT syndrome; 0 PR (PQ) al shortening to < 120 milliseconds or prolongation > 200 milliseconds (first degree atrioventricular block) ; 0 Intermittent second degree block (Wenckebach block while asleep is not ive) or third degree block, or atrioventricular iation; 0 QRS interval outside the range of 50-110 econds; 0 Known or suspected history of drug abuse within the past 3 years; 0 y of alcohol abuse or excessive intake of alcohol within the past 3 years; 0 t smoker (> 0 ttes per day); subjects are also excluded if they test positive for ne at screening; 0 ve screen for drugs of abuse at screening or admission to the study unit, or positive breath test for alcohol on admission to the study unit prior to the administration of investigational product. Subjects who utilize benzodiazepines for chronic anxiety or sleep disorders may be permitted to enter the study; History of severe allergy/hypersensitivity or ongoing ally important allergy/hypersensitivity; Whole blood or red blood cell donation, or any blood loss > 500 mL within 2 months prior to screening; Receipt of another new al entity (defined as a compound that has not been approved for marketing), or ipation in any other clinical study that included drug treatment within at least 30 days or 5 half-lives of the administration of investigational product in this study (whichever is longer). The period of exclusion to begin 30 days or 5 half-lives of igational product after the final dose, or after the last visit, whichever is longest. Subjects consented and screened, but not randomized into this study or a previous study, are not excluded; or Psychiatric illness such that subjects have been ted to an institution by way of official or judicial order After screening, 313 subjects were determined not to meet the inclusion/exclusion criteria. The remaining 48 subjects were randomized and received G933. The demographics of the 48 treated patients are shown in the table below.

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Based on PK/PD modeling conducted using al literature data on GPL-l and on modulators, the clinically efficacious dose of G933 was predicted to be in the range of 300 to 2000 ug/day. Thus, the dose increments of 5, 10, 30, 100, 300, 600, 1200, and 2000 pg G933 were selected. Safety data for each cohort was evaluated before dose escalation was allowed to proceed. To further enhance safety, the fold increases were greatest at the lower end of the proposed dose range and were reduced in a step-wise fashion so that the last dose increase was less than 2-fold. The maximum proposed dose level in this study was 2000 ug. The safety margin at the m proposed human dose of 2000 pg based on monkey AUC data was 0.8.

Subjects received 1 dose of G933 during the study. For all subjects, a screening visit was performed within 28 days prior to randomization (Day -29 to Day -2). Subjects were admitted to the unit on the g prior to receiving investigational product (Day - l) to allow for repeat assessment of ility criteria, to perform cardiac telemetry monitoring over a 4-hour period, and to standardize the level of physical activity before dosing the following morning. On Day 1, following an overnight fast for a minimum of 8 hours, baseline safety ments and blood samples were obtained, and the subject was administered a single subcutaneous (SC) dose of either G933 or placebo. Subjects remained in the study facility for timed assessments and safety monitoring (including cardiac monitoring by continuous telemetry and ittent digital electrocardiograms [ECG]) throughout Day 1 and Day 2, and remained housed until discharge. Subjects were discharged from the unit on the morning of Day 3 after safety ments were performed and blood samples collected. ts returned to the unit for outpatient visits on Days 4, 7, and 28.

Following screening, the study duration for each subject was approximately 29 days, which included an inpatient evaluation period and an outpatient follow-up period.

For pharmacodynamics evaluations, on Day 1, blood samples for measurement of glucose and insulin levels were drawn before each meal (breakfast, lunch, and evening meal) and 1 and 2 hours (i 15 minutes) after the start of each meal. The start of each meal on Day 1 was recorded and each meal was to be consumed within 30 minutes.

For immunological evaluations, anti-drug antibody (ADA) sampling occurred on Day 1, approximately 1 week post-dose, and approximately 1 month post-dose. \ For cokinetic (PK) evaluations, sampling times were pre-dose, and 1, 2, 4, 6, 8, 10, 12, 16, 24, 36, 48, and 72 hours post-dose (i 15 minutes for the first 2 hours and i 30 minutes for the remaining time points through 48 hours post-dose). On Day 1, PK samples were also to be drawn before each meal (breakfast, lunch, and evening meal), 1 and 2 hours (i 15 s) after the start of each meal.

(C) RESULTS Dose escalation ded from 5 to 300 [Lg as planned per the protocol; however, 300 [L g was not tolerated as evidenced by events of significant vomiting, and the dose was de-escalated to 150 [Lg for the final cohort. A flow diagram of the actual study is provided in Figure 9, and the subject disposition is shown in Figure 10.

No PK parameters could be derived for 5 [Lg G933 as only 3 subjects showed at least one quantifiable level in . Plasma concentrations of G933 were measurable from 2-4.5 hours until 16-24 hours in all ts in the 5 and 10 [Lg G933 groups. Plasma concentrations of G933 were measurable until 48 hours in all subjects in the 100 and 300 [Lg G933 groups. No subject had quantifiable plasma concentrations of G933 at 72 hours The mean tration-time profiles for all G933 doses displayed an apparent xponential decline following the peak, with a moderately rapid drop of concentration between the peak and 48 hours after dosing.

Single dose plasma G933 PK parameter estimates were listed by subject and treatment and are summarized by treatment in Table 19.

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The nt terminal elimination half-life (tug) of G933 was determined using at least 3 data points based on visual inspection over the 72-hour sampling period. T1/2 was not assessable in the ts of dose group 5 ug because of too low plasma concentrations. In the other doses group when %AUCex was higher than 20%, t1/2 and consequently AUC0_00 were also not reported. The geometric mean t1/2 value was about 10- 12 hr.

Inter-subject ility for maximum plasma concentration (Cmax) and AUC0_OO, as expressed by coefficient of variation (CV), was moderate (3-37%) for both parameters.

No subjects were ADA positive at baseline or post-baseline.

Blood re, pulse, food intake, and adverse events were monitored over the course of the study and are summarized in Table 20.

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The most nt TEAEs (incidence > 10% of subjects) were vomiting, nausea, dizziness, and headache. TEAEs of vomiting occurred only at the highest G933 doses (100, 150, and 300 ug). There were a total of 40 vomiting episodes for the 10 subjects who enced vomiting. Thirty episodes were from 5 subjects at the 300 ug dose, 9 episodes were from 4 subjects at the 150 ug dose, and 1 episode was from 1 subject at the 100 ug dose.

] Significant vomiting defined as 3 or more episodes of vomiting on a single day or across 2 consecutive days despite adjustment to diet, was reported for 2 of 6 subjects (33.3%) in the 150 ug G933 group and 5 of 6 ts (83.3%) in the 300 ug G933 group.

Due to the events of significant vomiting observed at the 300 ug dose, the dose of G933 was de-escalated to 150 ug for the final cohort.

At doses up to 150 ug the tolerability of G933 was acceptable with respect to nausea, vomiting, tachycardia and blood pressure. At a dose of 300 ug of G933, unacceptable levels of vomiting were seen, as was a significant rise in systolic blood pressure and pulse rate. Significant rises in pulse have been seen across the range of marketed GLP-l agonists after single dose, particularly in y volunteers, as has nausea and vomiting. Given that the gastrointestinal effects of G933 (vomiting) were seen at a single dose of 150 ug without any cardiovascular effects, a window exists were G933 can be safely administered to ts with type 2 diabetes mellitus at a dose expected to improve glycemic control and drive weight reduction, without a negative impact on the cardiovascular risk e. G933 met safety and tolerability endpoints in this single dose healthy eer study.

Example 5: Multiple-ascending-dose study part I (A) SUBJECTS Approximately 75 overweight or obese subjects with relatively well controlled type 2 diabetes mellitus (T2DM) are enrolled to participate in the study. The enrolled subjects meet the following inclusion and exclusion criteria.

Inclusion criteria: Male or female age 18 through 65 years at the time of screening; Body mass index 27 to 40 kg/m2 (inclusive); Diagnosis of T2DM and glucose control managed with min monotherapy where no icant dose change (increase or decrease 2 500 mg/day) has occurred in the 3 months prior to screening: (a) The screening hemoglobin AlC (glycated hemoglogin; HbAlc) value should be within the target range of 6.5% to 8.5%, or optionally 7.0% to 8.5%; (b) subjects prescribed a dipeptidyl peptidase-4 (DPPIV) inhibitor in addition to metformin erapy may be eligible to enter the study following a 4-week DPPIV inhibitor washout period prior to screening; (c) subjects prescribed less than 50% of the licensed dose of sulphonylurea in addition to metformin monotherapy may be eligible to enter the study following a 4-week sulphonylurea washout period; and (d) subjects prescribed a sodium- glucose nsporter 2 (SGLT2) tor in addition to min monotherapy may be eligible to enter the study ing a 4-week SGLT2 tor washout period; Venous access suitable for multiple cannulations; Cohort 4 subjects must be taking 2 10 mg daily dose of a statin for a period of at least 4 weeks prior to screening; and Cohort 4 ts must be willing and able to self-administer daily subcutaneous (SC) injections following an initial self-injection of placebo during the screening period.

Exclusion criteria: 0 Any condition that would interfere with evaluations of the investigational product or interpretation of subject safety or study results. Specific examples are: (a) past y of acute or chronic pancreatitis, or pancreatic amylase or lipase greater than twice the upper limit of normal (ULN) of the laboratory reference range at screening, (b) past history of gastroparesis ing treatment, (c) past history of surgery affecting the upper GI tract likely to affect the interpretation of safety and tolerability data (d) history of cholelithiasis g to episodes of acute ystitis not treated by cholecystectomy, or known biliary disease, and (e) Serum calcitonin suggestive of thyroid C—cell hyperplasia (calcitonin level > 50 ng/L), medullary thyroid carcinoma, or history or family history of multiple endocrine sia at screening; 0 History or presence of GI, renal, or hepatic disease (with the exception of Gilbert’s syndrome), or any other condition known to interfere with absorption, distribution, metabolism, or excretion of drugs; 0 History of cancer nally within the last 10 years), with the exception of non- melanoma skin cancer; 0 History or presence of diabetic foot ulcers; 0 Any clinically important illness (apart from T2DM for subjects with known diabetes), medical/surgical procedure, or trauma within 4 weeks prior to Day 1 dosing; 0 Symptoms of insulinopenia or poor blood glucose control (eg, significant thirst, nocturia, polyuria, polydipsia, or weight loss); 0 g blood glucose 2 200 mg/dL (l 1.11 mmol/L); 0 Positive hepatitis B e antigen or hepatitis C virus antibody serology at screening; 0 Positive human deficiency virus (HIV) test at screening or subject taking antiretroviral medications as ined by medical history or subject’s verbal report; 0 Aspartate transaminase (AST) 2 2.5 X ULN at screening; 0 Alanine transaminase (ALT) 2 2.5 X ULN at ing; 0 Total bilirubin 2 2 X ULN at screening; 0 Hemoglobin below the lower limit of the normal range at screening; 0 Neutrophils < 1.5 X 109/L at screening; 0 Thyroid-stimulating hormone (TSH) level above the normal range at screening; 0 Impaired renal on defined as glomerular filtration rate (GFR) S 60 mL/minute/1.73m2 (GFR estimated according to Modification in Renal Disease); 0 Persistent ed as documented on 2 2 prior occasions by the subject’s usual physician) macroalbuminuria (> 300 mg/L); 0 Significant late diabetic complications angiopathy with symptoms of congestive heart disease or peripheral arterial disease, microangiopathy with symptoms of neuropathy, gastroparesis, retinopathy, nephropathy); 0 Cardiac conduction defect (e.g., Wolff-Parkinson-White syndrome, sick sinus syndrome) during the screening period. 0 Abnormal vital signs, after 10 minutes of supine rest, defined as any of the following: (a) If < 60 years old, systolic BP < 90 mm Hg or 2140 mm Hg; if 2 60 years old, systolic BP < 90 mm Hg or 2 150 mm Hg, (b) Diastolic BP < 50 mm Hg or 2 90 mm Hg, or (c) HR < 45 or > 85 beats per minute; 0 Any clinically important abnormalities in rhythm, conduction or logy of the g ECG and any abnormalities in the d ECG that may interfere with the interpretation of QTc interval s, including abnormal ST-T-Wave morphology or left ventricular hypertrophy; 0 Prolonged QTcF > 450 msec (for both genders) or shortened QTcF < 340 msec, or family history of long QT syndrome; 0 PR (PQ) al shortening < 120 msec (PR < 120 msec but > 110 msec is able if there is no evidence of ventricular pre-excitation); 0 PR (PQ) interval prolongation (> 240 msec), intermittent second or third degree AV block, or AV iation; 0 QRS duration > 120 msec including persistent or intermittent bundle branch block 0 Implantable cardiac defibrillator or a permanent pacemaker, and symptomatic ventricular and/or atrial tachyarrhythmias; 0 Unstable angina pectoris or stable angina pectoris classified higher than Canadian Cardiovascular Society class II or a myocardial infarction or stroke; 0 History of hospitalization caused by heart failure or a sis of heart failure; Known or suspected history of drug abuse within the past 3 years; History of alcohol abuse or excessive intake of alcohol within the past 3 years; Positive screen for drugs of abuse at screening or admission to the study unit, or positive screen for alcohol on admission to the unit prior to the administration of investigational product. Patients who e benzodiazepines for chronic anxiety or sleep disorders may be permitted to enter the study; History of severe allergy/hypersensitivity or ongoing clinically important allergy/hypersensitivity; Whole blood or red blood cell donation, or any blood loss > 500 mL during the 2 months prior to screening; Received another new al entity (defined as a compound that has not been ed for marketing), or has participated in any other clinical study that included drug treatment within at least 30 days or 5 half-lives prior to the first stration of investigational product in this study ever is longer). The period of exclusion to begin 30 days or 5 half-lives of investigational product after the final dose, or after the last visit, ver is longest. Subjects consented and screened, but not randomized into this study or a previous Phase 1 study, are not Concurrent participation in another study of any kind; Use of any of the following medicinal products: (a) concurrent or previous use of a GLP-l agonist (b) use of systemic corticosteroids within 28 days prior to ing; (c) use of compounds known to g the QTc interval, or (d) use of any herbal preparations or medicinal products licensed for control of body weight or appetite within 1 week prior to Day 1; Psychiatric illness such that subjects have been committed to an institution by way of official or judicial order; and History of lactic acidosis or ketoacidosis.

(B) STUDY DESIGN A Phase 1/2, randomized, double-blind, multiple-ascending dose study is performed in two parts (A and B). A flow diagram of the study is provided in Figure 13.

Part A establishes a dose titration regimen for G933 (MEDIO382) and a maximal effective dose after titration, in T2DM subjects maintained on metformin therapy, over a period of approximately 12 days that is then be ed over an extended dosing period (Part B) to establish the efficacy of G933 on weight and glycemic control.

Part A consists of 3 cohorts. Cohort 1 consists of a stable dose (e.g., 100 ug) of investigational product administered daily for 7 days (9 subjects; 6 G933, 3 placebo).

Cohort 2 consists of an initial dose (e. g., 100 ug) of investigational product for 4 days and an up-titration step (titration dose 1; e.g., 150 ug) for 7 days (9 subjects; 6 G933, 3 placebo), and Cohort 3 consists of an initial dose (e. g., 100 ug ) of investigational product for 4 days, an up-titration step tion dose 1; e. g., 150 u g) for 4 days, and a second up- titration step (titration dose 2; e. g., 200 ug) for 7 days (9 subjects; 6 G933, 3 placebo).

Part B consists of l cohort. ts in this cohort (Cohort 4) are dosed at an initial dose (e.g., 100 ug) of investigational t for 4 days, an up-titration step tion dose 1; e.g., 150 ug) for 4 days, a second up-titration step (titration dose 2; e.g., 200 ug) for 4 days, and then titration dose 2 (e. g., 200 ug) for 28 days thereafter at home (48 subjects; 24 G933, 24 placebo).

The initial dose is 100 ug. Titration doses 1 and 2 do not exceed 300 ug. The study duration for each subject varies by cohort and consists of an inpatient leascending dose (MAD) or up-titration evaluation period ning on Day -2 and ending after Day 8 for Cohort 1, Day 12 for Cohort 2, Day 16 for Cohort 3, and Day 13 for Cohort 4) and an outpatient -up period. Additionally, only for Cohort 4, a 28-day period of at-home self-administration of investigational product (with weekly site visits) is included after the second ent up-titration step and before the end of study visit.

Subjects are admitted two evenings prior to receiving investigational product to allow for repeat assessment of eligibility criteria and baseline mixed-meal test (MMT) and to standardize the level of physical activity before dosing the following morning. On Day 1, following an overnight fast for a m of 8 hours, baseline blood s are drawn for safety, efficacy, pharmacokinetic (PK), pharmacodynamic (PD), and anti-drug antibody (ADA) laboratory tests. In addition, l2-lead ECGs are recorded, and the subject is administered a single SC dose of either G933 or placebo.

The subject remains in the facility for observation and safety tests including ECGs, telemetry, ring of vital signs, and assessments of the injection site for potential ons hout the day and during the treatment period. Additionally, for Cohort 4, 24-hour ambulatory blood pressure monitoring (ABPM) are performed.

Mixed-meal test (MMT) ures are performed on Day -1 and on the last day of the highest dose level achieved for all cohorts. onal time points include the first day at titration dose 1 (Cohort 2), the first day at titration dose 2 (Cohort 3), the day of discharge from the up-titration inpatient period (the fifth day of titration dose 2; Cohort 4), and the weekly site visits during the 28-day period of at-home self-administration (Cohort 4). For the MMT, the subject consumes a standardized meal (Ensure Plus®) within 5 minutes, and timed serial blood samples are obtained for measurement of e and ters related to glucose metabolism just before and through 240 minutes after consumption of the standardized meal. During the inpatient period, finger prick glucose samples are collected 15 minutes prior to and 2 hours after breakfast, mid-day, and evening meals, and prior to going to bed.

Cohort 4 subjects are re-admitted for an overnight inpatient stay at the end of the at-home self-administration period to collect the final endpoints.

Body weight is measured at multiple time points including (but not limited to) prior to the first dose, during the inpatient dosing period (and during the outpatient period for Cohort 4), at discharge from the unit, and at the 7- to 14-day follow-up visit.

Pharmacokinetics samples for G933 are obtained pre- and ose and at various time points by cohort up until 48 hours after the last dose of G933. Samples for min concentration are obtained prior to G933 . Antidrug antibodies samples are obtained pre-dose and at various time points up through 28 days post final dose.

Subjects are discharged from the unit the day after the final dose is administered in the study. A follow-up visit is performed for final safety assessments imately 28 days after the last dose of investigational product.

(C) EFFICACY ASSESSMENTS For evaluation of the impact on glucose control and weight after multiple doses of G933 compared to placebo, the percent change in MMT glucose AUC (up to 240 minutes post-MMT) and change in weight from baseline (Day -1) to the end of ent in Cohort 4 are compared between G933 and o groups using an analysis of covariance by adjusting for baseline measurement and ent group. Missing measurements at the end of treatment are replaced with the last available measurement. The comparison is conducted at a two-sided significance level of 0.1. A decrease in weight (e.g., between 1.3 and 2.0 kg over 4 weeks of repeat aily ) in the G933 treatment group as compared to the o group demonstrate that G933, e.g., at a dose of 100-300 ug, is effective to reduce weight.

] Change from baseline (Day -2) in hemoglobin Alc (glycated hemoglobin, HbAlc) and fructosamine, and percent change from baseline (Day -1) in 24-hour glucose AUC post-MMT, are analyzed in Cohort 4 similarly to the evaluation of weight. The 24- hour glucose AUC es include glucose measures from the pre-/post-MMT glucose metabolism panel as well as serum chemistry glucose levels and PD glucose samples, where those results are from unique time points. An increase in glucose control (e. g., at least 20% reduction in glucose area under the concentration-time curve (AUC) after a mixed-meal test (MMT) and/or as measured by hemoglobin Alc and fructosamine) in the G933 treatment group as compared to the placebo group trates that G933, e. g., at a dose of 0 ug, is effective to improve glucose control.

(D) RESULTS (i) PART A: s 1-3 G933 was successfully administered in doses up to 200 ug once a day (QD) for up to 15 days.

] Significant glucose reductions to normal levels with G933 were observed in both g plasma glucose and postprandial glucose from a mixed meal test. In the mixed- meal test, glucose area-under-the-curve (AUC) was reduced by >40% across Cohorts 1-3.

(Figure 14.) The change in baseline fasting glucose levels in Cohort 1 (on Day 7) and Cohort 3 (on Days 9 and 15) are shown in Figure 15.

A dose ent trend towards substantial weight loss up to 2 kg was observed.

The results are shown in Figure 16 and demonstrate that G933 substantially reduced body weight in overweight/obese patients with type 2 diabetes. In Cohort 3, a differential of more than 2 kg was observed.

G933 was well tolerated up to 200 ug with dose titration. The pharmacokinetics after repeat dosing are shown in Figure 17. The PK was linear and predictable. The half life was approximately 11 hours. There was minimal or no accumulation after multiple daily dosing, and steady state was achieved between Day 4 and Day 7. The maximal plasma concentrations in the dose range tested were 4.21 ng/mL and 18.90 ng/mL. The average daily exposure ranged between 2.89 ng/mL and 12.45 ng/mL.

] Only one sample was confirmed ve for anti-G933 antibody, post baseline.

Adverse events were manageable and in line with those expected of GLP-1 mono- agonists.

The vast majority of nausea and ng events reported occurred at the initiation of treatment, i.e., 100 ug dose level. Vomiting occurred in 26% (n25) of patients receiving G933 and none on placebo. In Cohort 1, no vomiting events were reported. In Cohort 2, three vomiting events were reported in two active patients. Both cases occurred on Day 1 of dosing at the 100 ug level, and both cases resolved without intervention. In Cohort 3, thirteen vomiting events were reported in three active patients. Two subjects vomited on Day 1, and these cases resolved without intervention. One subject vomited six times between Day 1 and Day 5. Intravenous saline was given on Day 3, and the subject was awn on Day 5.

Cardiac adverse events were reported in four (44.4%) of placebo patients and seven (36.8%) of patients treated with G933. Their frequency did not appear to increase with dose across active cohorts. An increase of about 10 bpm in pulse rate was observed in each cohort following treatment with G933. No significant change in systolic or diastolic blood pressure was observed, but a trend to a decline blood pressure was observed in all cohorts receiving either G933 or o. intestinal (GI) adverse events were reported in four (44.4%) of placebo patients and in thirteen (68.4%) of ts treated with G933. GI adverse events affected 3, 4, and 6 patients in Cohorts 1, 2, and 3, respectively. The most prevalent GI adverse event was nausea, which was reported in 6 (31.6%) of patients treated with G933.

Decreased appetite was reported in six ) of patients treated with G933 in Cohort 3.

One serious adverse event (SAE) was reported: asma pneumonia.

In summary, G933 was well tolerated, led to significant andial glucose reductions, and showed clinically significant dose-dependent weight loss. (ii) PART B: Cohort 4 G933 effectively lowered blood glucose and weight loss over a 41-day dosing period. The mean reduction in glucose area-under-the-curve (AUC) from baseline (day 1) to day 41 is shown in Figure 18. Over this time, G933 resulted in a 38.5% ion (90% CI. -47.1, , whereas o only resulted in a 16.1% reduction (90% CI. - 24.9, —8.0) (p—value: 0.001). G933 also improved glucose control as measured by HbAlc.

(See Figure 19.) After 41 days of treatment, HbA1c was reduced by 0.92 percent, from a baseline mean of 7.2% to a mean of 6.3% on day 42. In comparison, placebo only d HbA1c by 0.58 percent, from a baseline mean of 7.3% to a mean of 6.7%.

The effects of G933 on weight are shown in Figures 20 and 21. Figure 20 shows that after 41 days of treatment with G933, patients lost about 3.83 kg, whereas patients receiving o lost only about 1.71 kg (a difference of 2.12 kg between G933 and placebo; p<0.001). In addition, 44% of patients d with G933 lost at least 5 kg, whereas only 8% of patients receiving placebo lost at least 5 kg. Figure 21 shows that after 41 days of treatment with G933, patients lost about 4.18% of their weight, whereas patients receiving placebo only lost about 1.71% of their weight (a difference of 2.47% between G933 and placebo; p<0.001). In addition, 84% of patients treated with G933 lost at least 2% of their weight, whereas only 42% of patients receiving placebo lost at least 2% of their weight.

Treatment with G933 also ed in a decrease in liver fat. (See Figure 22). The mean relative reduction in liver fat percentage from baseline (95% CI) with G933 versus placebo was 20.5% (7.2, 33.9) (p=0.004). A icant reduction in liver volume was also ed in patients treated with G933 as compared to placebo (0.14 L (-0.24, -0.03), p=0.01).

Overall, adverse effects were balanced in patients treated with G933 and placebo.

The adverse effects are summarized in the table below. No deaths occurred.

Subject with Placebo 200 ug G933 N=26 N=25 At least one event 21 ) 22 ) At lease one G933-related event 15 (57.7%) 20 (80.0%) At least one event of 2 grade 3 severity 2 (7.7%) 0 Death (grade 5 severity) 0 0 At least one serious event 1 (3.8%) 0 WO 53575 _ 76 _ At least one s and/or 2 grade 3 severity event 2 (7.7%) 0 At least one G933-related serious event 1 (3.8%) 0 At least one event leading to discontinuation of G933 1 (3.8%) 3 (12.0%) A total of 17 patients reported nausea: 4 receiving placebo (15.4%) and 13 d with G933 (52.0%). A total of 8 patients vomited: 0 receiving placebo and 8 d with G933 (32.0%). Nausea and vomiting were more frequent during earlier doses. (See Figure 23.) No adverse changes in cardiac and hemodynamic parameters were observed. No increase in systolic or diastolic blood pressure was observed. An increase of 12.8 beats per minute (bpm) in heart rate was seen at Day 13, but this had fallen to 6.9 bpm by Day Overall, G933 was ive with respect to both blood glucose lowering and weight loss of the 41-day period. Glucose metabolism based on the mixed meal test normalized and was accompanied by a icant reduction in HbAlc. Remarkably, a greater point estimate for weight reduction was observed that what would be expected for treatment with liraglutide (3 mg).

G933 was well tolerated up to 41 days with up-titrating doses from 100 [Lg to 200 [Lg, confirming steady state achievement within one week of ent at 200 [Lg and minimal accumulation after multiple dosing. The maximal plasma trations observed at 200 [Lg dose levels ranged between 1.98 ng/mL and 34.30 ng/mL. e 6: Multiple-ascending-dose study part II (A) SUBJECTS Approximately 32 overweight or obese subjects with relatively well controlled type 2 diabetes mellitus (T2DM) are enrolled to participate in the study. The enrolled subjects meet the inclusion and exclusion criteria described in Example 5 with the following exceptions: Inclusion criteria: 0 Cohort 5 and 6 subjects must be g and able to self-administer daily subcutaneous (SC) injections following an initial self-injection of placebo (or normal saline) during the screening period.

Exclusion criteria: 0 Abnormal vital signs, after 10 minutes of supine rest, are defined as any of the following: (a) If systolic BP < 90 mm Hg or 2 140 mm Hg; (b) lic BP < 50 mm Hg or 2 90 mm Hg; or (c) HR <45 or > 85 bpm; 0 Unstable angina pectoris or stable angina pectoris classified higher than Canadian Cardiovascular Society class II or any previous medical history of dial infarction or stroke, or a history of a transient ischemic attack within the prior 12 months.

(B) STUDY DESIGN A ized, double-blind, multiple-ascending dose study is performed. A flow diagram of the study is provided in Figure 24. This study consists of 2 cohorts: cohort 5 and cohort 6. Cohort 5 consists of 16 subjects (12 active, 4 placebo) who begin dosing G933 382) at 100 ug for 5 days followed by 150 ug for 5 days, 200 ug for 5 days, and 300 ug for 7 days. Cohort 6 consists of a further 16 subjects (12 active, 4 placebo) who begin dosing G933 at 100 ug for 5 days followed by 200 ug for 5 days, and 300 ug for 7 days. s 5 and 6 have approximately 32 subjects total and are run in parallel.

The study consists of an inpatient up-titration evaluation period beginning on Day 3 for both Cohorts 5 and 6 and an outpatient follow-up period. The first 4 days and the final day of dosing at 300 ug are inpatient.

Subjects are admitted to the clinic as described in Example 5 for Cohorts 1-4.

However, they have an initial inpatient period ending on Day 3 followed by e self- administration of G933 with a clinic visit at each dose up-titration step until initiation of the 300 ug dose. Subjects have a second inpatient period for the initiation of the 300 ug dose ending on the fourth day at that dose followed by e self administration.

Mixed-meal test (MMT) ures are performed on Day -1, and on the last day of the highest does level achieved. Additional time points include before commencing the 300 ug dose (Day 16 for Cohort 5 and Day 11 for Cohort 6), and at the end of dosing (Day 22 for Cohort 5 and Day 17 for Cohort 6). The MMT are performed as described in Example 5.

Body weight is measured at le time points including (but not limited to) prior to the first dose, during the inpatient dosing period, and during the outpatient period, at discharge from the unit, at the 7- to 14-day follow-up visit, and at the 28-day End of Study Visit.

Pharmacokinetics samples for G933 are obtained pre- and post-dose and at various time points by cohort up until 48 hours after the last dose of G933. Samples for metformin concentration are ed prior to G933 dosing. Antidrug antibodies samples are obtained pre-dose and at various time points up through 28 days post final dose.

Subjects are discharged from the unit the day after the final dose is administered in the study. A follow-up visit is performed for final safety assessments approximately 28 days after the last dose of investigational t.

(C) RESULTS G933 effectively lowered e in Cohorts 5 and 6. The glucose AUC levels observed in Cohorts 5 and 6 are shown in Figures 25 and 26 respectively. In Cohort 5, ent with G933 resulted in a glucose AUC reduction of 41.7% (90% CI. —49.9, — 33.5), whereas treatment with o only resulted in a glucose reduction of 8.0% (90% CI. —19.6, 3.6) (p<0.001). In Cohort 6, treatment with G933 resulted in a glucose AUC reduction of 35.8% (90% CI. -43.3, , whereas treatment with placebo only ed in a glucose reduction of -6.9% (90% CI. -20.2, 6.3) (p: 0.002). The effect on glucose AUC was comparable with us cohorts, i.e. normalised about 40% reduction. The percent change from baseline glucose AUC in all cohorts in shown in Figure 27. G933 also effectively lowered fasting glucose levels in Cohorts 5 and 6 as shown in Figure 28.

G933 was also effective in reducing weight. As shown in Figure 29, after 22 days of treatment, patients treated with G933 in Cohort 5 lost about 3.09 kg, whereas patients d with placebo lost only about 0.98 kg (a difference of 2.11 kg). Similarly, after 17 days of treatment, patients treated with G933 in Cohort 6 lost about 2.23 kg, whereas patients treated with placebo lost only about 0.35 kg ( a difference of 1.88 kg). Cohorts 5 (2.11kg) and 6 (1.88kg) both showed comparable weight loss after 22 and 17 days to that observed during the 41 days of dosing in Cohort 4. _ 79 _ Change in weight and glucose levels are plotted er in Figure 30 and summarized in Table 22 below.

Table 22 Cohort Weight (kg) Glucose AUC Fasting Glucose N: active (mg/dl) l 100 ug n=6 —l.l —27.3 —28.00 2 150 ug n=6 —0.5 —8.9 —l7.90 3 200 ug n=6 —2.4 —3l.6 —43.20 4 200 ug n=25 —2.11 —22.5 —32.62 300 ug n=l2 —2.11 —33.7 —24.40 6 300 ug n=l2 —l.88 —28.9 —36.94 G933 was well tolerated in Cohorts 5 and 6, and the adverse event profile was in line with other marketed GLP-l ues such as utide. No deaths or serious adverse events were observed in either cohort.

Both titration schedules demonstrated that G933 up to a dose of 300 ug was well tolerated with respect to nausea and vomiting. In Cohort 5, a total of 4 patients reported 8 episodes of nausea: 1 patient (20%) who received placebo and 3 patients (27.3%) who received G933. A total of 3 ts ed 8 vomiting episodes: no patients who received placebo (0%) and 3 patients who received G933 (27.3%). In Cohort 6, a total of patients reported 6 episodes of : no patients who received o (0%) and 5 ts who received G933 (41.7%). One patient vomited twice: no patients who received placebo (0%) and 1 patient who received G933 (8.3%). Lower rates of nausea (8/24 2 33%) and vomiting (4/24 2 16%) were observed as subjects were out-patients 48 hours after the first dose.

Ambulatory blood pressure measurement showed no increase from baseline in systolic blood pressure/ diastolic blood pressure (SBP/DBP) in G933-treated patients, and a potential trend towards reduction. In Cohort 5, heart rate was increased by approximately 7.6 beats per minute (BPM) from baseline following 22 days administration of G933, in line with what has been observed previously and other GLP-l analogues and in Cohort 4. In Cohort 6, heart rate was increased by approximately 5.9 BPM from baseline ing 17 days administration of G933.

WO 53575 Plasma concentrations of G933 after 7 days at the highest dose of 300 [Lg obtained with both titration schemes are presented in Figure 3 l. The profiles pped especially once steady state was ed, suggesting that exposure at this dose level is independent of the titration scheme adopted. The maximal plasma tration observed at 300 [Lg dose ranged from 7.9 ng/mL to 30.9 ng/mL.

Summary of multiple-ascending-dose study results (parts I and II) Overall, in the dose range of 100-300 ug, G933 showed a linear PK with half-life of approximately 8-11 hours, minimal accumulation after daily repeat dose, and steady state achievement between day 4 and day 7 at all four dose levels tested. The results confirmed the results from the single dose study (Example 4). Plasma concentrations reached after 7 days at the highest dose of 300 ug were ndent of the ion scheme d. Between-subject variability in Cmax was approximately 20-30% in r cohorts, and 40-50% in larger Cohort 4. Maximal plasma concentrations in the dose range tested and with the titration scheme adopted, ranged between 1.98 ng/mL and 34.3 ng/mL, with an average daily plasma concentration ranging between 0.87ng/mL and 16.3ng/mL.

The safety and bility profile was comparable with other GLP-l mimetics, and significantly, G933 resulted in both weight loss and glucose control. *>l<>l< The disclosure is not to be limited in scope by the specific ments described which are intended as single rations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims (20)

WHAT IS CLAIMED IS:

1. A method of reducing body weight comprising administering to a human subject in need f 100-600 ug of a peptide comprising the amino acid sequence of HXzQGTFTSDX103X12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30; wherein X2 is G or S, X10 is Y or K, X12 is K, E, R, or S, X13 is K or Y, X15 is D or E, X16 is S or G, X17 is E, R, Q, or K, X18 is R, S, or A, X20 is R, K, or Q, X21 is D or E, X23 is V or I, X24 is A or Q, X27 is E or V, X28 is A or K, and X30 is G or R (SEQ ID NO:4).

2. A method of reducing body fat comprising administering to a human subject in need thereof 100-600 ug of a peptide sing the amino acid sequence of SEQ ID NO:4.

3. A method of treating obesity comprising administering to a human subject in need thereof 0 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

4. A method of treating or preventing a disease or condition caused or characterized by excess body weight sing administering to a human subject in need thereof 100-600 ug of a e comprising the amino acid sequence of SEQ ID NO:4.

5. A method of treating Nonalcoholic Steatohepatitis (NASH) comprising administering to a human subject in need thereof 100-600 ug of a peptide comprising the amino acid ce of SEQ ID NO:4.

6. A method of increasing lipid oxidation comprising administering to a human subject in need thereof 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

7. A method of reducing food intake sing administering to a human subject in need f 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

8. A method of lowering plasma glucose comprising administering to a human subject in need thereof 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID

9. The method of any one of 1-8, wherein the subject has diabetes.

10. The method of claim 9, wherein the es is type 2 diabetes mellitus.

11. A method of treating type 2 diabetes us comprising administering to a human a peptide comprising the amino acid ce of SEQ ID NO:4.

12. A method of improving glycemic control in a human subject with type 2 diabetes mellitus comprising administering to the subject 100-600 ug of a peptide comprising the amino acid sequence of SEQ ID NO:4.

13. The method of claim 11 or 12, wherein the administration reduces body weight.

14. The method of any one of claims 11-13, wherein the administration treats obesity.

15. The method of any one of claims 9-12, n the administration reduces body

16. The method of any one of claims 1-15, wherein X2 is G, X10 is K, X12 is E, R, or S, X13 is K, X17 is E 01‘ K, X18 is S, X20 is R, X27 is E, 01‘ X28 is A.

17. The method of any one of claims 1-16, wherein the peptide comprises, consists essentially of, or consists of SEQ ID NO: 19.

18. The method of any one of claims 1-17, wherein the stration comprises administering an initial dose for 3 to 10 days and a second higher dose thereafter.

19. The method of claim 18, wherein the initial dose is administered for 3 to 7 days.

20. The method of claim 18 or 19, wherein the initial dose is 100 ug of the peptide. _ 83