WO2019140025A1 - Combination therapy - Google Patents

Abstract

The disclosure describes a combination therapy for treating diabetes or chronic kidney disease by administering to a patient in need of such treatment an effective amount of an urocortin-2 compound (or a pharmaceutically acceptable salt thereof) with a oxyntomodulin analog (or a pharmaceutically acceptable salt thereof), and to methods of using the same to treat diabetes and chronic kidney disease.

Description

COMBINATION THERAPY

[0001] The disclosure relates to biology and medicine, and more particularly it relates to a combination of a urocortin-2 (UCN2) analog with an oxyntomodulin (OXM) analog, as well as to methods of using the same to treat diabetes and chronic kidney disease (CKD).

[0002] Type II diabetes (T2D) is the most common form of diabetes, as it accounts for about 90% of all diabetes. Over 300 million people worldwide are diagnosed with T2D, which is characterized by high blood glucose levels caused by insulin-resistance. The current standard of care for T2D includes diet and exercise as underlying adjunctive therapy along with available oral and injectable glucose lowering drugs. Nonetheless, individuals with T2D still remain whose symptoms are inadequately controlled. An alternative treatment for T2D is needed.

[0003] CKD is characterized by a progressive loss of kidney function. Individuals having CKD experience over time an increase in albuminuria, proteinuria, serum creatinine, and renal histopathological lesions. It eventually develops into end stage renal disease (ESRD) for many individuals and requires either dialysis or kidney transplant. CKD may be caused by several underlying conditions including, for example, diabetes (i.e., diabetic nephropathy). The current standard of care for kidney diseases includes angiotensin converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs). There remains a need for an alternative treatment for CKD.

[0004] ETCN2 is a 38-amino acid endogenous peptide (SEQ ID NO: 12). It is one of three known endogenous urocortins (UCN1 and UCN3 being the others) found in mammals and is part of the corticotropin-releasing hormone (CRH; also referred to as corticotropin releasing factor) family. UCN2 also has been associated with a reduction in blood pressure. See, Mackay et al. (2003) Euro. ./. Pharmacol. 469: 111-115 (2003).

[0005] Additionally, a number of peptides derived from pre-proglucagon, and analogs thereof, have been proposed as therapeutic agents for treating T2D and obesity, including OXM. For example, pre-proglucagon is a 158-amino acid precursor polypeptide that is differentially processed in the tissues to form a number of structurally related proglucagon- derived peptides, one of which is OXM. Some information about OXM can be found in US Patent Application Publication No. 2016/0368960. [0006] OXM is released along with glucagon -like-peptide- 1 (GLP-l) from the L-cells of the small intestine in proportion to nutrient ingestion. OXM activates both the glucagon (Gcg) and GLP-l receptors, with a slightly higher potency for the Gcg receptor over the GLP-l receptor. It is less potent than native Gcg and GLP-l on their respective receptors. Human Gcg also can activate both receptors, albeit with a strong preference for the Gcg receptor over the GLP-l receptor. GLP-l, however, is not capable of activating Gcg receptors.

[0007] OXM is involved in regulation of food intake and body weight and has been shown to suppress appetite and inhibit food intake in humans. In a 4-week study with overweight and obese subjects, three times daily preprandial subcutaneous administration of OXM produced a weight loss of 2.3 kg when compared to 0.5 kg in the placebo group. In this trial, nausea, the most common side-effect associated with GLP-l based therapy (such as exenatide and liraglutide), was less frequent. In another shorter study, OXM was shown to decrease caloric intake and to increase activity-related energy expenditure in overweight and obese subjects.

[0008] These studies suggest that OXM has a potential of being a well-tolerated anti diabetes/obesity agent. OXM, however, presents several challenges for development into a commercially-viable therapeutic agent. For one, endogenous OXM is quickly degraded in vivo by dipeptidyl peptidase IV (DPP4) and other peptidases, as well as being subject to rapid renal clearance due to its small size. It is therefore desirable to identify peptides that activate the Gcg and GLP-l receptors with improved metabolic stability and reduced rate of clearance.

[0009] OXM peptides with amino acid substitutions to improve stability and with additional modifications to slow clearance, such as PEGylation or lipidation, are known. Other peptides have been stated to bind to and activate both the Gcg receptor and the GLP- 1 receptor and to suppress body weight gain (see, e.g. , Int’l Patent Application Publication Nos. WO 2011/075393 and WO 2012/177444).

[0010] To date, both CKD and T2D remain significant health concerns, and clinicians are looking for alternative therapies treat them. Accordingly, this disclosure provides a new therapy for CKD and T2D, which includes a combination therapy allowing an individual in need thereof to receive benefits not only of UCN2 but also of an OXM analog. Such a combination of a UCN2 compound with an OXM analog is desired to provide treatment for CKD and T2D, which may be more effective than either therapeutic agent alone.

[0011] In view of the above, this disclosure describes a combination therapy that may be useful in treating diabetes or CKD. The combination therapy includes administering to an individual in need of such treatment an effective amount of a UCN2 analog according to Formula I (or a pharmaceutically acceptable salt of Formula I) as well as administering to the individual an effective amount of an OXM analog according to Formula II (or a pharmaceutically acceptable salt of Formula II).

[0012] In some instances, Formula I {i.e., the UCN2 analog) is as follows:

IVX_bbSLDVPIGLLQILX_ccEQEKQEKEKQQAKTNAX_ddlLAQV,

where I at position 1 is chemically modified by either acetylation or methylation at the N-terminus,

where X_bb is L or T,

where X_cc is L or I,

where X_dd is Q or E, and

where K at position 29 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy] -acetyl )₂-(yE)₂-CO- (CH₂)_Z-C0₂H and _z is 16 or 18 (SEQ ID NO:8).

[0013] In certain instances, pharmaceutically acceptable salts of Formula I may be used.

[0014] In some instances, as in SEQ ID NO: 8, the terminal V optionally may be amidated as a C-terminal primary amide.

[0015] In some instances, Formula II (i.e., the OXM analog) is as follows:

HX₂QGTFTSDYSKYLDEKKAK^*EFVEWLLX_IGGPSSG,

where Xi is S or E,

where X₂ is Aib,

where K^* at position 20 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(YGlu)_m- C0(CH₂)_n-C0₂H and _m is 1 or 2 and _n is 16 or 18, and the C-terminal amino acid optionally is amidated as a C-terminal primary amide (SEQ ID NO: 13).

[0016] In certain instances, pharmaceutically acceptable salts of Formula II may be used.

[0017] In particular instances, I at position 1 is modified by methylation at the N- terminus, X_bb is L, X_cc is L, X_dd is Q, and _z is 18 in Formula I (such that Formula I takes the form of SEQ ID NO:4). This instance of Formula I can be combined with any of the specific compounds that fit into Formula II that are exemplified in, for example, SEQ ID NO: 13. Alternatively, when Formula I takes the form of SEQ ID NO:4, it can be used in combination with any of the specific compounds of Formula II that are exemplified in SEQ ID NOS:9-l2. In either instance, pharmaceutically acceptable salts of Formula I and/or Formula II may be used.

[0018] In other particular instances, I at position 1 is modified by methylation at the N- terminus, X_bb is T, X_cc is I, X_dd is E, and _z is 18 in Formula I (such that Formula I takes the form of SEQ ID NO:7). This instance of Formula I can be combined with any of the specific compounds that fit into Formula II that are exemplified in, for example, SEQ ID NO: 13. Alternatively, when Formula I takes the form of SEQ ID NO: 7, it can be used in combination with any of the specific compounds of Formula II that are exemplified in SEQ ID NOS:9-l2. In these instances, pharmaceutically acceptable salts of Formula I and/or Formula II may be used.

[0019] In certain instances, Xi is E, _m is 1 and _n is 16 or 18 in Formula II (such that Formula II takes the form of SEQ ID NO:9 or SEQ ID NO: 10). These instances of Formula II may be combined with any of the specific UCN2 analgos that fit into Formula I that are exemplified in, for example, SEQ ID NOS:4 and 7. Specifically, the OXM analogs of SEQ ID NO:9 or SEQ ID NO: 10 can be combined with the UCN2 analogs of SEQ ID NO:4 or SEQ ID NO:7. In these instances, pharmaceutically acceptable salts of Formula I and/or Formula II may be used.

[0020] In other particular instances, Xi is S, _m is 1 or 2 and _n is 16 or 18 in Formula II (such that Formula II takes the form of SEQ ID NO: 11 or SEQ ID NO: 12). These instances of Formula II may be combined with any of the specific UCN2 analogs that fit into Formula I that are exemplified in, for example, SEQ ID NOS:4 and 7. Specifically, the OXM analogs of SEQ ID NO: 1 1 or SEQ ID NO: 12 can be combined with the UCN2 analogs of SEQ ID NO:4 or SEQ ID NO:7. In these instances, pharmaceutically acceptable salts of Formula I and/or Formula II may be used.

[0021] Also provided herein is a use of the combination therapy including an effective amount of a UCN2 analog according to Formula I or a pharmaceutically acceptable salt of Formula I and an effective amount of an OXM analog according to Formula II or a pharmaceutically acceptable salt of Formula II for treating CKD and/or diabetes. In some instances, the combination therapy is used to treat diabetes, especially T2D. Such a combination therapy may be combined with diet and exercise. In other instances, the combination therapy is used to treat CKD that may be caused by diabetic nephropathy. In these uses, the combination therapy and methods involve administering a UCN2 analog of Formula I and administering an OXM analog of Formula II or pharmaceutically acceptable salts thereof, which may be accomplished by subcutaneously administering one or both analogs.

[0022] Also provided herein is a method of treating CKD or diabetes (including T2D), where the method includes at least a step of administering to an individual in need thereof, an effective amount of a UCN2 analog of Formula I or pharmaceutically acceptable salts thereof including, for example, analogs of SEQ ID NO:4 or SEQ ID NO:7 in combination with an effective amount of an OXM analog of Formula II or pharmaceutically acceptable salts thereof. In some instances, the methods also can include administering an effective amount of one or more additional therapeutic agents.

[0023] Also provided herein is a pharmaceutical composition including (i) an effective amount of a UCN2 analog according to Formula I or a pharmaceutically acceptable salt thereof, and (ii) an effective amount of an OXM analog according to Formula II or a pharmaceutically acceptable salt thereof.

[0024] In some instances, the pharmaceutical composition also can include (iii) a pharmaceutically acceptable carrier, diluent or excipient and/or (iv) an additional therapeutic agent.

[0025] Also provided herein is a kit for treating CKD or diabetes (including T2D) including (i) an effective amount of a UCN2 analog according to Formula I or a pharmaceutically acceptable salt thereof, and (ii) an effective amount of an OXM analog according to Formula II or a pharmaceutically acceptable salt thereof.

[0026] Also provided herein is a UCN2 analog of Formula I or a pharmaceutically acceptable salt thereof for use in simultaneous, separate or sequential combination with an OXM analog of Formula II or a pharmaceutically acceptable salt thereof for use in treating CKD or diabetes (including T2D).

[0027] Also provided herein is a method of treating CKD or diabetes (including T2D), where the method includes at least a step of administering to an individual in need thereof an effective amount of a UCN2 analog of Formula I or a pharmaceutically acceptable salt thereof and an effective amount of an OXM analog of Formula II or a pharmaceutically acceptable salt thereof.

[0028] Also provided herein is a method of treating CKD, where the method includes at least a step of administering to an individual in need of such treatment an effective amount of a UCN2 analog of Formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an OXM analog of Formula II, or a pharmaceutically acceptable salt thereof. In some instances, the analogs of Formula I and Formula II may be subcutaneously administered.

[0029] Also provided herein is a method of treating T2D, where the method includes at least a step of administering to an individual in need thereof an effective amount of a UCN2 analog of Formula I or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an OXM analog of Formula II or a pharmaceutically acceptable salt thereof. Such a method may be combined with diet and exercise and/or may be combined with additional therapeutic agents. In some instances, the analogs of Formula I and Formula II may be subcutaneously administered.

[0030] Also provided herein is a UCN2 analog of Formula I or a pharmaceutically acceptable salt thereof for use in simultaneous, separate or sequential combination with an OXM analog of Formula II or a pharmaceutically acceptable salt thereof for treating CKD or diabetes (including T2D) and/or for preventing progression of diabetes into CKD.

[0031] Also provided herein is a UCN2 analog of SEQ ID NO:4 or SEQ ID NO:7 or a pharmaceutically acceptable salt thereof for use in simultaneous, separate or sequential combination with an OXM analog of Formula II or a pharmaceutically acceptable salt thereof for treating CKD or diabetes (including T2D).

[0032] Also provided herein is use of a UCN2 analog of Formula I or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating CKD or diabetes (including T2D), where the medicament can be administered simultaneously, separately or sequentially with an OXM analog of Formula II or a pharmaceutically acceptable salt thereof. In some instances, the UCN2 analog of Formula I is SEQ ID NO:4 or SEQ ID NO: 7 or a pharmaceutically acceptable salt thereof.

[0033] Also provided herein is an effective amount of a UCN2 analog of Formula I or a pharmaceutically acceptable salt thereof in combination with an effective amount of a OXM analog of Formula II or a pharmaceutically acceptable salt thereof for use in therapy, in particular, for treating CKD or diabetes (including T2D) and/or for preventing progression of diabetes into CKD.

[0034] It is contemplated that treating CKD and/or treating diabetes includes preventing progression of diabetes into CKD.

[0035] For purposes of clarity, the OXM analogs of Formula II may be made according to the methods disclosed in US Patent Application Publication No. 2016/0368960. In some instances, the OXM analogs of Formula II may have a C-terminal amino acid amidated as a C-terminal primary amide and/or may be made into a pharmaceutically acceptable salt. As noted above, and in some instances, the OXM analog of Formula II can have the following structure:

HAibQGTFTSD Y SKYLDEKKAK^*EF VEWLLEGGPS SG (SEQ ID NO: 10), where K^* at position 20 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(YGlu)_m- C0(CH₂)_n-C0₂H and _m is 1 or 2 and _n is 16 or 18; and the C-terminal amino acid may be amidated as a C-terminal primary amide.

[0036] In other instances, the OXM analog of Formula II can have the following structure:

H Aib Q GTF T SD Y SK YLDEKK AK^*EF VEWLL S GGP S SG (SEQ ID NO: 12), where K^* at position 20 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(YGlu)_m- C0(CH₂)_n-C0₂H and _m is 1 or 2 and _n is 18; and the C-terminal amino acid may be amidated as a C-terminal primary amide.

[0037] Exemplary OXM analogs are disclosed as Examples 1-4 of US Patent Application Publication No. 2016/0368960 and may be made in accordance with the teachings of that document and are disclosed as SEQ ID NOS:9-l2 herein.

[0038] More generally, some instances include a UCN2 analog of Formula I co formulated with a OXM analog of Formula II. Also, more generally, the UCN2 analgo of Formula I is administered simultaneously, separately or sequentially with the OXM analog of Formula II.

[0039] These and other advantages, effects, features and objects of this disclosure will become better understood from the detailed description of exemplary embodiments that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, details of the exemplary embodiments.

[0040] The analogs of Formula I can be synthesized using standard manual or automated solid-phase synthesis procedures. Automated peptide synthesizers are commercially available from, for example, Applied Biosystems (Foster City, CA) and Protein Technologies Inc. (Tucson, AZ). Reagents for solid-phase synthesis are readily available from commercial sources. Solid-phase synthesizers can be used according to the manufacturer’s instructions for blocking interfering groups, protecting amino acids during reaction, coupling, deprotecting, and capping of unreacted amino acids.

[0041] Typically, an N-a-carbamoyl protected amino acid and the N-terminal amino acid on the growing peptide chain attached to a resin are coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidone or methylene chloride in the presence of coupling agents such as diisopropyl -carbodiimide and l-hydroxybenzotri azole. The Na-carbamoyl protecting group is removed from the resulting peptide resin using a reagent such as trifluoroacetic acid (TFA) or piperidine, and the coupling reaction is repeated with the next desired Na- protected amino acid to be added to the peptide chain. Suitable amine protecting groups are well known in the art and are described in, for example, Green & Wuts,“Protecting Groups in Organic Synthesis,” (John Wiley & Sons, 1991). The most commonly used examples include tBoc and fluorenylmethoxycarbonyl (Fmoc). After completion of synthesis, peptides are cleaved from the solid-phase support with simultaneous side chain deprotection using standard treatment methods under acidic conditions.

[0042] One of skill in the art will appreciate that the peptide chain of the compounds of Formula I can be synthesized with a C-terminal carboxamide. For synthesizing C-terminal amide peptides, resins incorporating Rink amide MBHA or Rink amide AM linkers can be used with Fmoc synthesis, while MBHA resin can be used with tBoc synthesis.

[0043] Crude peptides can be purified using RP-HPLC on C8 or C18 columns using water-acetonitrile gradients in about 0.05% to about 0.1% TFA. Peptide purity can be verified by analytical RP-HPLC, and peptide identity can be verified by mass spectrometry. Peptides can be solubilized in aqueous buffers over a wide pH range.

[0044] The compounds of Formula I and Formula II as described herein can be formulated as pharmaceutical compositions administered by any route that makes the compound bioavailable. The route of administration may be varied in any way, limited by the physical properties of the therapeutic compounds and the convenience of the individual and the caregiver. In some instances, the compounds of Formulas I and II are for parenteral administration, such as intravenous or subcutaneous administration. Other embodiments may be designed in which one or more of such compounds (or pharmaceutically acceptable salts), is for oral, parenteral or transdermal administration, including intravenous or subcutaneous administration. Such pharmaceutical compositions and processes for preparing same are well known in the art. (See, e.g., Remington: The Science and Practice of Pharmacy (Troy, ed., 2l^st Ed., Lippincott, Williams & Wilkins, 2006).

[0045] The analogs of Formula I utilize a fatty acid of the formula C0-(CH₂)_Z-C0₂H, where _z is 16 or 18 that is chemically conjugated to an epsilon-amino group of a lysine side chain either by a direct bond or by a linker. These acids are“diacids” in that they have a carboxyl group on each end, where one end of the carboxyl is attached via an amide bond to a gE residue.

[0046] Moreover, the fatty acid suitable for use herein can be saturated or unsaturated. Examples of specific saturated fatty acids that are suitable for the compounds and uses thereof disclosed herein include octadecanedioic acid (Ci₈ diacid) or eicosanedioic acid (C20 diacid). In some instances, the fatty acid can be a saturated C ix diacid or a saturated C20 diacid as well as branched and substituted derivatives thereof.

[0047] The length and composition of the fatty acid impacts the half-life of the compound, the potency of the compound in in vivo animal models, and also impacts the solubility and stability of the compound. Conjugation of the ETCN2 analogs defined herein to a C18-C20 saturated fatty diacid results in compounds that exhibit desirable half-life, desirable potency in in vivo animal models, and also possess desired solubility and stability characteristics.

[0048] The analogs of Formula I and Formula II may react with any of a number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. See , e.g, Stahl el al. Handbook of Pharmaceutical Salts: Properties, Selection and ETse, 2^nd Revised Edition (Wiley-VCH, 2011); Berge et al. (1977) ./. Pharma Sci. 66: 1-19. Exemplary pharmaceutically acceptable salt for use herein include trifluoroacetate salts, acetate salts and hydrochloride salts among others. [0049] The analogs of Formula I or Formula II may be administered by a clinician physician or self-administered using an injection. It is understood that the gauge size and amount of injection volume can be readily determined by one of skill in the art. In some instances, the amount of injection volume is < about 2 ml, especially < about 1 ml. In some instances, the needle gauge is > about 27 G, especially > about 29 G.

[0050] The analogs of Formula I or Formula II (or pharmaceutically acceptable salts thereof) are generally effective over a wide dosage range. For example, dosages per day normally fall within a range of about 0.01 to about 50 mg/kg of body weight. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed with acceptable side effects, and therefore the above dosage range is not intended to limit the scope of the compositions and methods.

[0051] The disclosure also encompasses processes useful for synthesizing analogs of Formula I or Formula II, or a pharmaceutically acceptable salt thereof. The intermediates and analogs herein may be prepared by a variety of processes known in the art including via both chemical synthesis and recombinant technology. For example, the process using chemical synthesis is illustrated in the Examples below. The specific synthetic steps for each of the routes described may be combined in different ways to prepare compounds of Formula I or salts thereof. The reagents and starting materials are readily available to one of skill in the art. It is understood that the Examples are not intended to be limiting to the scope of the compositions and methods in any way.

[0052] As used herein,“about” means within a statistically meaningful range of a value or values such as, for example, a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature, volume, etc. Such a value or range can be within an order of magnitude typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by“about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.

[0053] As used herein,“Aib” means alpha amino isobutyric acid.

[0054] As used herein,“ AETC” means area under the curve. [0055] As used herein,“average molecular weight” means an average of the molecular weight of the different oligomer size components with a very narrow distribution and is determined by mass spectrometry techniques.

[0056] As used herein,“ECso” means a concentration of compound that results in 50% activation of the assay endpoint, e.g., cAMP.

[0057] As used herein, the term“ED50” means a concentration of compound that results in a 50% response in the in vivo assay endpoint, for example, plasma or blood glucose.

[0058] As used herein,“effective amount” means an amount or dose of the compound of Formula I, or a pharmaceutically acceptable salt thereof, and to an amount or dose of a compound of Formula II administered to the patient, that provides the desired effect in the patient under diagnosis or treatment. It is understood that the combination therapy of the present invention is carried out by administering a compound of Formula I, or a pharmaceutically acceptable salt thereof, together with the compound of Formula II (or a pharmaceutically acceptable salt thereof) in any manner which provides effective levels of the compound of Formula I (or a pharmaceutically acceptable salt thereof) and the compound of Formula II (or a pharmaceutically acceptable salt) in the body.

[0059] An effective amount can be readily determined by the attending clinician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for an individual, a number of factors are considered by the attending clinician, including, but not limited to the size, age and general health of the individual; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual; the particular compound administered; the mode/route of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0060] As used herein,“fatty acid” means a carboxylic acid with either 18 or 20 total carbon atoms.

[0061] As used herein,“in combination with” or“in combination” means administration of the compound of Formula I (or pharmaceutically acceptable salt), with a compound of Formula II (or pharmaceutically acceptable salt), simultaneously, or sequentially in any order, or any combination thereof. The two compounds may be administered either as part of the same pharmaceutical composition or in separate pharmaceutical compositions. The compound of Formula I (or pharmaceutically acceptable salt), can be administered prior to, at the same time as, or subsequent to administration of the compound of Formula II (or pharmaceutically acceptable salt),, or in some combination thereof. Where the compound of Formula I (or pharmaceutically acceptable salt), is administered at repeated intervals ( e.g ., during a standard course of treatment), the compound of Formula II (or pharmaceutically acceptable salt), can be administered prior to, at the same time as, or subsequent to, each administration of the compound of Formula I, or some combination thereof, or at different intervals in relation to therapy with the compound of Formula I, or in a single or series of dose(s) prior to, at any time during, or subsequent to the course of treatment with the compound of Formula I. Likewise, where the compound of Formula II (or pharmaceutically acceptable salt), is administered at repeated intervals (e.g., during a standard course of treatment), the compound of Formula I (or pharmaceutically acceptable salt), can be administered prior to, at the same time as, or subsequent to, each administration of the compound of Formula II, or some combination thereof, or at different intervals in relation to therapy with the compound of Formula II, or in a single or series of dose(s) prior to, at any time during, or subsequent to the course of treatment with the compound of Formula II. The compounds of Formula I or Formula II or pharmaceutically salts thereof are particularly useful in the methods of treatment, but certain modifications are preferred for such compounds. It will be understood that these preferences are applicable both to the methods of treatment and to the compounds described herein.

[0062] As used herein,“individual” means a mammal, such as a mouse, guinea pig, rat, dog, cat, or primate, especially a human.

[0063] As used herein,“saturated” means a fatty acid contains no carbon-carbon double or triple bonds.

[0064] As used herein, the term“treating” or“to treat” includes prohibiting, restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.

[0065] Certain abbreviations are defined as follows: “ACR” refers to urine albumin/urine creatinine ratio;“amu” refers to atomic mass unit;“Boc” refers to tert- butoxycarbonyl;“cAMP” refers to cyclic adenosine monophosphate;“DMSO” refers to dimethyl sulfoxide;“EIA/RIA” refers to enzyme immunoassay/radioimmunoassay;“hr” refers to hour;“HTRF” refers to homogenous time-resolved fluorescent;“i.v” refers to intravenous;“kDa” refers to kilodaltons;“LC-MS” refers to liquid chromatography-mass spectrometry;“MS” refers to mass spectrometry;“OtBu” refers to O-tert-butyl;“Pbf” refers to N -2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;“RP-HPLC” refers to reversed-phase high performance liquid chromatography;“s.c.” refers to subcutaneous; “SEM” refers to standard error of the mean;“TFA” refers to trifluoroacetic acid; and“Trt” refers to Trityl. Standard one- letter codes are used to represent the amino acid in the compounds of Formula I. All amino acids used in the Formula I and Formula II are L- amino acids. Standard three-letter codes may also be used to represent amino acids.

[0066] The following Examples provide compounds that may be used as the compound of Formula I.

[0067] EXAMPLE 1 :

[0068] IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi,

where I at position 1 is modified at the N-terminus by acetylation, X_bb is L, X_cc is L, X_dd is Q, and K at position 29 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂-CO- (CH₂)i₆-C0₂H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 1). The structure of this sequence is shown below.

[0069] The structure of this sequence contains the standard single letter amino acid code with exception of residues I at position 1 and K at position 29, where the structures of these amino acid residues have been expanded.

[0070] The peptide according to SEQ ID NO: l is generated by solid-phase peptide synthesis using a Fmoc/t-Bu strategy carried out on a Symphony Automated Peptide Synthesizer (PTI Protein Technologies Inc.; Tucson, AZ) starting from RAPP AM -Rink Amide Resin (H40023 Polystyrene AM RAM, Rapp Polymere GmbH) and with couplings using 6 equivalents of amino acid activated with diisopropylcarbodiimide (DIC) and Oxyma pure (1 : 1 : 1 molar ratio) in dimethylformamide (DMF) for 3 h at 25°C.

[0071] Extended coupling for Thr30 (10 h) is necessary to improve the quality of the crude peptide. A Fmoc-Lys(Alloc)-OH building block is used for K at position 29 coupling (orthogonal protecting group) to allow for site specific attachment of the fatty acid moiety later on in the synthetic process. The N-terminal residue (I at position 1) is acetylated using 10 equivalents of acetic acid with diisopropylcarbodiimide (DIC) and Oxyma pure (1 : 1 : 1 molar ratio) in dimethylformamide (DMF) for 1 h at 25°C.

[0072] After finishing the elongation of the peptide-resin described above, the Alloc protecting group present in the K at position 29 is removed using catalytic amounts of Pd(PPh₃)₄ in the presence of PhSiHi as a scavenger. Additional coupling/deprotection cycles is conducted using a Fmoc/t-Bu strategy to extend the K at position 29 side chain involved Fmoc-NH-PEG2-CH₂COOH (ChemPep Catalog#280102), Fmoc-Glu(OH)-OtBu (ChemPep Catalog# 100703) and HOOC-(CH₂)i₆-COOtBu. In all couplings, 3 equivalents of the building block are used with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 4h at 25°C.

[0073] Concomitant cleavage from the resin and side chain protecting group removal are carried out in a solution containing trifluoroacetic acid (TFA): triisopropylsilane : 1,2- ethanedithiol : methanol : thioanisole 80:5:5:5:5 (v/v) for 2 h at 25°C followed by precipitation with cold ether. Crude peptide is purified to > 99% purity (15-20% purified yield) by reversed-phase HPLC chromatography with water / acetonitrile (containing 0.1% v/v TFA) gradient on a Phenyl hexyl column (phenomenex, 5 micron, 100A), where suitable fractions are pooled and lyophilized.

[0074] In a synthesis performed essentially as described above, the purity of Example 1 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3H73 = 1718.8; calculated: M+3H73 = 1720.0; observed: M+4H74 = 1289.2; calculated: M+4H74 = 1290.3; observed: M+5H75 = 1031.5; calculated: M+5H75 = 1032.4).

[0075] EXAMPLE 2:

[0076] IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi,

where I at position 1 is modified at the N-terminus by acetylation, X_bb is L, X_cc is L, X_dd is Q, and K at position 29 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(YE)₂-CO- (CH₂)i₈-C0₂H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:2). The structure of this sequence is shown below.

[0077] The structure of this sequence contains the standard single letter amino acid code with exception of residues I at position 1 and K at position 29 where the structures of these amino acid residues have been expanded.

[0078] The peptide according to SEQ ID NO:2 is synthesized similarly as described above in Example 1. HOOC-(CH2)i8-COOtBu is incorporated using 3 equivalents of the building block with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 4 h at 25°C.

[0079] In a synthesis performed essentially as described above, the purity of Example 2 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3H73 = 1728.2; calculated: M+3H73 = 1729.4; observed: M+4H74 = 1296.3; calculated: M+4H74 = 1297.3; observed: M+5H75 = 1037.4; calculated: M+5H75 = 1038.0).

[0080] EXAMPLE 3:

[0081 ] IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi,

where I at position 1 is modified at the N-terminus by methylation, X_bb is L, X_cc is L, X_dd is Q, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂- C0-(CH₂)i₆-C0₂H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 3). The structure of this sequence is shown below.

[0082] The structure of this sequence contains the standard single letter amino acid code with exception of residues N-methyl isoleucine at position 1 and K at position 29, where the structures of these amino acid residues have been expanded.

[0083] The compound according to SEQ ID NO:3 is synthesized similarly as described above for Example 1. The N-terminal residue (N-methyl isoleucine at position 1) is incorporated as Boc-NMelle-OH using 6 equivalents of the building block with PyBOP (6 equiv) and DIEA (12 equiv) in DMF-DCM (1 : 1, v/v) for 15 h at 25°C. [0084] In a synthesis performed essentially as described above, the purity of Example 3 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3H73 = 1709.6; calculated: M+3H73 = 1710.7; observed: M+4H74 = 1282.2; calculated: M+4H74 = 1283.3; observed: M+5H75 = 1025.8; calculated: M+5H75 = 1026.8).

[0085] EXAMPLE 4:

[0086] IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi,

[0087] where I at position 1 is modified at the N-terminus by methylation, X_bb is L, X_cc is L, X_dd is Q, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂- C0-(CH₂)i₈-C0₂H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:4). The structure of this sequence is shown below.

[0088] The structure of this sequence contains the standard single letter amino acid code with exception of residues N-methyl isoleucine at position 1 and K at position 29, where the structures of these amino acid residues have been expanded.

[0089] The compound according to SEQ ID NO:4 is synthesized similarly as described above for Example 1. The N-terminal residue (N-methyl isoleucine at position 1) is incorporated as Boc-NMelle-OH using 6 equivalents of the building block with PyBOP (6 equiv) and DIEA (12 equiv) in DMF-DCM (1 : 1, v/v) for 15 h at 25°C. HOOC-(CH₂)i₈- COOtBu is incorporated using 3 equivalents of the building block with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 4 h at 25°C.

[0090] In a synthesis performed essentially as described above, the purity of Example 4 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3H73 = 1719.4; calculated: M+3H73 = 1720.1; observed: M+4H74 = 1289.8; calculated: M+4H74 = 1290.3; observed: M+5H75 = 1031.8; calculated: M+5H75 = 1032.4).

[0091] EXAMPLE 5:

[0092] IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi,

where I at position 1 is modified at the N-terminus by methylation, X_bb is T, X_cc is L, X_dd is E, and K at position 29 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(YE)₂-CO- (CH₂)i₈-C0₂H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:5). The structure of this sequence is shown below.

[0093] The structure of this sequence contains the standard single letter amino acid code with exception of residues N-methyl isoleucine at position 1, and K at position 29 where the structures of these amino acid residues have been expanded. [0094] The compound according to SEQ ID NO:5 is synthesized similarly as described above for Example 4.

[0095] In a synthesis performed essentially as described above, the purity of Example 5 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3H73 = 1715.7; calculated: M+3H73 = 1716.4; observed: M+4H74 = 1287.0; calculated: M+4H74 = 1287.5; observed: M+5H75 = 1029.7; calculated: M+5H75 = 1030.2).

[0096] EXAMPLE 6:

[0097] IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi,

where I at position 1 is modified at the N-terminus by methylation, X_bb is L, X_cc is L, X_dd is E, and K at position 29 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(YE)₂-CO- (CH₂)i₈-C0₂H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:6). The structure of this sequence is shown below.

[0098] The structure this compound contains the standard single letter amino acid code with exception of residues N-methyl isoleucine at position 1 and K at position 29 where the structures of these amino acid residues have been expanded. [0099] The compound according to SEQ ID NO:6 is synthesized similarly as described above for Example 4.

[0100] In a synthesis performed essentially as described above, the purity of Example 6 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3H⁺/3 = 1719.7; calculated: M+3H⁺/3 = 1720.4; observed: M+4H⁺/4 = 1289.8; calculated: M+4H⁺/4 = 1290.5; observed: M+5H⁺/5 = 1032.2; calculated: M+5H⁺/5 = 1032.6).

[0101] EXAMPLE 7:

[0102] IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi,

where I at position 1 is modified at the N-terminus by methylation, X_bb is T, X_cc is I, X_dd is E, and K at position 29 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(YE)₂-CO- (CH₂)i₈-C0₂H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:7). The structure of this sequence is shown below.

[0103] The structure of this sequence contains the standard single letter amino acid code with exception of residues N-methyl isoleucine at position 1 and K at position 29, where the structures of these amino acid residues have been expanded. [0104] The compound according to SEQ ID NO:7 is synthesized similarly as described above for Example 4.

[0105] In a synthesis performed essentially as described above, the purity of Example 7 is examined by analytical reversed-phase HPLC, and identity is confirmed using LC/MS (observed: M+3EE/3 = 1715.6; calculated: M+3EE/3 = 1716.4; observed: M+4EE/4 = 1286.8; calculated: M+4H⁺/4 = 1287.5; observed: M+5H⁺/5 = 1029.8; calculated M+5H⁺/5 = 1030.2).

[0106] It should be noted that, in addition to the methods of preparing the compounds described above, a convergent synthesis also may be used. For example, in this convergent synthesis, an acylated K side chain is constructed and/or obtained. This acylated K side chain fragment may have the acid fragments protected orthogonally as t-butyl esters or other protecting groups commonly known in peptide synthesis. It is believed that such a method of synthesis may produce the acylated side chain in high purity, >98%, which may reduce the downstream chromatography requirements, potentially leading to improved purity and increased process efficiency. For example, in an all linear build, the acylated K component (z.e., the fatty acid side chain having the amino-ethoxy moeities, etc.) typically is installed at the end of the synthesis, and this can create high levels of process impurities such as, but not limited to impurities have greater or fewer numbers of amino-ethoxy moieties which can be problematic to remove. ETsing the convergent synthesis approach (outlined herein) may de-risk an all linear synthetic build strategy, where a single mistake can result in a total loss. In addition, using a convergent synthesis approach may improve supply chain flexibility with comparable resourcing requirements to a standard all linear build. Moreover, a convergent synthesis approach also may be a means of lowering COPS (cost of product sold) and further improving robustness. Another benefit may be that the N-terminus N-methyl isoleucine residue frequently is a difficult coupling for a large peptide. Incorporation of N-methyl isoleucine onto a smaller fragment may be potentially a good means of de-risking this coupling issue.

[0107] Eising the compound of Example 4 as an example, the acylated K side chain is close to the C-terminus, a strategic retrosynthetic break for a convergent synthesis process may be between the A at position 28 and the K at position 29. Thus, this“fragment” will include the K at position 29 (and its accompanying side chain) along with the final 9 residues (leading up to the C-terminus). In some embodiments, this“fragment” may be the primary parent fragment produced on Rink Amide or Sieber Amide resin. Another retrosynthetic disconnection may be between G at position 10 and the L at position 11. Making a fragment of these sequences may ensure that such a sequence has no (or a lower) propensity for racemization. The third fragment of 18 amino acids ( e.g ., from the residue at position 11 to the A at position 28) also could be produced. This 18 residue fragment, along with the initial 10 amino acid fragment (e.g., the N-terminus to the G at position 10) both could be produced, for example, by a 2-CTC resin. The 2-CTC resin may be preferred for synthesis of most fragments as the resin can be orthogonally cleaved while leaving peptide protecting groups intact.

[0108] Thus, in summary, the following synthesis method for the compound of Example 4 is provided below:

1. Construct the fatty acid side chain that is connected to a K (e.g. , the K that will ultimately be K at position 29);

2. Construct a lO-amino acid fragment starting with the K with the fatty acid side chain (e.g, the K that will ultimately be K at position 29) and add the other amino acids to ending in the C-terminus after the final V;

3. Construct the 18-amino acid fragment, starting with the L at position 11 and ending with the A at position 28;

4. Construct the lO-amino acid fragment starting with the modified I at position 1 and ending with the G at position 10; and

5. The 18-amino acid fragment of step 3 could be coupled to the lO-amino acid fragment of step 4, and then this 28-residue construct could be coupled to the fragment of step 2 (having the side chain); alternatively the 18-amino acid fragment of step 3 could be coupled to the added to 10-amino acid fragment of step 2, and then this 28-residue construct could be coupled to the fragment of step 4.

[0109] Again, one of the benefits of using this“fragment” -based construction technique is that each fragment could be produced sequentially or simultaneously. Further, the smaller fragments of the peptides may be easier to purify and sometimes can be isolated in crystalline form which imparts high purity. Likewise, if an error is made in one of the fragment, only that fragment has to be discarded and re-created (rather than having to re create the entire sequence). Other strategic fragment breaks are posssible to further improve purity and efficiency such as but not limited to fragment condensation to produce the 18-amino acid residue.

[0110] In some embodiments, lyophilization may be incorporated as the strategy as a means of potentially de-risking potential physical property issues of the compound. Specifically, the compound may be constructed by in which it is purified via chromatography. Once purified, the solution may be concentrated and then isolated as a solid ( e.g ., dry powder) via lyophilization. In other instances, a solid may be obtained and isolated using a precipitation/filtration/drying/humidification procedure.

[0111] Lyophilization is the most commonly practiced (>80%) industrial means for production of solid peptide drug products for storage or reconstitution. In some instances, the primary drawback to precipitation is the extensive material and design space development necessary to assure a robust process. Precipitated compounds also may contain high density particles that tend to agglomerate, and frequently these precipated products may slowly dissolve with standard dissolution assays and/or drug product formulations. On the other hand, high surface area product produced by lyophilization may assure maximized dissolution rates in dissolution assays and/or pharmaceutical formulations. However, precipitation products also may be used, as this method tends to be less expensive for high volume products.

[0112] EXAMPLE 8:

[0113] ASSAYS

[0114] Provided below are the conditions and data for some of the above-recited Examples in several assays.

[0115] In Vitro Function and Selectivity:

[0116] CRHR agonistic activity is measured in a cell-based cAMP assay. Serial dilutions of the test peptides are made in assay buffer containing Hank’s Balanced Salt Solution (HBSS, without phenol red) supplemented with 20 mM HEPES and 0.05% lactalbumin enzymatic hydrolysate (LAH) (“assay buffer”). The highest concentration that is used starts from 1 mM in the human CRHR2b, whereas 100 pM starting concentration is used in the human CRHR1 assay. A one to three dilution of the test peptides is used in both assays.

[0117] A receptor over-expressing Chinese Hamster Ovary (CHO) cell line is used for the human CRHR2b assay. CHO cells are grown in DMEM supplemented with 10% fetal bovine serum at 37°C under suspension conditions and transiently transfected with cDNA constructs of human CRHR2b (Genbank Accession No.: AF011406.1). Forty-eight hours after the transfection, the cells are centrifuged to remove the culture media and re- suspended in fetal bovine serum containing 5% DMSO. They are cryofrozen and stored in vials in liquid nitrogen (20 x 10⁶ cells/ml/vial). On the day of the assay, cells are thawed and re-suspended in cold 30ml culture media supplemented with 20mM HEPES. The cells are then centrifuged to remove the media and washed once with HBSS supplemented with 20mM HEPES. Finally, following the last centrifugation, the cells are resuspended in assay buffer. Thirty thousand cells are used in the human CRHR2b assay for each treatment.

[0118] A human retinoblastoma cell line Y79 (ATCC #HTB- 18), which expresses endogenous human CRHR1, is used in the human CRHR1 assay. The cells are grown in RPMI 1640 (Hyclone, #SH30255) containing 20% fetal bovine serum and lOmM HEPES, in suspension culture. Cells are centrifuged to remove the culture media and washed once in HBSS supplemented with 20 mM HEPES. The cells are re-suspended in the assay buffer and 20,000 cells are used per treatment in the human CRHR1 assay.

[0119] The cells are dispensed into Costar 96-well black polystyrene half area EIA/RIA plates (Corning Incorporated, Coming, NY) followed by the addition of the diluted peptides, each at a volume of 20 pL. The agonist induced cAMP levels are detected using a HTRF cAMP Dynamic 2 kit (CisBio, Bedford, MA). After incubation at 37°C for 30 min, the assay is stopped by cell lysis via the addition of 20 pL of d2 -labeled cAMP and followed by 20 pL of cryptate-labeled anti-cAMP antibody, as described by the manufacturer. Cellular cAMP (as a result of agonist stimulation) competes with the d2- labeled cAMP for binding to the antibody. HTRF detection is performed on an Envision plate reader (Perkin Elmer Life and Analytical Sciences, Waltham, MA) by measuring ratiometric emission at 620 and 665 nm after excitation at 320 nm.

[0120] The data are converted to picomoles of cAMP using a standard curve obtained from the same assay performed with varying concentrations of unlabeled cAMP. Percent of the maximum activation of the cells is calculated using converted picomole cAMP data by comparing to the amount of cAMP produced by 1 pM human ETCN2 for the human CRHRZb or 1 pM human ETCN 1 for the human CRHR1 assay. The data are analyzed using a Curve Fitting Tool to calculate EC50. Numeric values shown below in Table 1 represent the mean of multiple runs (number of runs shown in parentheses) following the mean value

± SEM.

[0121] Table 1. In Vitro Activity for hCRHR2b and hCRHRl .

[0122] These data demonstrate that the compounds of Examples 1 to 7 have CRHR2 agonist activity in a cAMP assay. These data further demonstrate that the compounds of Examples 1 to 7 are selective for CRHR2, over CRHR1.

[0123] EXAMPLE 9:

[0124] COMBINATION STUDIES OF UCN2 ANALOG + OXM ANALOG

[0125] This example describes an effect of a long-acting UCN2 analog,“UCN2-X,” in combination with an OXM on metabolic parameters in DIO mice.

[0126] UCN2 analogs are proposed as a treatment not only for diabetes but also for metabolic syndrome, a collection of co-morbidities (dyslipidemia, obesity, hepatic steatosis, etc.) that are associated with insulin resistance and diabetes. In these studies, UCN2-X’ s mechanism of inducing body weight loss in combination with OXM compounds is investigated. “UCN2-X” refers to the compound of Example 7 above, and“OXM-Y” refers to the compound shown below.

[0127] The DIO model represents a pre-diabetic state that is more sensitive to insulin. These animals, although not diabetic, display insulin resistance, dyslipidemia, and hepatic steatosis, all characteristics of metabolic syndrome, after being placed on a high fat (60% Kcal from fat) diet for 12 weeks (Surwit et al. (1988) Diabetes 37: 1163-1167). [0128] The purpose of this example thereof is to assess the effects of a UCN2 compound of Formula I alone and in combination with an OXM-Y of Formula II on fasting glucose, fasting insulin, weight loss and body composition.

[0129] Here, C57/BL6 diet-induced obese (DIO) male mice (Taconic; Germantown, NY) weighing 41-50 g are individually housed in a temperature-controlled (24°C) facility with a 12 hour light/dark photoperiod (lights off at 10:00 AM and lights on at 10:00 PM), and have free access to food (TD95217) (Teklad; Indianapolis, IN) and water. After 2 week acclimatization to the facility, mice are randomized to treatment groups (n=6/group) based on body weight so each group has similar starting mean body weight. Animals are placed in PhenoMaster/LabMaster Calorimeter (TSE Systems; Chesterfield, MO) for 3-5 days of acclimatization Heat and respiratory Exchange Ratio (RER) are measured by indirect calorimetry as described using an open-circuit calorimetry system.

[0130] Groups:

1. Vehicle (20 mM Tris-HCl Buffer at pH 8.0, 10 ml/kg, s.c);

2. Oxyntomodulin (“OXM-Y”) at 10 nmol/kg, s.c., where OXM-Y has the following formula (that fits within the scope of Formula II):

H Aib Q GTF T SD Y SK YLDEKK AK^*EF VEWLLEGGP S S G,

where K^* at position 20 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-arnino-ethoxy)-ethoxy]-acetyl)2-(YGlu)_m- C0(CH₂)_n-C0₂H and _m is 1 and _n is 16, and the C-terminal amino acid is amidated as a C- terminal primary amide (SEQ ID NO: 10);

3. ETCN2-X (10 nmol/kg, s.c.); and

4. Combination (OXM-Y 10 nmol/kg, s.c. + ETCN2-X 10 nmol/kg, s.c.).

[0131] Treatments are subcutaneously administered to ad libitum fed DIO mice 30-90 minutes prior to the onset of the dark cycle every three days on Day 1, 4, 7, 10 and 13 for 15 days. During the course of the study, body weight and food intake is monitored daily, and body composition is assessed by quantitative nuclear magnetic resonance (QNMR) (EchoMRI LLC; Houston, TX) on Days -1 (one day prior to treatments) 8 and 15. At the end of the study, livers are harvested to measure liver triglyceride content.

[0132] Table 2 below shows data corresponding to each of the above measurements (two studies combined). All data are expressed as mean ± SEM of 5-6 mice per group. Area under the curve calculations of heat for 12 hour light versus dark photoperiod are made by using GraphPad Prism Software where baseline“y” values (heat) are started from“0”. Statistical analyses are assessed by two-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison test to compare treatment groups to vehicle group or each other. Significant differences are identified at p<0.05.

[0133] Table 2: Effect of Treatment With UCN2-X, OXM-Y or Combinations Thereof on Body Weight Change and Fat Mass Change.

[0134] Such data shows the combination achieves superior results than the agents alone.

[0135] SEP ID NO: 1

IVLSLDVPIGLLQILLEQEKQEKEKQQAKTNAQILAQV-NHi

wherein I at position 1 is modified at the N-terminus by acetylation, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(YE)₂-C0-(CH₂)i₆-C0₂H; and the C-terminal amino acid is amidated as a C-terminal primary amide.

[0136] SEP ID NO:2

IVLSLDVPIGLLQILLEQEKQEKEKQQAKTNAQILAQV-ME

where I at position 1 is modified at the N-terminus by acetylation, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂-CO-(CH₂)ix-C0₂H; _ancj the C-terminal amino acid is amidated as a C-terminal primary amide. [0137] SEP ID NO: 3

IVLSLDVPIGLLQILLEQEKQEKEKQQAKTNAQILAQV-NHi

where I at position 1 is modified at the N-terminus by methylation, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂-CO-(CH₂)i₆-C0₂H; _ancj the C-terminal amino acid is amidated as a C-terminal primary amide.

[0138] SEP ID NO:4

IVLSLDVPIGLLPILLEPEKPEKEKPPAKTNAPILAPV-NH2

where I at position 1 is modified at the N-terminus by methylation, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂-CO-(CH₂)ix-C0₂H; _ancj the C-terminal amino acid is amidated as a C-terminal primary amide.

[0139] SEP ID NO: 5

IVTSLDVPIGLLPILLEPEKPEKEKPPAKTNAEILAPV-NH2

where I at position 1 is modified at the N-terminus by methylation, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂-CO-(CH₂)ix-C0₂H; _ancj the C-terminal amino acid is amidated as a C-terminal primary amide.

[0140] SEP ID NO: 6

IVLSLDVPIGLLPILLEPEKPEKEKPPAKTNAEILAPV-NH2

where I at position 1 is modified at the N-terminus by methylation, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂-CO-(CH₂)ix-C0₂H; _ancj the C-terminal amino acid is amidated as a C-terminal primary amide.

[0141] SEP ID NO: 7

IVTSLDVPIGLLPILIEPEKPEKEKPPAKTNAEILAPV-NH2

where I at position 1 is modified at the N-terminus by methylation, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂-CO-(CH₂)ix-C0₂H; _ancj the C-terminal amino acid is amidated as a C-terminal primary amide.

[0142] SEP ID NO: 8

IVX_bbSLDVPIGLLQILX_ccEQEKQEKEKQQAKTNAX_ddlLAQV

where I at position 1 is modified by either acetylation or methylation at the N- terminus, X_bb is L or T, X_cc is L or I, X_dd is Q or E, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2- amino-ethoxy)-ethoxy]-acetyl)₂-(YE)₂-C0-(CH₂)_z-C0₂H and _z is 16 or 18, and the terminal V optionally may be amidated.

[0143] SEP ID NO: 9

HAibOGTFTSDYSKYLDEKKAK^*EFVEWLLEGGPSSG

where K^* at position 20 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(yGlu)_m- C0(CH₂)_n-C0₂H and _m is 1 and _n is 16 or 18; and the C-terminal amino acid may be amidated as a C-terminal primary amide.

[0144] SEP ID NO: 10

HAibOGTFTSD Y SKYLDEKKAK^*EF VEWLLEGGPS SG

where K^* at position 20 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(yGlu)_m- C0(CH₂)_n-C0₂H and _m is 2 and _n is 16 or 18; and the C-terminal amino acid may be amidated as a C-terminal primary amide.

[0145] SEP ID NO: 11

HAibOGTFTSDYSKYLDEKKAK^*EFVEWLLSGGPSSG

where K^* at position 20 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(yGlu)_m- C0(CH₂)_n-C0₂H and _m is 1 or 2 and _n is 16; and the C-terminal amino acid may be amidated as a C-terminal primary amide. [0146] SEP ID NO: 12

HAibQGTFTSDYSKYLDEKKAK^*EFVEWLLSGGPSSG

where K^* at position 20 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yGlu)_m- C0(CH₂)_n-C0₂H and _m is 1 or 2 and _n is 18; and the C-terminal amino acid may be amidated as a C-terminal primary amide.

[0147] SEP ID NO: 13

HX₂pGTFTSDYSKYLDEKKAK¾FVEWLLXiGGPSSG

where Xi is S or E, X₂ is Aib, and K^* at position 20 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)- ethoxy]-acetyl)₂-(YGlu)_m-C0(CH₂)_n-C0₂H and m is 1 or 2 and _n is 16 or 18; and the C- terminal amino acid may optionally be amidated as a C-terminal primary amide.

[0148] SEP ID NO: 14

IVLSLDVPIGLLpiLLEOARARAAREO ATTN ARIL ARV

Claims

CLAIMS The invention claimed is:

1. A combination therapy comprising:

administering to a patient in need of such treatment an effective amount of a compound according to Formula I:

IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi, wherein I at position 1 is chemically modified by either acetylation or methylation at its N-terminus,

wherein X_bb is L or T,

wherein X_cc is L or I,

wherein X_dd is Q or E, and

wherein K at position 29 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂- C0-(CH2)_Z-C0₂H and _z is 16 or 18, and wherein the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO:8), or a pharmaceutically acceptable salt of Formula I; and

administering to a patient in need of such treatment an effective amount of a compound according to Formula II:

HX₂QGTFTSDYSKYLDEKKAK¾FVEWLLX_IGGPSSG,

wherein Xi is S or E,

wherein X₂ is Aib,

wherein K^* at position 20 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(yGlu)m- C0(CH₂)_n-C0₂H and _m is 1 or 2 and _n is 16 or 18, and wherein the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 13), or a pharmaceutically acceptable salt of Formula II.

2. The combination therapy of Claim 1, wherein in Formula I, I at position 1 is modified by methylation at the N-terminus, X_bb is L, X_cc is L, X_dd is Q, and _z is 18 (SEQ ID NO:4).

3. The combination therapy of Claim 1, wherein in Formula II, Xi is E, and _m is 1 and _n is 16 or 18 (SEQ ID NOS:9 and 10).

4. The combination therapy of Claim 1, wherein the therapy is used to treat a disease selected from the group consisting of chronic kidney disease (CKD) and diabetes.

5. A method of treating a disease selected from the group consisting of diabetes and chronic kidney disease (CKD), the method comprising the step of:

administering to a patient in need thereof an effective amount of a compound of Formula I of Claim 1 and a compound of Formula II of Claim 1.

6. The method of Claim 5, further comprising the step of:

administering an effective amount of one or more additional therapeutic agents.

7. A method of treating type II diabetes in a patient, the method comprising the step of:

administering to a patient in need of such treatment an effective amount of a combination therapy of any one of Claims 1 to 4.

8. The method of Claim 7, wherein the administering step is combined with diet and exercise.

9. A method of treating chronic kidney disease (CKD) in a patient, the method comprising the step of:

administering to a patient in need of such treatment an effective amount of a combination therapy of any one of Claims 1 to 4.

10. The method according to any one of Claims 7 to 9, wherein the administering step is subcutaneous.

11. The method of Claim 10, wherein the C-terminal amino acid in both Formula I and Formula II is amidated.

12. A pharmaceutical composition comprising:

(i) an effective amount of a compound according to Formula I:

IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi, wherein I at position 1 is chemically modified by either acetylation or methylation at its N-terminus, X_bb is L or T, X_cc is L or I, X_dd is Q or E, and K at position 29 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂- (YE)₂-C0-(CH₂)_Z-C0₂H and _z is 16 or 18, and wherein the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 8), or a pharmaceutically acceptable salt of Formula I; and

(ii) an effective amount of a compound according to Formula II:

HX₂QGTFTSDYSKYLDEKKAK¾FVEWLLX_IGGPSSG, wherein Xi is S or E, X₂ is Aib, K^* at position 20 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2- (2-amino-ethoxy)-ethoxy]-acetyl)₂-(YGlu)_m-CO(CH₂)_n-C0₂H and _m is 1 or 2 and _n is 16 or 18, and wherein the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 13), or a pharmaceutically acceptable salt of Formula II.

13. The pharmaceutical composition of Claim 12 further comprising:

(iii) an additional therapeutic agent.

14. The pharmaceutical composition of Claim 13 further comprising:

(iv) a pharmaceutically acceptable carrier, diluent or excipient.

15. A kit for treating chronic kidney disease (CKD) or diabetes comprising:

(i) an effective amount of a compound according to Formula I:

IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NFh, wherein I at position 1 is chemically modified by either acetylation or methylation at its N-terminus, X_bb is L or T, X_cc is L or I, X_dd is Q or E, and K at position 29 is chemically modified through conjugation to an epsilon- amino group of a K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂- (YE)₂-C0-(CH₂)_Z-C0₂H and _z is 16 or 18, and wherein the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 8), or a pharmaceutically acceptable salt of Formula I; and

(ii) an effective amount of a compound according to Formula II:

HX₂QGTFTSDYSKYLDEKKAK¾FVEWLLX_IGGPSSG, wherein Xi is S or E, X₂ is Aib, K^* at position 20 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-

(2-amino-ethoxy)-ethoxy]-acetyl)2-₍ Glu)_m-CO(CH2)_n-CO2H _ancj _m i_s i _or

2 and _n is 16 or 18; and wherein the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 13), or a pharmaceutically acceptable salt of Formula II.

16. A compound useful for treating chronic kidney disease (CKD) or diabetes of the formula:

IVX_bbSLDVPIGLLQILX_ccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi,

wherein I at position 1 is chemically modified by either acetylation or methylation at its N-terminus, X_bb is L or T, X_cc is L or I, X_dd is Q or E, and K at position 29 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2- amino-ethoxy)-ethoxy]-acetyl)₂-(yE)₂-CO-(CH₂)_/-CO₂H and _z is 16 or 18, and wherein the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 8), or a pharmaceutically acceptable salt thereof,

for use in simultaneous, separate or sequential combination with a compound of the formula:

HX₂QGTFTSDYSKYLDEKKAK¾FVEWLLX_IGGPSSG,

wherein Xi is S or E, X₂ is Aib, K^* at position 20 is chemically modified through conjugation to an epsilon-amino group of a K side chain with ([2-(2-amino-ethoxy)- ethoxy]-acetyl)₂-(YGlu)_m-C0(CH₂)_n-C0₂H and _m is 1 or 2 and _n is 16 or 18; and wherein the C-terminal amino acid is optionally amidated as a C-terminal primary amide (SEQ ID NO: 13), or a pharmaceutically acceptable salt thereof.