WO2019140021A1 - Combination therapy - Google Patents

Abstract

The disclosure describes a combination therapy for treating diabetes or chronic kidney disease by administering to an individual in need of such treatment an effective amount of a urocortin-2 compound (or a pharmaceutically acceptable salt thereof) with a glucagon-like peptide analog (or a pharmaceutically acceptable salt thereof), such as dulaglutide, 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 a glucagon-like peptide (GLP) 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 accounting 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 the progressive loss of kidney function. Individuals having CKD experience overtime 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] Glucagon-like peptide 1 (GLP-l) is a 37-amino acid peptide that is secreted by the L-cells of the intestine in response to food ingestion that act as receptor agonists at a GLP-l receptor. Various GLP-l analogs and their uses for treating diabetes are known. One such example is dulaglutide. See , e.g., US Patent No. 7,452,966. Other GLP-l analogs are disclosed in US Patent Nos. 7,084,243; 7,271, 149; and 7,498,308.

[0006] To date, both T2D and CKD remain significant health concerns, and clinicians are looking for alternative therapies treat them. Accordingly, the present disclosure provides a new therapy for CKD and T2D, which is a combination therapy allowing an individual in need thereof to receive the benefits not only of UCN2 but also of a GLP-l . Such a combination of a UCN2 compound with a GLP-l analog is desired to provide treatment for diabetes and CKD, which may be more effective than either therapeutic agent alone.

[0007] 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 a GLP-l analog according to Formula II (or a pharmaceutically acceptable salt of Formula II).

[0008] 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).

[0009] In certain instances, pharmaceutically acceptable salts of Formula I may be used.

[0010] In some instances, as in SEQ ID NO: 8, the terminal V optionally may be amidated as a C-terminal primary amide.

[0011] In some instances, Formula II (i.e., the GLP-l analog) is as follows:

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGX_IGGGGGSGGGGSGGGGSX₂ ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV S VLTVLHQDWLN GKEYKCKV SNKGLPS SIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQ V S LT CL VKGF YP SDI A VEWE SN GQPENN YKTTPP VLD SDGSFFL Y SRLT VD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG,

where Xi is R or G, and X₂ is A or is deleted (SEQ ID NO:9). [0012] In certain instances, pharmaceutically acceptable salts of Formula II may be used.

[0013] 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), and Xi is G and X₂ is A in Formula II (such that Formula II takes the form of SEQ ID NO: 10). In other 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), and Xi is R, and X₂ is deleted in Formula II (such that Formula II takes the form of SEQ ID NO: 11). In either instance, pharmaceutically acceptable salts of Formula I and/or Formula II may be used.

[0014] Also provided herein is a use of the combination therapy including an effective amount of a compound according to Formula I or a pharmaceutically acceptable salt of Formula I and an effective amount of a compound 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 compound of Formula I and administering a compound of Formula II or pharmaceutically acceptable salts thereof, which may be accomplished by subcutaneously administering one or both compounds.

[0015] 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 compound of Formula I or pharmaceutically acceptable salts thereof including, for example, compounds of SEQ ID NO:4 or SEQ ID NO:7 in combination with an effective amount of a compound 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.

[0016] Also provided herein is a pharmaceutical composition including (i) an effective amount of a compound according to Formula I or a pharmaceutically acceptable salt thereof, and (ii) an effective amount of a compound according to Formula II or a pharmaceutically acceptable salt thereof. [0017] In some instances, this pharmaceutical composition also can include (iii) a pharmaceutically acceptable carrier, diluent or excipient and/or (iv) an additional therapeutic agent.

[0018] Also provided herein is a kit for treating CKD or diabetes (including T2D) including (i) an effective amount of a compound according to Formula I or a pharmaceutically acceptable salt thereof, and (ii) an effective amount of a compound according to Formula II or a pharmaceutically acceptable salt thereof.

[0019] Also provided herein is a compound of Formula I or a pharmaceutically acceptable salt thereof for use in simultaneous, separate or sequential combination with a compound of Formula II or a pharmaceutically acceptable salt thereof for use in treating CKD or diabetes (including T2D).

[0020] 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 in need thereof an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof and an effective amount of a compound of Formula II or a pharmaceutically acceptable salt thereof.

[0021] 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 compound of Formula I, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof. In some instances, the compounds of Formula I and Formula II may be subcutaneously administered.

[0022] 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 compound of Formula I or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an compound 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 compounds of Formula I and Formula II may be subcutaneously administered.

[0023] Also provided herein is a compound of Formula I or a pharmaceutically acceptable salt thereof, for use in simultaneous, separate or sequential combination with a compound 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.

[0024] Also provided herein is a compound 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 a compound of Formula II or a pharmaceutically acceptable salt thereof for treating CKD or diabetes (including T2D).

[0025] Also provided herein is use of a compound 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 a compound of Formula II or a pharmaceutically acceptable salt thereof. In some instances, the compound of Formula I is SEQ ID NO:4 or SEQ ID NO:7 or a pharmaceutically acceptable salt thereof.

[0026] Also provided herein is a compound of Formula I or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a compound 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.

[0027] It is contemplated that treating CKD and/or treating diabetes includes preventing the progression of diabetes into CKD.

[0028] For purposes of clarity, the GLP-l analogs of Formula II may be made according to the methods disclosed in US Patent No. 7,271, 149. Additional information about making GLP-l analogs may be found in US Patent Nos. 7,452,966; 7,084,243 and 7,498,308. As noted above, and in some instances, Formula II can have the following structure:

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGX1GGGGGSGGGGSGGGGSX2ESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVV S VLT VLHQDWLNGKEYKCKV SNKGLPS S IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENN YKTTPP VLD SDGSFFL Y SRLT VDK SRW QEGNVF S C S VMHE ALHNH YT QK S LSLSLG, where Xi is R or G; and X₂ is A or is deleted (SEQ ID NO:9).

[0029] In other instances, Formula II can have the following structure:

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGGSGGGGSGGGGSAESKYGPP

CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVF1NAKTKPREEQFNSTYRVV S VLTVLHQDWLNGKEYKCKV SNKGLPS SIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG (SEQ ID NO: 10).

[0030] In some instances, the GLP-l analog of Formula II is dulaglutide. In certain instances, it may be desirable to obtain dulaglutide that has been formulated and sold as Trulicity® by Eli Lilly and Company (Indianapolis, IN, ETSA). In this instances, Trulicity® is administered to the individual in combination with the compound of Formula I. Such administration of Trulicity® may be simultaneous, separate or sequential with the compound of Formula I. In particular instances, the compound of Formula I may be co- formulated with Trulicity®.

[0031] It is contemplated that GLP-l analogs of Formula II may be present as a dimer of that sequence joined by one or more disulfide bonds. See, e.g., ETS Patent No. 7,452,966.

[0032] More generally, some instances include the compound of Formula I co- formulated with the analog of Formula II. Also, more generally, the compound of Formula I is administered simultaneously, separately or sequentially with the analog of Formula II.

[0033] When Xi is G and X₂ is deleted in Formula II, the GLP-l analog has the following structure:

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSESKYGPPC PPCP APEAAGGP S VFLFPPKPKDTLMISRTPE VT C VVVD VSQEDPEV QFNW YVDG VEVHNAKTKPREEQFNST YRVV S VLTVLHQDWLNGKEYKCKV SNKGLP S SIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LG (SEQ ID NO: 11).

[0034] As shown in the foregoing structure above, saying X₂ is“deleted” means that S at position 46 of the peptide is attached directly to E at position 47 (as shown in SEQ ID NO: 11). Alternatively, when X₂ is“A”, then A is positioned after S at position 46 so that A is at position 47 and E is at position 48 (as shown in the structure of SEQ ID NO: 10).

[0035] SEQ ID NO: 11 is similar to the various“Gly⁸-Glu²²” analogs outlined in US Patent No. 7,271, 149 and may be constructed using the techniques outlined therein.

[0036] Stated differently, SEQ ID NO: 10 or SEQ ID NO: 11 ( i.e ., the GLP-l analog) may be used in combination with any of the compounds that fit within the scope of Formula I. For example, SEQ ID NO: 10 or SEQ ID NO: 11 {i.e., the GLP-l analog of Formula II) may be used in combination with any one of SEQ ID NOS: 1-7 {i.e., the UCN2 compound of Formula I). In certain instances, SEQ ID NO: 10 is used in combination with SEQ ID NO:4 {i.e., SEQ ID NO:4 constitutes the compound of Formula I and SEQ ID NO: 10 constitutes the analog of Formula II). In other instances, SEQ ID NO: 10 is used in combination with SEQ ID NO:7 ( i.e ., SEQ ID NO:7 constitutes the compound of Formula I and SEQ ID NO: 10 constitutes the analog of Formula II). Other instances may be designed in which SEQ ID NO: 11 is used in combination with SEQ ID NO:7 {i.e., SEQ ID NO:7 constitutes the compound of Formula I and SEQ ID NO: 11 constitutes the analog of Formula II). Still other instances may be designed in which SEQ ID NO: 11 is used in combination with SEQ ID NO:4 {i.e., SEQ ID NO:4 constitutes the compound of Formula I and SEQ ID NO: 11 constitutes the analog of Formula II).

[0037] These and other advantages, effects, features and objects of this disclosure will become better understood from the detailed description of exemplary embodiments that follows.

[0038] The compounds 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.

[0039] 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-hydroxybenzotriazole. 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.

[0040] 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.

[0041] Crude peptides can be purified using RP-HPLC on C8 or Cl 8 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.

[0042] 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).

[0043] The compounds 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.

[0044] 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. [0045] 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 UCN2 peptide 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.

[0046] The compounds 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 Use, 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.

[0047] The compounds 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.

[0048] The compounds 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.

[0049] The disclosure also encompasses processes useful for synthesizing compounds of Formula I or Formula II, or a pharmaceutically acceptable salt thereof. The intermediates and compounds 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.

[0050] 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.

[0051] As used herein,“Aib” means alpha amino isobutyric acid.

[0052] As used herein,“ AUC” means area under the curve.

[0053] 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.

[0054] As used herein,“ECso” means a concentration of compound that results in 50% activation of the assay endpoint, e.g., cAMP.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] As used herein,“fatty acid” means a carboxylic acid with either 18 or 20 total carbon atoms.

[0059] 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 I (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. [0060] As used herein,“individual” means a mammal, such as a mouse, guinea pig, rat, dog, cat, or primate, especially a human.

[0061] As used herein,“saturated” means a fatty acid contains no carbon-carbon double or triple bonds.

[0062] 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.

[0063] 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“Tit” 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.

[0064] The following Examples provide compounds that may be used as the compound of Formula I.

[0065] EXAMPLE 1 :

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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).

[0073] EXAMPLE 2:

[0074] 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 the 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:2). The structure of this sequence is shown below.

[0075] 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.

[0076] 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.

[0077] 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).

[0078] EXAMPLE 3:

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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).

[0083] EXAMPLE 4:

[0084] IVX_bbSLDVPIGLLQILXccEQEKQEKEKQQAKTNAX_ddlLAQV-NHi,

[0085] wherein 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.

[0086] 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.

[0087] 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.

[0088] 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).

[0089] EXAMPLE 5:

[0090] IVX_bbSLDVPIGLLQILX_ccEQEKQEKEKQQAKTNAX_ddILAQV-NH₂, 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.

[0091] 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.

[0092] The compound according to SEQ ID NO:5 is synthesized similarly as described above for Example 4.

[0093] 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).

[0094] EXAMPLE 6:

[0095] 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.

[0096] 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.

[0097] The compound according to SEQ ID NO:6 is synthesized similarly as described above for Example 4.

[0098] 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+3H73 = 1719.7; calculated: M+3H73 = 1720.4; observed: M+4H74 = 1289.8; calculated: M+4H74 = 1290.5; observed: M+5H75 = 1032.2; calculated: M+5H75 = 1032.6).

[0099] EXAMPLE 7:

[0100] 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.

[0101] 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.

[0102] The compound according to SEQ ID NO:7 is synthesized similarly as described above for Example 4.

[0103] 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).

[0104] 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 (i.e., the fatty acid side chain having the amino-ethoxy moieties, 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. Using 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.

[0105] Using 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.

[0106] 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 residue 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] EXAMPLE 8:

[0111] ASSAYS

[0112] Provided below are the conditions and data for some of the above-recited Examples in several assays.

[0113] In Vitro Function and Selectivity:

[0114] 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.

[0115] 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.

[0116] 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 20mM HEPES. The cells are re-suspended in the assay buffer and 20,000 cells are used per treatment in the human CRHR1 assay.

[0117] 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.

[0118] 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 CRHR2b 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.

[0119] Table 1. In Vitro Activity for hCRHR2b and hCRHRl .

[0120] 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.

[0121] EXAMPLE 9:

[0122] COMBINATION STUDIES OF UCN2 ANALOG + GLP- 1 ANALOG

[0123] This example describes an effect of a long-acting UCN2 analog,“UCN2-X,” in combination with a GLP-l analog on metabolic parameters in DIO mice.

[0124] 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 glucagon agonist analogs is investigated.“UCN2-X” refers to the compound of Example 7 above, and“GLP- 1 analog” refers to the compound of SEQ ID NO: 11 herein.

[0125] 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).

[0126] The purpose of this example thereof is to assess the effects of a UCN2 compound of Formula I alone and in combination with a GLP-l analog of Formula II on fasting glucose, fasting insulin, weight loss and body composition.

[0127] Here, male C57/BL6 mice (Jackson Laboratories; Bar Harbor, ME), 22 weeks old (on high fat diet since 6 weeks of age), are individually housed and maintained on D 12492 chow (60% lard high fat diet) (Research Diets; New Brunswick NJ) for 2 weeks in a vivarium and on a normal light cycle prior to experiment start. Animals are randomized by body weight to treatment groups using block randomization.

[0128] Groups:

1. vehicle + vehicle (phosphate buffered saline + 20 mM Tris-HCl Buffer at pH 8.0, 10 ml/kg, s.c);

2. GLP-l analog (“GLP-l Y”) at 1, 3, and 10 nmol/kg, where“GLP-l Y” is the compound of SEQ ID NO: 11;

3. Urocortin-II analog (“UCN2-X”) at 7.2, 24, and 72 nmol/kg, s.c.; and 4. Combinations of GLP-l Y and UCN2-X, at the following concentrations: a. GLP-l Y (1 nmol/kg, sc) + UCN2-X (7.2 nmol/kg, sc)

b. GLP-l Y (1 nmol/kg, sc) + UCN2-X (24 nmol/kg, sc)

c. GLP-l Y (1 nmol/kg, sc) + UCN2-X (72 nmol/kg, sc)

d. GLP-l Y (3 nmol/kg, sc + UCN2-X (7.2 nmol/kg, sc)

e. GLP-l Y (10 nmol/kg, sc) + UCN2-X (7.2 nmol/kg, sc).

[0129] On day 0 of the experiment, animals and food are weighed and recorded. For each study, animals are separated into two equal groups and started on separate days to simplify the logistics of the study. Data from two separate studies (four total cohorts) are combined. Animals are given a single subcutaneous injection (s.c.) of the indicated pretreatment and treatment in either 20 mM Tris-HCl ,pH 8 (for the UCN) or PBS (for the GLP-l analog) on days 0 (start), 3, 6, 9 and 12 at a volume of 10 ml/kg. Vehicle control animals are injected with a similar volume of this solution. The solutions are kept in sterile capped vials stored at 4°C for the duration of the study. Each treatment arm has an n of 6- 13 mice per group.

[0130] From day 0 to day 14, the animals and their food are weighed daily prior to dose administration. These data are used to calculate body weight gain and food consumption. The animals or the wire rack containing the food are placed in a weigh pan and the balance is allowed to stabilize. The weight is recorded.

[0131] On day 14, the animals are fasted overnight (about 16-18 hours) by placing them in a clean cage with a clean wire rack without food but allowed access to water, and on day 15 the tail of the animal is resected and blood and serum samples are collected. Blood glucose is measured using an Accu-Chek® Aviva® Blood Glucose Meter (Roche Diabetes Care; Indianapolis, IN). The serum samples are centrifuged in a micro hematocrit centrifuge at 9000 relative centrifugal force (ref) for 5 minutes. The serum is collected and analyzed for insulin using a Rat/Mouse Insulin Kit (Mesoscale Discovery; Rockville, MD).

[0132] On study day 0 and study day 14 (prior to fasting for the IPGTT measurement), body composition is analyzed using Quantitative Nuclear Magnetic Resonance EchoMRI analyzer (EMR-166-S, EchoMRI; Houston Tx). After calibrating the analyzer with a known amount of canola, the animals are placed in the analyzer, which measures fat and non-fat (lean) mass in grams. Change in mass is calculated by subtraction of the day 14 value from the day 0 value. [0133] Table 2 below shows data corresponding to each of the above measurements (two studies combined). The data are represented as the mean ± SEM. Statistical significance (*=p<0.05 vs. vehicle; one way ANOVA Dunnett’ s post hoc) is calculated using GraphPad

Prizm 7.00 software (La Jolla, Ca). i = excess over highest single dose (if maximum p- value of comparison of combination to each single agent is < 0.05) is determined by MIN test with Hommel step up for multiplicity. JMP 12.1.0 is used for excess over highest single dose ANOVA and SAS 9.2 for Hommel correction.

[0134] Table 2: Effect of Treatment With ETCN2-X, GLP-l Y or Combinations Thereof on Body Weight, Fat Mass and Fasting Blood Glucose and Fasting Serum Insulin.

[0135] Such data show that the combination provides better results than the individual agents.

[0136] SEP ID NO: 1

IVLSLDVPIGLLQILLEQEKQEKEKQQAKTNAQILAQV-NHi

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)i₆-C0₂H; _ancj the C-terminal amino acid is amidated as a C-terminal primary amide.

[0137] SEP ID NO:2

IVLSLDVPIGLLPILLEPEKPEKEKPPAKTNAPILAPV-NH2

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.

[0138] SEP ID NO: 3

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)i₆-C0₂H; _ancj the C-terminal amino acid is amidated as a C-terminal primary amide.

[0139] 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.

[0140] SEP ID NO: 5

IVTSLDVPIGLLQILLEQEKQEKEKQQAKTNAEILAQV-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)ix-C0₂H; _ancj the C-terminal amino acid is amidated as a C-terminal primary amide.

[0141] SEP ID NO: 6

IVLSLDVPIGLLOILLEOEKOEKEKOOAKTNAEILAOV-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: 7

IVTSLDVPIGLLOILIEOEKOEKEKOOAKTNAEILAOV-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.

[0143] SEP ID NO: 8

IVX_bbSLDVPIGLLOILX_ccEOEKOEKEKOOAKTNAX_ddlLAOV

where I at position 1 is chemically 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-( E)₂-CO-(CH₂)_z-CO₂F[ _ancj _z i_s 16 or 18, and where the terminal V optionally may be amidated.

[0144] SEP ID NO: 9

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGX1GGGGGSGGGGSGGGGSX2ESKYGP PCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNST YRVV S VLT VLHQDWLN GKEYKCK V SNKGLP S SIE KTISK AKGQPREPQ VYTLPP SQEEMTKN Q V SLT CLVKGF YP SDIAVEWESNGQPE NN YKTTPP VLD SDGSFFL Y SRLT VDK SRW QEGNVF S C S VMHE ALHNH YT QK SL S LSLG

where Xi is R or G, and X₂ is A or is deleted.

[0145] SEP ID NO: 10

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGGSGGGGSGGGGSAESKYGPP CPPCP APE AAGGP S VFLFPPKPKDTLMISRTPEVT C VVVD VSOEDPE V OFNW YVD GVEVE1NAKTKPREEQFNSTYRVV S VLTVLF1QDWLNGKEYKCKV SNKGLPS SIEK TISKAKGOPREPOVYTLPPSOEEMTKNOVSLTCLVKGFYPSDIAVEWESNGPPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWOEGNVFSCSVMHEALHNHYTOKSLSL SLG

[0146] SEP ID NO: 1 1

HGEGTFTSDVSSYLEEOAAKEFIAWLVKGGGGGGGSGGGGSGGGGSESKYGPPC PPCPAPEAAGGP S VFLFPPKPKDTLMISRTPEVT C VVVD VSOEDPEV OFNW YVDG VEVHNAKTKPREEQFNSTYRVV S VLT VLHQDWLN GKEYKCK V SNKGLP S SIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LG

[0147] SEP ID NO: 12

IVLSLDVPIGLLQILLEQARARAAREQ 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_bbSLDVPIGLLQILX_ccEQEKQEKEKQQAKTNAX_ddlLAQV,

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 the terminal V optionally may be amidated (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:

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGX1GGGGGSGGGGSGGGGSX2E

SKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PE V QFNW Y VD GVE VHNAKTKPREEQFN S T YRV V S VLT VLHQD WLN GKE

YKCKV SNKGLPS SIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQ V SLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE

GNVFSCSVMHEALHNHYTQKSLSLSLG,

wherein Xi is R or G, and

wherein X₂ is A or is deleted (SEQ ID NO:9), 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), and wherein in Formula II, Xi is G and X2 is A (SEQ ID NO: 10).

3. 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), and wherein in Formula II, Xi is R and X₂ is deleted (SEQ ID NO: 11).

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. A pharmaceutical composition comprising: (i) an effective amount of a compound according to Formula I:

IVX_bbSLDVPIGLLQILX_ccEQEKQEKEKQQAKTNAX_ddlLAQV, 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 the terminal V optionally may be amidated (SEQ ID NO:8); and

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

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGXiGGGGGSGGGGSGGG GSXiESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVD VSQEDPEVQFNW YVDGVEVHNAKTKPREEQFN ST YRVV S VL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP P S QEEMTKN Q VSLT CL VKGF YP SDI A VEWE SN GQPENNYKTTPP V LD SDGSFFL Y SRLTVDKSRW QEGNVF SC S VMHEALHNH YTQK SLS LSLG,

wherein Xi is R or G, and X2 is A or is deleted (SEQ ID NO:9), or a pharmaceutically acceptable salt of Formula II.

12. The pharmaceutical composition of Claim 11 further comprising;

(iii) an additional therapeutic agent.

13. The pharmaceutical composition of Claim 12 further comprising:

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

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

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

IVX_bbSLDVPIGLLQILX_ccEQEKQEKEKQQAKTNAX_ddlLAQV, 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 terminal V optionally may be amidated (SEQ ID NO:8), or a pharmaceutically acceptable salt of Formula I; and

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

HGEGTFTSDVSSYLEEQAAKEFIAWLVKGXiGGGGGSGGGGSGGG GSXiESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVD VSQEDPEVQFNW YVDGVEVHNAKTKPREEQFN ST YRVV S VL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP P S QEEMTKN Q VSLT CL VKGF YP SDI A VEWE SN GQPENNYKTTPP V LD SDGSFFL Y SRLTVDKSRW QEGNVF SC S VMHEALHNH YTQK SLS LSLG,

wherein Xi is R or G, and X2 is A or is deleted (SEQ ID NO:9), or a pharmaceutically acceptable salt of Formula II.

15 A compound useful for treating chronic kidney disease (CKD) or diabetes comprising the formula:

IVX_bbSLDVPIGLLQILX_ccEQEKQEKEKQQAKTNAX_ddlLAQV,

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)_z-CO₂H and _z is 16 or 18, and the terminal V optionally may be amidated (SEQ ID NO: 8), or a pharmaceutically acceptable salt thereof,

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

HGEGTFTSD V S S YLEEQ AAKEFIAWLVKGX1GGGGGSGGGGSGGGGSX2 ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVV S VLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRW QEGNVF S C S VMHEALHNH YT QK SL SL SLG, wherein Xi is R or G, and X₂ is A or is deleted (SEQ ID NO:9), or a pharmaceutically acceptable salt thereof.