OA16931A

OA16931A - Glucagon analogues.

Info

Publication number: OA16931A
Application number: OA1201400270
Authority: OA
Inventors: Dieter Wolfgang Hamprecht; Jakob Lind Tolborg; Ditte Riber
Original assignee: Zealand Pharma A/S; Boehringer Ingelheim International Gmbh
Priority date: 2011-12-23
Filing date: 2012-12-19
Publication date: 2016-01-25

Abstract

The invention provides glucagon analogue peptides and their use for promoting weight loss or preventing weight gain, and the treatment of obesity or excess body weight and associated conditions. The compounds may also be used to improve glycemic control and/or for the treatment of diabetes. The compounds may mediate their effect, inter alia, by having increased selectivity for the GLP-1 receptor as compared to human glucagon.

Description

The présent Invention relates to glucagon analogues and their medical use, for example in the treatment of excess food Intake, obesity and excess weight and associated conditions, and elevated cholestérol. The compounds may also be used to Improve glycaemic control and/or for the treatment of diabètes.

BACKGROUND OF THE INVENTION

Preproglucagon is a 158 amino acid precursor polypeptide that is differentially processed in the tissues to form a number of structurally related proglucagon-derived peptides, including glucagon (Glu), glucagon-like peptide-1 (GLP-1), glucagon-iike peptîde-2 (GLP-2), and oxyntomodulin (OXM). These molécules are involved in a wide variety of physiological fonctions, including glucose homeostasis, insulin sécrétion, gastric emptying and intestinal growth, as well as régulation of food Intake.

Glucagon is a 29-amino acid peptide that corresponds to amino acids 53 to 81 of pre-proglucagon and has the sequence His-Ser-GIn-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-ArgArg-Aia-GIn-Asp-Phe-Val-GIn-Trp-Leu-Met-Asn-Thr. Oxyntomodulin (OXM) is a 37 amino acid peptide which includes the complété 29 amino acid sequence of glucagon with an octapeptide carboxyterminal extension (amino acids 82 to 89 of pre-proglucagon, having the sequence Lys-ArgAsn-Arg-Asn-Asn-ile-Aia and termed “intervening peptide Γ or IP-1 ; the foil sequence of human oxyntomodulin is thus His-Ser-Gln-Giy-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-ArgArg-Ala-GIn-Asp-Phe-Val-GIn-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-lle-Ala). The major biologically active fragment of GLP-1 Is produced as a 30-amino add, C-terminally amidated peptide that corresponds to amino acids 98 to 127 of pre-progiucagon.

Glucagon helps maintain the level of glucose in the blood by binding to glucagon receptors on hépatocytes, causing the liver to release glucose - stored in the form of glycogen - through glycogenolysis. As these stores become depleted, glucagon stimulâtes the liver to synthesize additional glucose by gluconeogenesis. This glucose is released into the bloodstream, preventing the development of hypoglycémie.

OXM Is released into the blood in response to food ingestion and in proportion to meal calorie content. OXM has been shown to suppress appetite and inhibit food intake in humans (Cohen et al, Journal of Endocrinology and Metabolism, 88, 4696-4701, 2003; WO 2003/022304). In addition to those anorectic effects, which are similar to those of GLP-1, OXM must also affect body weight by another mechanlsm, since rats treated with oxyntomoduiin show less body weight gain than palrfed rats (Bioom, Endocrinology 2004,145, 2687). Treatment of obese rodents with OXM also improves their glucose tolérance (Parievliet et al, Am J Physïol Endocrinol Metab, 294, E142-7, 2008) and suppresses body weight gain (WO 2003/022304).

OXM activâtes both the giucagon and the GLP-1 receptors with a two-fold higher potency for the glucagon receptor over the GLP-1 receptor, but is less potent than native giucagon and GLP-1 on their respective receptors. Human glucagon is also capable of activating both receptors, though with a strong preference for the glucagon receptor over the GLP-1 receptor. GLP-1 on the other hand Is not capable of activating glucagon receptors. The mechanism of action of oxyntomoduiin is not well understood. In particular, it is not known whether some of the extrahepatic effects of the hormone are mediated through the GLP-1 and glucagon receptors, or through one or more unidentified receptors.

Other peptides hâve been shown to bind and activate both the giucagon and the GLP-1 receptor (Hjort et ai, Journal of Biological Chemistry, 269, 30121-30124,1994) and to suppress body weight gain and reduce food intake (WO 2006/134340, WO 2007/100535, WO 2008/10101, WO 2008/152403, WO 2009/155257 and WO 2009/155258).

Diabètes, especially type 2 diabètes, is establishing itself as an épidémie of the 21 ** century with an estimated 5% of the adult worid population suffering from the disease. The number of deaths attributable to diabètes is steadily growing, currentiy estimated at 3.8 million cases each year, reflecting the insufficient glycaemic control achieved with currentiy available treatments. Therefore, more effective therapeutics for glycaemic control are needed.

Obesity is a globally increasing health problem is associated with various diseases, particularly cardiovascular disease (CVD), type 2 diabètes, obstructive sleep apnea, certain types of cancer, and osteoarthritis. As a resuit, obesity has been found to reduce life expectancy. According to 2005 projections by the Worid Health Organization there are 400 million adults (âge > 15) classified as obèse worldwide. In the US, obesity Is now believed to be the second-leading cause of preventable death after smoking.

The rise in obesity drives an increase in diabètes, and approximately 90% of people with type 2 diabètes may be classifïed as obese. There are 246 million people worldwide with diabètes, and by 2025 It Is estimated that 380 million will hâve diabètes. Many hâve additional cardiovascular risk factors, including high/aberrant LDL and triglycérides and low HDL

Further conditions are associated with metabolic diseases, e.g. hypertension, atherogenic dyslipldemia, atheroscierosis, coronary heart disease, stroke and obesity iinked Inflammation. Accordingly, a treatment for the underlylng metabolic disease might hâve a positive Impact on follow-on conditions.

Accordingly, there Is a strong medical need for treating metabolic and associated diseases such as obesity, dyslipldemia and diabètes.

SUMMARY OF THE INVENTION

In a first aspect, the Invention provides a compound having the formula:

R¹-X-Z-R² wherein

R¹ is H, Cm alkyl, acetyl, formyl, benzoyi ortrifluoroacetyl;

R² Is OH or NH₂;

X Is a peptide which has the formula 1:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-Arg-Arg-Ala-X20-Asp-Phe-lleX24-Trp-Leu-X27-X28-X29 (I) wherein

X2 is selected from Ser, D-Ser and Afb;

X3 Is selected from Gin, His and Pro;

X12 Is selected from Lys and Y

X16 Is selected from Glu and Y;

X20 Is selected from Lys and Y;

X24 is selected from Glu and Y;

X27 Is selected from Leu and Y;

X28 Is selected from Ser and Y or is absent;

X29 is Ala or absent;

wherein at least one of X12, X16, X17, X20, X27 and X28 is Y;

wherein each residue Y Is Independently selected from Lys, Cys and Om;

wherein the side chain of at least one amino acid residue Y is conjugated to a lipophilie substituent having the formula:

(i) Z¹, wherein Z¹ Is a lipophilie molety conjugated directly to the side chain of Y; or (ii) Z’Z², wherein Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of Y via Z²;

and Z is absent or is a sequence of 1-20 amino acid units independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Om;

or a pharmaceutically acceptable sait thereof.

Peptide X may hâve the formula la:

His-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-T yr-Ser-Lys-T yr-Leu-Asp-X16-Arg-Arg-Ala-X20-Asp-Phe-lleX24-Trp-Leu-X27-X28-Ala (la) wherein

X2 Es selected from Ser, D-Ser and Aib;

X16 is selected from Glu and Y;

X20 is selected from Lys and Y;

X24 Is selected from Glu and Y;

X27 is selected from Leu and Y; and

X28 Is selected from Ser and Y.

Peptide X may hâve the sequence:

H-Aib-QGTFTSDYSKYLDKRRAKDFIEWLLSA;

H-Alb-QGTFTSDYSKYLDERRAKDFIEWLLSA;

H-Aib-QGTFTSDYSKYLDERRAKDFIKWLLSA;

HSQGTFTSDYSKYLDERRAKDFIKWLLSA;

H-Aib-QGTFTSDYSKYLDERRAKDFIEWLKSA; or

H-Aib-QGTFTSDYSKYLDERRAKDFIEWLLKA.

For example, peptide X may be:

H-Aib-QGTFTSDYSKYLDK*RRAKDFIEWLLSA;

H-Alb-QGTFTSDYSKYLDERRAK*DFIEWLLSA;

H-Alb-QGTFTSDYSKYLDERRAKDFiK*WLLSA;

HSQGTFTSDYSKYLDERRAKDFIKWLLSA;

H-Aib-QGTFTSDYSKYLDERRAKDFIEWLK*SA; or

H-Alb-QGTFTSDYSKYLDERRAKDFIEWLLK*A;

wherein K* indicates a Lys residue to which the lipophilie substituent is conjugated.

For example, the compound may be:

H-H-Alb-QGTFTSDYSKYLD-K(Hexadecanoyt-isoGlu)-RRAKDFIEWLLSA-NH₂ [Compound 1 ]; H-H-Alb-QGTFTSDYSKYLDERRA-K(Hexadecanoyl-isoGlu)-DFIEWLLSA-NH₂ [Compound 2]; H-H-Aib-QGTFTSDYSKYLDERRAKDFl-K(Hexadecanoy!-lsoGlu)-WLLSA-NH₂ [Compound 3]; H-HSQGTFTSDYSKYLDERRAKDFl-K(Hexadecanoyl-isoGlu)-WLLSA-NH₂ [Compound 4]; H-H-Aib-QGTFTSDYSKYLDERRAKDFlEWL-K(Hexadecanoyl-isoGlu)-SA-NH₂ [Compound 5]; or H-H-Alb-QGTFTSDYSKYLDERRAKDFIEWLL-K(Hexadecanoyl-isoGlu)-A-NH₂ [Compound 6]; or a pharmaceutically acceptable sait thereof.

In a second aspect, the Invention provides a compound having the formula

R’-X-Z-R² wherein

R’ Is H, Cm alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl;

R² Is OH or NH₂;

X is a peptide which has the formula II:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Arg-Ala-X20-Asp-Phe-lleX24-Trp-Leu-X27-X28-X29 (11) wherein

X2 is selected from Ser, D-Ser and Aib;

X3 is selected from Gin, His and Pro;

X12 is selected from Arg, Lys and Y;

X16 is selected from Glu and Y;

X17 Is selected from Arg and Y;

X20 is selected from Lys, Arg and Y;

X24 is selected from Glu and Y;

X27 is selected from Leu and Y;

X28 is selected from Ser and Y or absent;

X29 is Ala or absent;

wherein X12 and/or X20 Is Arg;

wherein at least one of X12, X16, X17, X20, X24, X27 and X28 is Y;

wherein each residue Y Is independently selected from Lys, Cys and Om;

(i) Z¹, wherein Z¹ is a lipophilie moiety conjugated directly to the side chain of Y; or (îi) Z¹Z², wherein Z¹ Is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of Y via Z²;

or a pharmaceutically acceptable sait thereof.

It may be désirable that X12 is Arg.

Peptide X may hâve the formula lia:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Arg-Tyr-Leu-Asp-X16-X17-Arg-Ala-X20-Asp-Phe-lleX24-Trp-Leu-Leu-Ser-Ala (lia) wherein

X2 is selected from Ser, D-Ser and Aib;

X3 is selected from Gin, His and Pro;

X16 Is selected from Glu and Y;

X17 Is selected from Arg and Y;

X20 Is selected from Arg and Lys; and

X24 Is selected from Glu and Y.

Peptide X may hâve the formula iib:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Arg-Tyr-Leu-Asp-Glu-X17-Arg-Aia-Arg-Asp-Phe-lleGlu-Trp-Leu-Leu-Ser-Ala (llb) wherein

X2 is selected from Ser, D-Ser and Aib;

X3 is selected from Gin, His and Pro; and

X17isY.

Peptide X may hâve the sequence:

HSQGTFTSDYSRYLDEKRARDFiEWLLSA;

H-DSer-QGTFTSDYSRYLDEKRARDFIEWLLSA;

H-Aib-QGTFTSDYSRYLDEKRARDFIEWLLSA;

HSHGTFTSDYSRYLDEKRARDFIEWLLSA;

H-DSer-HGTFTSDYSRYLDEKRARDFIEWLLSA;

H-Aib-GTFTSDYSRYLDEKRARDFIEWLLSA;

HSPGTFTSDYSRYLDEKRARDFIEWLLSA;

H-DSer-PGTFTSDYSRYLDEKRARDFIEWLLSA;

H-Aib-PGTFTSDYSRYLDEKRARDFIEWLLSA; or

H-Aib-QGTFTSDYSRYLDEKRAKDFIEWLLSA.

For example, X may be:

HSQGTFTSDYSRYLDEK*RARDFIEWLLSA; H-DSer-QGTFTSDYSRYLDEK*RARDFIEWLLSA; H-Aib-QGTFTSDYSRYLDEK*RARDFIEWLLSA; HSHGTFTSDYSRYLDEK*RARDFIEWLLSA; H-DSer-HGTFTSDYSRYLDEK*RARDFIEWLLSA; H-Aib-GTFTSDYSRYLDEK‘RARDFIEWLLSA; HSPGTFTSDYSRYLDEK*RARDFIEWLLSA; H-DSer-PGTFTSDYSRYLDEK*RARDFIEWLLSA; H-Aib-PGTFTSDYSRYLDEK*RARDFIEWLLSA; or H-Aib-QGTFTSDYSRYLDEK*RAKDFIEWLLSA.

The compound may be:

H-HSQGTFTSDYSRYLDE-K(Hexadecanoyl-lsoGlu)-RARDFIEWLLSA-NH₂ [Compound 7]; H-H-DSer-QGTFTSDYSRYLDE-K(Hexadecanoyl-lsoGlu)-RARDFIEWLLSA-NH₂ [Compound 8]; H-H-Alb-QGTFTSDYSRYLDE-K(Hexadecanoyl-isoGiu)-RARDFIEWLLSA-NH₂ [Compound 9]; H-HSHGTFTSDYSRYLDE-K(Hexadecanoyt-isoGlu)-RARDFIEWLLSA-NH₂ [Compound 10]; H-H-DSer-HGTFTSDYSRYLDE-K(Hexadecanoyl-lsoGlu)-RARDFIEWLLSA-NH₂ [Compound 11]; H-H-Alb-HGTFTSDYSRYLDE-K(Hexadecanoyl-lsoGlu)-RARDFIEWLLSA-NH₂ [Compound 12]; H-HSPGTFTSDYSRYLDE-K(Hexadecanoyl-isoGlu)-RARDFIEWLLSA-NH₂ [Compound 13]; H-H-DSer-PGTFTSDYSRYLDE-K(Hexadecanoyl-isoGlu)-RARDFIEWLLSA-NH₂ [Compound 14]; H-H-Aib-PGTFTSDYSRYLDE-K(Hexadecanoyl-isoGiu)-RARDFIEWLLSA-NH₂ [Compound 15]; or H-H-Aib-QGTFTSDYSRYLDE-K(Hexadecanoyl-lsoGlu)-RAKDFIEWLLSA-NH₂ [Compound 16];

or a pharmaceutically acceptable sait thereof.

In a third aspect, the Invention provides a compound having the formula

R’-X-Z-R² wherein

R¹ ls H, Cm alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R²lsOH orNH_a;

X ls a peptide which has the formula III:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Arg-Ala-X20-Asp-Phe-lleX24-Trp-Leu-X27-X28-X29 (III) wherein

X2 is selected from Ser, D-Ser and Aib;

X3 ls selected from Gin, His and Pro;

X12 ls selected from Lys and Y

X16 is selected from Glu and Y;

X17 ls selected from Arg and Y;

X20 is selected from Lys and Y;

X24 is selected from Glu and Y;

X27 is selected from Leu and Y;

X28 ls selected from Ser and Y or is absent:

X29 ls Ala or absent;

wherein X3 is His or Pro when X2 ls Ser or Aib, and X2 ls D-Ser when X3 ls Gin;

wherein at least one of X12, X16, X17, X20, X24, X27 and X28 Is Y;

wherein each residue Y is independently selected from Lys, Cys and Om;

wherein the side chain of at least one amino acid residue Y of X is conjugated to a lipophilie substituent having the formula:

(i) Z¹, wherein Z¹ is a lipophilie moiety conjugated directiy to the side chain of Y; or (il) Z¹Z², wherein Z¹ is a lipophilie moiety, Z² Is a spacer, and Z¹ is conjugated to the side chain of Y via Z²;

or a pharmaceutically acceptable sait thereof.

Peptide X may hâve the formula lila:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Arg-Ala-X20-Asp-Phe-lleX24-Trp-Leu-Leu-Ser-Ala (Ilia) wherein

X2 is selected from Ser, D-Ser and Aîb;

X3 is selected from Gin, His and Pro;

X12 is selected from Lys and Y

X16 is selected from Glu and Y;

X17 Is selected from Arg and Y;

X20 is selected from Lys and Y; and

X24 Is selected from Glu and Y.

Peptide X may hâve the formula lllb:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-X17-Arg-Ala-Lys-Asp-Phe-lleGlu-Trp-Leu-Leu-Ser-Ala (lllb) wherein

X2 is selected from Ser, D-Ser and Aib;

X3 is selected from Gin, His and Pro; and

X17isY.

Peptide X may hâve the sequence:

H-DSer-QGTFTSDYSKYLDEKRAKDFIEWLLSA;

HSHGTFTSDYSKYLDEKRAKDFIEWLLSA;

H-DSer-HGTFTSDYSKYLDEKRAKDFIEWLLSA;

HSPGTFTSDYSKYLDEKRAKDFIEWLLSA; or

H-DSer-PGTFTSDYSKYLDEKRAKDFIEWLLSA.

Peptide X may be:

H-DSer-QGTFTSDYSKYLDEK*RAKDFIEWLLSA;

HSHGTFTSDYSKYLDEK*RAKDFIEWLLSA;

H-DSer-HGTFTSDYSKYLDEK*RAKDFIEWLLSA;

HSPGTFTSDYSKYLDEK*RAKDFIEWLLSA; or

H-DSer-PGTFTSDYSKYLDEK‘RAKDFI EWLLSA;

The compound may be:

H-H-DSer-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-RAKDFlEWLLSA-NH2 [Compound 1 η; H-HSHGTFTSDYSKYLDE-K(Hexadecanoyl4soGlu)-RAKDFIEWLLSA-NH2 [Compound 18]; H-H-DSer-HGTFTSDYSKYLDE-K(Hexadecanoyl4soGlu)-RAKDFIEWLLSA-NH2 [Compound 19]; H-HSPGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu}-RAKDFlEWLLSA-NH2 [Compound 20]; or H-H-DSer-PGTFTSDYSKYLDE-K(HexadecanoyNsoGlu)-RAKDFlEWLLSA-NH2 [Compound 21]; or a pharmaceutically acceptable sait thereof.

ln any of the above aspects of the Invention, it may be désirable that peptide X contains only one residue Y.

Whether peptide X contains one or more than one residue Y, the or each residue Y may be Lys.

The Invention further provides an isoiated nucieic acid (which may be DNA or RNA) encoding a peptide X-Z as defined ln any of the three aspects of the Invention described above, I.e. the peptide backbone of any of these compounds of the invention, before addition of the lipophilie substituent to any residue Y. (Of course, this may only be appropriate when each residue In X-Z Is one of the 20 naturally occurring amino acids which can be incorporated Into protein by nucieic add translation.) Further provided Is an expression vector comprising such a nudeic add, and a host cell containing such a nucieic acid or expression vector.

In a fourth aspect, the invention provides the compounds:

H44-Aib-QGTFTSDYSKYLDE’K(Octadecanoyl-isoGlu)-RAKDFIEWLLSA-NH2 [Compound 22]; H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-RAKDFIEWLLSA-OH [Compound 23]; and H-H-Aib-QGTFTSDYSKYLDE-K(Octadecanoyl-isoGlu)-RAKDFIEWLLSA-OH [Compound 24],

The présent invention further provïdes a composition comprising a compound, nudeic add, expression vector or host cell of the invention in admixture with a carrier, ln preferred embodiments, the composition Is a pharmaceutically acceptable composition and the carrier is a pharmaceutically acceptable carrier. The composition may contain a pharmaceuticaliy acceptable sait of the compound of the invention.

in addition, the présent Invention provides a compound or composition of any aspect of the Invention as described above for use In a method of medical treatment

The compounds described find use, inter alia, In preventlng weight gain or promotlng weight loss. By preventing Is meant inhibiting or reducing when compared to the absence of treatment, and Is not necessarily meant to imply complété cessation of weight gain. The peptides may cause a decrease in food Intake and/or increased energy expenditure, resulting In the observed effect on body weight.

Independently of their effect on body weight, the compounds of the Invention may hâve a bénéficiai effect on circulating cholestérol levels, being capable of lowering drculating LDL levels and increasing HDL/LDL ratio.

The compounds may additionally hâve a bénéficiai effect on glycaemlc control, Independently of their effect on body weight. It is envlsaged thaï such compounds may be therapeutically useful in conditions which are not directly associated with or caused by excess weight or obesity, such as type I diabètes and gestational diabètes.

Of course, this does not preclude their use In conditions ultimately caused or exacerbated by obesity or excess weight. Indeed, their effect on glucose control and body weight may make them particularly suitable for treatment of such conditions.

Thus the compounds of the Invention can be used for direct or indirect therapy of any condition caused orcharacterised by excess bodyweight, such asthe treatmentand/orprévention of obesity, morbid obesity, obesity linked Inflammation, obesity linked gallbladder disease, obesity Induced sleep apnea. They may also be used for the prévention of metabolic syndrome, type I diabètes, type II diabètes, hypertension, atherogenlc dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, or stroke. Their effects In these conditions may be as a resuit of or associated with their effect on body weight, or may be Independent thereof.

Thus, the Invention provides a compound of the invention for use In a method of preventing weight gain or promoting weight loss in an individual in need thereof. Also provided ls the use of a compound of the Invention in the manufacture of a médicament for preventing weight gain or promoting weight loss In an individual. Also provided is a method of preventing weight gain or promoting weight loss in an individual in need thereof comprising administering a compound of the invention to the individual.

The Invention further provides a compound of the invention for use in a method of lowering circuiating LDL levels, and/or increasing HDL/LDL ratio in an individual in need thereof. Also provided is the use of a compound ofthe invention in the manufacture of a médicament for lowering circuiating LDL levels, and/or Increasing HDL/LDL ratio In an individual. Also provided is a method of lowering circuiating LDL levels, and/or increasing HDL/LDL ratio in an Individual In need thereof comprising administering a compound of the invention to the Individual.

The invention further provides a compound of the Invention for use in a method of prévention or treatment of a condition caused or characterised by excess body weight. Also provided is the use of a compound of the invention In the manufacture of a médicament for prévention or treatment of a condition caused or characterised by excess body weight. Also provided ls a method of prévention or treatment of a condition caused or characterised by excess body weight In an individual in need thereof comprising administering a compound of the invention to the individual.

The invention further provides a compound of the invention for use in a method of prévention and/or treatment of obesity, morbid obesity, morbid obesity prior to surgery, obesity linked inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, type I diabètes, type Il diabètes, metabolic syndrome, hypertension, atherogenic dyslipidemia, atherosclerois, arterioscierosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease In an Individual In need thereof. Also provided is the use of a compound of the invention in the manufacture of a médicament for prévention or treatment of such a condition. Also provided ls a method of prévention or treatment of such a condition in an individual In need thereof comprising administering a compound of the invention to the individual.

The invention further provides a compound of the invention for use in conjunction with an agent for treatment of obesity, dyslipidemia, diabètes, or hypertension. Also provided is the use of a compound of the Invention ln the manufacture of a médicament for use ln conjunction with an agent for treatment of obesity, dyslipidemia, diabètes, or hypertension. Also provided Is a method of treatment comprising administration of a compound of the invention in conjunction with an agent for treatment of obesity, dyslipidemia, diabètes, or hypertension to an Individuai ln need thereof. Also provided Is a pharmaceutical composition comprising a compound of the Invention and an agent for treatment of obesity, dyslipidemia, diabètes, or hypertension.

The agent for treatment of obesity may be a glucagon-like peptide receptor 1 agonist, peptide YY receptor agonist or analogue thereof, cannabinold receptor 1 antagonist, lipase Inhibitor, melanocortin receptor 4 agonist, or melanin concentrating hormone receptor 1 antagonist.

The agent for treatment of hypertension may be an angiotensin-converting enzyme inhibitor, angiotensln II receptor blocker, diuretic, beta-blocker, or calcium channel blocker.

The agent for treatment of dyslipidaemia may be a statin, a fibrate, a niacin and/or a cholestérol absorbtion Inhibitor.

The agent for treatment of diabètes may be metformin, a sulfonylurea, a glinlde, a DPP-1V inhibitor, a glitazone, a GLP-1 agonist, insulin or an Insulin analogue.

As already described, the Invention extends to expression vectors comprising the above-described nucleic acid sequence, optionally in combination with sequences to direct Its expression, and host cells containing the expression vectors. Preferably the host cells are capable of expressing and secreting the compound of the Invention, or the peptide backbone X-Z of the compound of the invention. In a still further aspect, the présent Invention provides a method of producing the compound, the method comprising culturing the host cells under conditions suitable for expressing the compound and purifying the compound thus produced. The method may comprise the further step of adding the lipophilie substituent at the appropriate amino acid position.

The Invention further provides a nucleic acid of the Invention, an expression vector of the Invention, or a host cell capable of expressing and secreting a compound of the invention, for use in a method of medical treatment. It will be understood that the nucleic acid, expression vector and host cells may be used for treatment of any of the disorders described herein which may be treated with the compounds of the invention themselves. Référencés to a therapeutic composition comprising a compound of the invention, administration of a compound of the invention, or any therapeutic use thereof, shouid therefore be construed to encompass the équivalent use of a nucleic add, expression vector or host cell of the invention, except where the context demands otherwise.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this spedfication, the conventional one letter and three letter codes for naturaily occurring amino adds are used, as well as generally accepted three letter codes for other amino acids, such as Aib (α-aminoisobutyric add), Om (omithine), Dbu (2,4 diaminobutyric acid), D-Ser (D-form of Ser) and Dpr (2,3-diaminopropanoic add).

The term native glucagon refers to native human glucagon having the sequence H-His-Ser-GlnGly-Thr-Phe-Thr-Ser-Asp-T yr-Ser-Lys-T yr-Leu-Asp-Ser-Arg-Arg-Ala-GIn-Asp-Phe-Val-GIn-T rp-LeuMet-Asn-Thr-OH (SEQ ID NO: 1).

The invention provides compounds as defined above. For the avoidance of doubt, In the définitions provided herein, it is generaliy intended that the sequence of peptide X can only be varied at those positions which are stated to allow variation, and only within the options stated. Amino adds within the sequence X can be considered to be numbered consecutively from 1 to 29 in the conventional N-terminal to C-terminal direction. Reference to a position within X should be construed accordingly, as should reference to positions within native human glucagon and other molécules.

Without wishing to be bound by any particular theory, the residues at positions 27,28 and 29 of native glucagon appear to provide slgnificant selectivity of the peptide for the glucagon receptor. The residues présent at these positions in the compounds of the invention may increase potency at and/or selectivity for the GLP-1 receptor, potentielly without slgnificant réduction of potency at the glucagon receptor.

Substitution of the naturally-occurring Met residue at position 27 (e.g. with Leu or Lys, espedally with Leu) also reduces the potential for oxidation, thereby Increasing the chemical stability of the compounds.

Substitution of the naturally-occurring Asn residue at position 28 (e.g. by Ser, Arg or Ala) also reduces the potential for deamidation in acidic solution, so Increasing the chemical stability of the compounds.

Substitution of one or both of the naturally-occurring Gin residues at positions 20 and 24 also reduces the potential for deamidation In acidic solution, thereby Increasing the chemical stability of the compounds.

Substitution of one or more of the naturally occurring amino acids at positions 12,16,17, 20, 24,27 and 28 with a suitable amino acid Y enables conjugatîon to a lipophilie substituent. The residue(s) Y at these positions may Independently be Cys, Om or Lys. More particularly, one or more of these residues may be Cys. Further, one or more of the residues at these positions may be Lys. Where the compound contalns more than one residue Y, they may be the same (ail Cys, ali Om, or ali Lys) or different. In some embodiments it may be désirable that each peptide X contains just one residue Y. The or each residue Y may be Lys.

As already disclosed, a compound of the Invention may comprise a C-terminal peptide sequence Z of 1-20 amino acids, for example to stabilise the conformation and/or secondary structure of the glucagon analogue peptide, and/or to render the glucagon analogue peptide more résistant to enzymatic hydroiysis, e.g. as described in WO99/46283.

When présent, Z represents a peptide sequence of 1-20 amino acid residues, e.g. in the range of 115, more preferably in the range of 1-10, in particular In the range of 1-7 amino acid residues, e.g., 1, 2, 3,4, 5, 6 or 7 amino acid residues, such as 6 amino acid residues. Each of the amino actd residues in the peptide sequence Z may Independently be selected from Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu (2,4 dîaminobutyric acid), Dpr (2,3-diaminopropanoic acid) and Om (omithine). Preferably, the amino acid residues are selected from Ser, Thr, Tyr, Glu, Lys, Arg, Dbu, Dpr and Om, more preferably selected exclusively from Glu, Lys, and Cys. The above-mentioned amino acids may hâve either D- or L-configuration, but preferably hâve an L-configuration. Particularly preferred sequences Z are sequences of three, four, five, six or seven consecutive lysine residues (i.e. Lys₃₁ Lys₄, Lys₅₁ Lyse or Lys₇), and particularly five or six consecutive lysine residues. Other exemplary sequences of Z are shown in WO 01/04156. Altematively the Cterminal residue of the sequence Z may be a Cys residue. This may assist In modification (e.g.

PEGylation, or conjugation to albumin) of the compound. ln such embodiments, the sequence Z may, for example, be only one amino acid ln length (i.e. Z = Cys) or may be two, three, four, five, six or even more amino acids in length. The other amino acids therefore serve as a spacer between the peptide X and the terminal Cys residue.

The peptide sequence Z has no more than 25% sequence identity with the corresponding sequence of the IP-1 portion of human OXM (which has the sequence Lys-Arg-Asn-Arg-Asn-AsnIle-Ala).

Percent (%) amino add sequence identity of a given peptide or polypeptide sequence with respect to another polypeptide sequence (e.g. IP-1 ) is calculated as the percentage of amino add residues In the given peptide sequence that are identical with correspondingly positioned amino add residues in the corresponding sequence of that other polypeptide when the two are aligned with one another, introducing gaps for optimal alignment if necessary. % identity values may be determined using WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)). WUBLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. A % amino add sequence identity value Is determined by the number of matching identical residues as determined by WU-BLAST-2, dîvided by the total number of residues ofthe reference sequence (gaps introduced by WU-BLAST-2 into the reference sequence to maximize the alignment score being ignored), muitîplied by 100.

Thus, when Z Is aligned optimally with the 8 amino acids of IP-1, it has no more than two amino adds which are identical with the corresponding amino adds of IP-1.

In some embodiments, Z is absent in the compound of the Invention.

One or more ofthe side chains ln amino add residue(s) Y of peptide X is conjugated to a lipophilie substituent Z¹ or Z’Z². Thus the lipophilie substituent Z¹ may be covaientiy bonded directly to an atom ln the amino add side chain, or altematively may comprise a lipophilie moiety Z¹ conjugated to the amino add side chain by a spacer Z². A lipophilie substituent Z¹ or Z’Z² may additionally be conjugated to a side chain of an amino add which Is part of the peptide Z if desired.

Without wishlng to be bound by any particular theory, it Is thought that the lipophilie substituent binds albumin In the blood stream, thus shielding the compounds of the Invention from enzymatic dégradation and thereby enhancing the half-life of the compounds. The spacer, when présent, Is used to provide spacing between the compound and the lipophilie substituent.

The lipophilie substituent (or moiety, as appropriate) may be attached to the amino acid side chain or to the spacer via an ester, a sulphonyi ester, a thioester, an amide or a sulphonamide, Accordingly it will be understood that preferably the lipophilie substituent Includes an acyl group, a sulphonyi group, an N atom, an O atom or an S atom which forms part of the ester, sulphonyi ester, thioester, amide or sulphonamide. Preferably, an acyl group In the lipophilie substituent forms part of an amide or ester with the amino acid side chain or the spacer.

The lipophilie substituent (or moiety) may be or may Include a hydrocarbon chaln having 4 to 30 C atoms. Preferably It has at least 8 or 12 C atoms, and preferably it has 24 C atoms or fewer, or 20 C atoms or fewer. The hydrocarbon chain may be linear or branched and may be saturated or unsaturated. It will be understood that the hydrocarbon chain is preferably substituted with a moiety which forms part of the attachment to the amino add side chain or the spacer, for example an acyl group, a sulphonyi group, an N atom, an O atom or an S atom. Most preferably the hydrocarbon chain is substituted with acyl, and accordingly the hydrocarbon chaln may be part of an alkanoyl group, for example decanoyl (caproyl), dodecanoyl (lauroyl), tetradecanoyl (myristoyl), hexadecanoyl (palmitoyl), heptadecanoyl, octadecanoyl (stearoyl), elcosanoyl or docosanoyl.

Thus, the or each Z¹ may be, or may comprise, a decanoyl (caproyl), dodecanoyl (lauroyl), tetradecanoyl (myristoyl), hexadecanoyl (palmitoyl), heptadecanoyl, octadecanoyl (stearoyl), eicosanoyl or docosanoyl group.

Accordingly, the lipophilie substituent may hâve the formula shown below:

A may be, for example, an acyl group, a sulphonyi group, NH, N-alkyl, an O atom or an S atom, preferably acyl. n is an integer from 3 to 29, preferably at least 7 or at least 11, and preferably 23 or less, more preferably 19 or less.

The hydrocarbon chain may be further substituted. For example, it may be further substituted with up to three substituents selected from NH₂₁ OH and COOH. If the hydrocarbon chain is further substituted, preferably it is further substituted with only one substituent. Alternatively or additionally, the hydrocarbon chain may include a cycloalkane or heterocycloalkane, for example as shown below:

Preferably the cycloalkane or heterocycloalkane is a six-membered ring. Most preferably, it is piperidine.

Alternatively, the lipophilie substituent may be based on a cyclopentanophenanthrene skeleton, which may be partially or fuily unsaturated, or saturated. The carbon atoms In the skeleton each may be substituted with Me or OH. For example, the lipophilie substituent may be cholyl, deoxycholyl or lithocholyl.

As mentioned above, the lipophilie substituent may be conjugated to the amino acid side chain by a spacer. When présent, the spacer is attached to the lipophilie substituent and to the amino acid side chain. The spacer may be attached to the lipophilie substituent and to the amino acid side chain independently by an ester, a sulphonyl ester, a thioester, an amide or a sulphonamide. Accordingly, it may include two moieties Independentiy selected from acyl, sulphonyl, an N atom, an O atom or an S atom. The spacer may hâve the formula:

wherein B and D are each independently selected from acyl, sulphonyl, NH, N-alkyl, an O atom and an S atom, preferably from acyl and NH. Preferably, n is an integer from 1 to 10, preferably from 1 to 5. The spacer may be further substituted with one or more substituents selected from Cm alkyl, Cm alkyl amine, Cm alkyl hydroxy and Cm alkyl carboxy.

Altematively, the spacer may hâve two or more repeat units of the formula above. B, D and n are each selected independently for each repeat unit. Adjacent repeat units may be covalently attached to each other via their respective B and D moleties. For example, the B and D moieties of the adjacent repeat units may together form an ester, a sulphony! ester, a thioester, an amlde or a sulphonamide. The free B and D units at each end of the spacer are attached to the amino acid side chain and the lipophilie substituent as described above.

Preferably the spacer has five or fewer, four or fewer or three or fewer repeat units. Most preferably the spacer has two repeat units, or Is a single unit.

The spacer (or one or more of the repeat units of the spacer, If it has repeat units) may be, for example, a natural or unnatural amino add. It will be understood that for amino adds having functionalised side chains, B and/or D may be a moiety within the side chain of the amino add. The spacer may be any naturally occurring or unnatural amino add. For example, the spacer (or one or more of the repeat units of the spacer, If it has repeat units) may be Gly, Pro, Ala, Val, Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gin, Asn, D-Glu, D-Glu, Asp, Ser, Thr, Gaba, Aib, D-Ala, 5-aminopentanoyt, 6-aminohexanoyl, 7-aminoheptanoyl, 8-amlnooctanoyl, 9-amlnononanoyl or 10aminodecanoyl.

For example, the spacer may be a single amino acid selected from DD-Glu, Gaba, β-Ala and □Glu.

The lipophilie substituent is conjugated to an amino add side chain of a Lys, Cys or Om residue. Preferably, the lipophilie substituent is conjugated to Lys.

An example of a lipophilie substituent and spacer is shown in the formula below:

which ls hexadecanoyl-iso-glutamic add.

Here, a Lys residue In the compound of the présent invention is covaîently attached to D-Glu (the spacer) via an amlde moiety. Hexadecanoyl (palmitoyl) ls covaîently attached to the U-Glu spacer via an amide moiety.

Altematively or additionally, one or more amino acid side chains in the compound of the invention may be conjugated to a polymeric moiety, for example, in order to increase solubility and/or half-life in vivo (e.g. in plasma) and/or bioavailability. Such modification ls also known to reduce clearance (e.g. rénal dearance) of therapeutic proteins and peptides.

The polymeric moiety is preferably water-soluble (amphrphilic or hydrophilic), non-toxic, and pharmaceutically înert. Suitable polymeric moleties include polyethylene glycol (PEG), homo- or co-polymers of PEG, a monomethyl-substituted polymer of PEG (mPEG), and polyoxyethylene glycerol (POG). See, for example, Int. J. Hematology 68:1 (1998): Bioconjugate Chem. 6:150 (1995); and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992),

Other suitable polymeric moieties include poly-amino adds such as poly-lysine, poly-aspartic add and poly-glutamic add (see for example Gombotz, et al. (1995), Bioconjugate Chem., vol. 6: 332351 ; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3,49-57; Tsukada, et al. (1984), J. Natl.

Cancer Inst., vol 73, : 721-729; and Pratesi, étal. (1985), Br. J. Cancer, vol. 52: 841-848).

The polymeric moiety may be stralght-chain or branched. It may hâve a molecular weight of 50040,000 Da, for example 500-10,000 Da, 1000-5000 Da, 10,000-20,000 Da, or 20,000-40,000 Da.

A compound of the invention may comprise two or more such moieties, in which case the total molecular weight of ail such moieties will generally fall within the ranges provided above.

The polymeric moiety may be coupled (by covalent linkage) to an amino, carboxy! or thiol group of an amino acid side chain. Prefened exampies are the thiol group of Cys residues and the epsilon amino group of Lys residues. The carboxy! groups of Asp and Glu residues may also be used.

The skilled reader will be well aware of suitable techniques that can be used to perform the coupling reaction. For example, a PEG moiety carrying a methoxy group can be coupled to a Cys thiol group by a maleimido linkage using reagents commerdally availabie from Nektar Therapeutics AL. See also WO 2008/101017, and the references cited above, for details of suitable chemîstry.

In another aspect, one or more ofthe amino acid side chains in a compound in the présent Invention, for example in peptide X, is/are conjugated to a polymeric moiety.

In a further aspect, the présent invention provïdes a composition comprising a compound of the invention as described herein, or a sait or dérivative thereof, in admixture with a carrier.

The term “dérivative thereor refers to a dérivative of any one ofthe compounds. Dérivatives include ali chemical modifications, ali conservative variants, ail prodrugs and ail métabolites of the compounds.

The Invention also provides the use of a compound ofthe présent invention in the préparation of a médicament for the treatment of a condition as described below.

The invention also provides a composition wherein the composition is a pharmaceutically acceptable composition, and the carrier Is a pharmaceutically acceptable carrier.

Peptide synthesis

The compounds of the présent Invention may be manufactured either by standard synthetic methods, recombinant expression Systems, or any other state of the art method. Thus the glucagon analogues may be synthesized in a number of ways, Including, for example, a method which comprises:

(a) synthesizing the peptide by means of solid-phase or liquid-phase methodology, either stepwise or by fragment assembly, and isolation and purifying of the final peptide product; or (b) expressing a nucleic acid construct that encodes the peptide X-Z (i.e. the peptide backbone of the compound of the Invention) In a host cell, and recovering the expression product from the host cell culture; or (c) effecting ceif-free In vitro expression of a nucleic acid construct that encodes the peptide X-Z (i.e. the peptide backbone of the compound of the invention), and recovering the expression product;

or any combination of methods of (a), (b), and (c) to obtain fragments of the peptide, subsequentiy ligating the fragments to obtain the peptide, and recovering the peptide. Typically, methods (b) and (c) wili be supplemented by adding the lipophilie substituent at the appropriate location within the peptide backbone after synthesis. In method (a), the derivatised amino acid may be incorporated directly during synthesis, or the lipophilie substituent may be added subsequentiy.

It Is preferred to synthesize the analogues of the invention by means of solid-phase or liquid-phase peptide synthesis. In this context, reference is made to WO 98/11125 and, among many others, Flelds, GB et al., 2002, Principles and practice of solid-phase peptide synthesis. In: Synthetic Peptides (2nd Edition), and the Examples herein.

For recombinant expression, the nucleic acid fragments of the Invention will normally be inserted In suitable vectors to form cloning or expression vectors carrying the nucleic acid fragments of the

Invention; such novel vectors are also part of the Invention. The vectors can, depending on purpose and type of application, be In the form of plasmids, phages, cosmids, mini-chromosomes, or virus, but also naked DNA which Is only expressed transiently in certain cells Is an important vector. Preferred cloning and expression vectors (plasmtd vectors) of the Invention are capable of autonomous réplication, thereby enabling high copy-numbers for the purposes of hlgh-ievel expression or high-level réplication for subséquent cloning.

In general outline, an expression vector comprises the following features In the 5'->3' direction and In opérable linkage: a promoter for driving expression of the nucleic acid fragment of the Invention, optionaily a nucleic add sequence encoding a leader peptide enabling sécrétion (to the extracellular phase or, where applicable, Into the peripiasma), the nudeic acid fragment encoding the peptide of the Invention, and optionaily a nudeic acid sequence encoding a terminator. They may comprise additional features such as selectable markers and origins of réplication. When operating with expression vectors In producer strains or cell lines it may be preferred that the vector is capable of Integrating into the host cell genome. The skilled person Is very familiar with suitable vectors and Is able to design one according to their spedfic requlrements.

The vectors of the Invention are used to transform host cells to produce the compound of the invention. Such transformed cells, which are also part of the invention, can be cultured cells or cell lines used for propagation of the nudeic add fragments and vectors of the invention, or used for recombinant production of the peptides of the invention.

Preferred transformed cells of the invention are mlcro-organisms such as bacteria [such as the spedes Escherichia (e.g. E. coll), Badllus (e.g. Badilus subtilis), Salmonella, or Mycobacterium (preferably non-pathogenlc, e.g. M. bovis BCG), yeasts (e.g., Saccharomyces cerevisiae and Pichia pastoris), and protozoans. Alternatively, the transformed ceils may be derived from a multicellular organism, i.e. it may be fungal cell, an insect cell, an algal cell, a plant cell, or an animal cell such as a mammalîan cell. For the purposes of donlng and/or optimised expression it Is preferred that the transformed cell Is capable of replicating the nudeic add fragment of the invention. Cells expressing the nudeic fragment are useful embodiments of the Invention; they can be used for small-scale or large-scale préparation of the peptides of the invention.

When produdng the peptide of the Invention by means of transformed cells, it is convenlent, aithough far from essential, that the expression product is secreted into the culture medium.

It will be understood that recombinatn expression of X-Z may only be appropriate when each residue In X-Z ls one of the 20 naturally occurring amino acids which can be incorporated into protein by conventional nucleic add translation. However, modified translation Systems are known which can introduce non-conventional amino acids and such Systems may be used if desired.

An exemplary synthesis route for a compound of the Invention is illustrated below. A person skilled in the art will be able to adapt the shown procedure as required in order to optimise the process for any compound of cholce.

Efficacy

Binding of the relevant compounds to GLP-1 or glucagon (Glu) receptors may be used as an indication of agonist activity, but In general it is preferred to use a biological assay which measures intracellular signalling caused by binding of the compound to the relevant receptor. For example, activation of the glucagon receptor by a glucagon agonist will stimulate cellular cyclic AMP (cAMP) formation. Simiiarly, activation of the GLP-1 receptor by a GLP-1 agonist will stimulate cellular cAMP formation. Thus, production of cAMP in suitable cells expressing one of these two receptors can be used to monitor the relevant receptor activity. Use of a suitable pair of cell types, each expressing one receptor but not the other, can hence be used to détermine agonist activity towards both types of receptor.

The skilled person will be aware of suitable assay formats, and examples are provided below. The GLP-1 receptor and/or the glucagon receptor may hâve the sequence of the receptors as described in the examples. For example, the assays may employ the human glucagon receptor (Glucagon-R) having primary accession number GI:4503947 and/or the human glucagon-like peptide 1 receptor (GLP-1 R) having primary accession number Gl:166795283. (in that where sequences of precursor proteins are referred to, it should of course be understood that assays may make use of the mature protein, lacklng the signal sequence).

ECso values may be used as a numerical measure of agonist potency at a given receptor. An EC» value is a measure of the concentration of a compound required to achteve half of that compound's maximal activity In a particular assay. Thus, for example, a compound having ECso[GLP-1] lower than the ECso[GLP-1] of glucagon in a particular assay may be considered to hâve higher GLP-1 receptor agonist potency than glucagon.

The compounds described in this spécification are typically Glu-GLP-1 dual agonists, as determined by the observation that they are capable of stimulating cAMP formation at both the glucagon receptor and the GLP-1 receptor. The stimulation of each receptor can be measured In Independent assays and afterwards compared to each other.

By comparing the EC_M value for the glucagon receptor (ECso [Glucagon-R]) with the ECso value for the GLP-1 receptor, (ECso [GLP-1 R]) for a given compound, the relative glucagon selectivity (%) of that compound can be found as follows:

Relative Glucagon-R selectivity [compound] = (1/ECso [Glucagon-R])x100 / (1/ECso [Glucagon-R] + 1/EC_m [GLP-1 R])

The relative GLP-1 R selectivity can likewise be found:

Relative GLP-1 R selectivity [compound] = (1/ECso [GLP-1 R])x100 / (1/ECso [Glucagon-R] + 1/ECso [GLP-1 R])

A compountfs relative selectivity allows its effect on the GLP-1 or glucagon receptor to be compared directly to Its effect on the other receptor. For exemple, the higher a compound's relative GLP-1 selectivity is, the more effective that compound is on the GLP-1 receptor as compared to the glucagon receptor.

Using the assays described beiow, we hâve found the relative GLP-1 selectivity for human glucagon to be approximately 5%.

The compounds of the invention hâve a higher relative GLP-1 R selectivity than human glucagon in that for a particular level of glucagon-R agonlst activity, the compound will display a higher level of

GLP-1R agonist activity (I.e. greater potency at the GLP-1 receptor) than glucagon. It will be understood that the absolute potency of a particular compound at the glucagon and GLP-1 receptors may be higher, lower or approximately equal to that of native human glucagon, as long as the appropriate relative GLP-1 R selectivity Is achieved.

Nevertheless, the compounds of this invention may hâve a lower EC» [GLP-1R] than human glucagon. The compounds may hâve a lower ECmIGLP-I-R] than glucagon while maintaining an EC50 [Glucagon-R] that Is less than 10-fold higher than that of human glucagon, less than 5-fold higher than that of human glucagon, or less than 2-fold higher than that of human glucagon.

The compounds of the Invention may hâve an EC₅₀ [Glucagon-R] that is less than two-fold that of human glucagon. The compounds may hâve an EC_M (Glucagon-R] that Is less than two-fold that of human glucagon and hâve an EC» [GLP-1R] that is less than half that of human glucagon, less than a fifth of that of human glucagon, or less than a tenth of that of human glucagon.

The relative GLP-1R selectivity of the compounds may be between 5% and 95%. For example, the compounds may hâve a relative selectivity of 5-20%, 10-30%, 20-50%, 30-70%, or 50-80%; or of 30-50%, 40-60,%, 50-70% or 75-95%.

The compounds ofthe Invention mayalso hâve effect on other Class B GPCR receptors, such as, but not limited to, Calcitonin gene-related peptide 1 (CGRP1), corticotropin-releasing factor 1 & 2 (CRF1 & CRF2), gastric Inhibitory polypeptide (GIP), glucagon-like peptide 1 & 2 (GLP-1 & GLP-2, glucagon (GCGR), secretin, gonadotropin releasing hormone (GnRH), parathyrold-hormone 1 & 2 (PTH1 & PTH2), vasoactive intestinal peptide (VPAC1 & VPAC2).

Therapeutic uses

The compounds ofthe invention may provide an attractive treatment option for, Inter alla metabolic diseases, including, obesity, dyslipidemia and diabètes mellitus (diabètes).

Metabolic syndrome is characterized by a group of metabolic risk factors In one person. They include abdominal obesity (excessive fat tissue around the abdominal Internai organs), atherogenic dyslipidemia (blood fat disorders including high triglycérides, low HDL cholestérol and/or high LDL cholestérol, which foster plaque buildup in artery walls), elevated blood pressure (hypertension), Insulin résistance and glucose intolérance, prothrombotic state (e.g. high fibrinogen or plasminogen activator Inhibitor—1 in the blood), and proinflammatory state (e.g., elevated C-reactive protein in the blood).

Individuels with the metabolic syndrome are at increased risk of coronary heart disease and other diseases related to other manifestations of arteriosclerosis (e.g., stroke and peripheral vascular disease). The dominant underlying risk factors for this syndrome appear to be abdominal obesity. Diabètes comprises a group of metabolic diseases characterized by hyperglycemia resulting from defects ln insulin sécrétion, insulin action, or both. Acute signs of diabètes include excessive urine production, resulting compensatory thirat and increased fluid Intake, blurred vision, unexplalned weight loss, lethargy, and changes in energy metabolism. The chronic hyperglycemia of diabètes is associated with long-term damage, dysfonction, and failure of various organs, notably the eyes, kldneys, nerves, heart and blood vessels. Diabètes 1s classified Into type 1 diabètes, type 2 diabètes and gestational diabètes on the basls on pathogenetic characteristics.

Type 1 diabètes accounts for 5-10% of ail diabètes cases and Is caused by auto-immune destruction of insulin-secreting pancreatic β-cells.

Type 2 diabètes accounts for 90-95% of diabètes cases and is a resuit of a complex set of metabolic disorders. Type 2 diabètes is the conséquence of endogenous Insulin production becoming insufficlent to maintain plasma glucose levels below the diagnostic thresholds.

Gestational diabètes refera to any degree of glucose intolérance Identified during pregnancy.

Pre-diabetes Includes lmpalred fastlng glucose and Impaired glucose tolérance and refera to those states that occur when blood glucose levels are elevated but below the levels that are established for the clinical dlagnosis for diabètes.

A large proportion of people with type 2 diabètes and pre-diabetes are at increased risk of morbldity and mortality due to the high prevalence of additional metabolic risk factors Including abdominal obesity (excessive fat tissue around the abdominal internai organs), atherogenlc dyslipidemia (blood fat disorders including high triglycérides, low HDL cholestérol and/or high LDL cholestérol, which foster plaque buildup in artery walls), elevated blood pressure (hypertension) a prothrombotic state (e.g. high fibrinogen or plasminogen activator inhibitor—1 in the blood), and proinflammatory state (e.g., elevated C-reactive protein in the blood).

Conversely, obesity confère an increased risk of developlng pre-diabetes, type 2 diabètes as well as e.g. certain types of cancer, obstructive sleep apnea and gall-blader disease.

Dyslipidaemia is associated with increased risk of cardiovascular diasese. High Density Lipoprotein (HDL) is of clinical importance since an inverse corrélation exists between plasma HDL concentrations and risk of atherosclerotic disease. The majority of cholestérol stored in atherosclerotic plaques originales from LDL and hence elevated concentrations Low Density Lipoproteins (LDL) is closely associated with atheroscierosls. The HDL/LDL ratio is a clinical risk indictor for atherosderosis and coronary atherosderosis in particular.

Without wishing to be bound by any particular theory, it is believed that the compounds of the invention act as GluGLP-1 dual agonists. The dual agonist combines the effect of glucagon on fat metabolism with the effects of GLP-1 on food intake. They might therefore act in a synergistic fashion to accelerate élimination of excessive fat déposition and induce sustainable weight loss. Certain of the compounds described may also hâve a bénéficiai effect on glucose control directly, independently of any effect on body weight.

The synergistic effect of dual GluGLP-1 agonists may also resuit in réduction of cardiovascular risk factors such as high cholestérol and LDL, which may be entirely independent of their effect on body weight.

The compounds of the présent invention may therefore be used as pharmaceutical agents for preventing weight gain, promoting weight loss, reducing excess body weight or treating obesity (e.g. by control of appetite, feeding, food intake, calorie intake, and/or energy expenditure), including morbid obesity, as well as associated diseases and health conditions caused or characterised by excess body weight. These indude but are not limited to obesity, morbid obesity, morbid obesity prior to surgery, obesity linked inflammation, obesity linked gallbladder disease and obesity induced sleep apnea. The compounds of the invention may also be used for treatment of metabolic syndrome, hypertension, type II diabètes, atherogenic dyslipidemia, atherosclerois, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke and microvascular disease. These are ail conditions which can be associated with obesity. However, the effects of the compounds of the invention on these conditions may be mediated In whole or in part via an effect on body weight, or may be independent thereof. Further, via their direct effect on glucose control, the compounds of the présent invention may be useful for treatment of any of the above conditions as well as others not necessarily associated with or caused by excess body weight, Including type I diabètes and gestational diabètes.

The compounds of the présent invention may further be used as pharmaceutical agents for lowering circulating LDL levels, and/or increasing HDL/LDL ratio.

Combination therapy

As noted above, it will be understood that reference in the following to a compound of the Invention also extends to a pharmaceutically acceptable sait or solvaté thereof as well as to a composition comprising more than one different compounds ofthe invention.

A compound of the invention may be administered as part of a combination therapy with an agent for treatment of obesity, hypertension dyslipidemla or diabètes.

In such cases, the two active agents may be given together or separately, and as part of the same pharmaceutical formulation or as separate formulations.

Thus a compound or sait thereof can further be used in combination with an anti-obesity agent, including but not limited to a glucagon-like peptide receptor 1 agonist, peptide YY or analogue thereof, cannabinoid receptor 1 antagonist, lipase Inhibitor, melanocortin receptor 4 agonist, or melanln concentrating hormone receptor 1 antagonist.

A compound of the Invention or sait thereof can be used in combination with an anti-hypertension agent, Including but not limited to an angiotensin-converting enzyme Inhibitor, angiotensin II receptor blocker, diuretics, beta-blocker, or calcium channei blocker.

A compound of the Invention or sait thereof can be used In combination with a dysiîpidaemia agent, including but not limited to a statîn, a fibrate, a niacin and/or a cholestérol absorbtion inhibitor.

Further, a compound of the Invention or sait thereof can be used in combination with an antidiabetic agent, Including but not Iimited to metformin, a sulfonylurea, a glinide, a DPP-IV Inhibitor, a glitazone, a different GLP-1 agonist or an Insulin. ln a preferred embodiment, the compound or sait thereof Is used in combination with Insulin, DPP-IV Inhibitor, sulfonylurea or metformin, partîcularly sulfonylurea or metformin, for achieving adéquate glycémie control. In an even more preferred embodiment the compound or sait thereof Is used ln combination with an insulin or an Insulin analogue for achieving adéquate glycémie control. Examples of Insulin analogues Include but are not Iimited to Lantus, Novorapid, Humalog, Novomix, and Actraphane HM.

Pharmaceutical compositions

The compounds of the présent Invention, or salts thereof, may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a therapeutically effective amount of a compound ofthe invention, or a sait thereof, ln a pharmaceutically acceptable carrier.

Such compositions comprise those of overall solid form as well as those of overall pasteous or liquid form, which can be selected and optimtsed with respect to the spécifie route of administration and/or needs of the patient. Such forms are perse known to a person skilled ln the art.

The therapeutically effective amount of a compound of the présent invention will dépend on the route of administration, the type of mammal being treated, and the physical characteristics of the spécifie mammal under considération. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may dépend on such factors as weight, diet, concurrent médication and other factors, well known to those skilled in the medical arts. The dosage slzes and dosing regimen most appropriate for human use may be gulded by the results obtained by the présent Invention, and may be confirmed ln properly designed clinical trials.

An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose ln laboratory animais and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into considération by a clinicien when determining an optimal dosage for a given subject. Such considérations are known to the skilled person.

The term “pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known In the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, stérile saline and phosphatebuffered saline at slightly acidic or physiologîcal pH may be used. pH buffering agents may be phosphate, citrate, acetate, tris/hydroxymethyt)amlnomethane (TRIS). N-Tris(hydroxymethyt)methyl -3- aminopropanesulphonlc acid (TAPS), ammonium bicarbonate, dïethanolamine, histidine, which Is a preferred buffer, arginine, lysine, or acetate or mixtures thereof. The term further encompases any agents listed in the US Pharmacopela for use in animais, including humans.

The term ‘pharmaceutically acceptable sait refers to a sait of any one of the compounds. Salts include pharmaceutically acceptable salts such as add addition salts and baslc salts. Examples of add addition salts indude hydrochloride salts, dtrate salts and acetate salts. Examples of baslc salts Include salts where the cation Is selected from alkali metals, such as sodium and potassium, alkaline earth metals, such as caldum, and ammonium Ions *N (R³) sfR*), where R³ and R⁴Independently désignâtes optionally substituted Ci^-alkyt, optionally substituted C₂^-alkenyl, optionally substituted aryl, or optionally substituted heteroaryl. Other examples of pharmaceutically acceptable salts are described in Remington's Pharmaceutical Sciences* ,17th édition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U.S.A., 1985 and more recent éditions, and In the Encyclopaedia of Pharmaceutical Technology.

Treatment Is an approach for obtaining benefidal or desired clinical results. For the purposes of this invention, benefidal or desired dlnical results Indude, but are not limited to, aliénation of symptoms, diminishment of extent of disease, stabilïzed (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and rémission (whether partial or total), whether detedable or undetectable. Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment* is an Intervention performed with the Intention of preventing the development or altering the pathology of a disorder. Accordingly, treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment indude those already with the disorder as well as those in which the disorder is to be prevented

The pharmaceutical compositions can be In unit dosage form. In such form, the composition is divided Into unit doses containing appropriate quantités of the active component. The unit dosage form can be a packaged préparation, the package containing discrète quantities ofthe préparations, for exampie, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet Itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen. In certain embodiments, packaged forms include a label or insert with instructions for use. Compositions may be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents Include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parentéral (including subcutaneous, Intramuscular, intravenous, intradermal, and transdermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

Subcutaneous or transdermal modes of administration may be particularly suitable for the compounds described herein.

Compositions of the invention may further be compounded In, or attached to, for example through covalent, hydrophobie and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the compound, increase bioavaïlabiiity, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof. Examples of carriers, drug delivery Systems and advanced drug delivery Systems include, but are not limited to, poiymers, for example cellulose and dérivatives, polysaccharides, for example dextran and dérivatives, starch and dérivatives, poly(vinyl alcohol), acrylate and méthacrylate poiymers, polylactic and polyglycolic add and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermogelling Systems, for example block copolymeric Systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions there of, well known to those skilled in the art of phase behaviour in lipid-water Systems, polymeric micelles, multiple émulsions, self-emulsifying, self-microemulsifying, cydodextrins and dérivatives thereof, and dendrimers.

METHODS

General synthesis of glucagon analogues

Solid phase peptide synthesis (SPPS) was performed on a microwave assisted synthesizer using standard Fmoc strategy in NMP on a polystyrène resin (TentaGel S Ram). HATU was used as coupling reagent together with DIPEA as base. Piperidine (20% in NMP) was used for deprotection. Pseudoprolines: Fmoo-Phe-Thr(.Psl. Me, Me pro)-OH and Fmoc-Asp-Ser(.Psi., Me, Me pro)-OH (purchased from NovaBiochem) were used where applicable.

Abbreviations employed are as follows:

IvDde:	1-(4,4-dimethy!-2,6-dioxocydohexylidene)3-methy!-butyl
Dde:	1-(4,4-dimethyi-2,6-dioxocyciohexyiidene)-ethyl
DCM:	dichloromethane
DMF:	N.N-dimethylformamide
DIPEA:	diisopropylethytamine
EtOH:	éthanol
EtîO:	diethyl ether
HATU:	N-[(dimethytamlno)-1 H-1,2,3-triazol[4,5-b]pyridine-1 -ylmethylene]-Nmethylmethanaminium hexafluorophosphate N-oxide
MeCN:	acetonitrile
NMP:	N-methylpyrroiidone
TFA:	trifluoroacetic add
TIS:	triisopropylsilane

Cleavage:

The crude peptide was cleaved from the resin by treatment with 95/2.5/2.5 % (v/v) TFA/TIS/ water at r.t. for 2 h. For peptides with a méthionine in the sequence a mixture of 95/5 % (v/v) TFA/EDT was used. Most of the TFA was removed at redueed pressure and the crude peptide was precipitated and washed with diethyl ethe r and allowed to dry to constant weight at ambient température.

General synthesis of acvlated glucaqon analogues

The peptide backbone was syntheslzed as described above for the general synthesis of glucagon analogues, with the exception that It was acylated on the side chain of a lysine residue with the peptide still attached to the resin and fully protected on the side chain groups, except the epsilonamine on the lysine to be acylated. The lysine to be acylated was incorporated with the use of Fmoc-Lys(ivDde)-OH or Fmoc-Lys(Dde)-OH. The N-terminal of the peptide was protected with a Boc group using B0C2O in NMP. While the peptide was still attached to the resin, the IvDde protecting group was selectlvely cleaved using 5 % hydtazïne hydrate in NMP. The unprotected lysine side chain was then first coupled with a spacer amino acid like Fmoc-Glu-OtBu, which was deprotected with piperidine and acylated with a fatty acid using standard peptide coupling methodology as described above. Altematively, the hlstidine at the N-terminal may be incorporated from the begînning as Boc-His(Boc)-OH. Cleavage from the resin and purification were performed as described above.

An alternative strategy Is to use Fmoc-Lys(Hexadecanoyl4soGlu(tBu))-OH for easy incorporation of the fatty acid and linker as part of the standard synthesis procedure.

Génération of cell lines exoresslnq human glucagon- and GLP-1 receptors

The cDNA encodîng either the human glucagon receptor (Glucagon-R) (primary accession number P47871 ) or the human giucagon-like peptide 1 receptor (GLP-1 R) (primary accession number P43220) were cloned from the cDNA clones BC104854 (MGC:132514/IMAGE:8143857) or BC112126 (MGC:138331/IMAGE:8327594), respectively. The DNA encodîng the Glucagon-R or the GLP-1-R was amplified by PCR using primers encodîng terminal restriction sites for subclonlng. The 5'-end primers additionally encoded a near Kozak consensus sequence to ensure efficient translation. The fidelîty of the DNA encodîng the Glucagon-R and the GLP-1-R was confirmed by DNA sequendng. The PCR products encodîng the Glucagon-R or the GLP-1-R were subcloned into a mammalian expression vector containing a neomycin (G418) résistance marker.

The mammalian expression vectors encodîng the Glucagon-R or the GLP-1-R were transfected into HEK293 cells by a standard calcium phosphate transfection method. 48 hr after transfection cells were seeded for limited dilution doning and selected with 1 mg/ml G418 in the culture medium. Three weeks laterl2 surviving colonies of Glucagon-R and GLP-1-R expressing cells were picked, propagated and tested In the Glucagon-R and GLP-1-R efficacy assays as described below. One Glucagon-R expressing done and one GLP-1-R expressïng done were chosen for compound profiling.

Glucagon receptor and GLP-1-receptor efficacy assavs

HEK293 cells expressing the human Glucagon-R, or human GLP-1-R were seeded at 40,000 cells per well in 96-well microtiter plates coated with 0.01 % poly-L-lysine and grown for 1 day in culture in 100 pl growth medium. On the day of analysis, growth medium was removed and the cells washed once with 200 μΙ Tyrode buffer. Cells were incubated in 100 μΙ Tyrode buffer containing increasing concentrations of test peptides, 100 μΜ IBMX, and 6 mM glucose for up 15 min at 37° C. The reaction was stopped by addition of 25 μ! 0.5 M HCl and incubated on ice for 60 min. The cAMP content was estimated using the FlashPIate® cAMP kit from Perkin-Elmer according to manufacturer Instructions. EC_W and relative efficacies compared to reference compounds (glucagon and GLP-1) were estimated by computer aided curve fitting.

Example 1: Synthesis of Compound 1 H-H-Aib-QGTFTSDYSKYLD-K(Hexadecanoy!-isoGlu)-RRAKDFIEWLLSA-NH2 (Compound 1 ) The peptide was synthesized on a CEM Uberty Peptide Synthesizer using TentaGel S Ram resin (1.04 g; 0.25 mmol/g) and Fmoc chemistry as described above using Fmoc-Phe-Thr(ip-Me,MePro)-OH and. Fmoc-Lys(Hexadecanoy!-isoGlu(tBu))-OH (Corden Pharma) was coupled manually using 396 mg dissolved in DMF/DCM (2:1,8 ml) with HATU (190 mg). The solution was added to the resin and then DlEA (86 μ!) was added. The resin was shaken gently for 4 hours and then washed with DMF (8x2 min).

The peptide was cleaved from the resin as described above. The crude peptide was purified on a Gemini column (5x25 cm; 10 pm; C18) with a 35 mt/min flow of a mixture of buffer A (0,1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 20% to 70% buffer B over 47 min, and fractions (9 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and iyophilised to give a white powder (88 mg; 95%). The mass was 3826.03 Da as determined by MS (Cale. 3826.05 Da).

Example 2: Activity at glucagon and GLP-1 receotors

Table 1. EC50 values for cAMP génération In HEK293 cells expressing GLP-1 receptor or Glucagon receptor

	EC50 (nM)
Compound	Glucagon receptor	GLP-1 receptor
1	0.15	0.12
2	0.35	0.36
4	0.79	1.31
7	0.06	0.06
8	0.20	0.20
g	0.10	0.05
10	0.06	0.47
11	0.09	0.15
12	0.14	0.06
14	0.19	0.12
15	0.42	0.06
16	0.11	0.06
17	0.05	0.07
18	0.09	0.09
21	0.21	0.08

Claims

1. A compound having the formula

R¹-X-Z-R² wherein

R¹ is H, Cm alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R²is OH orNH₂;

X Is a peptide which has the formula I:

X2 Is selected from Ser, D-Ser and Aib;

X3 Is selected from Gin, His and Pro;

X12 Is selected from Lys and Y

X16 is selected from Glu and Y;

X20 Is selected from Lys and Y;

X24 is selected from Glu and Y;

X27 is selected from Leu and Y;

X28 Is selected from Ser and Y or Is absent;

X29 is Ala or absent;

wherein at least one of X12, X16, X17, X20, X27 and X28 Is Y;

wherein each residue Y is independently selected from Lys, Cys and Om;

(i) Z¹, wherein Z¹1s a lipophilie moiety conjugated directly to the side chain of Y; or (ii) Z’Z³, wherein Z¹ is a lipophilie moiety, Z³ is a spacer, and Z¹ is conjugated to the side chain of Y via Z³;

and Z is absent or is a sequence of 1-20 amino add units Independentiy selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Om;

or a pharmaceutically acceptable sait thereof.

2. A compound according to daim 1 wherein X has the formula la:

His-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-X16-Arg-Arg-Aia-X20-Asp-Phe-ileX24-Trp-Leu-X27-X28-Ala (la) wherein

X2 is selected from Ser, D-Ser and Aib;

X16 Is selected from Glu and Y;

X20 is selected from Lys and Y;

X24 is selected from Glu and Y;

X27 is selected from Leu and Y; and

X28 is selected from Ser and Y.

3. A compound according to daim 1 or daim 2 wherein X has the sequence:

H-Aib-QGTFTSDYSKYLDKRRAKDFIEWLLSA;

H-Alb-QGTFTSDYSKYLDERRAKDFIEWLLSA:

H-Aib-QGTFTSDYSKYLDERRAKDFIKWLLSA;

HSQGTFTSDYSKYLDERRAKDFIKWLLSA;

H-Aib-QGTFTSDYSKYLDERRAKDFIEWLKSA; or

H-Aib-QGTFTSDYSKYLDERRAKDFIEWLLKA.

4. A compound according to claim 3 wherein X is:

H-Aib-QGTFTSDYSKYLDK*RRAKDFIEWLLSA;

H-Aib-QGTFTSDYSKYLDERRAK*DFIEWLLSA;

H-Aib-QGTFTSDYSKYLDERRAKDFIK*WLLSA;

HSQGTFTSDYSKYLDERRAKDFIK*WLLSA;

H-Aib-QGTFTSDYSKYLDERRAKDFIEWLK*SA; or

H-Aib-QGTFTSDYSKYLDERRAKDFIEWLLK‘A;

5. A compound having the formula

R¹-X-Z-R² wherein

R¹ Es H, C_M alkyî, acetyl, formyl, benzoyl or trifluoroacetyl;

R’isOH orNH₂;

X is a peptide which has the formula II:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Arg-Ala-X20-Asp-Phe-lleX24-Trp-Leu-X27-X28-X29 (II) wherein

X2 is selected from Ser, D-Ser and Aib;

X3 Is selected from Gin, His and Pro;

X12 Is selected from Arg, Lys and Y;

X16 Is selected from Glu and Y;

X17 Is selected from Arg and Y;

X20 Is selected from Lys, Arg and Y;

X24 Is selected from Glu and Y;

X27 Is selected from Leu and Y;

X28 Is selected from Ser and Y or absent;

X29 Is Ala or absent;

wherein X12 and/or X20 Is Arg;

wherein at least one of X12, X16, X17, X20, X24, X27 and X28 Is Y;

wherein each residue Y Is independently selected from Lys, Cys and Om;

wherein the side chain of at least one amino add residue Y is conjugated to a lipophilie substituent having the formula:

(i) Z¹, wherein Z¹ Is a lipophilie moiety conjugated directly to the side chain of Y; or (ii) Z’Z², wherein Z¹ is a lipophilie moiety, Z² Is a spacer, and Z¹ Is conjugated to the side chain of Y via Z²;

and Z is absent or Is a sequence of 1-20 amino acid unlts Independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Om;

or a pharmaceutically acceptable sait thereof.

6. A compound according to daim 5 wherein X12 ls Arg.

7. A compound according to claim 5 wherein X has the formula lia:

X2 ls selected from Ser, D-Ser and Aîb;

X3 ls selected from Gin, His and Pro;

X16 is selected from Glu and Y;

X17 ls selected from Arg and Y;

X20 ls selected from Arg and Lys; and

X24 ls selected from Glu and Y.

8. A compound according to claim 7 wherein X has the formula llb:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Arg-Tyr-Leu-Asp-Giu-X17-Arg-Aia-Arg-Asp-Phe-lieGlu-Trp-Leu-Leu-Ser-Aia (llb) wherein

X2 ls selected from Ser, D-Ser and Aib;

X3 ls selected from Gin, His and Pro; and

X17 ls Y.

9. A compound according to any one of daims 5 to 8 wherein X has the sequence:

HSQGTFTSDYSRYLDEKRARDFIEWLLSA;

H-DSer-QGTFTSDYSRYLDEKRARDFi EWLLSA;

H-Aib-QGTFTSDYSRYLDEKRARDFIEWLLSA;

HSHGTFTSDYSRYLDEKRARDFIEWLLSA;

H-DSer-HGTFTSDYSRYLDEKRARDFIEWLLSA; H-Aib-GTFTSDYSRYLDEKRARDFIEWLLSA;

HSPGTFTSDYSRYLDEKRARDFIEWLLSA; H-DSer-PGTFTSDYSRYLDEKRARDFIEWLLSA; H-Aib-PGTFTSDYSRYLDEKRARDFIEWLLSA; or H-Aib-QGTFTSDYSRYLDEKRAKDFIEWLLSA.

10. A compound according to claim 9 wherein X is:

HSQGTFTSDYSRYLDEK*RARDFIEWLLSA;

H-DSer-QGTFTSDYSRYLDEK*RARDFIEWLLSA; H-Alb-QGTFTSDYSRYLDEK*RARDFIEWLLSA; HSHGTFTSDYSRYLDEKRARDFIEWLLSA; H-DSer-HGTFTSDYSRYLDEK*RARDFIEWLLSA; H-Aib-GTFTSDYSRYLDEKRARDFIEWLLSA; HSPGTFTSDYSRYLDEKRARDFIEWLLSA; H-DSer-PGTFTSDYSRYLDEK*RARDFIEWLLSA; H-Aib-PGTFTSDYSRYLDEK*RARDFIEWLLSA; or H-Aib-QGTFTSDYSRYLDEKRAKDFIEWLLSA;

11. A compound having the formula

R¹-X-Z-R² wherein

R¹ Is H, Cm alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² Is OH orNH₂;

X Is a peptide which has the formula III:

X2 is selected from Ser, D-Ser and Aïb;

X3 Is selected from Gin, His and Pro;

X12 is selected from Lys and Y

X16 is selected from Glu and Y;

X17 is selected from Arg and Y;

X20 is selected from Lys and Y;

X24 Is selected from Glu and Y;

X27 is selected from Leu and Y;

X28 is selected from Ser and Y or is absent;

X29 Is Ala or absent;

wherein X3 is His or Pro when X2 is Ser or Aib, and X2 is D-Ser when X3 Is Gin;

wherein at least one of X12, X16, X17, X20, X24, X27 and X28 is Y;

wherein each residue Y is independently selected from Lys, Cys and Om;

wherein the side chain of at least one amino 8cld residue Y of X is conjugated to a iïpophiiïc substituent having the formula:

(i) Z¹, wherein Z¹ Is a lipophilie moiety conjugated directly to the side chain of Y; or (ii) Z¹Z², wherein Z¹ Is a lipophilie moiety, Z² Is a spacer, and Z¹ is conjugated to the side chain of Y via Z²;

or a pharmaceutically acceptable sait thereof.

12. A compound according to claim 11 wherein X has the formula Ilia:

X2 is selected from Ser, D-Ser and Aib;

X3 is selected from Gin, His and Pro;

X12 is selected from Lys and Y

X16 is selected from Glu and Y;

X17 is selected from Arg and Y;

X20 Is selected from Lys and Y; and

X24 is selected from Glu and Y.

13. A compound according to claim 12 wherein X has the formula lllb:

His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-X17-Arg-Ala-Lys-Asp-Phe-ileGlu-Trp-Leu-Leu-Ser-Aia (ülb) wherein

X2 is selected from Ser, D-Ser and Aib;

X3 is selected from Gin, His and Pro; and

X17ls Y.

14. A compound according to any one of claims 11 to 13 wherein X has the sequence:

H-DSer-QGTFTSDYSKYLDEKRAKDFiEWLLSA;

HSHGTFTSDYSKYLDEKRAKDFIEWLLSA;

H-DSer-HGTFTSDYSKYLDEKRAKDFIEWLLSA;

HSPGTFTSDYSKYLDEKRAKDFIEWLLSA; or H-DSer-PGTFTSDYSKYLDEKRAKDFIEWLLSA.

15. A compound according to claim 14 wherein X Is:

H-DSer-QGTFTSDYSKYLDEKRAKDFIEWLLSA;

HSHGTFTSDYSKYLDEKRAKDFIEWLLSA;

H-DSer-HGTFTSDYSKYLDEKRAKDFIEWLLSA;

HSPGTFTSDYSKYLDEKRAKDFIEWLLSA; or

H-DSer-PGTFTSDYSKYLDEKRAKDFIEWLLSA;

wherein K indicates a Lys residue to which the lipophilie substituent is conjugated.

16. A compound according to any one of the preceding claims wherein peptide X contains only one residue Y.

17. A compound according to any one of the preceding claims wherein the or each residue Y is Lys-

18. A compound according to any one of the preceding claims wherein Z is selected from Lys₃, Lys₄, Lys₅. Lyse and Lys₇.

19. A compound according to any one of claims 1 to 17 wherein Z 1s absent.

20. A compound according to any one of the preceding claims wherein the or each Z¹comprises a hexadecanoyl or octadecanoyl moiety.

21. A compound according to claim 20 wherein the or each lipophilie substituent Is hexadecanoyl-lsoGlu or octadecanoyl-iso-Giu.

22. A compound according to any one of the preceding claims wherein R¹ is H.

23. A compound according to any one of the preceding claims wherein R² is NH₂.

24. A compound according to any one of the preceding claims wherein one or more of the amino acid side chains in the compound Is conjugated to a polymeric moiety.

25. A compound according to claim 24 wherein one or more of the amino acid side chains in peptide X Is conjugated to a polymeric moiety.

26. A compound according to claim 1 which Is:

H-H-Aib-QGTFTSDYSKYLD-K(Hexadecanoyl-isoGlu)-RRAKDFIEWLLSA-NH₂ [Compound 1]; H-H-Aib-QGTFTSDYSKYLDERRA-K(Hexadecanoyl-isoGlu)-DFIEWLLSA-NH₂ [Compound 2]; H-H-Aib-QGTFTSDYSKYLDERRAKDFI-K(Hexadecanoyl-isoGlu)-WLLSA-NH₂ [Compound 3]; H-HSQGTFTSDYSKYLDERRAKDFI-K(Hexadecanoyl-isoGlu)-WLLSA-NH₂ [Compound 4];

H-H-Aib-QGTFTSDYSKYLDERRAKDFiEWL·K(Hexadecanoyl-lsoGlu)-SA-NH₂ [Compound 5J: or H-H-Aib-QGTFTSDYSKYLDERRAKDFIEWLL-K(Hexadecanoyl-lsoGlu)-A-NH₂ [Compound 6]; or a pharmaceutically acceptable sait thereof.

27. A compound according to claim 5 which is:

H-HSQGTFTSDYSRYLDE-K(Hexadecanoyl-isoGiu)-RARDFiEWLLSA-NH₂ [Compound η;

H-H-DSer-QGTFTSDYSRYLDE-K(Hexadecanoyi-lsoGlu)-RARDFIEWLLSA-NH₂ [Compound 8]; H-H-Aib-QGTFTSDYSRYLDE-K(Hexadecanoy1-lsoGlu)-RARDFIEWLLSA-NH₂ [Compound 9]; H-HSHGTFTSDYSRYLDE-K(HexadecanoyHsoGlu)-RARDFIEWLLSA-NH₂ [Compound 10]; H-H-DSer-HGTFTSDYSRYLDE-K(Hexadecanoyl-lsoGlu)-RARDFIEWLLSA-NH₂ [Compound 11]; H-H-Aib-HGTFTSDYSRYLDE-K(Hexadecanoyi-lsoGlu)-RARDFIEWLLSA-NH₂ [Compound 12]; H-HSPGTFTSDYSRYLDE-K(Hexadecanoyl-lsoGlu)-RARDFIEWLLSA-NH₂ [Compound 13]; H-H-DSer-PGTFTSDYSRYLDE-K(Hexadecanoyi-isoGlu)-RARDFIEWLLSA-NH₂ [Compound 14]; H-H-Aib-PGTFTSDYSRYLDE-K(Hexadecanoy1-lsoGlu)-RARDFIEWLLSA’NH₂ [Compound 15] or H-H-Aib-QGTFTSDYSRYLDE-K(Hexadecanoyl-lsoGiu)-RAKDFIEWLLSA-NH₂ [Compound 16]; or a pharmaceutically acceptable sait thereof.

28. A compound according to claim 11 which Is:

H-H-DSer-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-RAKDFIEWLLSA-NH2 [Compound 17]; H-HSHGTFTSDYSKYLDE-K(Hexadecanoyl-lsoGlu)-RAKDFIEWLLSA-NH2 [Compound 18]; H-H-DSer-HGTFTSDYSKYl_DE-K(Hexadecanoyi-lsoGlu)-RAKDFIEWLLSA-NH2 [Compound 19]; H-HSPGTFTSDYSKYLDE-K(Hexadecanoyl-lsoGlu)-RAKDFIEWLLSA-NH2 [Compound 20]; or H-H-DSer-PGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-RAKDFIEWLLSA-NH2 [Compound 21]; or a pharmaceutically acceptable sait thereof.

29. A compound selected from the group: H-H-Aib-QGTFTSDYSKYLDE-K(Octadecanoy1-lsoGlu)-RAKDFIEWLLSA-NH₂ [Compound 22]; H-H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-RAKDFIEWLLSA-OH [Compound 23]; and H-H-Aib-QGTFTSDYSKYLDE-K(Octadecanoyl-lsoGlu)-RAKDFIEWLLSA-OH [Compound 24];

or a pharmaceutically acceptable sait thereof.

30. A composition comprising a compound according to any one of the preceding daims, or a sait or dérivative thereof, In admlxture with a carrier.

31. A composition according to claim 30 wherein the composition is a pharmaceutically acceptable composition, and the carrier is a pharmaceutically acceptable carrier.

32. An Isolated nucleic add encoding a peptide X-Z as defined ln any one of ciaims 1 to 19.

33. A vector comprising a nucleic add according to claim 32.

34. A host cell comprising a nucleic acid according to claim 32 or a vector according to daim

33.

35. A compound according to any one of daims 1 to 29 for use In a method of medical treatment.

36. A compound according to any one of daims 1 to 29 for use ln a method of preventing weight gain or promoting weight loss ln an Individual ln need thereof.

37. A compound according to any one of daims 1 to 29 for use in a method of lowering circuiating LDL levels, and/or increaslng HDL/LDL ratio in an individual in need thereof.

38. A compound according to any one of daims 1 to 29 for use In a method of treatment of a condition caused or characterised by excess body weight.

39. A compound according to any one of daims 1 to 29 for use in a method of prévention and/or treatment of obesity, morbid obesity, morbid obesity prior to surgery, obesity linked Inflammation, obesity linked gallbladder disease, obesity induced sleep apnea, type I diabètes, type 11 diabètes, gestational diabètes, metabolic syndrome, hypertension, atherogenic dyslipidemia, atherosderois, arteriosderosls, coronary heart disease, peripheral artery disease, stroke or microvascuiar disease ln an individual ln need thereof.

40. A compound according to any one of ciaims 35 to 39 wherein the compound Is administered as part of a combination therapy together with an agent for treatment of obesity, dyslipidemia, diabètes, or hypertension.

41. A compound according to claim 40, wherein the agent for treatment of obesity Is a glucagon-like peptide receptor 1 agonlst, peptide YY receptor agonist or analogue thereof, cannabinoid receptor 1 antagonist, lipase Inhibitor, melanocortin receptor 4 agonist, ormelanln concentrating hormone receptor 1 antagonist.

42. A compound according to claim 40 wherein the agent for treatment of hypertension Is an angiotensin-converting enzyme Inhibitor, angiotensin II receptor blocker, diuretic, beta-blocker, or calcium channel blocker.

43. A compound according to claim 40 wherein the agent for treatment of dyslipidaemia Is a statin, a fibrate, a niadn and/or a cholestérol absorbtîon inhibitor.

44. A compound according to daim 40 wherein the agent for treatment of diabètes Is metformin, a sulfonylurea, a glinide, a DPP-IV Inhibitor, a glitazone, a GLP-1 agonist, Insulin or an Insulin analogue.