OA16677A

OA16677A - Combination of acylated glucagon analogues with insulin analogues.

Info

Publication number: OA16677A
Application number: OA1201300296
Authority: OA
Inventors: Keld Fosgerau; Ditte Riber
Original assignee: Zealand Pharma A/S
Priority date: 2011-01-20
Filing date: 2012-01-20
Publication date: 2015-12-07

Abstract

The invention relates to methods for treating metabolic disorders, including diabetes by using a combination of an acylated glucagon analogue and an insulin analogue. The invention also features a kit that includes an acylated glucagon analogue and an insuline analogue.

Description

The présent invention relates to combinations of an acylated glucagon analogue with an insulin analogue and their medical use, for example, in the treatment of obesity and diabètes.

BACKGROUND OF THE INVENTION

Obesity and diabètes are globally increasing health problems and are associated with various diseases, particularly cardiovascular disease (CVD), obstructive sleep apnea, stroke, peripheral artery disease, microvascular complications and osteoarthritis.

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.

Cardiovascular disease accounts for about 50% of the mortality in people with diabètes and the morbidity and mortality rates relating to obesity and diabètes underscore the medical need for efficacious treatment options.

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-like peptide-2 (GLP-2), and oxyntomodulin (OXM). These molécules are involved in a wide variety of physiological functions, 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-Ala-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-lle-Ala and termed “intervening peptide Γ or IP-1 ; the full sequence of human oxyntomodulin is thus His-Ser-GIn-Gly-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 acid, C-terminally amidated peptide that corresponds to amino acids 98 to 127 of pre-proglucagon.

Glucagon helps maintaîn 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 addltional glucose by gluconeogenesis. This glucose is released into the bloodstream, preventing the development of hypoglycemia. Additionally, glucagon has been demonstrated to increase lipolysis and decrease body weight.

GLP-1 decreases elevated blood glucose levels by improving glucose-stimulated insulin sécrétion and promûtes weight loss chiefly through decreasing food intake.

Oxyntomodulin is released into the blood in response to food ingestion and in proportion to meal calorie content. The mechanism of action of oxyntomodulin is not well understood. In particular, it is not known whether the effects of the hormone are mediated exclusively through the glucagon receptor and the GLP-1 receptor, or through one or more as-yet unidentifïed receptors.

Other peptides hâve been shown to bind and activate both the glucagon and the GLP-1 receptor (Hjort et al, 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 2008/101017, WO 2008/152403, WO 2009/155257 and WO 2009/155258).

Stabilization of peptides has been shown to provide a better pharmacokinetic profile for several drugs. In particular addition of one or more polyethylene glycol (PEG) or acyl group has been shown to prolong half-life of peptides such as GLP-1 and other peptides with short plasma stability.

In WO 00/55184A1 and WO 00/55119 are disclosed methods for acylation of a range of peptides, in particular GLP-1. Madsen et al (J. Med. Chem. 2007, 50, 6126-6132) describe GLP-1 acylated at position 20 (Liraglutide) and provide data on its stability.

Stabilization of OXM by PEGylation and C-terminal acylation has also been shown to improve the pharmacokinetic profile of selected analogues in W02007/100535, W008/071972 and in Endocrinology 2009, 150(4), 1712-1721 by Druce, M R et al.

It has recently been shown that PEGylation of glucagon analogues has a significant effect on the pharmacokinetic profile of the tested compounds (W02008/101017) but also interfères with the potency of these compounds.

SUMMARY OF THE INVENTION

In an first aspect, the invention features a combination of compounds for use in a method of treatment, a use, and a method for preventing or reducing weight gain; promoting weight loss; improving circulating glucose levels, glucose tolérance or circulating cholestérol levels; lowering circulating LDL levels; increasing HDL/LDL ratio; or treating a condition caused or characterized by excess body weight (e.g., obesity, morbid obesity, obesity-linked inflammation, obesity-linked gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, pre-diabetes, insulin résistance, glucose intolérance, type 2 diabètes, type I diabètes, hypertension, atherogenic dyslipidaemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke or microvascular disease). The combination of compounds for use in a method of treatment, a use, and a method employs administering to a mammalian (e.g., human) subject (e.g„ having type I or type II diabètes) a combination of compounds including (a) a compound having the formula R¹-Z-R², where R’ is H, Ci.4 alkyl, acetyl, formyl, benzoyl, ortrifluoroacetyl; R² is OH or NH2; and Z is a peptide having the formula I: His-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-Phe-Val-X24-Trp-Leu-X27-X28-Ala-X30; (I), where X2 is selected from Aib and Ser; X12 is selected from Lys, Arg, or Leu; X16 is selected from Arg and X; X17 is selected from Arg and X; X20 is selected from Arg, His, and X; X21 is selected from Asp and Glu; X24 is selected from Ala and X; X27 is selected from Leu and X; X28 is selected from Arg and X; X30 is X or is absent; where at least one of X16, X17, X20, X24, X27, X28, and X30 is X; and where each residue X is independently selected from the group consisting of Glu, Lys, Ser, Cys, Dbu, Dpr, and Orn (e.g., Lys, Glu, and Cys); where the side chain of at least one residue X is conjugated to a lipophilie substituent having the formula (i) Z¹, where Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z’Z², where Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z²; and (b) an insulin analogue (e.g., insulin glulisine (Apidra™), insulin lispro (Humalog™), Degludec, LY2963016, LY2605541, pegylated insulin Lispro, insulin glargine (Lantus™, Glaritus, Basalin, Basalog, Glarvia, BIOD-620), insulin detemir (Levemir™) Humulin, Huminsulin, insulin isophane (Humulin N, Insulatard, Novolin N), insulin and insulin isophane (Humulin 70/30, Humulin 50/50, Mixtard 30, Actraphane™ HM), insulin degludec and insulin aspart (DegludecPlus/NN-5401), insulin aspart (Novolog), insulin aspart and insulin protamine (Novolog mix, Novolog mix 70/30), insulin (NN-1953, IN-105, HinsBet, Capsulin, Nasulin, Afrezza, ORMD-0801, SuliXen, Humulin R), insulin buccal (Oral-lyn) and hyaluronidase insulin (Analog-PH20)). The combination of (a) and (b) may be administered in amounts that together are effective. The component (a) and (b), respectively, may be administered within one month (e.g., within three, two, or one weeks; six, five, four, three, two, or one days; or 18,12, 8, 6, 4, 3, 2, or 1 hours) of each other. The combination of compounds for use in a method of treatment, a use, and a method may prevent or reduce weight gain, may promote weight loss, or may improve circulating glucose levels.

In certain embodiments, X16 is selected from Glu, Lys, and Ser; X17 is selected from Lys and Cys; X20 is selected from His, Lys, Arg, and Cys; X24 is selected from Lys, Glu, and Ala; X27 is selected from Leu and Lys; and/or X28 is selected from Ser, Arg, and Lys. The peptide of formula I may include one or more of the following combinations of residues: X2 is Aib and X17 is Lys; X2 is Aib and X17 is Cys; X2 is Aib and X20 is Cys; X2 is Aib and X28 is Lys; X12 is Arg and X17 is Lys; X12 is Leu and X17 is Lys; X12 is Lys and X20 is Lys; X12 is Lys and X17 is Lys; X16 is Lys and X17 is Lys; X16 is Ser and X17 is Lys; X17 is Lys and X20 is Lys; X17 is Lys and X21 is Asp; X17 is Lys and X24 is Glu; X17 is Lys and X27 is Leu; X17 is Lys and X27 is Lys; X17 is Lys and X28 is Ser; X17 is Lys and X28 is Arg; X20 is Lys and X27 is Leu; X21 is Asp and X27 is Leu; X2 is Aib, X12 is Lys, and X16 is Ser; X12 is Lys, X17 is Lys, and X16 is Ser; X12 is Arg, X17 is Lys, and X16 is Glu; X16 is Glu, X17 is Lys, and X20 is Lys; X16 is Ser, X21 is Asp, and X24 is Glu; X17 is Lys, X24 îs Glu, and X28 is Arg; X17 is Lys, X24 is Glu, and X28 is Lys; X17 is Lys, X27 is Leu, and X28 is Ser; X17 is Lys, X27 is Leu, and X28 is Arg; X20 is Lys, X24 is Glu, and X27 is Leu; X20 is Lys, X27 is Leu, and X28 is Ser; X20 is Lys, X27 is Leu, and X28 is Arg; X16 is Ser, X20 is His, X24 îs Glu, and X27 is Leu; X17 is Lys, X20 is His, X24 is Glu, and X28 is Ser; X17 is Lys, X20 is Lys, X24 is Glu, and X27 is Leu; or X17 is Cys, X20 îs Lys, X24 is Glu, and X27 is Leu. The peptide of formula I may contain only one amino acid of the type conjugated to the lipophilie substituent (e.g., only one Lys residue, only one Cys residue, or only one Glu residue, where the lipophilie substituent is conjugated to that residue). The peptide sequence of formula I may include one or more intramolecular bridges (e.g., a sait bridge or a lactam ring), for example, where the intramolecular bridge is formed between the side chains of two amino acid residues which are separated by three amino acids (e.g., between the side chains of residue pairs 16 and 20, 17 and 21, 20 and 24, or 24 and 28) in the linear amino acid sequence of formula I. The intramolecular bridge may involve a pair of residues selected from the group consisting of; X16 is Glu and X20 is Lys; X16 is Glu and X20 is Arg; X16 îs Lys and X20 is Glu; X16 is Arg and X20 is Glu; X17 is Arg and X21 is Glu; X17 is Lys and X21 is Glu; X17 is Arg and X21 is Asp; X17 is Lys and X21 is Asp;

X20 is Glu and X24 îs Lys; X20 is Glu and X24 is Arg; X20 is Lys and X24 is Glu; X20 is Arg and X24 is Glu; X24 is Glu and X28 is Lys; X24 îs Glu and X28 is Arg; X24 is Lys and X28 is Glu; and X24 is Arg and X28 is Glu.

In certain embodiments of any of the combinations of compounds for use in methods of treatment, uses, and methods described above, at least one of X16, X17, X20, and Χ28 is conjugated to a lipophilie substituent. X30 may be absent or X30 may be présent and may be conjugated to a lipophilie substituent, for example, only one lipophilie substituent (e.g., at position 16,17, 20, 24, 27, 28 or 30; position 16, 17 or 20, or at position 17) or exactly two lipophilie substituents, e.g., each at one of positions 16,17, 20, 24, 27, 28, and 30 (e.g., at positions 16 and 17, 16 and 20, 16 and 24, 16 and 27, 16 and 28, 16 and 30,17 and 20,17 and 24,17 and 27,17 and 28, 17 and 30, 20 and 24, 20 and 27, 20 and 28, 20 and 30, 24 and 27, 24 and 28, 24 and 30, 27 and 28, 27 and 30, or 28 and 30).

In certain embodiments of any of the combinations of compounds for use in methods of treatment, uses, and methods described above, the compound has the formula: R¹-Z-R², where R¹ is H, CM alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl; R² is OH or NH2; and Z is a peptide having the formula lia: His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20X21-Phe-Val-X24-Trp-Leu-Leu-X28-Ala; (lia); where X12 is selected from Lys, Arg, and Leu; X16 is selected from Ser and X; X17 is X; X20 is selected from His and X; X21 is selected from Asp and Glu; X24 is selected from Ala and Glu; X28 is selected from Ser, Lys, and Arg; and where each residue X is independently selected from the group consisting of Glu, Lys, and Cys; where the side chain of at least one residue X is conjugated to a lipophilie substituent having the formula (i) Z¹, where Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z’Z², where Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z².

In other embodiments of the above combinations of compounds for use in methods of treatment, uses, and methods, the compound has the formula R’-Z-R², where R¹ is H, CM alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl; R² is OH or NH2; and Z is a peptide having the formula llb: HisSer-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21 -Phe-ValX24-Trp-Leu-Leu-X28-Ala; (llb); where X12 îs selected from Lys, Arg, and Leu; X16 is selected from Ser and X; X17 is X; X20 is selected from His and X; X21 is selected from Asp and Glu; X24 is selected from Ala and Glu; X28 is selected from Ser, Lys, and Arg; and where each residue X is independently selected from the group consisting of Glu, Lys, and Cys; where the side chain of at least one residue X is conjugated to a lipophilie substituent having the formula (i) Z¹, where Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z¹Z², where Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z².

In partîcular embodiments, the compound has the formula R’-Z-R², where R¹ is H, Cm alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl; R² is OH or NH₂; and Z is a peptide having the formula Ilia:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-X20-X21-PheVal-X24-Trp-Leu-Leu-X28-Ala; (Ilia); where X12 is selected from Lys And Arg; X17 is X; X20 is selected from His and X; X21 is selected from Asp and Glu; X24 is selected from Ala and Glu; X28 is selected from Ser, Lys, and Arg; and where each residue X is independently selected from Glu, Lys, and Cys; where the side chain of at least one residue X is conjugated to a lipophilie substituent having the formula (I) Z¹, where Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z’Z², where Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z².

In particular embodiments, the compound has the formula R’-Z-R², where R’ is H, C,.4 alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl; R² is OH or NH₂; and Z is a peptide having the formula 11Ib: HisSer-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-X20-X21 -Phe-ValX24-Trp-Leu-Leu-X28-Ala; (lllb); where X12 Is selected from Lys and Arg; X17 is X; X20 is selected from His and X; X21 is selected from Asp and Glu; X24 is selected from Ala and Glu; X28 is selected from Ser, Lys, and Arg; and where each residue X is independently selected from Glu, Lys, and Cys; where the side chain of at least one residue X is conjugated to a lipophilie substituent having the formula: (i) Z¹, where Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z’Z², where Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z².

In other particular embodiments, the compound has the formula: R’-Z-R², where R¹ is H, Cm alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl; R² is OH or NH₂; and Z is a peptide having the formula IVa: His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-His-X21Phe-Val-X24-Trp-Leu-Leu-X28-Ala; (IVa); where X12 is selected from Lys and Arg; X17 is X; X21 is selected from Asp and Glu; X24 is selected from Ala and Glu; X28 is selected from Ser, Lys, and Arg; where X is selected from the group consisting of Glu, Lys, and Cys; and where the side chain of X is conjugated to a lipophilie substituent having the formula (i) Z¹, where Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z¹Z², where Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z².

In still other particular embodiments, the compound has the formula R’-Z-R², where R¹ is H, Cm alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl; R² is OH or NH₂; and Z is a peptide having the formula IVb: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-Ser-X17-Ala-Ala-HisX21-Phe-Val-X24-Trp-Leu-Leu-X28-Ala; (IVb); where X12 is selected from Lys and Arg; X17 îs X; X21 is selected from Asp and Glu; X24 is selected from Ala and Glu; X28 is selected from Ser, Lys, and Arg; where X is selected from the group consisting of Glu, Lys, and Cys; and where the side chain of X is conjugated to a lipophilie substituent having the formula (i) Z¹, where Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z¹Z², where Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z².

In any of the above combinations of compounds for use in methods of treatment, uses, and methods, the peptide Z may hâve the sequence HSQGTFTSDYSKYLDSKAAHDFVEWLLRA; HSQGTFTSDYSKYLDKKAAHDFVEWLLRA;

HSQGTFTSDYSKYLDSKAAKDFVEWLLRA;

HSQGTFTSDYSKYLDSKAAHDFVEWLKRA;

HSQGTFTSDYSKYLDSKAAHDFVEWLLKA;

HSQGTFTSDYSRYLDSKAAHDFVEWLLRA;

HSQGTFTSDYSLYLDSKAAHDFVEWLLRA;

HSQGTFTSDYSKYLDSKAAHDFVEWLLRAK; HSQGTFTSDYSKYLDSKAAHDFVEWLLSAK;

HSQGTFTSDYSKYLDSKAAHDFVEWLKSA;

HSQGTFTSDYSKYLDSKAAHDFVKWLLRA;

HSQGTFTSDYSKYLDSCAAHDFVEWLLRA;

HSQGTFTSDYSKYLDSCAAHDFVEWLLSA;

HSQGTFTSDYSKYLDSKAACDFVEWLLRA;

HSQGTFTSDYSKYLDKSAAHDFVEWLLRA; H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLSA;

H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLSAK; H-Aib-QGTFTSDYSKYLDSKAARDFVAWLLRA; H-Aib-QGTFTSDYSKYLDSKAAKDFVAWLLRA; H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLRA; H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLKA;

H-Aîb-QGTFTSDYSKYLDSKAAKDFVAWLLSA; H-Aib-QGTFTSDYSKYLDSKAAHDFVAWLLKA; H-Aib-QGTFTSDYSKYLDKKAAHDFVAWLLRA;

H-Aib-QGTFTSDYSRYLDSKAAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDSKAAHDFVKWLLSA;

H-Aib-QGTFTSDYSLYLDSKAAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDSCAAHDFVEWLLSA;

H-Aib-QGTFTSDYSKYLDSKAACDFVEWLLRA; H-Aib-QGTFTSDYSKYLDK()KAAE()DFVEWLLRA·, H-Aib-QGTFTSDYSKYLDSKAAHDFVE()WLLK()A;

H-Aib-QGTFTSDYSKYLDSKAAK()DFVE()WLLRA; H-Aib-QGTFTSDYSKYLDSK()AAHE()FVEWLLKA; or H-Aib-QGTFTSDYSKYLDSK()AAKE()FVEWLLRA.

In other embodiments, the peptide Z has the formula HSQGTFTSDYSKYLDS-K*-AAHDFVEWLLRA; HSQGTFTSDYSKYLD-K*-KAAHDFVEWLLRA; HSQGTFTSDYSKYLDSKAA-K*-DFVEWLLRA; HSQGTFTSDYSKYLDSKAAHDFVEWL-K‘-RA; HSQGTFTSDYSKYLDSKAAHDFVEWLL-K*-A; HSQGTFTSDYSRYLDS-K*-AAHDFVEWLLRA; HSQGTFTSDYSLYLDS-K*-AAHDFVEWLLRA; HSQGTFTSDYSKYLDSKAAHDFVEWLLRA-K*; HSQGTFTSDYSKYLDSKAAHDFVEWLLSA-K*; HSQGTFTSDYSKYLDSKAAHDFVEWL-K*-SA; HSQGTFTSDYSKYLDSKAAHDFV-K*-WLLRA; HSQGTFTSDYSKYLDS-C*-AAHDFVEWLLRA; HSQGTFTSDYSKYLDS-C*-AAHDFVEWLLSA; HSQGTFTSDYSKYLDSKAA-C*-DFVEWLLRA; HSQGTFTSDYSKYLD-K*-SAAHDFVEWLLRA; H-Aib-QGTFTSDYSKYLDS-K*-AAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLLSA-K·; H-Aib-QGTFTSDYSKYLDS-K*-AARDF\/AWLLRA; H-Aib-QGTFTSDYSKYLDSKAA-K*-DFVAWLLRA; H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLL-K*-A; H-Aib-QGTFTSDYSKYLDS-K*-AAHDFVEWLLRA; H-Aib-QGTFTSDYSKYLDS-K*-AAHDFVEWLLKA; H-Aib-QGTFTSDYSKYLDSKAA-K*-DFVAWLLSA; H-Aib-QGTFTSDYSKYLDSKAAHDFVAWLL-K*-A; H-Aib-QGTFTSDYSKYLD-K*-KAAHDFVAWLLRA; H-Aib-QGTFTSDYSRYLDS-K*-AAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDSKAAHDFV-K*-WLLSA; H-Aib-QGTFTSDYSLYLDS-K*-AAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDS-C*-AAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDSKAA-C*-DFVEWLLRA; H-Aib-QGTFTSDYSKYLD-S*-KAAHDFVEWLLSA;

H-Aib-QGTFTSDYSKYLDK()K*AAE()DFVEWLLRA; H-Aib-QGTFTSDYSKYLDSK*AAHDFVE()WLLK()A; H-Aib-QGTFTSDYSKYLDSK*AAK()DFVE()WLLRA; H-Aib-QGTFTSDYSKYLDSK()AAHE()FVEWLLK*A; or H-Aîb-QGTFTSDYSKYLDSK()AAK*E()FVEWLLRA, where indicates the position of a lipophilie substituent.

In any of the above combinations of compounds for use in methods of treatment, uses, and methods, Z¹ may include a hydrocarbon chain having 10 to 24 C atoms, 10 to 22 C atoms, or 10 to 20 C atoms (e.g., a dodecanoyl, 2-butyloctanoyl, tetradecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, or eicosanoyl moiety) and/or Z² may be or may include one or more amino acid residues, for example, a γ-Glu, Glu, β-Ala or ε-Lys residue, or a 3-aminopropanoyl, 4aminobutanoyl, 8-aminooctanoyl, or 8-amino-3,6-dioxaoctanoyl moiety (e.g., where the lipophilie substituent is selected from the group consisting of dodecanoyl-y-Glu, hexadecanoly- γ-Glu, hexadecanoyl-Glu, hexadecanoyl-[3-aminopropanoyl], hexadecanoyl-[8-aminooctanoyl], hexadecanoyl-e-Lys, 2-butyloctanoyl- γ-Glu, octadecanoyl-Y-Glu, and hexadecanoyl-[4aminobutanoyl]). In particular embodiments, Z has the formula: HSQGTFTSDYSKYLD-K(Hexadecanoyl-Y-Glu)-KAAHDFVEWLLRA; HSQGTFTSDYSKYLDSKAAHDFVEWL-K(Hexadecanoyl-Y-Glu)-RA; HSQGTFTSDYSKYLDSKAA-K(Hexadecanoyl-Y-Glu)-DFVEWLLRA; HSQGTFTSDYSKYLDSKAAHDFVEWLL-K(Hexadecanoyl-Y-Glu)-A; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-Y-Glu)-AAHDFVEWLLRA; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-Y-Glu)-AARDFVAWLLRA;

H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-Y-Glu)-AAHDFVEWLLSA (Compound X); H-Aib-QGTFTSDYSKYLDSKAAHDFVEWLL-K(Hexadecanoyl-Y-Glu)-A; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-Y-Glu)-AAHDFVEWLLKA; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-Y-Glu)-AAHDFVE()WLLK()A; HSQGTFTSDYSKYLDS-K(Hexadecanoyl-Y-Glu)-AAHDFVEWLLRA; H-AibQGTFTSDYSKYLDSKAA-K(Hexadecanoyl-Y-Glu)-DFVAWLLRA;

H-Aib-QGTFTSDYSKYLDS-K(Dodecanoyl-Y-Glu)-AAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[3-aminopropanoyl])-AAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[8-aminooctanoyl])-AAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-c-Lys)-AAHDFVEWLLSA;

HSQGTFTSDYSKYLDS-K(Hexadecanoyl)-AAHDFVEWLLSA; HSQGTFTSDYSKYLDS-K(Octadecanoyl- Y-Glu)-AAHDFVEWLLSA; HSQGTFTSDYSKYLDS-K([2-Butyloctanoyl]-Y-Glu)-AAHDFVEWLLSA;

HSQGTFTSDYSKYLDS-K(Hexadecanoyl-[4-Aminobutanoyl])-AAHDFVEWLLSA; HSQGTFTSDYSKYLDS-K(Octadecanoyl- y-Glu)-AAHDFVEWLLSA; HSQGTFTSDYSKYLDS-K(Hexadecanoyl-E)-AAHDFVEWLLSA;

H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl)-AAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDS-K(Octadecanoyl- y-GIu)-AAHDFVEWLLSA; H-AibQGTFTSDYSKYLDS-K{[2-Butyloctanoyl]-Y-Glu)-AAHDFVEWLLSA;

H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-[4-Aminobutanoyl])-AAHDFVEWLLSA; H-Aib'QGTFTSDYSKYLDS-K(Octadecanoyl- Y-Glu)-AAHDFVEWLLSA; or H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-E)-AAHDFVEWLLSA;

where residues marked “()” participate in an intramolecular bond.

In other particular embodiments, Z has the formula: H-Aib-QGTFTSDYS-K(Hexadecanoyl-isoGlu)-YLDSKAAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLD-K(Hexadecanoyl-isoGlu)-KAAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDSKAA-K(Hexadecanoyl-isoGlu)-DFVEWLLSA; H-Aib-QGTFTSDYSKYLDSKAAHDFV-K(Hexadecanoyl-isoGlu)-WLLSA; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoLys)-AARDFVAWLLRA; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAKDFVEWLLSA; H-Aib-QGTFTSDYSKYLDE-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHEFVEWLLSA; H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAEDFVEWLLSA; or H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLEA.

In another aspect, the invention features a combination of compounds for use in a method of treatment, a use, and a method for preventing or reducing weight gain; promoting weight loss; improving circulating glucose levels, glucose tolérance or circulating cholestérol levels; lowering circulating LDL levels; increasing HDL/LDL ratio; or treating a condition caused or characterized by excess body weight. The method includes administering to a mammalian (e.g., human) subject (e.g., having type 1 or type 2 diabètes) a combination of compounds including:

(a) a compound having the formula: R¹-Z-R², where R¹ is H, Cm aikyl, acetyl, formyl, benzoyl, or trifluoroacetyl; R² is OH or NH₂; and Z is a peptide having the formula V: His-Aib-GIn-Gly-Thr-PheThr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-X17-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-X28; (V), where: X17 is X; X28 is Ser or absent; where X is selected from the group consisting of Glu, Lys, and Cys; and where the side chain of X is conjugated to a lipophilie substituent having the formula (i) Z¹, where Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z¹Z^z, where Z¹ ts a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z²; and (b) an insulin analogue (e.g., insulin glulisîne (Apidra™), insulin lispro (Humalog™), Degludec, LY2963016, LY2605541, pegylated insulin Lispro, insulin glargine (Lantus™, Glaritus, Basalin, Basalog, Glarvia, BIOD-620), insulin detemir (Levemir™) Humulin, Huminsulin, insulin isophane (Humulin N, Insulatard, Novolin N), insulin and insulin isophane (Humulin 70/30, Humulin 50/50, Mixtard 30, Actraphane™ HM), insulin degludec and insulin aspart (DegludecPlus/NN-5401), insulin aspart (Novolog), insulin aspart and insulin protamine (Novolog mix, Novolog mix 70/30), insulin (NN-1953, IN-105, HinsBet, Capsulin, Nasulin, Afrezza, ORMD-0801, SuliXen, Humulin R), insulin buccal (Oral-lyn) and hyaluronidase insulin (Analog-PH20)). The combination of (a) and (b) may be administered in amounts that together are effective. The combination of (a) and (b) may be administered within one month (e.g., within three, two, or one weeks; six, five, four, three, two, or one days; or 18,12, 8, 6, 4, 3, 2, or 1 hours) of each other. The condition caused or characterized by excess body weight may be selected from the group consisting of obesity, morbid obesity, obesity-linked inflammation, obesity-linked gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, pre-diabetes, insulin résistance, glucose intolérance, type 2 diabètes, type I diabètes, hypertension, atherogenic dyslipidaemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, and microvascular disease. The combination of compounds for use in a method of treatment, a use, and a method may prevent or may reduce weight gain, may promote weight loss, and/or may improve circulating glucose levels. In certain embodiments, Z has the formula H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)AAHDFVEWLLS or H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLL.

In another aspect, the invention features a combination of compounds for use in a method of treatment, a use, and a method for preventing or reducing weight gain; promoting weight loss; improving circulating glucose levels, glucose tolérance or circulating cholestérol levels; lowering circulating LDL levels; increasing HDL/LDL ratio; or treating a condition caused or characterized by excess body weight, the method including administering to a mammalian (e.g., human) subject (e.g., having type 1 or type 2 diabètes) a combination of compounds including (a) a compound having the formula: R¹-Z-R², where R¹ is H, C,_4 alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl; R²îs OH or NH₂; and Z is a peptide having the formula VI: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-TyrSer-Lys-Tyr-Leu-Asp-Ser-X17-Ala-Ala-His-Asp'Phe-Val-Glu-Trp-Leu-Leu-Ser-Ala; (VI) where X17 is X; where X is selected from the group consisting of Glu, Lys, and Cys; and where the side chain of X is conjugated to a lipophilie substituent having the formula: (i) Z¹, where Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z’Z², where Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z²; and (b) an insulin analogue (e.g., insulin glulisine (Apidra™), insulin lispro (Humalog™), Degludec, LY2963016, LY2605541, pegylated insulin Lispro, insulin glargine (Lantus™, Glaritus, Basalin, Basalog, Glarvia, BIOD-620), insulin detemir (Levemir™) Humulin, Huminsulin, insulin isophane (Humulin N, Insulatard, Novolin N), insulin and insulin isophane (Humulin 70/30, Humulin 50/50, Mixtard 30, Actraphane™ HM), insulin degludec and insulin aspart (DegludecPlus/NN-5401), insulin aspart (Novolog), insulin aspart and insulin protamine (Novolog mix, Novolog mix 70/30), insulin (NN-1953, IN-105, HinsBet, Capsulin, Nasulin, Afrezza, ORMD-0801, SuliXen, Humulin R), insulin buccal (Oral-lyn) and hyaluronidase insulin (Analog-PH20)). The combination of (a) and (b) may be administered in amounts that together are effective. The combination of (a) and (b) may be administered within one month (e.g., within three, two, or one weeks; six, five, four, three, two, or one days; or 18,12, 8, 6, 4, 3, 2, or 1 hours) of each other. The condition caused or characterized by excess body weight is selected from the group consisting of obesity, morbid obesity, obesity-linked inflammation, obesity-linked gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, pre-diabetes, insulin résistance, glucose intolérance, type 2 diabètes, type I diabètes, hypertension, atherogenic dyslipidaemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, and microvascular disease. The combination of compounds for use in a method of treatment, a use, and a method may prevent or reduce weight gain, may promote weight loss, or may improve circulating glucose levels. In particular embodiments, Z has the formula: H-AibEGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA.

In a combination of compounds for use in a method of treatment, a use, and a method ofthe first aspect, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS-K(HexadecanoylisoGlu)-AAHDFVEWLLSA-NH₂ and insulin glargine; H-H-Aib-QGTFTSDYSKYLDSK(Hexadecanoyl-isoGlu)-AAHDF\/EWLLSA-NH₂ and insulin detemir; H-H-AibQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and glulisine (Apidra™); HH-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDF\/EWLLSA-NH₂ and insulin lispro (Humalog™); H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and degludec; H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and Actraphane HM;

H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and LY2963016; H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and LY2605541; or H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and pegylated insulin Lispro.

In a particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDSK(Hexadecanoyl-isoGIu)-AAHDFVEWLLSA-NH₂ and insulin glargine, and the disease being treated is type 2 diabètes. In another particular embodiment, the combination of (a) and (b) includes H-H'Aib-QGTFTSDYSKYLDS-KiHexadecanoyl'isoGlui-AAHDFVEWLLSA-NHî and insulin detemir, and the disease being treated is type 2 diabètes. In another particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)AAHDFVEWLLSA-NH₂ and glulisine (Apidra™), and the disease being treated is type 2 diabètes. In another particular embodiment, the combination of (a) and (b) includes H-H-AibQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and insulin lispro (Humalog™), and the disease being treated is type 2 diabètes. In another particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)AAHDFVEWLLSA-NH₂ and degludec, and the disease being treated is type 2 diabètes. In a particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDSK(Hexadecanoyl-isoGlu)-AAHDF\/EWLLSA-NH2 and Actraphane HM, and the disease being treated is type 2 diabètes. In another particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and LY2963016, and the disease being treated is type 2 diabètes. In another particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)AAHDFVEWLLSA-NH₂ and LY2605541, and the disease being treated is type 2 diabètes. In another particular embodiment, the combination of (a) and (b) includes H-H-AibQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and pegylated insulin Lispro, and the disease being treated is type 2 diabètes.

In a particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDSK(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and insulin glargine, and the administration results in weight loss (e.g., in an overweight or obese subject). In another particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)AAHDFVEWLLSA-NH₂ and insulin detemir, and the administration results in weight loss (e.g., in an overweight or obese subject). In another particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and glulisine (Apidra™), and the administration results in weight loss (e.g., in an overweight or obese subject). In another particular embodiment, the combination of (a) and (b) includes H-H-AibQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and insulin lispro (Humalog™), and the administration results in weight loss (e.g., in an overweight or obese subject). In another particular embodiment, the combination of (a) and (b) includes H-H-AibQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and degludec, and the administration results in weight loss (e.g., in an overweight or obese subject). In another particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS13

K(Hexadecanoyl-isoGlu)-AAHDF\/EWLLSA-NH₂ and Actraphane HM, and the administration results in weight loss (e.g., in an overweight or obese subject). In another particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)AAHDFVEWLLSA-NH₂ and LY2963016, and the administration results in weight loss (e.g., in an overweight or obese subject). In another particular embodiment, the combination of (a) and (b) includes H-H-Aib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and LY2605541, and the administration results in weight loss (e.g., in an overweight or obese subject). In another particular embodiment, the combination of (a) and (b) includes H-H-AibQGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂ and pegylated insulin Lispro, and the administration results in weight loss (e.g., in an overweight or obese subject).

In any of the above aspects, the combination of (a) and (b) are administered within one week, three days, two days, one day, 12 hours, or six hours of each other.

In a further aspect, the invention features a combination of compounds for use in a method of treatment, a use, and a method for preventing or reducing weight gain; promoting weight loss; improving circulating glucose levels, glucose tolérance or circulating cholestérol levels; lowering circulating LDL levels; increasing HDL/LDL ratio; or treating a condition caused or characterized by excess body weight in a mammaiian subject (e.g., having type 1 or type 2 diabètes) that is receiving an insulin analogue (e.g., insulin glulisine (Apidra™), insulin lispro (Humalog™), Degludec, LY2963016, LY2605541, pegylated insulin Lispro, insulin glargine (Lantus™, Glaritus, Basalin, Basalog, Glarvia, BIOD-620), insulin detemir (Levemir™) Humulin, Huminsulin, insulin isophane (Humulin N, Insulatard, Novolin N), insulin and insulin îsophane (Humulin 70/30, Humulin 50/50, Mixtard 30, Actraphane™ HM), insulin degludec and insulin aspart (DegludecPlus/NN-5401), insulin aspart (Novolog), insulin aspart and insulin protamine (Novolog mix, Novolog mix 70/30), insulin (NN-1953, IN-105, HinsBet, Capsulin, Nasulin, Afrezza, ORMD-0801, SuliXen, Humulin R), insulin buccal (Oral-lyn) and hyaluronidase insulin (Analog-PH20)), the method including administering to the subject a compound of the présent invention in an effective amount. The condition caused or characterized by excess body weight may be selected from the group consisting of obesity, morbid obesity, obesity-linked inflammation, obesity-linked galibladder disease, obesity-induced sleep apnea, metabolic syndrome, pre-diabetes, insulin résistance, glucose intolérance, type 2 diabètes, type I diabètes, hypertension, atherogenic dyslipidaemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, and microvascular disease. The combination of compounds for use in a method of treatment, a use, and a method may prevent or reduce weight gain, may promote weight loss, or may improve circulating glucose levels.

In any of the above aspects, the compound may be part of a composition including the compound, or a sait or dérivative thereof, in admixture with a carrier. The composition may be a pharmaceutically acceptable composition, and the carrier may be a pharmaceutically acceptable carrier. The compound may be administered in a dosage of 0.1 nmol/kg body weight to 1 pmol/kg body weight (e.g., 3 nmol/kg to 30 nmol/kg). The insulin analogue may be administered in a dosage of 0.02 U/kg to 20 U/kg (e.g., 0.1 U/kg to 0.3 U/kg or about 0.2 U/kg). The compound may be administered every other week, weekly, every other day, daily, twice daily, or three times daily. The insulin analogue may be administered weekly, every other day, daily, twice daily, or three times daily.

The combination of compounds may be administered in an amount sufficient to reduce food intake in the subject by at least 5%, 10%, 15%, 20%, 25%, 30%, or 50%. The combination of compounds may be administered in an amount sufficient to reduce the subject’s fasting blood glucose level by at least 1, 2, 3, 4, 5, 6, 8,10,11,12,15, or 20 mM. The combination of compounds may be administered in an amount sufficient to reduce the subject’s HbA1c level by at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, 1.5%, or 2.0%. The administration of the combination of compounds may resuit in a body weight réduction of at least 3%, 5%, 8%, 10%, 12%, 15% or 20% within 1 year of starting administration. The administration of the combination of compounds may resuit in a body weight réduction of at least 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10% or 15% within six months of administration. The administration of the combination of compounds may resuit in a body weight réduction of at least 0,5%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10% or 15% within three months of administration.

In any of the above aspects, the compound or insulin analogue may be administered subcutaneously, întravenously, intramuscularly, by inhalation, rectally, buccally, intraperitoneally, Intraarticularly, or orally. The subject may be a human.

In another aspect, the invention features a kit including (a) a compound as recited in any of the above aspects; and (b) an insulin analogue (e.g., insulin glulisine (Apidra™), insulin lispro (Humalog™), Degludec, LY2963016, LY2605541, pegylated insulin Lispro, insulin glargine (Lantus™, Glaritus, Basalin, Basalog, Glarvia, BIOD-620), insulin detemir (Levemir™) Humulin, Huminsulin, insulin isophane (Humulin N, Insulatard, Novolin N), insulin and insulin isophane (Humulin 70/30, Humulin 50/50, Mixtard 30, Actraphane™ HM), insulin degludec and insulin aspart (DegludecPlus/NN-5401), insulin aspart (Novolog), insulin aspart and insulin protamine (Novolog mix, Novolog mix 70/30), insulin (NN-1953, IN-105, HinsBet, Capsulin, Nasulin, Afrezza, ORMD

0801, SuliXen, Humulin R), insulin buccal (Oral-lyn) and hyaluronidase insulin (Analog-PH20)), optionally including (c) instructions for administering (a) and (b) to a mammalian subject in need of preventing or reducing weight gain; promoting weight loss; improving circulating glucose levels, glucose tolérance or circulating cholestérol levels; lowering circulating LDL levels; increasing HDL/LDL ratio; or treatment for a condition caused or characterized by excess body weight.

Embodiments of the présent invention will now be described by way of example and not limitation with reference to the accompanying figures. However, various further aspects and embodiments of the présent invention will be apparent to those skilled in the art in view of the présent disclosure.

“and/or” where used herein is to be taken as spécifiedisclosure of each ofthe two specified features or components with or without the other. For example “A and/or B is to be taken as spécifie disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictâtes otherwise, the descriptions and définitions ofthefeatures set out above are not limited to any particular aspect or embodiment of the invention and apply equally to ail aspects and embodiments which are described.

DESCRIPTION OF THE FIGURES

Figure 1. Effect of treatment of 21 days s.c. administration of Lantus, Levemir, Compound X (H-HAib-QGTFTSDYSKYLDS-K(Hexadecanoyl-isoGlu)-AAHDFVEWLLSA-NH₂) and combinations thereof on body weight change (g). Data are averages +/- SEM with n=9-11. Data are compared by 2-way ANOVA vs. vehicle, ***p<0.001.

Figure 2. Effect of 21 days s.c. administration of Lantus, Levemir, Compound X and combinations thereof on daily food intake and accumulated food intake and daily food intake. Data are averages +/- SEM with n=9-11. Data are compared by 2-way ANOVA vs. vehicle, ***p<0.001.

Figure 3. Effect of 21 days s.c. administration of Lantus, Levemir, Compound X and combinations thereof on daily water intake and accumulated water intake. Data are averages +/- SEM with n=911. Data are compared by 2-way ANOVA vs. vehicle, ***p<0.001

Figure 4. Effect of 21 days s.c. administration of Lantus, Levemir, Compound X and combinations thereof on delta-Blood Glucose (d-BG). Data are averages +/- SEM with n=9-11.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this spécification, the conventional one letter and three letter codes for naturally occurring amino acids are used, as well as generally accepted three letter codes for other amino acids, including Aib (α-aminoisobutyric acid), Orn (omithine), Dbu (2,4 diaminobutyric acid) and Dpr (2,3-diaminopropanoic acid).

Unless otherwise indicated, the L-isomeric forms of naturally occurring amino acids are reffered to.

The term “native glucagon refers to native human glucagon having the sequence H-His-Ser-GInGly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-GIn-Asp-Phe-Val-GIn-Trp-LeuMet-Asn-Thr-OH.

Unless otherwise indicated, the L-isomeric forms of naturally occurring amino acids are referred to. The peptide sequence of a compound employed according to the invention differs from that of native glucagon at least at positions 18, 20, 24, 27, 28 and 29. In addition, it may differ from that of native glucagon at one or more of positions 12, 16 and 17.

Native glucagon has Arg at position 18. The compound employed in accordance with the invention has the small hydrophobie residue Ala at position 18 which is believed to increase potency at both glucagon and GLP-1 receptors but particularly the GLP-1 receptor.

The residues at positions 27, 28 and 29 of native glucagon appear to provide significant selectivity for the glucagon receptor. The substitutions at these positions with respect to the native glucagon sequence, particularly the Ala at position 29, may increase potency at and/or selectivity for the GLP-1 receptor, potentially without significant réduction of potency at the glucagon receptor. Further examples which may be included in the compounds to be employed in the invention include Leu at position 27 and Arg at position 28. Furthermore, Arg at position 28 may be particularly preferred when there is a Glu at position 24 with which it can form an intramolecular bridge, since this may increase its effect on potency at the GLP-1 receptor.

Substitution of the naturally occurring Met residue at position 27 (e.g., with Leu, Lys or Glu) 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 Arg or Ser) also reduces the potential for deamidation in acidic solution, thereby increasing the chemical stability of the compounds.

Potency and/or selectivity at the GLP-1 receptor, potentially without significant loss of potency at the glucagon receptor, may also be increased by introducing residues that are likely to stabilise an alpha-helical structure in the C-terminal portion of the peptide. It may be désirable, but is not believed essential, for this helical portion of the molécule to hâve an amphipathtc character. Introduction of residues such as Leu at position 12 and/or Ala at position 24 may assist. Additionally or alternatively charged residues may be introduced at one or more of positions 16, 20, 24, and 28. Thus the residues of positions 24 and 28 may ail be charged, the residues at positions 20, 24, and 28 may ail be charged, or the residues at positions 16,20, 24, and 28 may ail be charged. For example, the residue at position 20 may be His or Arg, particularly His. The residue at position 24 may be Glu, Lys or Arg, particularly Glu. The residue at position 28 may be Arg, Introduction of an intramolecular bridge in this portion of the molécule, as discussed above, may also contribute to stabilising the helical character, e.g., between positions 24 and 28.

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, so increasing the chemical stability of the compounds.

A substitution relative to the native glucagon sequence at position 12 (i.e., of Arg or Leu) may increase potency at both receptors and/or selectivity at the GLP-1 receptor.

C-terminal truncation of the peptide does not reduce potency of both receptors and/or selectivity of the GLP-1 receptor. In particular, truncation of position 29 or truncation of both position 28 and 29 does not reduce the receptor potency to any of the two receptors.

The side chain of one or more of the residues designated X (i.e., positions 16, 17, 20, 24, 27 and 28, and/or 30 if présent) is conjugated to a lipophilie substituent. It will be appreciated that conjugation of the lipophilie substituent to a particular side chain may affect (e.g., reduce) certain of the benefits which the unconjugated side chain may provide at that position. The inventors hâve found that compounds of the invention provide a balance between the benefits of acylation and the benefits of particular substitutions relative to the native glucagon sequence.

Compositions employed in accordance with 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 bioavailability, 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, polymers, for example cellulose and dérivatives, polysaccharides, for example dextran and dérivatives, starch and dérivatives, poly(vinyl alcohol), acrylate and méthacrylate polymers, polylactic and polyglycolic acid and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermogelling Systems, for example block co-polymerîc 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 behavlour in lîpid-water Systems, polymeric micelles, multiple émulsions, self-emulsifying, self-microemulsifyïng, cyclodextrins and dérivatives thereof, and dendrimers.

Other groups hâve attempted to prolong the half life of GluGLP-1 dual agonist compounds by derivatisation with PEG (W02008/101017). However such derivatisation appears to be most effective when applied to the C-terminus of the molécule rather than in the central core of the peptide backbone, and potency of these compounds is still decreased compared to the corresponding unmodifîed peptide.

By contrast, the compounds employed in the présent invention retain high potency at both the glucagon and GLP-1 receptors while having significantly protracted pharmacokinetic profiles compared to the corresponding unmodifîed peptides.

Native glucagon has Ser at position 16. Substitution with Ala, Gly or Thr has been shown to reduce adenylate cyclase activation at the glucagon receptor significantly (Unson et al., Proc. Natl. Acad. Sci. 1994, 91,454-458). Hence, derivatisation with a lipophilie substituent at position 16 would not hâve been expected to yield compounds retarning potency at the glucagon receptor, as is surprisingly shown by the compounds described in this spécification. In W02008/101017 a negatively charged residue was found to be désirable at position 16 to minimise loss of potency.

The presence of basic amino acids at positions 17 and 18 is generally believed to be necessary for full glucagon receptor activation (Unson et al., J. Biol. Chem. 1998, 273, 10308-10312). The présent inventors hâve found that, when position 18 is alanine, substitution with a hydrophobie amino acid in position 17 can still yield a highly potent compound. Even compounds in which the amino acid in position 17 is derivatised with a lipophilie substituent retain almost full potency at both glucagon and GLP-1 receptors, as well as displaying a significantly protracted pharmacokinetic profile. This is so even when a lysine at position 17 is derivatised, converting the basic amine side chain into a neutral amide group.

The présent inventors hâve also found that compounds with acylation at position 20 are still highly active dual agonists, despite indications from other studies that substitution in position 20 should be a basic amino acid having a side chain of 4-6 atoms in length to enhance GLP-1 receptor activity compared to glucagon (W02008/101017). The compounds described herein retain both GLP-1 and glucagon receptor activity when position 20 is substituted with lysine and acylated.

Peptide synthesis

The peptide component of the compounds of the invention may be manufactured by standard solid or liquid phase synthetic methods, recombinant expression Systems, or any other suitable method. Thus the peptides 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 , isolation and purification of the final peptide product;

(b) expressing a nucleic acid construct that encodes the peptide in a host cell and recovering the expression product from the host cell culture; or (c) effecting cell-free in vitro expression of a nucleic acid construct that encodes the peptide and recovering the expression product;

or any combination of methods of (a), (b), and (c) to obtain fragments of the peptide, subsequently ligating the fragments to obtain the peptide, and recovering the peptide.

It may be preferred to synthesize the analogues of the invention by means of solid phase or liquid phase peptide synthesis. In this context, reference is given to WO 98/11125 and, amongst many others, Fields, GB et al., 2002, “Principles and practice of solid-phase peptide synthesis. In: Synthetic Peptides (2nd Edition) and the examples herein.

Lipophilie substituent

One or more of the amino acid side chains in the compound employed in the invention is conjugated to a lipophilie substituent Z¹. Without wishing to be bound by theory, it is thought that the lipophilie substituent binds albumin in the blood stream, thus shielding the compounds of the invention from enzymatic dégradation which can enhance the half-life of the compounds. It may also modulate the potency of the compound, e.g., with respect to the glucagon receptor and/or the GLP-1 receptor.

In certain embodiments, only one amino acid side chain is conjugated to a lipophilie substituent. In other embodiments, two amino acid side chains are each conjugated to a lipophilie substituent. In yet further embodiments, three or even more amino acid side chains are each conjugated to a lipophilie substituent. When a compound contains two or more lipophilie substituents, they may be the same or different.

The lipophilie substituent Z¹ may be covalently bonded to an atom in the amino acid side chain, or alternatively may be conjugated to the amino acid side chain by a spacer Z².

The term ‘‘conjugated is used here to describe the physical attachment of one identifiable chemical moiety to another, and the structural relationship between such moieties. It should not be taken to imply any particular method of synthesis.

The spacer Z², when présent, is used to provide a spacing between the compound and the lipophilie moiety.

The lipophilie substituent may be attached to the amino acid side chain or to the spacer via an ester, a sulphonyl ester, a thioester, an amide or a sulphonamide. Accordingly it will be understood that preferably the lipophilie substituent includes an acyl group, a sulphonyl group, an N atom, an O atom or an S atom which forms part of the ester, sulphonyl 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 may include a hydrocarbon chain having 10 to 24 C atoms, e.g. 10 to 22 C atoms, e.g. 10 to 20 C atoms. Preferably it has at least 11 C atoms, and preferably it has 18 C atoms or fewer. For example, the hydrocarbon chain may contain 12, 13, 14,15, 16,17 or 18 carbon atoms. The hydrocarbon chain may be linear or branched and may be saturated or unsaturated. From the discussion above it will be understood that the hydrocarbon chain is preferably substituted with a moiety which forms part of the attachment to the amino acid side chain or the spacer, for example an acyl group, a sulphonyl group, an N atom, an O atom or an S atom. Most preferably the hydrocarbon chain is substituted with acyl, and accordingly the hydrocarbon chain may be part of an alkanoyl group, for example a dodecanoyl, 2-butyloctanoyl, tetradecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl oreicosanoyl group.

As mentioned above, the lipophilie substituent Z¹ may be conjugated to the amino acid side chain by a spacer Z². 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 sulphonamïde. Accordingly, it may include two moieties independently selected from acyl, sulphonyl, an N atom, an O atom or an S atom. The spacer may consist of a linear Cmo hydrocarbon chain or more preferably a linear C^s hydrocarbon chain. Furthermore the spacer can be substituted with one or more substituents selected from alkyl, Ci.₆ alkyl amine, Ci.₆ alkyl hydroxy and C_v6 alkyl carboxy.

The spacer may be, for example, a residue of any naturally occurring or unnatural amino acid. For example, the spacer may be a residue of Gly, Pro, Ala, Val, Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gin, Asn, D-Glu, D-Glu, ε-Lys, Asp, Ser, Thr, Gaba, Aib, D-Ala (i.e. 3-aminopropanoyl), 4aminobutanoyl, 5-aminopentanoyl, 6-aminohexanoyl, 7-aminoheptanoyl, 8-aminooctanoyl, 9aminononanoyl, 10-aminodecanoyl or 8-amino-3,6-dioxaoctanoyl. In certain embodiments, the spacer is a residue of Glu, D-Glu, ε-Lys, D-Ala (i.e. 3-aminopropanoyl), 4-aminobutanoyl, 8aminooctanoyl or 8-amino-3,6-dioxaoctanoyl. In the présent invention, D-Glu and isoGlu are used interchangeably.

The amino acid side chain to which the lipophilie substituent is conjugated is a side chain of a Glu, Lys, Ser, Cys, Dbu, Dpr or Orn residue. For example it may be a side chain of a Lys, Glu or Cys residue. Where two or more side chains carry a lipophilie substituent, they may be independently selected from these residues. Thus the amino acid side chain includes an carboxy, hydroxyl, thiol, amide or amine group, for forming an ester, a sulphonyl ester, a thioester, an amide or a sulphonamïde with the spacer or lipophilie substituent.

An example of a lipophilie substituent comprising a lipophilie moiety Z¹ and spacer Z² is shown in the formula below:

Here, the side chain of a Lys residue from the peptide of formula I is covalently attached to an y-Glu spacer (Z²) via an amide lînkage. A hexadecanoyl group (Z¹) is covalently attached to the y-Glu spacer via an amide lînkage. This combination of lipophilie moiety and spacer, conjugated to a Lys residue, may be referred to by the short-hand notation K(Hexadecanoyl-y-Glu), e.g., when shown in formulae of spécifie compounds. γ-Glu can also be referred to as isoGlu, and a hexadecanoyl group as a palmitoyl group. Thus it will be apparent that the notation (Hexadecanoyl-y-Glu) is équivalent to the notations (isoGlu(Palm)) or (isoGlu(Palmitoyl)) as used for example in PCT/GB2008/004121.

The skilled person will be well aware of suitable techniques for preparing the compounds employed in the invention. For examples of suitable chemistry, see WO98/08871, WOOO/55184, WOOO/55119, Madsen et al (J. Med. Chem. 2007, 50, 6126-32), and Knudsen étal. 2000 (J. Med Chem. 43,1664-1669).

PEGylated and/or acylation hâve a short half-life (T%), which gives rise to burst increases of GluGLP-1 agonist concentrations. The glucagon receptor is thus being subjected to burst exposure to the glucagon agonism once (or twice) daily throughout the treatment period.

Without being bound to any theory repeated burst exposure of GluR to glucagon agonism seems to bring havoc to the lipid and free fatty acid trafficking between the liver and adipose tissue with the resuit that fat accumulâtes in the liver.

Constant exposure of GluR to glucagon agonism blocks accumulation of fat in the liver

It has thus been found, that repeated treatment with glucagon or short acting dual GluGLP-1 agonists give rise to enlarged liver due to fat and glycogen accumulation (Chan et al., 1984. Exp. Mol. Path. 40, 320-327).

Repeated treatment with long-actrng acylated dual GluGLP-1 agonists do not give rise to change in liver size (enlarged or shrunken) in normal weight subjects, but normalize liver lipid content (Day et al., 2009; Nat.Chem.Biol. 5, 749 - 57).

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. Similarly, 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 make use the human glucagon receptor (GlucagonR) having primary accession number Gl: 4503947 (NP_000151.1 ) and/or the human glucagon-like peptide 1 receptor (GLP-1 R) having primary accession number Gl:166795283 (NP_002053.3). (Where sequences of precursor proteins are referred to, it should of course be understood that assays may make use of the mature protein, lacking the signal sequence).

ECm values may be used as a numerical measure of agonist potency at a given receptor. An ΕΟ₅₀value is a measure of the concentration of a compound required to achieve half of that compound's maximal activity in a particular assay. Thus, for example, a compound having ΕΟ₅₀ [GLP-1 R] lower than the EC₅₀ [GLP-1 R] of native glucagon in a particular assay may be considered to hâve higher potency at the GLP-1 R than glucagon.

The compounds described in this spécification are typically Glu-GLP-1 dual agonists, i.e,, they are capable of stimulating cAMP formation at both the glucagon receptor and the GLP-1 R. The stimulation of each receptor can be measured in independent assays and afterwards compared to each other.

By comparing the EC₅₀ value for the glucagon receptor (EC50 [Glucagon-R]) with the ECso value for the GLP-1 receptor (ECæ [GLP-1 R]) for a given compound the relative glucagon selectivity (%) of that compound can be found:

Relative Glucagon-R selectivity [Compound] = (1/ECso [Glucagon-R])x100% / (I/EC50 [Glucagon-R] + 1/EC_So [GLP-1 R])

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

Relative GLP-1 R selectivity [Compound] = (1/ECm [GLP-1 R])x100% / (1/ECsq [Glucagon-R] + 1/EC_S0 [GLP-1 R])

A compound’s relative selectivity allows its effect on the GLP-1 or glucagon receptor to be compared directly to its effect on the other receptor. For example, 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 below, we hâve found the relative GLP-1 selectivity for human glucagon to be approximately 5%.

The compounds employed in the invention hâve a higher relative GLP-1 R selectivity than human glucagon. Thus, for a particular level of glucagon-R agonist activity, the compound will display a higher level of GLP-1 R 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 employed in this invention may hâve a lower EC_S0 [GLP-1 R] than human glucagon. The compounds may hâve a lower ECso [GLP-1 R] than glucagon while maintaîning 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.

It may be désirable that EC_So of any given compound for both the Glucagon-R and GLP-1 R should be less than 1 nM.

The compounds employed in the invention may hâve an ECso [Glucagon-R] that is less than twofold that of human glucagon. The compounds may hâve an EC₅₀ [Glucagon-R] that is less than two-fold that of human glucagon and hâve an EC» [GLP-1 R] 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-1 selectivity of the compounds may be greater than 5% and less than 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%.

Improvinq circulating glucose levels, glucose tolérance or circulating cholestérol levels Normal blood sugar levels fluctuate depending on duration after last meal. A normal blood glucose level range for fasting individuals should be below 100 mg/dl and their level should be below 130140 mg/dl or so around an hour after eating.

Ideally the fasting blood glucose levels should be around 90 mg/dl. Diabètes are diagnosed when fasting blood glucose levels are approaching 120 mg/dl or higher.

Blood sugar levels outside the normal range may be an indicator of a medical condition. A persistently high level is referred to as hyperglycemia; low levels are referred to as hypoglycemia. Diabètes mellitus is characterized by persistent hyperglycemia from any of several causes, and is the most prominent disease related to failure of blood sugar régulation. A temporarily elevated blood sugar level may also resuit from severe stress, such as trauma, stroke, myocardial infarction, surgery, or illness. Intake of alcohol causes an initial surge in blood sugar, and later tends to cause levels to fall. Also, certain drugs can increase or decrease glucose levels.

If blood sugar levels drop too low, a potentially fatal condition called hypoglycemia develops. Symptoms may include lethargy, impaired mental functioning; irritability; shaking, twitching, weakness in arm and leg muscles; pale complexion; sweating; paranoid or aggressive mentality and loss of consciousness. Brain damage is even possible.

If levels remain too high, appetite is suppressed over the short term. Long-term hyperglycémie causes many of the long-term health problems associated with diabètes, including eye, kidney, heart disease and nerve damage.

Type 1 diabètes is a lifelong condition that can be controlled with lifestyle adjustments and medical treatments. Keeping blood glucose levels under control can prevent or minimïze complications. Insulin treatment is one component of a diabètes treatment plan for people with type 1 diabètes.

Insulin treatment replaces or suppléments the body's own insulin, restoring normal or near-normal blood sugar levels. Many different types of insulin treatment can successfully control blood sugar levels: the best option dépends upon a variety of individual factors. With a little extra planning, people with diabètes who take insulin can lead a full life and keep their blood sugar under control.

The central problem for those requiring external insulin is picking the right dose of insulin and the right timing.

Physiological régulation of blood glucose, as in the ηοπ-diabetic, would be best. Increased blood glucose levels after a meal is a stimulus for prompt release of insulin from the pancréas. The increased insulin level causes glucose absorption and storage in cells, reduces glycogen to glucose conversion, reducing blood glucose levels, and so reducing insulin release. The resuit is that the blood glucose level rises somewhat after eating, and within an hour or so, returns to the normal ’fasting' level. Even the best dîabetic treatment with synthetic human insulin or even insulin analogs, however administered, falls far short of normal glucose control in the non-diabetic.

Complicating matters is that the composition of the food eaten affects intestinal absorption rates. Glucose from some foods is absorbed more (or less) rapidly than the same amount of glucose in other foods. In addition, fats and proteins cause delays in absorption of glucose from carbohydrates eaten at the same time.

It is a well known fact that insulin causes weight gain in patients with type 2 diabètes. Insulin is a hormone secreted by the pancréas in response to glucose intake usually in the diet. Its rôle is to drive glucose into the cells of the body where it is used as a source of energy (measured in calories). Insulin therefore pumps calories into cells. If this energy (glucose) is not used by the cells or is more than is needed, it is converted into an energy storage form known as fat. Because of these actions insulin is called an anabolic hormone.

The word “anabolic means building up tissue. If a person is using his or her muscles and is physically active, the extra energy is converted into new (larger and/or stronger) muscles rather than fat. In a sense, a person who is sedentary, not using his muscles, getting more calories than he needs and taking insulin is in the midst of a “perfect (metabolîc) storm that will resuit in weight gain. The issue of insulin causing weight gain has long been a troubling aspect of the treatment for type 2 diabètes. It is not a problem in type 1 diabètes where patients have virtualJy no circulating insulin and need to receive it from an external source.

In type 2 diabètes the physiology is quite different. Here the body does make insulin, but the tissues are “résistant to its effects, In fact, in the early stages of type 2 diabètes insulin levels can actually be high. This occurs because the tissues are résistant to insulin and higher insulin levels become necessary to drive sugar (glucose) into the cells and thereby drop the sugar level in the blood. The cause of insulin résistance is complex and is still a very active area of research. It appears that a certain type of fat tissue, fat that is contained in the abdomen (also called viscéral adipose tissue), produces certain hormones and other substances that together cause insulin résistance. This was a major surprise in medicine when it was discovered only 10 or 15 years ago. Prior to that fat tissue was considered to be “metabolically inert, which means that it was just a storage tissue and didn’t affect metabolism. This was very far from the truth and viscéral fat Is now considered to be very active and complex metabolically. It produces a host of hormones (for example leptin, ghrelin and adiponectin) and other factors (cytokines) that have major influences on metabolism.

The discovery that insulin résistance was the central “lésion in type 2 diabètes led to a whole area of research that resulted in linking type 2 diabètes to high blood pressure, truncal or abdominal obesity, abnormal blood lipids (elevated triglycérides and low HDL cholestérol) and high waist to hip ratio (the “apple” body type).

Using insulin to treat type 2 diabètes is problematic. The person with type 2 diabètes is usually overweight and circulating insulin levels may already be high. Adding additional insulin will certainly cause weight gain and this can actually make the insulin résistance worse. The usual justification is that using insulin will protect the remarning insulin-producing beta cells in the pancréas from having to work overtime. However, only a few months ago this issue was reviewed by one of the leading diabètes authorities in the world: Dr. Ralph DeFronzo. DeFronzo recently gave the prestigious Banting Lecture and it was published in the April 2009 issue of Diabètes. DeFronzo suggests that the American Diabètes Association guidelines for treatment of type 2 diabètes may be misguided and in need of révision.

Regarding insulin-induced weight gain, he notes that when insulin is added to the treatment regimen, “ali of these insulin-based add-on studies have been associated with a high incidence of 28 hypoglycemia [low blood sugar] and major weight gain (range 4.2-19.2 Ibs, mean 8.5 Ibs within 612 months or less)....Moreover it is unclear why one would initiate insulin before exenatide [a newer non-insulin drug] since insulin rarely decreases A1C to <7% and is associated with significant weight gain... (Diabètes, Journal ofthe American Diabètes Association, April 2009, vol 58(4), page 786).Other potentially serious side-effects and related long term complications often associated with insulin treatment are well known. In particular, risk of developing hypoglycemia, allergy, résistance, and edema and related insulin side effects are well known short and longerterm side-effects of insulin treatment.

Glu-GLP-1 dual agonists of the présent invention activâtes the GLP-1 receptor, a membrane-bound cell-surface receptor coupled to adenylyl cyclase by the stimulatory G-protein, Gs, in pancreatic beta cells. Glu-GLP-1 dual agonists of the présent invention increases intracellular cyclic AMP (cAMP), leading to insulin release in the presence of elevated glucose concentrations. This insulin sécrétion subsides as blood glucose concentrations decrease and approach euglycemia, Glu-GLP1 dual agonists of the présent invention also decreases glucagon sécrétion in a glucose-dependent manner. The mechanism of blood glucose lowering also involves a delay in gastric emptying. GLP-1 (7-37) has a half-life of 1.5-2 minutes due to dégradation by the ubiquitous endogenous enzymes, dipeptidyl peptidase IV (DPP-IV) and neutral endopeptidases (NEP). Unlike native GLP1, Glu-GLP-1 dual agonists ofthe présent invention are stable against metabolic dégradation by both peptidases and has a prolonged plasma half-life after subcutaneous administration. The pharmacokinetic profile of Glu-GLP-1 dual agonists of the présent invention, which makes them suitable for once daily administration, is a resuit of self-association that delays absorption, plasma protein binding and stability against metabolic dégradation by DPP-IV and NEP.

Combination of Glu-GLP-1 dual agonists of the présent invention with insulin may hâve advantages over current type2 diabètes thérapies:

. The combination acts in a glucose-dependent manner, meaning it will stimulate insulin sécrétion only when blood glucose levels are higher than normal. Consequently, it shows negligîble risk of hypoglycemia.

• The combination has the potential for inhibiting apoptosis and stimulating régénération of beta cells (seen in animal studies).

• The combination decreases appetite and maintains body weight, as shown in a head-tohead study versus glimepiride.

• The combination lowers blood triglycéride levels.

• The combination has only mild and transient side effects, mainly gastrointestinal

For treatment of type 2 diabètes condition and in partîcular late stage type 2 diabètes condition, use of Glu-GLP-1 dual agonists in combination with insulin may further improve e.g. normalize circulating glucose levels, glucose tolérance or circulating cholestérol levels.

In one embodiment, the présent invention is directed to treatment of diabètes melitus where a GluGLP-1 dual agonists of the présent invention is co-administred with an insulin to improve the circulating glucose levels, glucose tolérance or circulating cholestérol levels.

In another embodiment, the présent invention is directed to treatment of type 2 diabètes where a Glu-GLP-1 dual agonists of the présent invention is co-administred with an insulin to improve the circulating glucose levels, glucose tolérance or circulating cholestérol levels.

Insulin analogues

The methods, kits, and compounds of the invention may use any insulin analogue known in the art.

Such insulin analogues comprise wild type insulin molécules, preferably of human genetic origin, as well as those which are modified chemically, e.g. by the exchange of single amino acids and/or the addition of side chains and/or the coupling with one or more medium sized molécules or polymers. Such insulin analogues also comprise compositions of such non-modified or modified insulins with other chemical substances which make them apt e.g. for the incorporation into spécifie medical compositions and/or mixtures with other insulin analogues.

In the context of this invention, human wildytype insulin is preferably produced recombinantly, which technique is per se known to the person skilled in the art. Such recombinant human insulins are also called Normal insulin. Products comprising recombinant human insulins are sold e.g. by the company Eli Lilly (Indianapolis, IN, USA) under the product names Humulin™, Huminsulin™, Huminsulin™ basal, Humulin™ N, Humulin™ R, Humulin™ 70/30 and Humulin™ 50/50; or by the company Novo Nordisk (Bagsværd, Denmark) under the product names Novolin™, Actrapid / Novolin™ and Actraphane™; or by the company Sanofi-Aventis (Schiltigheim, France) under the product names Insuman™ and Insuman™ basal.

This invention further pertains to genetically modified insulins. They are also preferably produced recombinantly. These modifications are intended to adapt the stability and/or absorption profile in the patients body. An example for a genetically modified human insulin is Insulin aspart, which is characterized by the exchange of proline in position B28 against aspartic acid. It is marketed e.g. by Novo Nordisk, depending on further admixtures under the trade names NovoRapid™,

Novolog™, Novolog™ mix, Novolog™ mix 70/30, NovoMix™ etc. Another example of a genetically modifed insulin included herewith, is human insulin characterized by the two exchanges of (ï) asparagine in position B3 against lysine and (ii) lysine in position B29 against glutamic acid. It was developed by Sanofi-Aventis and is sold e.g. under the trade name Apidra™ by this provider.

This invention further pertains to insulins modified or further modified by the covalent binding of chemical compounds. Such a modification leads to a spécifie absorption profile in the patient’s body. One example is so-called Insulin detemir (Detemir) which is characterized by a fatty acid, esp. myristic acid, bound to the lysine amino acid at position B29 of human insulin. This spécifie myristylated insulin is markted under the trade name Levemir™ by Novo Nordisk. Another example is Insulin degludec™, developed by Novo Nordisk and described to be an ultralong-acting basal insulin. It is characterized by the délétion of the aminoaeîd alanin in position B30 and a carboxypentadecanoyl rest linked via a glutamic acid linker to position 29 of the same modified Bchain (N⁶⁰²⁹-^ -(15-carboxypentadecanoyl)-L-Y-glutamyl]-des-B30-L-threonine-insulin human; CAS no. 844439-96-9). Spécial préparations of it are sold under the names Degludec™ and DegludecPlus™ the latter being a combination product of Insulin degludec™ and Insulin aspart.

Other chemical substances according to the invention to be mixed with insulins, comprise ail chemical substances appropriate for the incorporation in medical compositions without being covalently bound to insulin. In the context of this invention, it is preferred that they interact with insulin and/or improve its intended physiological effect. Such chemical substances are per se known to the person skilled in the art. For example they comprise nuclear proteins like protamine or dérivatives thereof, preferably Neutral Protamine Hagedorn (NPH). They can be used e.g. for the modification of the onset and/or the duration of the insulin action. Such insulines are e.g. marketed by Eli Lilly under the product names Insulin NPH or Insulin isophane or under the name NPH insulin by Novo Nordisk. Further examples are the above mentioned products Humulin™ N, Humulin™ R, Humulin™ 70/30 and Humulin™ 50/50.

Insulin Glargine (marketed by Sanofi-Aventis under the name Lantus™) is described below as the subject of one preferred mode of the invention. Alternatives and/or generic versions of this insulin, also included hereby, are e.g. the ones that are commercially availabe under the trade names Glaritus, Basalin and Basalog/Glarvia.

Further forms of insulins according to the invention can be characterized by their application route. For example they can be applied orally, nasaly or by inhalation. Examples are NN-1953, IN-105, Nasulin™ (developed by CPEX Pharmaceuticals; Wilmington, DE, USA), Afrezza, BIOD-620, Oral lyn, HinsBet, Capsulin, Analog-PH20, ORMD-0801, SuliXen. Preferred are NN-1953, IN-105, BIOD-620 and Analog-PH20.

Examples of particular insulin analogues include insulin glulisine (Apidra™), glargine (Lantus™), Novorapid™, insulin lispro (Humalog™), Novomix™, Actraphane™ HM, insulin detemir (Levemir™), insulin glulisin (Apidra™), Degludec, LY2963016, LY2605541, and pegylated insulin Lispro, insulin glargine (Lantus™, Glaritus, Basalin, Basalog, Glarvia, BIOD-620), insulin detemir (Levemir™) Humulin, Huminsulin, insulin isophane (Humulin N, Insulatard, Novolin N), insulin and insulin isophane (Humulin 70/30, Humulin 50/50, Mixtard 30, Actraphane™ HM), insulin degludec and insulin aspart (DegludecPlus/NN-5401), insulin aspart (Novolog), insulin aspart and insulin protamine (Novolog mix, Novolog mix 70/30), insulin (NN-1953, IN-105, HinsBet, Capsulin, Nasulin, Afrezza, ORMD-0801, SuliXen, Humulin R), insulin buccal (Oral-lyn) and hyaluronidase insulin (Analog-PH20).

Further exemplary insulin analogues are described in detail below.

Insulin glargine (Lantus™)

Insulin glargine is an insulin analogue containing a substitution in the asparagine at position 21, along with the addition of two arginines to the carboxy terminal of the B chain. It is indicated for once-daily administration by injected subcutaneous injection and maintains a long duration of action and no pronounced peak concentration. Insulin glargine and related compounds and compositions are described in U.S. Patent Nos. 5,656,722, 7,476,652, and 7,713,930. Exemplary compounds related to insulin glargine are described in U.S. Patent No. 5,656,722 and hâve the sequence Asp^A2'-Human insulin-Arg^B31-OH; Glu^A21-Human insulin-Arg^B31-OH; Gly^’-Human insulin-Arg^B31OH; Ser^’-Human insulin-Arg^B31-OH; Thr^’-Human insulin-Arg^B31-OH; Ala^A21-Human insulinArg^B31-OH; Asp^’-Human insulin-Arg^B31-Arg^B32-OH; Glu^’-Human insulin-Arg^B31-Arg^B32-OH; Gl/²¹-Human insulin-Arg^B31-Arg^B32-OH; Ser^’-Human insulin-Arg^B31-Arg^B32-OH; Thr^’-Human insulin-Arg^B31--Arg^B32-OH; Ala^A21-Human insulin-Arg⁸³¹-Arg^B32-OH; Asp^A21-Asn^B10-Human insulinArg^B31-OH; Glu^A21-Asn^B10-Human insulin-Arg^B31-OH; Gly^A21-Asn^B10-Human insulin-Arg^B31-OH; Ser^A21-Asn^B10-Human insulin-Arg^B31-OH; Thr^A21-Asn^B10-Human insulin-Arg^B31-OH; Ala^A21-Asn^B1°Human insulin-Arg^B31-OH; Asp^A21-Asn^B10-Human insulin-Arg^B31-Arg^B32-OH; Glu^A21-Asn^BW-Human insulin-Arg^B31-Arg^B32-OH; Gly^A21-Asn^B10-Human insulin-Arg^B31-Arg^B32-OH; Ser^AZ1-Asn^B10-Human insulin-Arg^B31-Arg^B32-OH; Thr^A21-Asn^B10-Human insulin-Arg^B31-Arg^B32-OH; and Ala^A21-Asn^B1°Human insulin-Arg^B31-Arg^B32-OH.

Insulin detemir (Levemir™)

Insulin detemir is a long-acting analogue of human insulin that has a C14 fatty acid chain (myristic acid) bound to the lysine at position B29 and the threonine at position 30 is omitted. Analogues of insulin detemir are described in US Patent Nos. 5,750,497; 5,866,538; 6,011,007; and 6,869,930, and hâve the formula

A-Chiin SS

I 7|

Gly—De— Val—Ghi—Gin—Cys—Cys—Thr—Ser—Ile— Cys—Ser—

3'4561 8 9 10 1112

S

I

B-ChainS

I

Xii—Val—Xaa—Gin—His—Leu—Cys—Gly—Ser—His—Leu—-Val—

23456789 10 1112

A-Chain (contd.)

Leu—Tyr—Gin—Leu—Glu—Asn—Tyr—Cys—Xaa (SEQ ID NO: 1 )

14 15 16 17 18 19 I 21 r^s

B-Chain (contd.)S

I

Glu—Ala—Leu—Tyr—Leu—Val—Cys—Gly—Glu—Arg—Gly—Phe—

14 15 16 17 18 19 20 21 22 2324

Β-Chain (contd.)

Phe—Tyr—Thr—Pro—Lys—Xaa (SEQ ID NO: 2)

26 27 28 2930

Xaa at positions A21 and B3 are, independently, any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; Xaa at position B1 is Phe or is deleted; Xaa at position B30 is (a) a non-codable, lipophilie amino acid having from 10 to 24 carbon atoms, in which case an acyl group of a carboxylic acid with up to 5 carbon atoms is bound to the ε-amino group of Lys^B29, (b) any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys, in which case the ε-amino group of Lys⁰²⁹ has a lipophilie substituent or (c) deleted, in which case the ε-amino group of Lys⁸²⁹ has a lipophilie substituent; and any Zn²⁺ complexes thereof, provided that when Xaa at position B30 is Thr or Ala, Xaa at positions A21 and B3 are both Asn, and Xaa at position B1 is Phe, then the insulin dérivative is a Zn²⁺ complex.

In one preferred embodiment, the invention employs to a human insulin dérivative in which the B30 amino acid residue is deleted or is any amino acid residue coded for by the genetic code except Lys, Arg, and Cys; the^A21 and the⁰³ amino acid residues are, independently, any amino acid residues which can be coded for by the genetic code except Lys, Arg and Cys; Phe⁰¹ may be deleted; the ε-amino group of Lys ^B29 has a lipophilie substituent which comprises at least 6 carbon atoms; and 2-4 Zn²⁺ ions may be bound to each insulin hexamer with the proviso that when B30 is Thr or Ala and A21 and B3 are both Asn, and Phe⁰¹ is not deleted, then 2-4 Zn²⁺ ions are bound to each hexamer of the insulin dérivative.

In another preferred embodiment, the invention employs to a human insulin dérivative in which the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; the A21 and the B3 amino acid residues are, independently, any amino acid residues which can be coded for by the genetic code except Lys, Arg and Cys, with the proviso that if the B30 amino acid residue is Ala or Thr, then at least one of the residues A21 and B3 is different from Asn; Phe ⁰¹ may be deleted; and the ε-amino group of Lys⁰²⁰ has a lipophilie substituent which comprises at least 6 carbon atoms.

In another preferred embodiment, the invention employs to a human insulin dérivative in which the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; the A21 and the B3 amino acid residues are, independently, any amino acid residues which can be coded for by the genetic code except Lys, Arg and Cys; Phe⁰¹ may be deleted; the ε-amino group of Lys⁰²⁰ has a lipophilie substituent which comprises at least 6 carbon atoms; and 2-4 Zn²⁺ ions are bound to each insulin hexamer.

In another embodiments,B30 amino acid residue is deleted, Asp, Glu, Thr, a lipophilie amino acid having at least 10 carbon atoms, a lipophilie α-amino acid having from 10 to 24 carbon atoms. In another preferred embodiment, the B30 amino acid is a straight chain, saturated, aliphatic a-amino acid having from 10 to 24 carbon atoms. In other preferred embodiments, the B30 amino acid is Dor L-N ε-dodecanoyllysine, α-amino decanoic acid, α-amino undecanoic acid, α-amino dodecanoic acid, α-amino tridecanoic acid, cc-amino tetradecanoic acid, α-amino pentadecanoic acid, a-amino hexadecanoic acid, or an α-amino acid. In other preferred embodiments, the A21 amino acid residue is Ala, Gin, Gly, or Ser. In other preferred embodiments, the B3 amino acid residue is Asp, Gin, or Thr. In another preferred embodiment, the ε-amino group of Lys⁰²⁰ has a lipophilie substituent which is an acyl group corresponding to a carboxylic acid having at least 6 carbon atoms. in another preferred embodiment, the ε-amino group of Lys⁰²⁰ has a lipophilie substituent which is an acyl group, branched or unbranched, which corresponds to a carboxylic acid having a chain of carbon atoms 8 to 24 atoms long. In another preferred embodiment, the ε-amino group of Lys⁸²⁰ has a lipophilie substituent which is an acyl group corresponding to a fatty acid having at least 6 carbon atoms. In another preferred embodiment, the ε-amino group of Lys⁰²⁰ has a lipophilie substituent which is an acyl group corresponding to a linear, saturated carboxylic acid having from 6 to 24 carbon atoms. In another preferred embodiment, the ε-amino group of Lys^B29has a lipophilie substituent which is an acyl group corresponding to a linear, saturated carboxylic acid having from 8 to 12 carbon atoms. In another preferred embodiment, the ε-amino group of Lys⁸²⁹ has a lipophilie substituent which is an acyl group corresponding to a linear, saturated carboxylic acid having from 10 to 16 carbon atoms. In another preferred embodiment, the ε-amino group of Lys⁸²⁹ has a lipophilie substituent which is an oligo oxyethylene group comprising up to 10, preferably up to 5, oxyethylene units. In another preferred embodiment, the ε-amino group of Lys⁸²⁹ has a lipophilie substituent which is an oligo oxypropylene group comprising up to 10, preferably up to 5, oxypropylene units. In other preferred embodiments, each insulin hexamer binds 2 Zn²⁺ ions, 3 Zn²⁺ ions, or 4 Zn²⁺ ions.

Examples of preferred human insulin dérivatives for use according to the présent invention in which no Zn²⁺ ions are bound are the following: N^tB29-tridecanoyl des(B30) human insulin, N^t829tetradecanoyl des(B30) human insulin, Nⁱ⁸²⁹-decanoyl des(B30) human insulin, N^EB29-dodecanoyl des(B30) human insulin, N^EB29-tridecanoyl Gly*²¹ des(B30) human insulin, N^EB29-tetradecanoyl Gly*²¹des(B30) human insulin, N^EB29-decanoyl Gly*²¹ des(B30) human insulin, N^E829-dodecanoyl Gly*²¹des(B30) human insulin, N^£B29-trîdecanoyl Gly*²¹ Gin⁸³ des(B30) human insulin, N^EB29-tetradecanoyl Gly*²¹ Gin⁸³ des(B30) human insulin, N^tB29-decanoyl Gly*²¹ Gin⁸³ des(B30) human insulin, N^eBZ9dodecanoyl Gly*²¹ Gin⁸³ des(B30) human insulin, N^E029-tridecanoyl Ala*²¹ des(B30) human insulin, N^EB2fl-tetradecanoyl Ala*²¹ des(B30) human insulin, N^tB29-decanoyl Ala*²¹ des(B30) human insulin, N^E829-dodecanoyl Ala*²¹ des(B30) human insulin, N^cB29-tridecanoyl Ala*²¹ Gln⁸³des(B30) human insulin, N^£B29-tetradecanoyl Ala*²¹ Gln^B3des(B30) human insulin, N'⁸²⁹-decanoyl Ala*²¹ Gin⁸³des(B30) human insulin, N^eB29 -tridecanoyl Gln^B3des(B30) human insulin, N^E029 -dodecanoyl Ala*²¹Gln^B3 des(B30) human insulin, εΒ29 -decanoyl Gln⁸³des(B30) human insulin; N^£B29-tetradecanoy! Gln⁸³des(B30) human insulin, N^tB29-dodecanoyl Gln^B3des(B30) human insulin, N^EB29-tridecanoyl Gly*²¹ human insulin, N^EB29-tetradecanoyl Gly*²¹ human insulin, N^EB29-decanoyl Gly*²¹ human insulin, N^£B29-dodecanoyl Gly*²¹ human insulin, N^tB29-tridecanoyl Gly*²¹ Gin⁰³human insulin, N^E829tetradecanoyl Gly*²¹ Gln^B3 human insulin, N^EB29-decanoyl Gly*²¹ Gin⁶³ human insulin, N^E029dodecanoyl Gly*²¹ Gin⁶³ human insulin, N^EB29-tridecanoyl Ala*²¹ human insulin, N^E029-tridecanoyl Ala*²¹ human insulin, N^E829-decanoyl Ala*²¹ human insulin, N^iB29-dodecanoyl Ala*²¹ human insulin, N^E029-tridecanoyl Ala*²¹ Gin⁸³ human insulin, N^E029-tetradecanoyl Ala*²¹ Gin⁸³ human insulin, N^e829decanoyl Ala*²¹ Gin⁸³ human insulin, N^t829-dodecanoyl Ala*²¹ Gin⁸³ human insulin, N^EB29-tridecanoyl Gin⁸³ human insulin, N^cB29-tetradecanoyl Gin⁸³ human insulin. N^E029-decanoyl Gin⁰³ human insulin, N^£0Z9-dodecanoyl Gin⁸³ human insulin, N^E029-tridecanoyl Gin⁸³⁰ human insulin, N^cB29-tetradecanoyl Gin⁰³⁰ human insulin, N^t829-decanoyl Gin⁸³⁰ human insulin, N^EB29-dodecanoyl Gin⁰³⁰ human insulin, N^£0zs-tridecanoyl Gly*²¹ Glu⁸³⁰ human insulin, N^EB29-telradecanoyl Gly*²¹ Glu⁸³⁰ human insulin, N^eBZ9 decanoyl Gly*²¹ Glu⁰³⁰ human insulin, N^EB29-dodecanoyl Gly*²¹ Glu^B3° human insulin, N^EBZ9tridecanoyl Gly*²¹ Gin⁸³ Glu⁰³⁰ human insulin, N^E029-tetradecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin, N^E029-decanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin, N^EB29-dodecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin, N^cB29-tridecanoyl Ala*²¹ Glu⁰³⁰ human insulin, N^E029-tetradecanoyl Ala*²¹ Glu⁰³⁰ human insulin, N^cBZ9-decanoyl Ala*²¹ Glu⁰³⁰ human insulin, N^E029-dodecanoyl Ala*²¹ Glu⁰³⁰ human insulin, N^E02B-tridecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulin, N^E029-tetradecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulin, N^EBZ9-decanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulin, N^tB29-dodecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰human insulin, N^E029-tridecanoyl Gin⁰³ Glu⁰³⁰ human insulin, N^E029-tetradecanoyl Gin⁰³ Glu⁰³⁰ human insulin, N^E0Z9-decanoyl Gin⁰³ Glu⁸³⁰ human insulin, N^eBZ9-dodecanoyl Gin⁰³ Glu⁰³⁰ human insulin.

Examples of preferred human insulin dérivatives for use according to the présent invention in which Zn²⁺ ions are bound per insulin hexamer are the following: (N^E0z9-tridecanoyl des(B30) human insulin)6, 2Zn^z+, (N^E029-tetradecanoyl des(B30) human insulin)0, 2Zn^z+, (N^EB29-decanoyl des(B30) human insulin)e, 2Zn²⁺, (N^eB29-dodecanoyl des(B30) human insulin)61 2Zn²⁺, (N^EB29-tridecanoyl Gly*²¹des{B30) human insulin)e, 2Zn^z+, (N^E029-tetradecanoyl Gly*²¹ des(B30) human insulin)0. 2Zn²⁺, (N^EB29-decanoyl Gly*²¹ des(B30) human insulin)6, 2Zn²⁺, (N^E0Z9-dodecanoyl Gly*²¹ des(B30) human insulin)e, 2Zn²⁺, (N^tB29-tridecanoyl Gly*²¹ Gln^B3des(B30) human insulin)0, 2Zn²⁺, (N^E028-tetradecanoyl Gly*²¹ Gln⁰³des(B3O) human insulin)6, 2Zn^z+, {N^EB29-decanoyl Gly*²¹ Gln⁰³des(B3O) human insulin)6, 2Zn^z+, (N^tBZ9-dodecanoyl Gly*²¹ Gln^B3des(B30) human insulin)0, 2Zn^a+, (N^E029-tridecanoyl Ala*²¹des(B30) human insulin)0, 2Zn^z+, (N^E029-tetradecanoyl Ala*²¹ des(B30) human insulin)6, 2Zn^2t, (N^E029-decanoyl Ala*²¹ des(B30) human insulin)e, 2Zn²⁺, (N^EB29-dodecanoyl Ala*²¹ des(B30) human insulin)a, 2Zn²⁺, (N^tB29-tridecanoyl Ala*²¹ Gln^B3des(B30) human insulin)0, 2Zn²*, (N^tB29-tetradecanoyl Ala*²¹ Gln⁰³des(B3O) human insulin)6, 2Zn^z+, (N^E029-decanoyl Ala*²¹ Gln^B3des(B30) human insulin)6, 2Zn²⁺, (N^EB29-dodecanoyl Ala*²¹ Gln^B3des(B30) human insulin)e. 2Zn²⁺, (N^EBZ9-tridecanoyl Gin⁰³des(B30) human insulin)6, 2Zn²*, (N^E029-tetradecanoyl Gln^B3des(B30) human insulin)0, 2Zn²⁺, (N^eB2Bdecanoyl Gln^B3des(B30) human insulin)6, 2Zn²⁺, (N^tBZ9-dodecanoyl Gln^B3des(B30) human insulin)6, 2Zn²⁺, (N^EB29-tridecanoyl human insulin)0, 2Zn^z+, (N^E0z9-tetradecanoyl human insulin)0, 2Zn²⁺, (N^EBZ9decanoyl human insulin)6, 2Zn²⁺, (N^E029-dodecanoyl human insulin)6, 2Zn²⁺. (N^E029-tridecanoyl Gly*²¹human insulin)e, 2Zn²⁺, (N^E029-tetradecanoyl Gly*²¹ human insulin)6, 2Zn²⁺, (N^EB29-decanoyl Gly*²¹human insulin)6, 2Zn²⁺, (N^E029-dodecanoyl Gly*²¹ human insulin)0, 2Zn²⁺, (N^c029-tridecanoyl Gly*²¹Gin⁰³ human insulin)6, 2Zn²⁺, (N^EB29-tetradecanoyl Gly*²¹ Gin⁰³ human insulin)6, 2Zn²⁺, (N^E029decanoyl Gly*²¹ Gin⁰³ human insulin)0, 2Zn^z+, (N^tB29-dodecanoyl Gly*²¹ Gin⁰³ human insulin)0, 2Zn^2t, (N^E0z9-tridecanoyl Ala*²¹ human insulîn)6, 2Zn²*, (N^E029-tetradecanoyl Ala*²¹ human insulîn)0, 2Zn²⁺, (N^E0Z0-decanoyl Ala*²¹ human insulin)6, 2Zn²’, (N^tB2B-dodecanoyl Ala*²¹ human insulin)6, 2Zn^Zt, (N^EB29-tridecanoyl Ala*²¹ Gin⁰³ human insulin)0, 2Zn^z+, (N^E029-tetradecanoyl Ala*²¹ Gin⁰³ human insulin)0, 2Zn²*, (N^E02B-decanoyl Ala*²¹ Gin⁰³ human insulin)₆₁ 2Zn^2t, (N^E029-dodecanoyl Ala*²¹Gln^B3 human insulîn)e> 2Zn²\ (N^E029-tridecanoyl Gin⁰³ human insulinje. 2Zn^z+, (N^E02B-tetradecanoyl Gin⁰³human insulinje. 2Zn²*, (N^E029-decanoyl Gin⁰³ human insulinje, 2Zn^z\ (N^EB29-dodecanoyl Gin⁰³human insulinje, 2Zn²*, {N^E029-tridecanoyl Gin⁰³⁰ human insulin)6, 2Zn^z+, (N^iB29-tetradecanoyl Glu⁰³⁰human insulinje, 2Zn²*, (N^E029-decanoyl Glu⁰³⁰ human insulinje, 2Zn^z+, (N^tB29-dodecanoyl Glu⁰³⁰human insulinje, 2Zn²*, (N^EB29-tridecanoyl Gly*²¹ Glu⁰³⁰ human insulin)e, 2Zn²*, (N^E020-tetradecanoyl Gly*²¹ Glu⁰³⁰ human insulinje, 2Zn²*, (N^E029-decanoyl Gly*²¹ Glu⁰³⁰ human insulinje, 2Zn^z+, (N^t029dodecanoyl Gly*²¹ Glu⁰³⁰ human insulinje, 2Zn²⁺, (N^E029-tridecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulinje, 2Zn²⁺, (N^E029-tetradecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6, 2Zn^z+, (N^lB29-decanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulinje, 2Zn^z\ (N^E029-dodecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insu1în)e, 2Zn^z+, (N^E029-tridecanoyl Ala*²¹ Glu⁰³⁰ human insulinje, 2Zn²⁺, (N^E029-tetradecanoyl Ala*²¹ Glu⁰³⁰human insulinje, 2Zn^z+, (N^EB29-decanoyl Ala*²¹ Glu⁰³⁰ human insulinje, 2Zn^z+, (N^E029-dodecanoyl Ala*²¹ Glu⁰³⁰ human insulîn)B, 2Zn²⁺, (N^E029-tridecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulinj6, 2Zn²⁺, (N^lBZ9-tetradecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulinje, 2Zn^z+, (N^tBZ9-decanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰human insulinje, 2Zn²⁺, (N^E029-dodecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6, 2Zn^z+, (N^EB29tridecanoyl Gin⁰³ Glu⁰³⁰ human insulinje, 2Zn²⁺, (N^E029-tetradecanoyl Gin⁰³ Glu⁰³⁰ human insulinje, 2Zn²*, (N^E029-decanoyl Gin⁰³ Glu⁰³⁰ human insulinje, 2Zn²⁺, (N^E029-dodecanoyl Gin⁰³ Glu⁰³⁰ human insulin)_B, 2Zn²⁺.

Examples of preferred human insulin dérivatives for use according to the présent invention in which three Zn²⁺ ions are bound per insulin hexamer are the following: (N^E029-tridecanoyl des(B30) human insulinje, 3Zn²⁺, (N^E029-tetradecanoyl des(B30) human insulinje, 3Zn²*, (N^E029-decanoyl des(B30J human insulinje, 3Zn^z+, (N'^B29-dodecanoyl des(B30J human insulinje, 3Zn^z+, (N^EB2B-tridecanoyl Gly*²¹des(B30) human insulinje, 3Zn^z+, (N^E029-tetradecanoyl Gly*²¹ des(B30) human insulinje, 3Zn²⁺, (N^tB29-decanoyl Gly*²¹ des(B30) human insulinje, 3Zn^z+, (N^t029-dodecanoyl Gly*²¹ des(B30) human insulinje, 3Zn²⁺, (N^E029-tridecanoyl Gly*²¹ Gln^B3des(B30) human insulinje, 3Zn^z+, (N^EB29-tetradecanoyl Gly*²¹ Gln⁰³des(B3O) human insulinje, 3Zn²⁺, (N^t029-decanoyl Gly*²¹ Gin⁸³ des(B30) human insulinje, 3Zn^z+, (N^E0Za-dodecanoyl Gly*²¹ Gln⁰³des(B3O) human insulinje, 3Zn²⁺, (N^E029-tridecanoyl Ala*²¹des(B30) human insulinje, 3Zn^z+, (N^cB2B-tetradecanoyl Ala*²¹ des(B30J human insulinje, 3Zn²⁺, (N^E029-decanoyl Ala*²¹ des(B30J human insulinje, 3Zn²⁺, (N^E029-dodecanoyl Ala*²¹ des(B30) human insulinje, 3Zn²\ (N^EB29-tridecanoyl Ala*²¹ Gln^B3des(B30) human insulinje, 3Zn²⁺, (N^E029-tetradecanoyl Ala*²¹ Gln^B3des(B30) human insulinje, 3Zn²⁺, (N^EB29-decanoyl Ala*²¹ Gln^B3des(B30) human insulinje, 3Zn^z+, (N^E029-dodecanoyl Ala*²¹ Gln^B3des(B30J human insulinje, 3Zn^z+, (N^tB29-tridecanoyl Gin⁰³des(B30) human insulinje, 3Zn²\ (N^cB29-tetradecanoyl Gln^B3des(B30) human insulinje, 3Zn²⁺, (N^eB2Bdecanoyl Gln⁰³des(B3O) human insulinje, 3Zn^z+, (N^E029-dodecanoyl Gln⁰³des(B3OJ human insulinje, 3Zn^z+, (N^tB29-tridecanoyl human insulinje, 3Zn²⁺, (N^E0ze-tetradecanoyl human insulinje, 3Zn²⁺, (N^eB2Bdecanoyl human insulinje, 3Zn²⁺, (N^E029-dodecanoyl human insulinje, 3Zn²⁺, (N^E029-tridecanoyl Gly*²¹37 human insulin)₆, 3Zn²⁺, (N^£029-tetradecanoyl Gly*²¹ human insulin)6. 3Zn²\ (N^£029-decanoyl Gly*²¹human insulin)6, 3Zn²*, (N^£029-dodecanoy! Gly*²¹ human insulin)e. 3Zn²⁺, (N^£02S-tridecanoyl Gly*²¹Gln^B3 human insulin)61 3Zn^z+, (N^EB29-tetradecanoyl Gly*²¹ Gin⁰³ human insulin)e, 3Zn^z+, (N^E029decanoyl Gly*²¹ Gin⁶³ human insulin)6, 3Zn²⁺, (N^E029-dodecanoyl Gly*²¹ Gin⁰³ human insulin)6, 3Zn²⁺, (N^£029-tridecanoyl Ala*²¹ human insulin)e, 3Zn^z+, (N^£B29-tetradecanoyl Ala*²¹ human insulin)6, 3Zn²⁺, (N^E029-decanoyl Ala*²¹ human insulin)6, 3Zn²⁺, (N^EB29-dodecanoyl Ala*²¹ human insulin)6. 3Zn^z+, (N^E029-tridecanoyl Ala*²¹ Gin⁰³ human insulin)e, 3Zn^z+, (N^tBZ9-tetradecanoyl Ala*²¹ Gln^B3 human insulin)6( 3Zn^z+, (N^E029-decanoyl Ala*²¹ Gin⁰³ human insulin)6, 3Zn²⁺, (N^£B29-dodecanoyl Ala*²¹ Gin⁰³human insulin)61 3Zn^z+, (N^£B29-tridecanoyl Gin⁰³ human insulin)6, 3Zn²⁺, (N^£0Z9-tetradecanoyl Gin⁰³human insulin)e. 3Zn²⁺, (N^£029-decanoyl Gin⁰³ human insulin)6, 3Zn²⁺, (N'^B29-dodecanoyl Gin⁰³human insulin)6. 3Zn^z+, (N^£B29-tridecanoyl Glu⁰³⁰ human insulin)6, 3Zn²⁺, (N^E029-tetradecanoyl Glu⁰³⁰human insulin)s, 3Zn²⁺, (N^£029-decanoyl Glu⁰³⁰ human insulin)6, 3Zn²*, (N^E029-dodecanoyl Glu⁰³⁰human insulin)6, 3Zn²⁺, (N^£029-tridecanoyl Gly*²¹ Glu⁰³⁰ human insulin)e, 3Zn²⁺, (N^£029-tetradecanoyl Gly*²¹ Glu⁰³⁰ human insulin)e, 3Zn²⁺, (N^E029-decanoyl Gly*²¹ Glu⁰³⁰ human insulin)81 3Zn²⁺, (N^eB2Bdodecanoyl Gly*²¹ Glu⁰³⁰ human insulin)e, 3Zn^z+, (N^£029-tridecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6, 3Zn^2t, (N^£0Z9-tetradecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin)e, 3Zn²*, (N^E029-decanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulîn)6. 3Zn^z+, (N^£029-dodecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6r 3Zn²⁺, (N^E029-tridecanoyl Ala*²¹ Glu⁶³⁰ human insulin)6> 3Zn²⁺, (N^£029-tetradecanoyl Ala*²¹ Glu⁰³⁰human insulin)6( 3Zn²⁺, (N^£029-decanoyl Ala*²¹ Glu⁰³⁰ human insulin)6, 3Zn²⁺, (N^£029-dodecanoyl Ala*²¹ Glu⁰³⁰ human insulin)e, 3Zn²*, (N^£029-tridecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6, 3Zn²⁺, (N^£029-tetradecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulin)fl, 3Zn^z+, (N^E029-decanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰human insulin)6l 3Zn²⁺, (N^£029-dodecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6, 3Zn^z+, (N^£B29tridecanoyl Gin⁰³ Glu⁰³⁰ human insulin)61 3Zn²⁺, (N^E029-tetradecanoyl Gin⁰³ Glu⁰³⁰ human insulin)6, 3Zn²⁺, (N^£B29-decanoyl Gin⁰³ Glu⁰³⁰ human insulin)6, 3Zn²⁺, (N^E029-dodecanoyl Gin⁰³ Glu⁰³⁰ human insulin)₆, 3Zn²⁺.

Examples of preferred human insulin dérivatives for use according to the présent invention in which fourZn²⁺ ions are bound per insulin hexamer are the following: (N^EB29-tridecanoyl des(B30) human insulin)6, 4Zn²⁺, (N^£B29-tetradecanoyl des(B30) human insulin)6, 4Zn²⁺, (N^E0Z9-decanoyl des(B30) human insulin)6, 4Zn²⁺, (N^E029-dodecanoyl des(B30) human insulin)B, 4Zn²⁺, (N^£029-tridecanoyl Gly*²¹des(B30) human insulin)6, 4Zn²⁺, (N^£BZ9-tetradecanoyl Gly*²¹ des(B30) human insulin)6l 4Zn²*, (N^£029-decanoyl Gly*²¹ des(B30) human insulin)6, 4Zn²⁺, (N^£029-dodecanoyl Gly*²¹ des(B30) human insulin)6, 4Zn²⁺, (N^£029-tridecanoyl Gly*²¹ Gln^B3des(B30) human insulin)6, 4Zn²\ (N^E029-tetradecanoyl Gly*²¹ Gln⁰³des(B3O) human insulin)0, 4Zn^2t, (N^£029-decanoyl Gly*²¹ Gln^B3des(B30) human insulin)61 4Zn²⁺, (N^tB29-dodecanoyl Gly*²¹ Gln⁰³des(B3O) human insulin)6< 4Zn²\ (N^EBZ9-tridecanoyl Ala*²¹des(B30) human insulin)0, 4Zn²⁺, (N^£029-tetradecanoyl Ala*²¹ des(B30) human insulin)0, 4Zn^z\ (N^£029-decanoyl Ala*²¹ des(B30) human insulin)6.4Zn²⁺, (N^£B29-dodecanoyl Ala*²¹ des(B30) human insulin)61 4Zn²⁺, (N^eB29-tridecanoyl Ala*²¹ Gln^B3des(B30) human insulin)e, 4Zn²⁺, (N^E029-tetradecanoyl Ala*²¹ Gln^B3des(B30) human insulin)6, 4Zn²⁺, (N^tB29-decanoyl Ala*²¹ Gln^B3des(B30) human insulin)61 4Zn^2t, (N^£029-dodecanoyl Ala*²¹ Gln⁰³des(B3O) human insulin)61 4Zn²‘, (N^tB29-tridecanoyl Gin⁰³des(B30) human insu!in)a, 4Zn²⁺, (N^£B29-tetradecanoyl Gln^B3des(B30) human insulin)61 4Zn^2t, (N^EB29decanoyl Gln^S3des(B30) human insulin)e, 4Zn²⁺, (N^tB29-dodecanoyl Gln^B3des(B30) human insulin)6, 4Zn^z+, (N^E029-tridecanoyl human insulin)6, 4Zn²⁺, (N^tB29-tetradecanoyl human insulin)6, 4Zn²*, (N^tB29decanoyl human insulin)e, 4Zn²*, (N^E029-dodecanoyl human rnsulin)e, 4Zn²\ (N^E029-tridecanoyl Gly*²¹human insulin)6, 4Zn^2t, (N^£B29-tetradecanoyl Gly*²¹ human insulin)6, 4Zn²\ (N^tB29-decanoyl Gly*²¹human insulin)6, 4Zn²\ (N^EB29-dodecanoyl Gly*²¹ human insulin)6, 4Zn²⁺, (N^EB29-tridecanoyl Gly*²¹Gin⁰³ human insulin)6, 4Zn²\ (N^E02s-tetradecanoyl Gly*²¹ Gin⁰³ human insulin)6, 4Zn²*, (N^E029decanoyl Gly*²¹ Gin⁰³ human insulin)6, 4Zn²⁺, (N^E029-dodecanoyl Gly*²¹ Gin⁰³ human insulin)6, 4Zn^z+, (N^£029-tridecanoyl Ala*²¹ human insulin)e, 4Zn²*, (N^EB29-tetradecanoyl Ala*²¹ human insulin)6. 4Zn²‘, (N^E0Z9-decanoyl Ala*²¹ human insulin)6, 4Zn²⁺, (N^E029-dodecanoyl Ala*²¹ human insulin)6, 4Zn^z+, (N^E0294ridecanoyl Ala*²¹ Gin⁰³ human insulin)6, 4Zn^2t, (N^£B29-tetradecanoyl Ala*²¹ Gin⁰³ human insulin)6, 4Zn²⁺, (N^tB29-decanoyl Ala*²¹ Gin⁰³ human insulin)e, 4Zn²*, (N^t029-dodecanoyl Ala*²¹ Gin⁰³human insulin)6, 4Zn²⁺, (N^E029-tridecanoyl Gin⁰³ human insulin)6( 4Zn²⁺, (N^£B29-tetradecanoyl Gin⁰³human insulin)e, 4Zn²⁺, (N^cB29-decanoyl Gin⁰³ human insulin)6, 4Zn²⁺, (N^£029-dodecanoyl Gin⁰³human insulin)6, 4Zn²⁺, (N^cB29-tridecanoyl Glu⁰³⁰ human insulin)&, 4Zn²⁺, (N^£B29-tetradecanoyl Glu⁰³⁰human insulin)ei 4Zn²⁺, (N^£029-decanoyl Glu⁰³⁰ human insulin)6, 4Zn²⁺, (N^EB29-dodecanoyl Glu⁰³⁰human insulin)6, 4Zn²⁺, (N^E029-tridecanoyl Gly*²¹ Glu⁰³⁰ human insulin)6, 4Zn²⁺, (N^£029-tetradecanoyl Gly*²¹ Glu⁰³⁰ human insulin)6, 4Zn²⁺, (N^E029-decanoyl Gly*²¹ Glu⁰³⁰ human insulin)B, 4Zn^z+, (N^tB29dodecanoyl Gly*²¹ Glu⁰³⁰ human insulin)61 4Zn²⁺, (N^£029-tridecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6, 4Zn²⁺, (N^£029-tetradecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6, 4Zn²*, (N^E029-decanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6, 4Zn^z+, (N^Efl29-dodecanoyl Gly*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6. 4Zn^z+, (N^£B29-tridecanoyl Ala*²¹ Glu⁰³⁰ human lnsulin)61 4Zn²*, (N^£029-tetradecanoyl Ala*²¹ Glu⁰³⁰human insulin)6, 4Zn²⁺, (N^E029-decanoyl Ala*²¹ Glu⁰³⁰ human insulin)61 4Zn²⁺, (N^£029-dodecanoyl Ala*²¹ Glu⁰³⁰ human insulin)6, 4Zn²⁺, (N^E029-tridecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human însulin)6, 4Zn^z+, (N^tB29-tetradecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulin)s. 4Zn²⁺, (N^£029-decanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰human insulin)6, 4Zn²⁺, (N^E029-dodecanoyl Ala*²¹ Gin⁰³ Glu⁰³⁰ human insulin)6, 4Zn²⁺, (N^EB29tridecanoyl Gin⁰³ Glu⁰³⁰ human insulin)61 4Zn²⁺, (N^EB29-tetradecanoyl Gin⁰³ Glu⁰³⁰ human insulin)6, 4Zn²⁺, (N^£B29-decanoyl Gin⁰³ Glu⁰³⁰ human insulin)51 4Zn²⁺, (N^tB29-dodecanoyl Gin⁰³ Glu⁰³⁰ human insulin)₆₁ 4Zn²*,

Insulin qlulisine (Apidra™)

Insuiin glulisine is a human insulin analogue in which the asparagine at position B3 is replaced by lysine and the lysine in position B29 is replaced by glutamic acid (3^fl-lysine 29^B-glutamic acidhuman insulin). Analogues of insuling glulisine are described in U.S. Patent No. 6,221,633 and hâve the formula:

SS

II (Al-A5)-Cys-Cys-A8-A9-A10-Cys-(A12-A19)-C|s-A21

S---S SS

II

Bl-Val-B3-Glu-His-Leu-Cys-(B8-B18)-Cys-(B20-B26)-B27-B28-B29-B30, where (A1-A5) are the amino acid residues in the positions A1 to A5 of the A chain of human insulin or animal insulin, (A12-A19) are the amino acid residues in the positions A12 to A19 of the A chain of human insulin or animal insulin, (B8-B18) are the amino acid residues in the positions B8 to B18 of the B chain of human insulin or animal insulin, (B20-B26) are the amino acid residues in the positions B20 to B26 of the B chain of human insulin or animal insulin, A8, A9, A10 are the amino acid residues in the positions A8, A9 and A10 of the A chain of human insulin or animal insulin, A21 is Asn, Asp, Gly, Ser, Thr or Ala, B30 is -OH or the amino acid residue in position B30 of the B chain of human insulin or animal insulin, B1 is Phe or a hydrogen atom, B3 is a naturally occurring basic amino acid residue, B27, B28 and B29 are the amino acid residues in the positions B27, B28 and B29 of the B chain of human insulin or animal insulin or in each case are another naturally occurring amino acid residue, where at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is replaced by another naturally occurring amino acid residue.

Of the twenty naturally occurring amino acids which are genetically encodable, the amino acids Gly, Ala, Val, Leu, Ile, Ser, Thr, Cys, Met, Asn, Gin, Phe, Tyr, Trp and Pro are designated here as neutral amino acids, the amino acids Arg, Lys and His are designated as basic amino acids and the amino acids Asp and Glu are designated as acidic amino acids.

Preferably, the insulin dérivative or its physiologically tolerable sait for use according to the présent invention is a dérivative of bovine insulin. porcine insulin or human insulin, namely an insulin dérivative or a physiologically tolerable sait thereof of the formula 1, which is distinguished in that A8 is (Ala), A9 is Ser, A10 is Val and B30 is Ala (amino acid residues A8 to A10 and B30 of bovine insulin), A8 is Thr, A9 is Ser and A10 is Ile (amino acid residues A8 to A10 of the insulins of man or pig), where B30 is Ala (amino acid residue B30 of porcine insulin) or B30 is Thr (amino acid residue B30 of human insulin). Particularly preferably, an insulin dérivative or a physiologically tolerable sait thereof of the formula I with the amino acid residues A8 to A10 and B30 of human insulin is furthermore distinguished in that (A1-A5) are the amino acid residues in the positions A1 to A5 of the A chain of human insulîn, (A12-A19) are the amino acid residues in the positions A12 to A19 of the A chain of human insulin, (B8-B18) are the amino acid residues in the positions B8 to B18 of the B chain of human insufin, and (B20-B26) are the amino acid residues in the positions B20 to B26 of the B chain of human insulin. Further preferred embodiments of the présent invention are an insulin dérivative or a physiologically tolerable sait thereof of the formula 1, wherein the amino acid residue in position B1 of the B chain is Phe or an insulin dérivative or a physiologically tolerable sait thereof of the formula 1, wherein the amino acid residue in position B3 of the B chain is a His, Lys or Arg.

Further preferred embodiments for use in the présent invention are an insulin dérivative or a physiologically tolerable sait thereof of the formula 1, wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is replaced by a naturally occurring amino acid residue which is selected from the group consisting of the neutral or of the acidic amino acids, an insulin dérivative or a physiologically tolerable sait thereof of the formula I, wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is a naturally occurring amino acid residue which is selected from the group consisting of Ile, Asp and Glu, preferably wherein at least one of the amino acid residues in the positions B27, B28 of the B chain is replaced by a naturally occurring amino acid residue which is selected from the group consisting of the neutral amino acids, or particularly preferably wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is Ile, or an insulin dérivative or a physiologically tolerable sait thereof of the formula I, wherein at least one ofthe amino acid residues in the positions B27, B28 and B29 of the B chain is a naturally occurring amino acid residue which is selected from the group consisting of the acidic amino acids, preferably wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is Asp, preferably wherein the amino acid residue in position B27 or B28 of the B chain is Asp, or wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is Glu.

A preferred embodiment for use in the présent invention is also an insulin dérivative or a physiologically tolerable sait thereof of the formula I, wherein the amino acid residue in position B29 of the B chain is Asp. Further preferred embodiments are an insulin dérivative or a physiologically tolerable sait thereof of the formula I, wherein the amino acid residue in position B27 of the B chain is Glu, an insulin dérivative or a physiologically tolerable sait thereof ofthe formula I, wherein the amino acid residue in position B28 of the B chain is Glu, or an insulin dérivative or a physiologically tolerable sait thereof of the formula I, wherein the amino acid residue in position B29 of the B chain is Glu.

Very particularly preferably, an insulin dérivative or a physiologically tolerable sait thereof is one which is distinguished in that the B chain has the sequence Phe Val Lys Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr, for example Lys(B3), Glu(B29)-human insulin, or an insulin dérivative or a physiologically tolerable sait thereof which is distinguished in that the amino acid residue in position B27 of the B chain is Ile, preferably an insulin dérivative or a physiologically tolerable sait thereof which is distinguished in that the B chain has the sequence Phe Val Lys Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Ile Pro Lys Thr, for example Lys (B3), Ile (B27)human insulin, or an insulin dérivative or a physiologically tolerable sait thereof of the formula I, wherein the amino acid residue in position B28 of the B chain is Ile, preferably an insulin dérivative or a physiologically tolerable sait thereof which is distinguished in that the B chain has the sequence Phe Val Lys Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Ile Lys Thr, for example Lys (B3), Ile (B28)-human insulin,

Particularly preferably, an insulin dérivative or a physiologically tolerable sait thereof is distinguished in that the amino acid residue in position B28 of the B chain is Ile and the amino acid residue in position A21 is Asp, is preferably one wherein the A chain has the sequence Gly Ile Val Glu Gin Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gin Leu Tyr Gin Leu Glu Asn Tyr Cys Asp and the B chain has the sequence Phe Val Lys Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Pyr Thr Ile Lys Thr (Lys(B3), lle(B28), Asp(A21)-human insulin).

Insulin Lispro and pegylated forms thereof

Insulin Lispro is a fast acting insulin analogue having in which the penultimate lysine and proline residues on the C-terminal end of the B-chain are reversed (Lys^B28Pro^B2B human insulin). This compound is described in U.S. Patent No. 5,461,031.

Pegylated lispro is described, for example, in PCT Publication WO/2009/152128 and hâve the formula P-[(A)-(B)], or a pharmaceutically acceptable sait thereof, where A is the A-chain of insulin lispro; B is the B-chain of insulin lispro; and P is a PEG having a molecular weight in the range from about 20 kDa to about 40 kDa, and wherein the A and B are properly cross-linked and P is attached either directly or indirectly via a covalent bond to the alpha-amino group of the glycine at position 1 of A, the alpha-amino group of the phenylalanine at position 1 of B, or the epsilon-amino group of the lysine at position 28 of B. The présent invention may also employ compositions comprising a plurality of mono- and di-PEGylated insulin lispro compounds wherein greater than about 75% of the PEGylated insulin lispro compounds in the composition are mono-PEGylated compounds of the formula. The présent invention may also employ compositions comprising mono-PEGylated insulin compounds of the formula wherein greater than about 50% of the mono-PEGylated compounds in the composition hâve a PEG covalently attached either directly or indirectly to the epsilon-amino group ofthe lysine at position 28 ofthe B-chain.

Degludec

Degludec is a human insulin analogue having the formula:

Η-Gly—Ile-Val-Glu-GIn-Cys-Cys-Thr-Ser-lle-Cys-Ser-Leu-Tyr-Gin—Leu—

Glu-Asn-Tyr-Cys-Asn-OH 20]

Η-Phe-Val-Asn-GIn-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr— 10

Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-OH

Degludec is indicated for thrice weekly injection and has a long half life. Also included is DegludecPlus (NN-5401).

Actraphane™

Actraphane is a range of insulin suspensions for injection. These include Actraphane 10 (soluble insulin 10% and isophane insulin 90%), Actraphane 20 (soluble insulin 20% and isophane insulin 80% ), Actraphane 30 (soluble insulin 30% and isophane insulin 70%); Actraphane 40 (soluble insulin 40% and isophane insulin 60%), and Actraphane 50 (soluble insulin 50% and isophane insulin 50%).

LY2963016

LY2963016,_a new insulin glargin analogue, is described, for example, in the PCT publications

WO 2004096854, WO 2003053460, WO 2003053339, WO 2010080609, WO 2010080606,

WO 2010014946, WO 2010002283, WO 2009132129, WO 2009129250, WO 2007081824, the US publication No. 20100099601, the Chinese publication CN 101519446, orthe Australian Publication No. AU 2008326324.

LY2605541

LY2605541, a new insulin analogue, is described, for example, in the PCT publications

WO 2004096854, WO 2003053460, WO 2003053339, WO 2010080609, WO 2010080606,

WO 2010014946, WO 2010002283, WO 2009132129, WO 2009129250,WO 2007081824, the US publication No. 20100099601, the Chinese patent No. CN 101519446, or the Australïan publication AU 2008326324.

Additional insulin analogues and dérivatives

New insulin dérivatives with an extremely delayed time effect profile for use in the treatment of diabètes are described, for example, in the PCT publications WO 2009087081, WO 2009087082 and the German publications DE 102008003568 and DE 102008003566.

These analogues have a B chain modified with a termina! amidated basic amino acid (arginine or lysine), an N-terminal arginine or lysine on the A-chain, position 8 of the A-chain substituted (A8) with histîdine and position 21 of the A (A21 ) chain substituted with a glycine. Acidic amino acids at positions A5, A15, A18, B-1, B0, and B1-B4 are also substituted. The prolonged time-action profile allows these variants to be used without the risk of inducing hypoglycemia.

Also, the isoelectric point of the insulin is changed by addition or substitution of négative and positive charged amino acid residues and by an amidation of the C-terminal carboxy group of the B chain and histîdine in position 8 of the insulin A chain. The prolonged time-action profile allows these variants to be used without the risk of inducing hypoglycemia.

Further forms of insulin

Insulins applied orally, nasaly or by inhalation includes but is not limited to NN-1953, IN-105, Nasulin, Afrezza, BIOD-620, Oral-lyn, HinsBet, Capsulin, Analog-PH20, ORMD-0801 and SuliXen. In a preferred embodiment is included NN-1953, IN-105, BIOD-620 and Analog-PH20.

Therapeutic uses

The methods, kits, and compounds of the invention may provide an attractive treatment option for metabolic diseases including obesity and diabètes mellitus (diabètes).

Diabètes comprises a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin sécrétion, insulin action, or both. Acute signs of diabètes include excessive urine production, resulting compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss, lethargy, and changes in energy metabolism. The chronic hyperglycemia of diabètes is associated with long-term damage, dysfunction, and failure of various organs. notably the eyes, kidneys, nerves, heart and blood vessels. Diabètes is classified into type 1 diabètes, type 2 diabètes and gestational diabètes on the basis 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 insufficient to maintain plasma glucose levels below the diagnostic thresholds.

Gestational diabètes refers to any degree of glucose intolérance identifîed during pregnancy.

Pre-diabetes includes impaired fasting glucose and impatred glucose tolérance and refers to those states that occur when blood glucose levels are elevated but below the levels that are established for the clinical diagnosis for diabètes.

A large proportion of people with type 2 diabètes and pre-diabetes are at increased risk of morbidity and mortality due to the high prevalence of additional metabolic risk factors including 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) a prothrombotic state (e.g., high fibrinogen or plasminogen activator inhibitor—1 in the blood), hypertriglyceridemia, hypercholesterolemia and proinflammatory state (e.g., elevated C-reactive protein in the blood).

Conversely, obesity confers an increased risk of developing 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 cardîovascular 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 originates from LDL and hence elevated concentrations Low Density Lipoproteins (LDL) is closely associated with atherosclerosis. The HDL/LDL ratio is a clinical risk indictor for atherosclerosis and coronary atherosclerosis in partîcular.

Without wishing to be bound by any partîcular theory, it is believed that the compounds employed in the invention act as GluGLP-1 dual agonists. The dual agonist may combine the effect of glucagon, e.g., on fat metabolism with the effect of GLP-1, e.g., on blood glucose levels and food intake. They might therefore act to accelerate élimination of excessive adipose tissue, induce sustainable weight loss, and improve glycaemic control. Dual GluGLP-1 agonists might also act to 45 reduce cardiovascular risk factors such as high cholestérol and LDL-cholesterol. Dual GluGLP-1 agonists might also act to reduce circulating triacylglycerol levels and lowering circulating free fatty acids.

The compounds employed in the présent invention can 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 including but not limited to obesity linked inflammation, obesity linked gallbladder disease and obesity induced sleep apnea. The compounds employed in the invention may also be used for treatment of insulin résistance, glucose intolérance, pre-diabetes, increased fasting glucose, type 2 diabètes, hypertension, dyslipidemia (or a combination of these metabolîc risk factors), atherosclerois, arteriosclerosis, coronary heart disease, peripheral artery disease and stroke. 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.

Pharmaceutical compositions

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

The therapeutically effective amount of a compound employed in 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 sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the présent invention, and may be confirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in 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 clinician 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 Remingtoris Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, stérile saline and phosphatebuffered saline at slightly acidic or physiological pH may be used. pH buffering agents may be phosphate, citrate, acetate, tris/hydroxymethyl)aminomethane (TRIS), NTris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, which is a preferred buffer, arginine, lysine, or acetate or mixtures thereof. The term further encompases any agents listed in the US Pharmacopeia for use in animais, including humans.

The term pharmaceutically acceptable sait refers to the sait of the compounds. Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Examples of acid addition salts include hydrochloride salts, citrate salts and acetate salts. Examples of basic salts include salts where the cation is selected from alkali metals, such as sodium and potassium, alkaline earth metals such as calcium, and ammonium ions *N(R³)3(R⁴), where R³ and R⁴independently désignâtes optionally substituted Cve-alkyl, optionally substituted C₂.₆-alkenyl, optionally substituted aryl, or optionally substituted heteroaryl. Other examples of pharmaceutically acceptable salts are described in Remingtoris 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 bénéficiai or desired clinical results. For the purposes of this invention, bénéficiai or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether détectable 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 include those already with the disorder as well as those in which the disorder is to be prevented. By treatment is meant inhibiting or reducing an increase in pathology or symptoms (e.g., weight gain, hyperglycaemia) when compared to the absence of treatment, and is not necessarily meant to imply complété cessation of the relevant condition.

The pharmaceutical compositions can be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component. The unît dosage form can be a packaged préparation, the package containing discrète quantities of the préparations, for example, 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. 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 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.

Combination therapy

The methods and kits of the invention include administration a combination therapy of a compound described herein with an insulin analog together with a further active agent for treatment of diseases including diabètes, obesity, dyslipidaemia, and hypertension.

In such cases, the three or further active agents may be given together or separately.

Thus the compound of the invention (or the sait thereof) can be used in a further combination with an anti-diabetic agent including but not limited to metformin, a sulfonylurea, a glinide, a DPP-IV inhibitor, a glitazone, or insulin. In a preferred embodiment the compound or sait thereof is used in combination with insulin, DPP-IV inhibitor, sulfonylurea or metformin, particularly sulfonylurea or metformin, for achieving adéquate glycémie control.

The compound or sait thereof can further be used in a further 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 melanin concentrating hormone receptor 1 antagonist.

The compound or sait thereof can be used in a further 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 channel blocker.

The compound or sait thereof can be used in a further combination with an anti-dyslipidaemia agent including but not limited to a statin, a fibrate, a niacin and/or a cholestérol absorbtion inhibitor.

METHODS

Materials

Test substances

SPrUg Name h	MW	Peptide¹'	.'Purityⁱ	Solvent .
	(g/mol)	/Content Calculated^l;’		'··. A Λ
	; .·. i V.li? . . .-		S®?.':...
Compound X	3669.2	88.9	94	PBS

³BS: Phosphate buffered saline Gibco (#10010, pH=7.4). The molar équivalents of peptide used are calculated from the mass of the lyophilized compound, the experimentally determined purity, and the peptide content (calculated or experimentally determined)¹.

Compound X was produced internally at Zealand Pharma A/S. Lantus (Insulin glargine, Sanofi Aventis) and Levemir (Insulin detemir, Novo Nordisk) were purchased from the local pharmacy (Glostrup Apotek, Denmark). Both insulins are delivered as containers with 3 ml and 100 U/ml. These préparations of insulin are used directly (un-diluted). For dosing of Lantus the standard pen system Optipen is used, with a minimum dosing of 1 U. For dosing of Levemir the pen system Junior demi is used, with a minimum dosing of 0.5U.

Animais

Eighty (80) db/db (BKS.Cg-m +/+ Lepr^/S) female mice aged 7 weeks were obtained from Charles River, US. The mice were acclimatized in their new environment and ailowed free access to normal chow (Altromin 1324, Brogaarden A/S, Gentofte, Denmark) and domestic quality tap water added citric acid to pH -3.6, except as indicated. The mice were group-housed with 3-4 mice per ¹ The équation used to calculate the molar équivalents of peptide is: n_pepHdB = (m_{lyophliwa canpound} * (% purity/100) * (% peptide content/lOOfj/MWpgpwv· cage in a light-, température-, and humidity-controlled room (12-hour light: 12-hourdark cycle, with lights on at 06.00 AM to 06.00 PM hour; 24°C; 50% relative humidity).

Procedure

Pre-screen

Prior to treatment, in weeks 1-3, a tail-blood sample for the détermination of blood glucose was obtained on non-fasted animais to détermine diabetic state and to identify outliers, which were excluded. The inclusion criteria of BG > 16 mM glucose was applied,

Stratification

Stratification of the animais was based on HbA1c levels (primary) and BW as measured at baseline (day -4). Thus, on day -3, the 66 mice were selected based on the pre-screen and baseline measurements into 6 study groups of 11 mice (3-4 mice/cage).

Dosing, body weight, food, and water intake

Ail mice were mock treated for at 3 days (BID, SC, 100 μΙ vehicle) to acclimatize the animais to handling and injections. Dosing (day 0, mice at âge 12 weeks as in pilot study) started in the afternoon on that day, and the mice were treated twice daily with 2 SC injections according to Table 1 for a total of 21 days (4 injections per day). Thus, last day of dosing was day 21 in the morning. The daily injections took place between 7:00 and 8:00 and between 14:00 and 15:00 with fresh solutions prepared in the morning (only Compound X). Insulin was kept in the refrigerator.

Table 1

Groups (n=11/ group)	Substance	Route	Substance 1 (U/animal)	Substance 2 (nmol/kg)	Approx. daily Use of substance (mg/day)²
Group 1	Saline+PBS		0	0	-
Group 2	Lantus+PBS	S.C.	3	0	-
Group 3	Levemir+PBS	BID	6	0	-
Group 4	Saline+Compoun d X		0	10	0.0628

² Daily use of substance per day calculated as: Dose (nmol/kg/day) * MW (g/mol) * 0.05kg/mouse * 11 mouse/group * 1.3 (spill-factor)

Groups (n=11/ group)	Substance	Route	Substance 1 (U/animal)	Substance 2 (nmol/kg)	Approx. daily Use of substance (mg/day)²
Group 5	Lantus + Compound X		3	10	0.0628
Group 6	Levemir + Compound X		6	10	0.0628
S.C = subcu	aneous, BID = bi-daily

Dosing solutions of Compound X for the weekend were prepared the Friday before. One vial was prepared for every dosing. Injection volume (Compound X or PBS): 5 ml/kg. Throughout the study (day -2 to day 21 ) body weights (BW) were recorded daily and used to administer the body weightcorrected doses of substances. Food and water intake (Fl, Wl) was measured every day (day -3 to day 21 ) as cage averages.

Blood samples

On day -4 (before starting treatment) in 8h fasted mice, a blood sample (150 μΙ) was obtained from the orbital plexus using an EDTA coated micro-pipette taken into EDTA coated tubes kept on ice. From that sample, a drop was used for analysis of blood glucose (BG) (sticks).

Also, 30 μΙ sample of blood was transferred to a new tube fortesting of HbA1c. Stored samples for HbA1c analysis were kept at 4 °C for no more than 48 hours before analysis. The remaining blood was centrifuged, and the resulting plasma (approximately 50 μΙ) was stored (at -80°C) for later analysis of plasma insulin level.

On day 21 (before termination) in 8h fasted mice a blood sample (350 μΙ) was taken, and BG, HbA1c, and p-insulin were measured as described above. In addition a plasma sample (at least 100 μI) was stored (at -80°C) for later analysis of exposure.

Termination

The study was terminated on day 21. Ail animais were sacrificed immediately following the last blood sampling by CO₂ anesthésia followed by cervical dislocation.

Analysis

The whole blood glucose level was analyzed on tail-blood samples by the immobilized glucose oxidase method (Elite Auto analyser, Bayer, Denmark) following the manufacturées protocol. Blood samples (sample size 30 pl) were analyzed for HbA1c using the Cobas c111 analyzer (Roche Diagnostics, Mannheim, Germany) in single déterminations by Department of Molecular Pharmacology. Plasma (sample size 5 μΙ) and insulin content was measured using an insulin alpha-LISA assay in triplicate by Department of Molecular Pharmacology. A measure of peptide exposure in plasma (sample size 100 μΙ) will be determined by the Department of Bioanalysis and Pharmacokinetics.

Data analysis

Statistical analyses will be performed using GraphPad Prism version 5. The measured parameters will be compared using a one way and/or two-way ANOVA and relevant post-hoc analyses will be conducted. Différences will be considered statistically significant at p < 0.05. Possible outliers will be evaluated by Grubbs outlier test.

Génération of cell lines expressinq human qlucaqon- and GLP-1 receptors

The cDNA encoding either the human glucagon receptor (Glucagon-R) (primary accession number P47871) orthe human glucagon-like peptide 1 receptor (GLP-1 R) (primary accession number P43220) were cloned from the cDNA clones BC104854 (MGC:132514/lMAGE:8143857) or BC112126 (MGC:138331/IMAGE:8327594), respectively. The DNA encoding the Glucagon-R or the GLP-1 R was amplified by PCR using primers encoding terminal restriction sites for subcloning. The 5'-end primers additionally encoded a near Kozak consensus sequence to ensure efficient translation. The fidelity of the DNA encoding the Glucagon-R and the GLP-1 R was confirmed by DNA sequencing. The PCR products encoding 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 encoding 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 cloning and selected with 1 mg/ml G418 in the culture medium. Three weeks later 12 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 clone and one GLP-1 R expressing clone were chosen for compound profiling.

Glucagon receptor and GLP-1 Receptor efficacv assays

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 15 min at 37° C. The reaction was stopped by addition of 25 μΙ 0.5 M HCl and incubated on ice for 60 min. The cAMP content was estimated using the FlashPIate® cAMP kit from Perkin-Elmer. EC₅q and relative efficacies compared to reference compounds (glucagon and GLP-1) were estimated by computer aided curve fitting.

In Vivo: female db/db mice aged 10-11 weeks were treated for 21 days with bi-daily s.c. injections. Groups: vehicle (PBS), Lantus (3U), Levemir{6U), COMPOUND X (10nmol/kg), Lantus (3U)+COMPOUND X (10nmol/kg), Levemir (6U)+COMPOUND X (10nmol/kg). Fasting blood glucose (BG) was measured before and after 21 days of treatment.

EXAMPLES

Example 1: Réduction of weight gain by the compound Compound X in mice receiving insulin analogues

As shown in Figure 1, we observed a significant increase in body weight in mice treated with either Lantus or Levemir, while treatment with Compound X caused a significant decrease in BW.

Interestingly, BW in mice treated with both Compound X and Lantus or Levemir was similar to that of vehicle control. Our results indicate that combination of a long-acting insulin and GluGLP-1 dual agonist Compound X may improve glycémie control while avoiding the undesirable weight gain of conventional insulin treatment, or promote a overall weight-loss while improving glycémie control.

Food intake was reduced in mice receiving Compound X in combination with either Lantus or Levemir as compared to mice receiving Lantus or Levemir alone alone, as shown in Figure 2. Similarly, intake of water in mice receiving Compound X combination with either Lantus or Levemir was reduced, as compared to mice receiving either Latnus or Levemire alone. These results are shown in Figure 3.

Example 2: Efficacy on GLP-1 and Glucagon receptors

Figure 4 shows the delta-BG. When mice were treated with Lantus alone or in combination with the glucagon-GLP-1 dual agonist Compound X, in contrast to vehicle control we observed a decrease in delta-BG over the course of the 21-day experiment (mM, -9.6±1.9 vs. -10.9±1.1, Lantus vs.

Lantus+ Compound X; p=ns). In animais treated with Levemir, we also observed a decrease in delta-BG, which was more pronounced when combined with Compound X (mM, -2.1 ±1.6 vs. 9.8±2.8, Levemir vs. Levemir+ Compound X, p<0.05).

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

Ail publications, patent applications, and patents mentioned in this spécification are herein incorporated by référencé to the same extent as if each independent publication, patent application, or patent was specifically and individually indicated to be incorporated by référencé.

Claims

1. A combination of compounds for use in a method for preventing or reducing weight gain; promoting weight loss; improving circulating glucose levels, glucose tolérance or circulating cholestérol levels; lowering circulating LDL levels; increasing HDL/LDL ratio; or treating a condition caused or characterized by excess body weight, wherein said method comprises administering to a mammalian subject a combination of compounds comprising:

(a) a compound having the formula:

R¹-Z-R² wherein R¹ is H, Cv₄ alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl;

R² is OH or NH₂;

and Z is a peptide having the formula I:

His-X2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-PheVal-X24-Trp-Leu-X27-X28-Ala-X30; (I) wherein

X2 is selected from Aib and Ser;

X12 is selected from Lys, Arg, or Leu;

X16 is selected from Arg and X;

X17 is selected from Arg and X;

X20 is selected from Arg, His, and X;

X21 is selected from Asp and Glu;

X24 is selected from Ala and X;

X27 is selected from Leu and X;

X28 is selected from Arg and X;

X30 is X or is absent;

wherein at least one of X16, X17, X20, X24, X27, X28, and X30 is X;

and wherein each residue X is independently selected from the group consisting of Glu, Lys, Ser, Cys, Dbu, Dpr, and Orn;

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

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

and (b) an insulin analogue.

2. A compound for use in a method for preventing or reducing weight gain; promoting weight loss; improving circulating glucose levels, glucose tolérance or circulating cholestérol levels; lowering circulating LDL levels; increasing HDL/LDL ratio; or treating a condition caused or characterized by excess body weight in a mammalian subject that is receiving an insulin analogue, said compound having the formula:

R¹-Z-R² wherein R1 is H, C1-4 alkyl, acetyl, formyl, benzoyl, ortrifluoroacetyl;

R2 is OH or NH2;

and Z is a peptide having the formula I:

X2 is selected from Ai b and Ser;

X12 is selected from Lys, Arg, or Leu;

X16 is selected from Arg and X;

X17 is selected from Arg and X;

X20 is selected from Arg, His, and X;

X21 is selected from Asp and Glu;

X24 is selected from Ala and X;

X27 is selected from Leu and X;

X28 is selected from Arg and X;

X30 is X or is absent;

wherein at least one of X16, X17, X20, X24, X27, X28, and X30 is X;

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

3. The combination of compounds for use of claim 1 or the compound for use of claim 2, wherein the compound having formula R’-Z-R² and the insulin analogue are formulated for simultaneous or sequential administration and/or wherein the compound having formula R’-Z-R² and the insulin analogue are formulated as separate médicaments.

4. The combination of compounds for use or the compound for use according to any one of claims 1-3, wherein said condition caused or characterized by excess body weight is selected from the group consisting of obesity, morbid obesity, obesity-linked inflammation, obesity-linked gallbladder disease, obesity-induced sleep apnea, metabolic syndrome, pre-diabetes, insulin résistance, glucose intolérance, type 2 diabètes, type I diabètes, hypertension, atherogenic dyslipidaemia, atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, and microvascular disease.

5. The combination of compounds for use or the compound for use of any preceding claim, wherein said subject has type 1 or type 2 diabètes and/or wherein the subject is human.

6. The combination of compounds for use or the compound for use of any preceding claim, wherein one or more of said residues X is independently selected from Lys, Glu and Cys; and/or wherein

X16 is selected from Glu, Lys, and Ser;

X17 is selected from Lys and Cys;

X20 is selected from His, Lys, Arg, and Cys;

X24 is selected from Lys, Glu, and Ala;

X27 is selected from Leu and Lys; and/or

X28 is selected from Ser, Arg, and Lys; and/or wherein the peptide of formula I includes one or more ofthe following combinations of residues:

X2 is Aib and X17 is Lys;

X2 is Aib and X17 is Cys;

X2 îs Aib and X20 is Cys;

X2 is Aib and Χ2Θ is Lys;

X12 is Arg and X17 is Lys;

X12 is Leu and X17 is Lys;

X12 is Lys and X20 is Lys;

X12 is Lys and X17 is Lys;

X16 is Lys and X17 is Lys;

X16 is Ser and X17 is Lys;

X17 is Lys and X20 is Lys;

X17 is Lys and X21 is Asp;

X17is Lys and X24 is Glu;

X17 is Lys and X27 is Leu;

X17 is Lys and X27 is Lys;

X17 îs Lys and X28 is Ser;

X17 is Lys and X28 is Arg;

X20 is Lys and X27 is Leu;

X21 is Asp and X27 is Leu;

X2 is Aib, X12 is Lys, and X16 is Ser;

X12 is Lys, X17 is Lys, and X16 is Ser;

X12 is Arg, X17 is Lys, and X16 is Glu;

X16 is Glu, X17 is Lys, and X20 is Lys;

X16 is Ser, X21 is Asp, and X24 is Glu;

X17 is Lys, X24 is Glu, and X28 is Arg;

X17 is Lys, X24 is Glu, and X28 is Lys;

X17 is Lys, X27 is Leu, and X28 is Ser;

X17 is Lys, X27 is Leu, and X28 is Arg;

X20 is Lys, X24 is Glu, and X27 is Leu;

X20 is Lys, X27 is Leu, and X28 is Ser;

X20 is Lys, X27 is Leu, and X28 is Arg;

X16 is Ser, X20 is His, X24 is Glu, and X27 is Leu;

X17 is Lys, X20 is His, X24 is Glu, and X28 is Ser;

X17 is Lys, X20 is Lys, X24 is Glu, and X27 is Leu; or

X17 is Cys, X20 is Lys, X24 is Glu, and X27 is Leu.

7. The combination of compounds for use or the compound for use of any preceding claim, wherein the peptide of formula I contains only one amino acid of the type conjugated to the lipophilie substituent, optionally wherein the peptide of formula I contains only one Lys residue, only one Cys residue, or only one Glu residue, and wherein the lipophilie substituent is conjugated to that residue.

8. The combination of compounds for use or the compound for use of any preceding claim, wherein the peptide sequence of formula I comprises one or more intramolecular bridges, optionally wherein the intramolecular bridge is formed between the side chains of two amino acid residues which are separated by three amino acids in the linear amino acid sequence of formula I, optionally wherein the intramolecular bridge is formed between the side chains of residue pairs 16 and 20, 17 and 21, 20 and 24, or 24 and 28, optionally wherein the intramolecular bridge involves a pair of residues selected from the group consisting of:

X16 is Glu and X20 is Lys;

X16 is Glu and X20 is Arg;

X16 is Lys and X20 is Glu;

X16 is Arg and X20 is Glu;

X17 is Arg and X21 is Glu;

X17 is Lys and X21 is Glu;

X17 is Arg and X21 is Asp;

X17 is Lys and X21 is Asp;

X20 is Glu and X24 is Lys;

X20 is Glu and X24 is Arg;

X20 is Lys and X24 is Glu;

X20 is Arg and X24 is Glu;

X24 is Glu and X28 is Lys;

X24 is Glu and X28 is Arg;

X24 is Lys and X28 is Glu; and

X24 is Arg and X28 is Glu and/or optionally wherein the intramolecular bridge is a sait bridge or a lactam ring.

9. The combination of compounds for use or the compound for use of any preceding claim, wherein: a) at least one of X16, X17, X20, and X28 is conjugated to a lipophilie substituent;

b) X30 is absent; or

c) wherein X30 is présent and is conjugated to a lipophilie substituent.

10. The combination of compounds for use or the compound for use of any preceding claim, wherein

a) the compound has just one lipophilie substituent, at position 16, 17, 20, 24, 27, 28 or 30, preferably at position 16,17 or 20, particularly at position 17; or

b) the compound has precisely two lipophilie substituents, each at one of positions 16, 17, 20, 24, 27, 28, and 30; or

c) the compound has lipophilie substituents at positions 16 and 17, 16 and 20,16 and 24, 16 and 27, 16 and 28, 16 and 30, 17 and 20, 17 and 24,17 and 27, 17 and 28,17 and 30, 20 and 24, 20 and 27, 20 and 28, 20 and 30, 24 and 27, 24 and 28, 24 and 30, 27 and 28, 27 and 30, or 28 and 30.

11. The combination of compounds for use or the compound for use of any of claims 1 to 5, wherein said compound has the formula:

R¹-Z-R² wherein R¹ is H, C_1J( alkyl, acetyl, formyl, benzoyl, or trifluoroacetyl;

R² is OH or NH₂;

and Z is a peptide having the formula:

His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-PheVal-X24-Trp-Leu-Leu-X28-Ala;

wherein:

X12 is selected from Lys, Arg, and Leu;

X16 is selected from Ser and X;

X17isX;

X20 is selected from His and X;

X21 is selected from Asp and Glu;

X24 is selected from Ala and Glu;

X28 is selected from Ser, Lys, and Arg;

and wherein each residue X is independently selected from the group consisting of Glu, Lys, and Cys;

optionally wherein X12 is selected from Lys and Arg and X16 is Ser; or optionally wherein X12 is selected from Lys and Arg, X16 is Ser and X20 is His.

12. The combination of compounds for use or the compound for use of any of claims 1 to 5, wherein said compound has the formula:

R² is OH or NH₂;

and Z is a peptide having the formula:

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp'Tyr-Ser-X12-Tyr-Leu-Asp-X16-X17-Ala-Ala-X20-X21-PheVal-X24-Trp-Leu-Leu-X28-Ala;

wherein:

X12 is selected from Lys, Arg, and Leu;

X16 îs selected from Ser and X;

X17isX;

X20 is selected from His and X;

X21 is selected from Asp and Glu;

X24 is selected from Ala and Glu;

X28 is selected from Ser, Lys, and Arg;

(i) Z¹, wherein Z¹ is a lipophilie moiety conjugated directly to the side chain of X; or (ii) Z¹Z², wherein Z¹ is a lipophilie moiety, Z² is a spacer, and Z¹ is conjugated to the side chain of X via Z^z, optionally wherein X12 is selected from Lys and Arg and X16 is Ser; or optionally wherein X12 is selected from Lys and Arg, X16 is Ser and X20 is His.

13. The combination of compounds for use or the compound for use of any preceding claim, wherein said peptide Z has the sequence: