WO2005080412A1 - Modulators of fatty acid-stimulated insulin secretion and uses thereof - Google Patents

Abstract

Peptide compounds are described, and methods and uses thereof for reducing insulin secretion, for preventing or treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion and hyperlipidemia) and/or for preventing or treating a condition associated with defective glycemic control in a subject (e.g. a diabetic subject).

Description

MODULATORS OF PATTY ACID-STIMULATED INSULIN SECRETION AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of US provisional application No. 60/546,189 titled "Modulators of Fatty Acid-Stimulated Insulin Secretion and Uses Thereof" filed February 23, 2004, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION The invention relates to compounds and uses thereof for modulation of insulin levels, and particularly relates to the modulation of fatty acid-stimulated or induced insulin secretion and for preventing or treating related conditions.

BACKGROUND OF THE INVENTION Given the central role of insulin in the regulation of glucose levels in physiological systems, such systems have developed complex regulatory mechanisms to modulate insulin levels in response to environmental stimuli. Given the importance of such regulation, any defects in such regulatory mechanisms often leads to illness, such as diabetes and insulin-resistant states.

Fatty acids (FFA) are potent inducers of insulin secretion. In fact, circulating FFA play an integral role in the sensitization of beta cells to glucose challenge (Stein DT et al. 1996. J Clin Invest. 97(12): 2728-35). In addition, chronic hyperinsulinemia in insulin resistant states is often associated with hyperlipidemia and soon leads to beta cell exhaustion. Beta cell exhaustion can ultimately lead to beta cell death, which can also cause further diabetic complications .

There thus remains a need for agents and methods for the modulation and regulation of insulin levels and in turn the prevention and treatment of related conditions.

SUMMARY OF THE INVENTION

The invention relates to agents such as peptide compounds and methods and uses thereof, for the regulation of insulin secretion/levels and the prevention and/or treatment of related conditions.

By convention and as used herein, amino acids can be represented by their single-letter abbreviations, where uppercase letters denote L-amino acids and lowercase letters denote D-amino acids, with the exception of glycine which is neither D nor L. Herein, where amino acids are represented by their three-letter abbreviations, they can be either D- amino acids or L-amino acids, with the exception of glycine which is neither D nor L.

Accordingly, in an aspect, the invention provides a substantially pure peptide compound comprising a peptide domain having an amino acid sequence selected from:

(a) ' an amino acid sequence selected from the group consisting of

Val-Tyr-Leu-Gly-Phe-Ser-Leu-Gln (SEQ ID NO:9); Leu-Lys-Ala-Val-Glu-Ala-Leu-Ala-Ser (SEQ ID NO : 10) ;

Trp-Ala-Gly-Ser-Ala-Leu-Ala-Glu (SEQ ID NO: 11); and Tyr-Asn-Ala- Ser-Asn-Val-Ala-Ser-Phe (SEQ ID NO : 12 ) ;

(b) an amino acid sequence that is 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of (a) and which modulates insulin levels in a subject; and

(c) an amino acid sequence comprising a fragment of at least 5 contiguous amino acids of the amino acid sequence of (a) and which modulates insulin levels in a subject;

wherein:

- said peptide domain optionally further comprises an additional 1-3 amino acids at its carboxy terminus;

- each amino acid of said peptide domain is independently a D-amino acid or an L-amino acid;

- the amino terminal H₂N- group of said peptide domain is optionally modified to R(CO)HN-; and

- the carboxy terminal -C(0)OH group of said peptide domain is optionally modified to -C(0)NH₂, -C(0)R, -C(0)OR, -C(0)NHR, or -C(0)NRR;

where R at each occurrence is independently selected from substituted or unsubstituted (Cι-C₆) alkyl , substituted or unsubstituted (Cx-Cg) alkenyl, substituted or unsubstituted (Cι-C₆) alkynyl, substituted or unsubstituted (C₆-Cι₄) aryl, and substituted or unsubstituted (C;_L-C₆) alkyl (C₆-Cι₄) aryl ; and

"-^N is a covalent linkage. In embodiments, the peptide domain contains at least one D-amino acid or contains only D-amino acids. In embodiments, the 1-3 additional amino acids are selected from the group consisting of Gly-Lys and Gly-Lys-Lys.

The invention further provides a substantially pure peptide compound of Formula I : Z¹-X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-Z² I

wherein:

X¹ is selected from the group consisting of Val, Leu, Trp, Tyr and related amino acids;

X² is selected from the group consisting of Tyr, Lys, Ala, Asn and related amino acids;

X³ is selected from the group consisting of Leu, Ala, Gly and related amino acids having a non-polar side chain;

X⁴ is selected from the group consisting of Gly, Val, Ser and related amino acids;

X^s is selected from the group consisting of Phe, Glu, Ala, Asn and related amino acids;

X⁶ is selected from the group consisting of Ser, Ala, Leu, Val and related amino acids;

X⁷ is selected from the group consisting of Leu, Ala and related amino acids having a non-polar side chain;

X⁸ is selected from the group consisting of Gin, Ala, Glu, Ser and related amino acids;

X⁹ is absent or is selected from the group consisting of Ser, Phe and related amino acids; Z¹ is an N-terminal group of the formula H₂N- or R(CO)HN-;

Z² is a C-terminal group of the formula -C (O) OH, -C(0)NH₂, -C(0)R, -C(0)OR, -C(0)NHR, -C(0)NRR or 1-3 amino acids followed by a C-terminal group of the formula -C(0)OH, -C(0)NH₂, -C(0)R, -C(0)OR, -C(0)NHR, -C(0)NRR;

R at each occurrence is independently selected from (Cι-C₆) alkyl, (Cι-C₆) alkenyl, (Cx-Cs) alkynyl, substituted (C_.-C₆) alkyl, substituted (Cι-C₆) alkenyl, or substituted (Cι-C₆) alkynyl ; and

"-" is a covalent linkage.

The invention further provides a substantially pure synthetic peptide compound having a peptide domain of Formula II :

X¹-X²-X³-X⁴-X⁵-X^s-X⁷-X⁸-X⁹ II

wherein X¹ to X⁹ and "-" are as defined above.

In an embodiment, each of X¹ through X⁹ are independently selected from the group consisting of D-amino acids and L-amino acids. In a further embodiment, each of X¹ through X⁹ are D-amino acids.

In an embodiment, the 1-3 amino acids of Z² are selected from the group consisting of Gly-Lys and Gly-Lys- Lys .

In embodiments, the peptide compound is selected from the group of peptides consisting of:

(a) vylgfslqGK (SEQ ID NO:l); (b) lkavealasGK (SEQ ID NO: 2) ;

(c) wagsalaeGK (SEQ ID NO:3);

(d) ynasnvasfGK (SEQ ID NO: 4) ;

(e) vylgfslqGKK (SEQ ID NO: 5) ; (f) lkavealasGKK (SEQ ID NO:6);

(g) wagsalaeG K (SEQ ID NO:7);

(h) ynasnvasfGKK (SEQ ID NO: 8);

(i) vylgfslq (SEQ ID NO:9);

(j) lkavealas (SEQ ID NO: 10); (k) wagsalae(SEQ ID NO:ll);

(1) ynasnvasf (SEQ ID NO: 12); and

(m) a peptide comprising the amino acid sequence of any one of (a) to (1) with conservative amino acid substitutions;

wherein lowercase symbols denote D-amino acids and uppercase symbols denote L-amino acids.

The invention further provides a pharmaceutical composition comprising the above-mentioned peptide compound and a pharmaceutically acceptable carrier. In an embodiment the composition comprises about lOOμg to about lOOmg of said peptide compound.

The invention further provides a method for reducing insulin secretion and levels, for preventing and/or treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or for preventing and/or treating a deterioration of glycemic control in a subject (e.g. a diabetic subject), the method comprising administering to said subject an effective amount of the above-mentioned compound or composition.

The invention further provides a use of the above- mentioned compound to prepare a medicament . In an embodiment, the medicament is for reducing insulin secretion and levels, for preventing and/or treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or for preventing and/or treating a deterioration of glycemic control in a subject (e.g. a diabetic subject) .

The invention further provides a use of the above- mentioned compound for reducing insulin secretion and levels, for preventing and/or treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or for preventing and/or treating a deterioration of glycemic control in a subject (e.g. a diabetic subject).

The invention further provides a package comprising the above-mentioned compound or composition together with instructions for its use. In an embodiment, the use is for reducing insulin secretion and levels, for preventing and/or treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or for preventing and/or treating a deterioration of glycemic control in a subject (e.g. a diabetic subject) .

In an embodiment, the condition is selected from the group consisting of diabetes, insulin resistance syndrome and hyperglycemia .

In an embodiment, the subject is a mammal, in a further embodiment, a human.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Plasma insulin levels in overnight fasted Sprague- Dawley rats that were administered glucose (0.4 g/kg iv) alone or glucose in mixture with 2 ml/kg of intralipids (Soybean oil; the composition is given in Table 4), as described in Example 2. Four animals per group; data presented as mean + SEM.

Figure 2 : Effect on plasma insulin levels in overnight fasted Sprague-Dawley rats that were administered a mixture containing glucose (0.4 g/kg iv) and intralipids (2 ml/kg) of peptides 1 to 4 (SEQ ID NOS.-1-4), as described in Example 2. Four animals per group; data expressed as mean + SEM.

Figure 3: Effect of peptides 1 to 4 (SEQ ID NOS: 1-4) (0.3 mg/kg iv 5 min prior t'o challenge with glucose and intralipid mixture) on plasma glucagon levels in overnight fasted Sprague-Dawley rats that were administered a mixture containing glucose (0.4 g/kg iv) and intralipids (2 ml/kg), as described in Example 2. Four animals per group; data expressed as mean ± SEM.

Figure 4: Effects of continuous infusion of peptide #4 (SEQ ID NO: 4) or saline to C57BL/ks db/db mice on (A) weight gain (day 1 to day 25) ; (B) Area Under the Curve (AUC) of glucose levels in C57BL/ks db/db mice measured at 1 pm from day 15 to 25; (C) post-prandial glucose clearance on the 25^th day following a 30 min meal; (D) AUC over 360 minutes of the post-prandial glucose levels following a 30 min meal, as described in Example 3. The animals were continuously infused with the peptides for 25 days using Alzet™ minipumps surgically implanted subcutaneously on the backs of the animals (saline, n=6; peptide #4 (SEQ ID N0:4), n=4 ; data expressed as mean ± SEM) .

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawings, which is exemplary and should not be interpreted as limiting the scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are peptide compounds and results demonstrating their effectiveness in reducing insulin secretion and blood glucose levels in vivo .

A set of short synthetic, peptides 1 to 4 (SEQ ID N0s:l-4) were found to inhibit insulin secretion elicited by FFA in fasted normal Sprague-Dawley rats. Moreover, one of the peptides, peptide 4 (SEQ ID N0:4), when continuously infused for 25 days to C57db/db mice (a well characterized diabetic animal model) reduced overall glycemia, fasting glucose and postprandial glucose levels at the end of the treatment period. Accordingly, in an embodiment, the invention provides the peptides vylgfslqGK (peptide#l; SEQ ID NO:l); lkavealasGK (peptide #2; SEQ ID NO:2); wagsalaeGK (peptide #3; SEQ ID NO : 3 ) and ynasnvasfGK (peptide #4; SEQ ID NO: 4) . Charged amino acids can be added to either or both the amino-terminal or carboxy-terminal of peptides of the invention to increase solubility. For example, peptides 9-12 (SEQ ID NOS: 9-12) can be modified by adding carboxy-terminal amino acids GK to increase solubility, thereby providing peptides 1 to 4 (SEQ ID NOs:l-4), with G acting as a spacer and K providing a charged moiety. Similarly, adding GKK to peptides 9-12 (SEQ ID NOs:9-12) provides peptides 5-8 (SEQ ID NOS: 5-8) and increases solubility.

Accordingly, in embodiments the invention provides the following peptides:

Peptide No: Exemplary Peptide SEQ ID NO:

1 vylgfslqGK 1

2 lkavealasGK 2

3 wagsalaeGK 3

4 ynasnvasfGK 4

5 vylgfslqGKK 5

6 lkavealasGKK 6

7 wagsalaeGKK 7

8 ynasnvasfGKK 8

9 vylgfslq 9

10 lkavealas 10

11 wagsalae 11

12 ynasnvasf 12 For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below.

The term "amino acid" as used herein includes both and D isomers of the naturally occurring amino acids (Table 1) as well as other nonproteinaceous amino acids used in peptide chemistry to prepare synthetic analogs of peptides. Examples of naturally-occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc. whereas nonproteinaceous amino acids are norleucine, norvaline, cyclohexyl alanine, biphenyl alanine, homophenyl alanine, naphthyl alanine, pyridyl alanine, phenyl alanines substituted at the ortho, para and meta positions with alkoxy, halogen or nitro groups etc. These compounds are known to persons versed in the art of peptide chemistry.

Table 1: Amino acids and corresponding 3- and 1-letter representations .

The term "modulation" as used herein refers to both upregulation (i.e., activation or stimulation (e.g., by agonizing or potentiating)) and downregulation (i.e. inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting) . In the present context, the term "peptide" is a compound comprising a linear polymer containing at least 2 amino acids to a maximum of 50 amino acids joined by peptide bonds. For example, the peptide can contain 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids and specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids .

In the present context, a "peptide compound" is a compound that has a peptide domain, which is optionally modified by chemical techniques or natural processes. Modifications can occur anywhere in the peptide domain, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a he e moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation₍ of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, N- and 0-linked glycosylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. See, e.g., Creighton, T.E., Proteins - Structure and Molecular Properties 2nd Ed., .H. Freeman and Company, New York

(1993) ; Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983). Mention is made of amino-terminal modification via addition of a blocking group (e.g. carbamate, an acyl group comprising a hydrophobic moeity such as cyclohexyl, phenyl , benzyl, short chain linear and branched alkyl groups of 1-6 carbon atoms) . Mention is made of carboxy-terminal modification via addition of an amine group (e.g. -NH₂) or an alkylaryl such as benzylamine, phenylethylamine, phenylpropylamine, and aliphatic amines possessing short chain linear and branched alkyl groups of 1 to 6 carbon atoms.

If desired, polar or charged amino acids can be added to either or both of the amino-terminal or carboxy- terminal of the peptide domain to increase solubility of a peptide compound of the invention. For example, GK or GKK can be added to the carboxy-terminal to increase solubility, with G acting as a spacer and K providing a charged moiety.

Preferably, the peptide domain contains at least one D-amino acid to decrease the rate of proteolytic degradation of the peptide compound. Peptide compounds comprising fragments of the amino acid sequences of SEQ ID NOs : 1-12 may also be useful for modulating blood glucose levels. Such fragments can have 5, 6, 7, 8 or more contiguous amino acids of an amino acid sequence of SEQ ID NOs : 1-12. The term "peptidomimetic" refers to a molecule that mimics the structural and/or functional features of a peptide. Persons skilled in the art use variety of methods to derive peptidomimetics of a peptide: substitutions of individual amino acids with synthetic chemical entities, nonproteinaceous amino acid analogues, deletions, additions of amino acids, replacing one or more of amino acids in the peptide with scaffolds such as beta turn mimetics, or with known pharmacophores . A description of the general methods are given in Peptidomimetic protocols (Methods in molecular medicine Vol . 23 W. M. Kazmierski (ed.), Humana Press and Advances in Amino Acid Mimetics and Peptidomimetics, Vols . 1 & 2 A. Abell (Ed) ) .

A compound is "substantially pure" or "is'olated" when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally 75% or over 90% , by weight, of the total material in a sample. Thus, for example, a peptide that is chemically synthesised will generally be substantially pure insofar as it is free from its naturally associated components. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl , isopentyl, hexyl , etc. The alkyl groups can be (C_ι-C₃) alkyl, or (C₁-C₃) alkyl. A "substituted alkyl" has substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, ketones (including alkylcarbonyl and arylcarbonyl groups) , and esters (including alkyloxycarbonyl and aryloxycarbonyl groups)), thiocarbonyl , acyloxy, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido, amidine, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, heterocyclyl , aralkyl , or an aromatic or heteroaromatic moiety. The moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls (including phosphonates and phosphinates) , sulfonyls (including sulfates, sulfonamidos, sulfamoyls and sulfonates) , and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF₃, -CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF₃, -CN, and the like.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. An "alkenyl" is an unsaturated branched, straight chain, or cyclic hydrocarbon radical with at least one carbon-carbon double bond. The radical can be in either the cis or trans conformation about the double bond(s) . Typical alkenyl groups include, but are not limited to, ethenyl , propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl , pentenyl, hexenyl, etc. An "alkynyl" is an unsaturated branched, straight chain, or cyclic hydrocarbon radical with at least one carbon-carbon triple bond. Typical alkynyl groups include, but are not limited to, ethynyl , propynyl , butynyl , isobutynyl, pentynyl, hexynyl, etc. "aryl" refers to aromatic radicals having 6 to 14 ring carbon atoms, wherein one or more ring carbon atoms is optionally substituted with a heteroatom (i.e. N, 0 or S) ; "substituted aryl" refers to aryl radicals further bearing one or more substituents as set forth above.

"alkyl (aryl) " refers to aryl-substituted alkyl radicals; and "substituted alkyl (aryl) " refers to alkyl (aryl) radicals further bearing one or more substituents as set forth above . A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reducing insulin secretion and levels, treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or treating a deterioration of glycemic control in a subject (e.g. a diabetic subject) . A therapeutically effective amount of a compound of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing an increase in insulin secretion and levels, preventing a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or preventing a deterioration of glycemic control in a subject (e.g. a diabetic subject) . A prophylactically effective amount can be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration.

Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions .

The invention also provides for reduction of the fragments of peptide compounds of the invention to generate mimetics, e.g., peptide or non-peptide agents, such as small molecules . The invention further relates to variants of amino acid sequences of SEQ ID NOS: 1-12 and their exemplary peptides 1 to 12. In the present context, the term "variant" is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identical to an amino acid sequence of SEQ ID NO : 1 to 12 and their exemplary peptides # 1 to 12. Preferably, the variant is conservatively substituted relative to the peptide compound of the invention.

Thus, the lead peptide compounds described herein may be modified or varied to improve their therapeutic utility. For example, variant peptide compounds can be made by conservatively substituting at least one amino acid residue in an amino acid sequence of SEQ ID NO: 1-12 with a different amino acid related by either structure or side chain function: aromatic, aliphatic, positively- or negatively-charged. Such substitutions preferably are made in accordance with the following list as presented in Table 2.

Table 2 : Examples of related amino acids

Alternatively, another group of substitutions of the peptide compounds of the present invention are those in which at least one amino acid residue has been removed and a different residue inserted in its place according to the following Table 3. Another group of substitutions are defined herein as exchanges within one of the following five groups:

Table 3 : Relations among amino acids

Small aliphatic, nonpolar or Ala, Ser, Thr, slightly polar residues (Pro , Gly)

Polar, negatively charged Asp, Asn, Glu, residues and their amides Gin

Polar, positively charged His, Arg, Lys residues

Large aliphatic, nonpolar Met, Leu, He, residues Val, (Cys)

Aromatic residues Phe, Tyr, Trp

The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking any side chain and thus imparts flexibility to the chain. This however tends to promote the formation of secondary structure other than alpha-helical. Pro, because of its unusual geometry, tightly constrains the chain. It generally tends to promote beta turn-like structures. Cys is capable of participating in disulfide bond formation. Tyr, because of its hydrogen bonding potential, has significant kinship with Ser, and Thr, etc.

In addition, any amino acid representing a component of the peptide compounds can be replaced by the same amino acid but of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration may be replaced with an amino acid of the same chemical structural type, but of the opposite chirality, generally referred to as the D- amino acid, depending upon its composition and chemical configuration. Additional variations include beta and gamma amino acids that provide different spatial arrangement of chemical groups .

In addition to the substitutions outlined above, synthetic amino acids that provide similar side chain functionality can be introduced into the peptide compound.

For example, aromatic amino acids may be replaced with D- or L-naphthylalanine, D- or L-Phenylglycine, D- or L-2- thienylalanine, D- or L-1-, 2-, 3- or 4-pyrenylalanine, D- or L-3-thienylalanine, D- or L- (2-pyridinyl) -alanine, D- or L- (3-pyridinyl) -alanine, D- or L- (2-pyrazinyl) -alanine, D- or L- (4-isopropyl) -phenylglycine, D- (trifluoromethyl) - phenylglycine, D- (trifluoromethyl) -phenylalanine, D-p- fluorophenylalanine, D- or L-p-biphenylalanine D- or L-p- methoxybiphenylalanine, D- or L-2-indole (alkyl) alanines, and D- or L-alkylalanines where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl , butyl, pentyl, isopropyl, iso-butyl, iso-pentyl groups. Non-carboxyl te amino acids can be made to possess negative charge, such as the non-limiting examples of phosphono- or sulfated (e.g. - S0₃H) amino acids . Other substitutions may include unnatural alkylated amino acids which are made by combining an alkyl group with any natural amino acid. Basic natural amino acids such as lysine, arginine may be substituted with alkyl groups at NH₂. Others are nitrile derivatives (e.g., containing the CN- moiety in place of CONH₂) of asparagine or glutamine, and sulfoxide derivative of methionine. In addition, any amide linkage in the peptide domain can be replaced by a ketomethylene, hydroxyethyl, ethyl/reduced amide, thioamide or reversed amide moieties, e.g. (-C=0) -CH₂-) , (-CHOH) -CH₂-) , (CH₂-CH₂-) , (-C=S)-NH-), or (-NH- (-C=0) for (-C=0)-NH-).

Compounds of the invention can be prepared, for example, by replacing, deleting, or inserting an amino acid residue of a peptide compound or peptide domain of the invention, with other conservative amino acid residues, i.e., residues having similar physical, biological, or chemical properties, and screening for biological function. It is well known in the art that some modifications and changes can be made in the structure of a peptide or polypeptide without substantially altering its biological function, to obtain a biologically equivalent peptide or polypeptide. Thus, the peptide compounds and peptide domains of the present invention also extend to biologically equivalent peptide compounds and peptide domains that differ from a portion of the sequence of SEQ ID NOs: 1-12 by conservative amino acid substitutions. As used herein, the term "conserved amino acid substitutions" refers to the substitution of one amino acid for another at a given location in the peptide compound or domain, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide compound by routine testing. In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6)s are assigned to amino acid residues (as detailed in United States Patent No. 4,554,101, incorporated herein by reference) : Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (- 0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); He (- 1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0) . In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: He (+4.5); Val (+4.2) Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8) Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3) Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, He, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gin, Tyr. Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally- occurring, non-genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include beta-alanine, 3-amino-propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine (sarcosine) , hydroxyproline, ornithine, citrulline, t-butylalanine, t-butylglycine, N- methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, norvaline, 2 -napthylalanine, pyridylalanine, 3- benzothienyl alanine, 4 -chlorophenylalanine, 2- fluorophenylalanine, 3 -fluorophenylalanine, 4- fluorophenylalanine, penicillamine, 1 , 2 , 3 , 4-tetrahydro- isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2 -amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyric acid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, cysteic acid, epsilon-amino hexanoic acid, delta- amino valeric acid, or 2 , 3-diaminobutyric acid. In alternative embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al . (J. Mol . Bio. 179:125-142, 1984) . Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, He, Pro, Met and Trp, and genetically encoded hydrophilic amino acids include Thr, His, Glu, Gin, Asp, Arg, Ser, and Lys. Non-genetically encoded hydrophobic amino acids include t- butylalanme, while non-genetically encoded hydrophilic amino acids include citrulline and homocysteine .

Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents such as -OH, -SH, -CN, -F, -CI, -Br, -I, -N0₂, - NO, -NH₂, -NHR, -NRR, -C(0)R, -C(0)OH, -C(0)OR, -C(0)NH₂, - C(0)NHR, -C(0)NRR, etc., where R is independently (Cι-C₆) alkyl, substituted (Cχ-C₆) alkyl, (Cι-C₃) alkenyl, substituted (Cx-Cg) alkenyl, (Cx-C ) alkynyl, substituted (Cχ-C₃) alkynyl, (C₅-C₂o) aryl, substituted (C₃-C₂o) aryl, (C_e-C₂g) alkaryl, substituted (C₃-C₂6) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl or substituted 6-26 membered alkheteroaryl . Genetically encoded aromatic amino acids include Phe, Tyr, and Tryp, while non-genetically encoded aromatic amino acids include phenylglycine, 2 -napthylalanine, beta-2-thienylalanine, 1,2,3, 4-tetrahydro-isoquinoline-3- carboxylic acid, 4-chlorophenylalanine, 2- fluorophenylalanine3-fluorophenylalanine, and 4- fluorophenylalanine .

An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar) . Genetically encoded apolar amino acids include Gly, Leu, Val, He, Ala, and Met, while non-genetically encoded apolar amino acids include cyclohexylalanine. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala, Leu, Val, and He, while non-genetically encoded aliphatic amino acids include norleucine. A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and Gin, while non-genetically encoded polar amino acids include citrulline, N-acetyl lysine, and methionine sulfoxide .

An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His, while non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2 , 3 , -diaminopropionic acid, 2 , 4-diaminobutyric acid, and ho oarginine .

The above classifications are not absolute and an amino acid may be classified in more than one category. In addition, amino acids can be classified based on known behaviour and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids. Amino acids can also include bifunctional moieties having amino acid-like side chains .

Conservative changes can also include the substitution of a chemically derivatised moiety for a non- derivatised residue, by for example, reaction of a functional side group of an amino acid. Thus, these substitutions can include compounds whose free amino groups have been derivatised to amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Similarly, free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides, and side chains can be derivatized to form O-acyl or 0-alkyl derivatives for free hydroxyl groups or N-im-benzylhistidine for the i idazole nitrogen of histidine. Peptide analogues also include amino acids that have been chemically altered, for example, by methylation, by amidation of the C-terminal amino acid by an alkylamine such as ethylamine, ethanolamine, or ethylene diamine, or acylation or methylation of an amino acid side chain (such as acylation of the epsilon amino group of lysine) . Peptide analogues can also include replacement of the amide linkage in the peptide with a substituted amide (for example, groups of the formula -C (0) -NR, where R is (C ~ C₆) alkyl, (Cχ-C₆) alkenyl, (Cχ-C₆) alkynyl, substituted (Cχ-C₆) alkyl, substituted (Cι-C₃) alkenyl, or substituted (Cχ-C₆) alkynyl) or isostere of an amide linkage (for example, -CH₂NH- , -CH₂S, -CH₂CH₂-, -CH=CH- (cis and trans), -C(0)CH₂-, - CH(0H)CH₂-, or -CH₂S0-) .

In order to improve the pharmaceutical characteristics of a peptide compound of the invention, the size of the peptide domain can be reduced by deleting one or more amino acids and use amino acid mimetics or dipeptide mimics containing non-peptide bonds. Examples of using molecular scaffolds such as benzodiazepine, azepine, substituted gamma lactam rings, keto-methylene pseudopeptides, β-turn dipeptide cores and β-aminoalcohols for these purposes are known to peptide chemists and are described in in Peptidomimetic protocols (Methods in molecular medicine Vol . 23 W. M. Kazmierski (ed.), Humana Press and Advances in Amino Acid Mimetics and Peptidomimetics, Vols . 1 & 2 A. Abell (Ed)).

Covalent modifications of the peptide compound or peptide domain are thus included within the scope of the present invention. Such modifications may be introduced into the peptide domains by reacting targeted amino acid residues of the peptide domain with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. The following examples of chemical derivatives are provided by way of illustration and not by way of limitation. Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines) , such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e.g., at pH 5.5- 7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Other suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoesters/e . g. as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal , 2 , 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin according to known method steps. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. The specific modification of tyrosinyl residues per se is well-known, such as for introducing spectral labels into tyrosinyl residues by reaction with aromatic diazonium compounds or tetranitromethane . N-acetylimidazol and tetranitromethane may be used to form O-acetyl tyrosinyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R'~ N=C=N-R') such as l-cyclohexyl-3- (2-morpholinyl- ( -ethyl) carbodiimide or l-ethyl-3- (4-azonia-4 , 4- dimethylpentyl) carbodiimide . Furthermore aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues may be frequently deamidated to the corresponding glutamyl and aspartyl residues. Other modifications of the peptide compounds in the present invention may include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains acetylation of the N-terminal amine, methylation of main chain amide residues (or substitution with N-methyl amino acids) and, in some instances, amidation of the C- terminal carboxyl groups, according to known method steps.

Covalent attachment of fatty acids (C6-C18) to the peptide compounds confer additional biological properties such as protease resistance, plasma protein binding, increased plasma half life, intracellular penetration etc. The above description of modification of a peptide compound does not limit the scope of the approaches nor the possible modifications that can be engineered.

Peptides (e.g. peptide domains of the compounds of the invention) or peptide analogues can be synthesised by standard chemical techniques, for example, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques well known in the art. Peptides and peptide analogues containing only L-amino acids can also be prepared using recombinant DNA technology using standard methods . Accordingly, the invention further provides nucleic acids that encode peptide domains of the compounds of the invention. Such nucleic acids may be introduced into cells for expression using standard recombinant techniques for stable or transient expression. Nucleic acid molecules of the invention may include any chain of two or more nucleotides including naturally occurring or non-naturally occurring nucleotides or nucleotide analogues.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For topical administration, peptide compounds of the invention are formulated into solutions, ointments, salves, gels, or creams as generally known in the art .

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol , propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, a peptide compound of the invention can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG) . Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. In accordance with an alternative aspect of the invention, peptide compound may be formulated with one or more additional compounds that enhance the solubility of the peptide compound.

Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like. Salt forms of peptide compounds of the invention are particularly preferred. Of course, when the compounds of this invention are used for therapeutic purposes, those compounds may also be in the form of a salt, but the salt must be pharmaceutically acceptable.

The invention thus further provides a composition of a pharmaceutical preparation in which one or more peptide compounds of the invention alone or in combination with one or more pharmaceutically suitable excipient or excipients.

The present invention also provides pharmaceutical compositions comprising a peptide compound of the present invention in combination with a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art, and are administered individually or in combination with other therapeutic agents, preferably via parenteral routes. Especially preferred routes include intramuscular and subcutaneous administration. Parenteral daily dosages, preferably a single, daily dose, are in the range from about 100 μg to about 100 mg of body weight, although lower or higher dosages may be administered. The required dosage will depend upon the severity of the condition of the patient and upon such criteria as the patient's height, weight, sex, age, and medical history.

In making the compositions of the present invention, the active ingredient, which comprises at least one peptide compound of the present invention, is usually mixed with an excipient or diluted by an excipient. When an excipient is used as a diluent, it may be a solid, semi- solid, or liquid material which acts as a vehicle, carrier, or medium for the active ingredient.

Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, mannitol, starches, gum acacia, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include lubricating agents such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxybenzoates, sweetening agents or flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art .

The compositions are preferably formulated in a unit dosage form with each dosage normally containing from about 100 μg to about 100 mg . The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient.

Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved by the use of polymers to complex or absorb a compound of the present invention. The controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose, carboxymethylcellulose, and protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Such teachings are disclosed in Remington's Pharmaceutical Sciences (1980).

Another possible method to control the duration of action by controlled release preparations is to incorporate a peptide compound of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers .

Similarly, the present invention provides a method for treating conditions associated with insulin activity or regulation in a mammal, preferably a human, in need of such treatment comprising administering an effective amount of a peptide compound of the invention or a composition of the present invention, to such a mammal .

The invention further provides a pharmaceutical preparation containing 1 μg - 100 mg of the peptide compound of the invention.

The invention further provides a method in which said pharmaceutical preparations are administered to a patient diagnosed with elevated insulin levels with the objective of reducing the adverse effects of elevated insulin levels .

The invention further provides a use of the said pharmaceutical preparation in a treatment designed for a diabetic patient with the objective of reducing the adverse effects of elevated insulin levels.

In embodiments, the therapeutic method may be used in conjunction with a diagnostic method. For example, a subject suffering from a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or a deterioration of glycemic control (e.g. diabetes) may be identified or diagnosed using a diagnostic method and then subsequently treated using a therapeutic method. Further, the therapeutic method may be used for treatment in conjunction with the diagnostic or prognostic method which is used to monitor the progress of the treatment .

In accordance with another aspect of the invention, therapeutic compositions of the present invention, comprising a peptide compound of the invention, may be provided in containers or commercial packages which further comprise instructions for its use for reducing insulin secretion and levels, for preventing and/or treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or for preventing and/or treating a deterioration of glycemic control in a subject (e.g. a diabetic subject).

Accordingly, the invention further provides a commercial package comprising a peptide compound of the invention or the above-mentioned composition together with instructions for reducing insulin secretion and levels, for preventing and/or treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or for preventing and/or treating a deterioration of glycemic control in a subject (e.g. a diabetic subject) .

The invention further provides a use of the above- noted peptide compounds and compositions for reducing insulin secretion and levels, for preventing and/or treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or for preventing and/or treating a deterioration of glycemic control in a subject (e.g. a diabetic subject) .

The invention further provides a use of the above- noted peptide compounds and compositions for the preparation of a medicament. In an embodiment the medicament is for reducing insulin secretion and levels, for preventing and/or treating a condition associated with defective glycemic control (e.g. diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion, hyperlipidemia) and/or for preventing and/or treating a deterioration of glycemic control in a subject (e.g. a diabetic subject) .

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to" . The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.

EXAMPLES

EXAMPLE 1: Synthesis of peptides

Peptides were made using solid phase peptide synthesis approach using Fluorenylmethoxycarbonyl-protected L-amino acids according to standard methods (Peptide synthesis protocols: Methods in molecular biology Vol. 35. M.W. Pennington and B.M. Dunn (Eds). Humana Press. 1994.). Completion of coupling was monitored by Kaiser color test. Crude peptides were further purified by preparative HPLC on Vydac C₁₈-columns using acetonitrile gradient in 0.1% TFA. The peptides were vacuum-dried to remove acetonitrile and lyophilized from 0.1% TFA. Purity was assessed by analytical HPLC and masses were determined by MALDI-TOF using Voyager Biospectrometry Workstation (Perspective Systems) . Peptides were prepared as TFA salts and dissolved in saline for administering to animals.

EXAMPLE 2: In vivo rodent model to study FFA-glucose stimulated insulin and glucagon secretion

All rats were fed ad libidum on standard laboratory chow and were maintained on a 12h light .-dark cycle. Animals were fasted overnight prior to the experiment . Rats were anesthetized (isoflurane 2%) and catheters for iv injections (jugular vein; PE-90, drugs and/or glucose injections) and blood withdrawal (carotid artery; PE-90) were installed. The animals received a glucose bolus (0.6 ml/ rat; i.e. 0.4 g/kg prepared in saline) in the presence or absence of intralipids (Sigma 1-141; phospholipid stabilized soybean oil; 20% emulsion) . No heparin was injected with the formulated intralipids. Insulin and glucagon measurements were made using a radioimmunoassay kits (LINGO) . Results are shown in Figures 1 to 3.

Free fatty acid (FFA) supplementation produced a significant increase in insulin levels both C_maχ and AUC during the observation period (1 h) (Figure 1) . These results suggested that FFA could potently amplify glucose-stimulated insulin secretion (GSIS) and this model could be used to test peptides for antagonistic function.

Peptides 1 to 4 (SEQ ID NOs : 1-4) reduced FFA- stimulation of GSIS to levels observed with glucose alone (Figure 2) . These peptides blocked the contribution of FFA to the insulin kinetics after a glucose challenge.

The peptides did not affect glucagon kinetics upon glucose and intralipid challenge (Figure 3) .

Table 4: Fatty acid composition (%) of soybean oil

EXAMPLE 3 : Evaluation of the effects of continuous delivery of peptide #4 (SEQ ID NO: 4) on the development of diabetes in the C57BL/ks db/db mice. All mice were fed ad libidum on standard laboratory chow and were maintained on a 12h light: dark cycle. 6-7 weeks old animals were anesthetized (isoflurane 2%) and an Alzet™ minipump was installed subcutaneously on the back of the animals. The pumps delivered approximately 20-40 μg/kg/hr (as per the pump description) of peptide #4 (SEQ ID NO: 4) for 25 days. Weight and blood glucose levels were measured throughout the experimental period and a fed glucose (12 hours fasting, 30 minutes feeding) profile established for saline- (n=6) and peptide #4 (SEQ ID NO : 4) -treated (n=4) animals. Results are shown in Figure 4.

The results showed surprisingly significant effects of peptide #4 (SEQ ID NO: 4) on the overall glycemic control in these diabetic animals. First the weight loss seen in advanced diabetes is prevented and the treated animals retained more weight than the saline treated controls (Fig 4A) . The daily glucose levels from days 15-25 are reduced due to the effect of peptide #4 (SEQ ID NO -.4) (Fig 4B) . Fasting glucose levels are 5 mmol lower in peptide #4 (SEQ ID NO:4)- treated group compared to the controls (Fig. 4C) and the postprandial glucose levels in peptide #4 (SEQ ID NO:4)- treated mice were also found to be lower (Fig. 4C) as also reflected in the total AUC of postprandial glucose levels for 6h (Fig. 4D) . The above results show that peptides described herein decreased FFA-stimulated GSIS and this proved to have a curative effect on the overall glycemic control in db/db mice. Hence these peptides and their variants could be of potential use in medicaments to treat diabetic patients and insulin resistant obese subjects in order to increase overall insulin sensitivity and achieve adequate glycemic control. These measures have proven to delay and reduce the consequences of diabetes and insulin resistance that lead to morbidity and mortality in diabetic patients.

Throughout this application, various references are referred to describe more fully the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure .

Claims

WHAT IS CLAIMED IS:

1. A substantially pure peptide compound of Formula I:

Z¹-X¹-X²-X³-X⁴-X^s-X^s-X⁷-X⁸-X⁹-Z² I

wherein:

X¹ is selected from the group consisting of Val, Leu, Trp, Tyr and related amino acids;

X² is selected from the group consisting of Tyr, Lys, Ala, Asn and related amino acids;

X³ is selected from the group consisting of Leu, Ala, Gly and related amino acids having a non-polar side chain;

X⁴ is selected from the group consisting of Gly, Val, Ser and related amino acids;

X^s is selected from the group consisting of Phe, Glu, Ala, Asn and related amino acids;

X⁶ is selected from the group consisting of Ser, Ala, Leu, Val and related amino acids;

X⁷ is selected from the group consisting of Leu, Ala and related amino acids having a non-polar side chain;

X⁸ is selected from the group consisting of Gin, Ala, Glu, Ser and related amino acids;

X⁹ is absent or is selected from the group consisting of Ser, Phe and related amino acids;

Z¹ is an N-terminal group of the formula H₂N-or R(CO)HN-; Z² is a C-terminal group of the formula -C(0)OH, -C(0)NH₂, -C(0)R, -C(0)0R, -C(0)NHR, -C(0)NRR or 1-3 amino acids followed by a C-terminal group of the formula -C(0)OH, -C(0)NH₂, -C(0)R, -C(0)OR, -C(0)NHR, -C(0)NRR;

R at each occurrence is independently selected from (C-C₆) alkyl, (Cχ-C₃) alkenyl, (Cχ-C₃) alkynyl, substituted (Cχ-C₆) alkyl, substituted (C -C₃) alkenyl, or substituted (Cχ-C₆) alkynyl ; and

"-" is a covalent linkage.

2. A substantially pure synthetic peptide compound having a peptide domain of Formula II:

X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ II

wherein:

X¹ is selected from the group consisting of Val, Leu, Trp, Tyr and related amino acids;

X² is selected from the group consisting of Tyr, Lys, Ala, Asn and related amino acids;

X³ is selected from the group consisting of Leu, Ala, Gly and related amino acids having a non-polar side chain;

X⁴ is selected from the group consisting of Gly, Val, Ser and related amino acids;

X⁵ is selected from the group consisting of Phe, Glu, Ala, Asn and related amino acids;

X^s is selected from the group consisting of Ser, Ala, Leu, Val and related amino acids; X⁷ is selected from the group consisting of Leu, Ala and related amino acids having a non-polar side chain;

X⁸ is selected from the group consisting of Gin, Ala, Glu, Ser and related amino acids;

X⁹ is absent or is selected from the group consisting of Ser, Phe and related amino acids; and

"-" is a covalent linkage.

3. The peptide compound of claim 1 or 2 , wherein each of X¹ through X⁹ are independently selected from the group consisting of D-amino acids and L-amino acids.

4. The peptide compound of claim 1 or 2 , wherein each of X¹ through X⁹ are D-amino acids. ₍

5. The peptide compound of claim 1, wherein said 1-3 amino acids of Z² are selected from the group consisting of Gly-Lys and Gly-Lys-Lys.

6. The peptide compound of claim 2, wherein said peptide domain of Formula II is selected from the group consisting of:

Val-Tyr-Leu-Gly-Phe-Ser-Leu-Gln (SEQ ID NO: 9); Leu-Lys-Ala-Val-Glu-Ala-Leu-Ala-Ser (SEQ ID N0:10);

Trp-Ala-Gly-Ser-Ala-Leu-Ala-Glu (SEQ ID NO: 11); and

Tyr-Asn-Ala-Ser-Asn-Val-Ala-Ser-Phe (SEQ ID NO:12);

where each amino acid is independently a D-amino acid and an L- amino acid.

7. The peptide compound of claim 2, wherein said peptide domain of Formula II is selected from the group consisting of:

(a) vylgfslqGK (SEQ ID NO:l); (b) lkavealasGK (SEQ ID NO: 2) ;

(c) wagsalaeGK (SEQ ID NO:3);

(d) ynasnvasfGK (SEQ ID NO:4);

(e) vylgfslqGKK (SEQ ID NO:5);

(f) lkavealasGKK (SEQ ID NO:6); (g) wagsalaeGKK (SEQ ID NO:7);

(h) ynasnvasfGKK (SEQ ID NO: 8);

(i) vylgfslq (SEQ ID NO:9);

(j) lkavealas (SEQ ID NO:10);

(k) wagsalae(SEQ ID NO: 11); (1) ynasnvasf (SEQ ID NO: 12); and

(m) a peptide domain comprising the amino acid sequence of any one of (a) to (1) with conservative amino acid substitutions;

wherein lowercase symbols denote D-amino acids and uppercase symbols denote L-amino acids.

8. A substantially pure peptide compound comprising a peptide domain having an amino acid sequence selected from: (a) an amino acid sequence selected from the group consisting of

Val-Tyr-Leu-Gly-Phe-Ser-Leu-Gln (SEQ ID NO:9);

Leu-Lys-Ala-Val-Glu-Ala-Leu-Ala-Ser (SEQ ID NO:10); Trp-Ala-Gly-Ser-Ala-Leu-Ala-Glu (SEQ ID NO:ll); and

Tyr-Asn-Ala-Ser-Asn-Val-Ala-Ser-Phe (SEQ ID NO:12);

(b) an amino acid sequence that is 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of (a) and which modulates insulin levels in a subject; and (c) an amino acid sequence comprising a fragment of at least 5 contiguous amino acids of the amino acid sequence of (a) and which modulates insulin levels in a subject;

wherein:

- said peptide domain optionally further comprises an additional 1-3 amino acids at its carboxy terminus;

- each amino acid of said peptide domain is independently a D-amino acid or an L-amino acid;

- the amino terminal H₂N- group of said peptide domain is optionally modified to R(CO)HN-; and

- the carboxy terminal -C(0)OH group of said peptide domain is optionally modified to -C(0)NH₂, -C(0)R, -C (O) OR, -C(0)NHR, or -C(0)NRR;

where R at each occurrence is independently selected from substituted or unsubstituted (C -C₆) alkyl , substituted or unsubstituted (Cχ-C₆) alkenyl, substituted or unsubstituted (Cι-C₆) alkynyl, substituted or unsubstituted (C₆-Cι₄) aryl, and substituted or unsubstituted (Cι-C₆) alkyl (C₆-Cι₄) aryl; and

"-" is a covalent linkage.

9. The peptide compound of claim 8 wherein said peptide domain has an amino acid sequence selected from the group consisting of

Val-Tyr-Leu-Gly-Phe-Ser-Leu-Gln (SEQ ID NO: 9);

Leu-Lys-Ala-Val-Glu-Ala-Leu-Ala-Ser (SEQ ID NO: 10);

Trp-Ala-Gly-Ser-Ala-Leu-Ala-Glu (SEQ ID NO-.ll); and Tyr-Asn-Ala-Ser-Asn-Val-Ala-Ser-Phe (SEQ ID NO.-12);

where each amino acid is independently a D-amino acid and an L- amino acid.

10. The peptide compound of claim 8 or 9, wherein said peptide domain comprises at least one D-amino acid.

11. The peptide compound of claim 8 or 9, wherein said peptide domain comprises only D-amino acids.

12. The peptide compound of any one of claims 8 to 11, wherein said peptide domain further comprises 1-3 amino acids selected from the group consisting of Gly-Lys and Gly-Lys-Lys at the C-terminus of said peptide domain.

13. The peptide compound of claim 12 which is selected from the group of peptides consisting of:

(a) vylgfslqGK (SEQ ID NO:l);

(b) lkavealasGK (SEQ ID NO:2); (c) wagsalaeGK (SEQ ID NO: 3);

(d) ynasnvasfGK (SEQ ID NO:4);

(e) vylgfslqGKK (SEQ ID NO: 5);

(f) IkavealasGKK (SEQ ID NO: 6); (g) wagsalaeGKK (SEQ ID NO:7); and

(h) ynasnvasfGKK (SEQ ID NO:8);

wherein lowercase symbols denote D-amino acids and uppercase symbols denote L-amino acids.

14. A pharmaceutical composition comprising the peptide compound of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. The composition of claim 14, wherein said composition comprises about lOOμg to about lOOmg of said peptide compound.

16. A method for reducing insulin secretion or for preventing or treating a condition associated with defective glycemic control in a subject, said method comprising administering to said subject an effective amount of the peptide compound of any one of claims 1 to 13.

17. The method of claim 16, wherein said condition is selected from the group consisting of diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion and hyperlipidemia.

18. The method of claim 16 or 17, wherein the subject is a mammal.

19. The method of claim 18, wherein the mammal is a human.

20. Use of the peptide compound of any one of claims 1 to 13 in the preparation of a medicament for reducing insulin secretion or for preventing or treating a condition associated with defective glycemic control in a subject.

21. Use of the peptide compound of any one of claims 1 to 13 for reducing insulin secretion or for preventing or treating a condition associated with defective glycemic control in a subject.

22. The use of claim 20 or 21, wherein said condition is selected from the group consisting of diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion and hyperlipidemia.

23. The use of claim 22 , wherein the subject is a mammal .

24. The use of claim 23, wherein the mammal is a human.

25. A package comprising the peptide compound of any one of claims 1 to 13, together with instructions for reducing insulin secretion or for preventing or treating a condition associated with defective glycemic control in a subject .

26. The package of claim 25, wherein said condition is selected from the group consisting of diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion and hyperlipidemia. 50

18. The method of claim 17, wherein the mammal is a human .

19. Use of the peptide compound of any one of claims 1 to 12 in the preparation of a medicament for reducing insulin secretion or for preventing or treating a condition associated with defective glycemic control in a subject.

20. Use of the peptide compound of any one of claims 1 to 12 for reducing insulin secretion or for preventing or treating a condition associated with defective glycemic control in a subject.

21. The use of claim 19 or 20, wherein said condition is selected from the group consisting of diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion and hyperlipidemia.

22. The use of claim 21 , wherein the subject is a mammal .

23. The use of claim 22, wherein the mammal is a human.

24. A package comprising the peptide compound of any one of claims 1 to 12 , together with instructions for reducing insulin secretion or for preventing or treating a condition associated with defective glycemic control in a subject .

25. The package of claim 24, wherein said condition is selected from the group consisting of diabetes, hyperinsulinism, insulin resistance syndrome, beta cell exhaustion and hyperlipidemia.