JP2008539735A - Glucagon-like peptide 1 (GLP-1) receptor antagonists and methods for their pharmacological use - Google Patents

Abstract

  The present invention provides peptides with novel modifications that provide suitable derivatization sites for improving the pharmacokinetic properties of the peptides. These GLP-1 modified peptides function as GLP-1 receptor agonists in vivo. The peptides of the present invention provide new therapies for patients with reduced endogenous insulin secretion, eg, type 2 diabetes.

Description

  This application claims the benefit of US Provisional Application No. 60 / 678,723, filed May 6, 2005, the contents of which are hereby incorporated by reference in their entirety.

  The present invention relates to novel modifications that provide suitable derivatization sites for improving the pharmacokinetic properties of peptides. Such N-terminal modification of the main chain amino group of the first amino acid residue or C-terminal modification of the main chain carboxylic acid group of the last amino acid residue may be aliphatic, C3-C7 cycloalkyl, aryl, or one or more Mono- or bicyclic heteroaromatics containing the above nitrogen, oxygen and / or sulfur heteroatoms may be included. In addition, the N-terminal or C-terminal modification may provide suitable derivatization sites, including but not limited to amino and thiol groups. The modified peptides of the present invention are useful in stimulating the release of insulin from pancreatic beta cells in a glucose-dependent manner, thereby helping individuals suffering from metabolic disorders such as diabetes or impaired glucose tolerance (pre-diabetic state). Provide treatment options.

  Diabetes is characterized by abnormal glucose metabolism that manifests itself by elevated blood glucose levels, especially in diabetic patients. The underlying defects are two large groups, type 1 diabetes or insulin-dependent diabetes (IDDM) that occurs when patients lack beta cells that produce insulin in their pancreatic islets of Langerhans; It leads to the classification of diabetes into type 2 diabetes, or non-insulin dependent diabetes mellitus (NIDDM), which occurs in patients with altered cellular function and insulin action.

  Type 1 diabetes is currently treated with insulin, while the majority of type 2 diabetic patients are treated with agents that stimulate β-cell function or increase the patient's tissue sensitivity to insulin. Over time, nearly half of Type 2 diabetic subjects have lost their response to these agents and must then be placed on insulin therapy. The drugs currently used to treat type 2 diabetes are described below.

[alpha] -Glucosidase inhibitors (e.g. Precose ^(R) , Voglibose ^{( TM )} and Miglitol ^(R) ) reduce postprandial glucose excursions by delaying the absorption of glucose from the intestinal tract. These drugs are safe and provide treatment to mild to moderately affected diabetic subjects. However, gastrointestinal side effects have been reported in the literature.

Insulin sensitizers are drugs that enhance the body's response to insulin. Thiozolidinediones, such as Avandia ^™ (rosiglitazone) and Actos ^™ (pioglitazone), activate peroxisome proliferator-activated receptor (PPAR) gamma subtypes and are a poorly described set of Regulates gene activity. The first drug in this class, Rezulin ^™ (troglitazone), was recovered due to elevated liver enzyme levels and drug-induced hepatotoxicity. These liver effects do not appear to be a major problem in patients using Avandia ^™ and Actos ^™ . Even so, hepatic enzyme testing is recommended every two months for the first year of treatment and thereafter. Avandia ^™ and Actos ^™ appear to be associated with fluid retention and edema. Another potential side effect is weight gain. Avandia ^™ is not indicated for use with insulin due to concerns about congestive heart failure.

  Insulin secretagogues, such as sulfonylurea (SFU) and other agents that act through ATP-dependent K + channels, are another drug type currently used to treat type 2 diabetes. SFU is the standard treatment for patients with type 2 diabetes who have mild to moderate fasting hyperglycemia. SFU has limitations that include the potential to induce hypoglycemia, weight gain, and high primary and secondary failure rates. 10-20% of patients initially treated fail to show a significant treatment effect (primary failure). Secondary failure is indicated by an additional 20-30% loss of treatment effect 6 months after taking SFU. Insulin treatment is required in 50% of SFU responders after 5-7 years of therapy (Non-Patent Document 1).

Glucophage ^™ (Metformin HCl) is a biguanide that lowers blood glucose by reducing hepatic glucose output and increasing peripheral glucose uptake and utilization. The drug is effective in lowering blood glucose in subjects affected mildly and moderately and has no side effects of being able to induce weight gain or hypoglycemia. However, Glucophage ^™ has a number of side effects including gastrointestinal upset and the possibility of lactic acidosis. Glucophage ^™ is contraindicated in diabetic patients older than 70 years and in subjects with impaired renal or liver function. Finally, Glucophage ^™ has a primary and secondary failure rate similar to SFU.

  Insulin treatment is performed after diet, exercise and oral medication fail to adequately control blood glucose. This treatment has the disadvantage that it is an injection, it can cause hypoglycemia, and it causes weight gain.

  Problems with current treatments require new therapies to treat type 2 diabetes. In particular, new treatments are needed that maintain normal (glucose-dependent) insulin secretion. These new drugs depend on glucose to promote insulin secretion (ie, produce insulin secretion only in the presence of elevated blood glucose); low primary and secondary failure rates; and Should have preservation of islet cell function. Strategies for developing new therapies disclosed herein are based on the cyclic adenosine monophosphate (cAMP) signaling mechanism and its effect on insulin secretion.

Cyclic AMP is a major regulator of the insulin secretion process. This increase in signaling molecules promotes K + channel closure after activation of the protein kinase A pathway. Closure of the K ⁺⁺ channel causes cellular depolarization and subsequent opening of the Ca ⁺⁺ channel, which in turn leads to endocytosis of the insulin granules. Even a slight effect on insulin secretion occurs in the absence of low glucose concentrations (Non-Patent Document 2). Secretagogues such as PACAP (pituitary adenylate cyclase activating peptide), VIP (vasoactive intestinal peptide), GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide 1) are: The cAMP system is used to regulate insulin secretion in a glucose-dependent manner (Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5). Insulin secretagogues that act by increasing cAMP, such as GLP-1, VIP, GIP and PACAP, can increase insulin synthesis in addition to insulin release (Non-Patent Document 6; Non-Patent Document 7). ).

GLP-1 is released from intestinal L cells after a meal and functions as an incretin hormone (ie, it enhances glucose-induced insulin release from pancreatic β cells). It is a 37 amino acid peptide that is differentially expressed by the glucagon gene depending on the tissue type. Clinical data was collected with GLP-1 to support the beneficial effects of increasing cAMP levels in β cells. Infusions of GLP-1 in poorly controlled patients with type 2 diabetes normalized their fasting blood glucose levels (8), and longer infusions improved beta cell function to that of normal subjects (Non-patent document 9). Recent reports have shown that GLP-1 improves the ability of β cells to respond to glucose in subjects with impaired glucose tolerance (Non-Patent Document 10). All of these effects, however, have a short duration due to the short half-life of the peptide.

  Amylin Pharmaceuticals is conducting a Phase III study using a 39 amino acid peptide, exendin-4 (AC2993), originally identified by the American Monster Lizard (Gila Monster). Amylin reported that clinical trials showed improved glycemic control in patients with type 2 diabetes treated with exendin-4. However, the incidence of nausea and vomiting was significant.

  Glucose-dependent insulinotropic peptide (GIP) is a 42-residue intestinal peptide involved in the regulation of fat and glucose metabolism, and insulin secretory function resides at residues 1-30. GIP is degraded by proteolysis of dipeptidyl peptidase IV (DPPIV) and excreted by renal excretion. Limited clinical data was collected with GIP. Intravenous (IV) or continuous administration in patients with type 2 diabetes caused an acute increase in plasma insulin levels (Non-Patent Document 11; Non-Patent Document 12). These effects, however, have a short duration due to the short half-life of the peptide.

  PACAP is a potent stimulator of glucose-dependent insulin secretion from pancreatic β cells. Three different PACAP receptor types (PAC1, VPAC1 and VPAC2) have been described (Non-Patent Document 13; Non-Patent Document 14). PACAP does not exhibit receptor selectivity and has comparable activity and potency with all three receptors. PAC1 is primarily located in the CNS, while VPAC1 and VPAC2 are more widely distributed. VPAC1 is located in the CNS and in the liver, lungs and intestines. VPAC2 is located in the CNS, pancreas, skeletal muscle, heart, kidney, adipose tissue, testis and stomach. Recent studies argue that VPAC2 is responsible for insulin secretion from β cells (Non-Patent Document 15; Non-Patent Document 16). This insulinotropic action of PACAP is mediated by the GTP binding protein Gs. Intracellular cAMP accumulation, in turn, activates non-selective cation channels in β-cells to increase [Ca ++] and promote exocytosis of secretory granules containing insulin.

  GLP-1 is a member of a family of structurally related peptide hormones, the glucagon / secretin family. Within this family, GLP-1 (7-36) and GLP-1 (7-37) (30 and 31 amino acids, respectively) are the same precursor preproglucagon, as is glucagon (30 amino acids). Depart from. By tissue-specific processing, preproglucagon is converted primarily to GLP-1 in the gut and to glucagon in the pancreas. The GLP-1 receptor belongs to the family of G protein coupled receptors.

  GLP-1 reduces plasma glucose concentrations mediated by glucose-dependent insulin secretion. Considering the important role of GLP-1 in maintaining normoglycemia, there has been considerable interest in identifying GLP-1 receptor agonists. Clinical trials have shown the ability of GLP-1 infusion to promote insulin secretion and normalize plasma glucose in diabetic subjects. However, GLP-1 is rapidly degraded in the body and has a very short half-life. Furthermore, GLP-1 causes intestinal motility side effects at or near its therapeutic dose. Thus, GLP-1 itself has significant limitations as a therapeutic agent, and modified versions of the peptide with enhanced stability are being followed. To date, non-peptide agonists of the GLP-1 receptor have not been described.

Recent studies have shown a variety of biological effects of GLP-1. GLP-1 causes glucose-dependent insulin secretion from the pancreas (Non-patent Document 17). GLP-1 also increases satiety and reduces food intake (Non-patent Document 18). In addition, GLP-1 protects ischemia and reperfused myocardium (Non-Patent Document 19) and improves endothelial dysfunction (Non-Patent Document 20). GLP-1 was also associated with improved learning and exhibited a neuroprotective effect (21).

  Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide initially isolated from porcine upper small intestine (Non-patent Document 22; Patent Document 1). This peptide belongs to a family of small structurally related polypeptides, including herodermin, secretin, somatostatins and glucagon. The biological effects of VIP are mediated by activation of membrane bound receptor proteins that are coupled to the intracellular cAMP signaling system. These receptors were originally known as VIP-R1 and VIP-R2, however, they were later found to be the same receptors as VPAC1 and VPAC2. VIP exhibits comparable activity and potency with VPAC1 and VPAC2.

  In order to improve the stability of GLP-1, efforts have been made to alter the sequence of the native peptide to improve half-life and exposure, or to non-human proteins that are GLP-1 receptor agonists (exendin-4 ) Concentrated on using. These products include exenatide, a peptide from the saliva of the American monster monster (Gila monster), currently in Phase III clinical trials as an injection twice a day. Another GLP-1 analogue is currently phase II acylated form of natural GLP-1, Liragglutide (NN-2211), as a once-daily injection. Other reported modifications include conjugation to albumin and other techniques for improving in vivo half-life.

  PACAP, VIP, GIP, GLP-1 or exendin-4 has glucose-dependent insulinotropic activity, but with fewer side effects, and is preferably stable in the formulation and has a long plasma half-life in vivo There is a need for improved peptides having a phase. Such improved in vivo half-life results from peptides with both reduced disappearance and reduced sensitivity to proteolysis. In addition, more reliable management of plasma glucose levels may prevent long-term diabetic complications. Thus, new diabetes drugs should provide patients with an improved quality of life.

U.S. Pat. No. 3,879,371 Schen et al., Diabetes Res. Clin. Pract. 6: 533-543, 1989 Weinhaus et al., Diabetes 47: 146-1435, 1998 Komatsu et al., Diabetes 46: 1928-1938, 1997. Filippsson et al., Diabetes 50: 1959-1969, 2001. Drucker, Endocrinology 142: 521-527, 2001 Skogland et al., Diabetes 49: 1156-1164, 2000 Borboni et al., Endocrinology 140: 5530-5537, 1999 Gutniak et al., New Eng. J. et al. Med. 326: 1316-132, 1992 Rachman et al., Diabetes 45: 1524-1530, 1996. Byrne et al., Diabetes 47: 1259-1265, 1998. Kindmark et al. Clin. Endocrinol. Metab. 86: 2015-2019, 2001 Meier et al., Diabetes 53 (Suppl 3): S220-S224, 2004. Harmar et al., Pharmacol. Reviews 50: 265-270, 1998 Vaudry et al., Pharmacol. Reviews 52: 269-324, 2000 Inagaki et al., Proc. Natl. Acad. Sci. USA 91: 2679-2683, 1994. Tsutsumi et al., Diabetes 51: 1453-1460, 2002. Holst, Curr. Opin. Edocrinol. Diabetes 12: 56-62, 2005 Flint et al. Clin. Invest. 101: 515-520, 1998 Nikoladis et al., Circ. 109: 962-965, 2004 Nystrom, Am. J. et al. Physiol. Endocrinol. Metab. 287: E1209-E1215 During et al., Nat. Med. 9: 1173-1179, 2003 Said and Mutt, Science 169: 1217-1218, 1970

[Summary of Invention]
The present invention provides novel modifications that provide suitable derivatization sites for improving the pharmacokinetic properties of peptides. Such an N-terminal modification of the amino group of the first peptide residue may be aliphatic, C3-C7 cycloalkyl, aryl, or mono- or bicyclic hetero, containing one or more nitrogen, oxygen and / or sulfur heteroatoms. Aromatics can be included. In addition, N-terminal modifications can provide suitable derivatization sites, including but not limited to amino and thiol groups. Some examples of such N-terminal modifications include, but are not limited to, 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 4-amino-2-chloro-benzoic acid, 4-amino -3-methoxy-benzoic acid, 4-amino-3-methyl-benzoic acid, 1-amino-cyclopentane-3-carboxylic acid, trans-3-aminocyclohexanecarboxylic acid, D-pipecolic acid, 4-amino-1 -Methyl-1H-imidazole-2-carboxylic acid, 4-methylthiobenzoic acid, 2-methylthiobenzoic acid, 2-methylthionicotinic acid, proline, 6-aminohexanoic acid, benzoic acid, (S) -tetrahydroisoquinoline acetic acid, indoline -2-carboxylic acid, cis-3-aminocyclohexanecarboxylic acid, L-pipecolic acid, 9-glutenylmethoxycar Nyl, 2-thio-polyethylene glycol benzoic acid, 2-thio-polyethylene glycol nicotinic acid, 4-amino-1-methyl-1H-imidazole-2-carboxylic acid, 1-amino-cyclopentane-3-carboxylic acid, 4 -Amino-1-methyl-1H-imidazole-2-carboxylic acid, 1-amino-cyclopentane-3-carboxylic acid, 1-amino-cyclopentane-3-carboxylic acid, (2-mercapto-1H-benzimidazole -1-yl) acetic acid, 2- (tritylthio) ethyl] amino} nicotinate, 2-{[2- (tritylthio) ethyl] amino} nicotinate, 1- [2- (tritylthio) ethyl] -1H-imidazole-2- Carboxylate, 4-{[2- (tritylthio) ethyl] amino} pyrimidine-5-carboxylate 1- [2- (tritylthio) ethyl] -1H-benzimidazole-2-carboxylate, 2-mercapto-1H-imidazol-1-yl) acetic acid, ({1- [2- (tritylthio) ethyl] -1H- Imidazol-2-yl} thio) acetate, 3- (2-tritylsulfanylethylamino) pyrazine-2-carboxylate, 4-mercaptothiazole-5-carboxylic acid, 2-mercaptothiazole-5-carboxylic acid, 2- ( 2-tritylsulfanylethylamino) thiazole-5-carboxylate, 2-mercapto-6-methylpyrimidine-4-carboxylic acid, 5-mercaptonicotinic acid, 5-isopropyl-2-mercaptothiazole-4-carboxylic acid, 1- Hexadecyl-1H-benzimidazol-2-ylsulfanyl) acetic acid And 2- (2-tert-butoxycarbonylaminoethylamino) thiazole-5-carboxylic acid.

The present invention relates to a compound of formula (I)
Z1-A1-A2-A3-Gly-A5-Phe-Thr-A8-Asp-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-Phe-A23-A24- A25-A26-A27-A28-A29-A30-A31-A32-A33-A34-A35-A36-A37-A38-A39-A40-Z2 (SEQ ID NO: 1)
For the peptide
Where
A1 is His, Phe, Tyr, d-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N ^α -acetylhistidine, α-fluoromethyl-histidine, α-methyl-histidine , 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
A2 is Ser, Gly, Ala, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1 -Aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid or (1-aminocyclooctyl) carboxylic acid;
A3 is Glu, Gln or Ala;
A5 is Thr or Ala;
A8 is Ser or Ala;
A10 is Val, Leu, Tyr or Ala;
A11 is Ser, Ala, Arg, Gly, Lys or Thr;
A12 is Lys, Ser, Arg, Ala, Asn, His, or Gln;
A13 is Tyr or Gln;
A14 is Leu, Met or Ala;
A15 is Asp or Glu;
A16 is Gly, Glu, Ala, Ser, Phe, Trp, Thr, His, Lys, Arg, Val, Ile, Leu, Met, Asn, Gln or Tyr;
A17 is Gln, Glu, Arg, Ala, Lys, Leu, Met, Val, Phe, Ile, Trp, Asn, Asp, His, Ser, Thr, Gly or Tyr;
A18 is Ala, Arg, Lys, Phe, Ile, Leu, Tyr, Val, Met, Gly or His;
A19 is Ala or Val;
A20 is Lys, Arg, Ala, Gln or Cys;
A21 is Glu, Leu, Ala or Asp;
A23 is Ile or Val;
A24 is Ala, Glu, Lys, Gln, Asn or Lys-X;
A25 is Trp or Ala;
A26 is Leu or Ala;
A27 is Val, Lys, Ala or Met;
A28 is Lys, Asn, Arg, Ala, Gln or Cys;
A29 is Gly, Ala, Lys, Thr or Cys;
A30 is Arg, Gly, Ala, Cys or has been deleted;
A31 is Gly Pro, Lys, Cys, Lys-X or has been deleted;
A32 is Ser, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A33 is Ser, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A34 is Gly, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A35 is Ala, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A36 is Pro, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A37 is Pro, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A38 is Pro, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A39 is Ser, Lys, Cys, Lys-X, Cys-PEG or is deleted; and A40 is Lys-X, Cys-PEG, or is deleted.

Lys-X is Lys modified with a fatty acid exemplified by CH ₃ (CH ₂ ) _n COOH (where n ranges from 0 to about 24) with N ^ε .

  Z1 is

Selected from.

Z2 can be a hydroxyl group such that the peptide has an unmodified carboxylic acid C-terminus, or Z2 can be a modification of the C-terminal carboxylic acid group. Z2 can be a modification such as amidation, or Z2 can also be an unnatural amino acid or amide. Z2 is not limited,

Can be mentioned.

  For the peptide of formula (I), the N-terminal modification can be linked via an amide bond to the α-amino group of the first amino acid of said peptide. The C-terminal modification can be linked via an amide bond to the main chain carboxylic acid group of the last amino acid of the peptide. Examples of peptides of formula (I) can be found in the peptides described in Table 1 (for example, SEQ ID NOs: 1 to 56), but are not limited thereto.

  A derivative of the invention has been fused with another compound, such as a compound (eg, polyethylene glycol, “PEG”) to increase the half-life of the peptide and / or reduce the potential immunogenicity of the peptide Peptides can be included. For example, PEGylated peptides typically have a longer half-life in vivo (Greenwald, Adv. Drug. Del. Rev. 55: 217-250, 2003).

In the case of pegylation, fusion of the peptide to PEG can be accomplished by any means known to those skilled in the art. For example, pegylation can be accomplished by first introducing a cysteine mutation in the peptide to provide a linker that attaches to PEG, followed by site-specific derivatization with PEG-maleimide. Alternatively, the N-terminal modification can incorporate a reactive moiety for attachment to PEG, as exemplified by the amine, mercapto or carboxylic acid groups of the N-terminal modified compounds disclosed above. For example, pegylation first introduces a mercapto moiety into the peptide via an N-terminal modifying group to provide a linker that attaches to the PEG, then, for example, Nektar Therapeutics (San Carlos, Calif., USA) and / or It can be achieved by site-specific derivatization with a methoxy-PEG-maleimide reagent supplied by either NOF (Tokyo, Japan). In addition to maleimide, a number of Cys reactive groups are known to those skilled in the art of protein crosslinking, such as the use of alkyl halides and vinyl sulfones (see, for example, Proteins, Structure and Molecular Properties , 2nd edition, T.E. Creighton). , WH Freeman and Company, New York, 1993). In addition, PEG can be introduced by direct attachment to a C-terminal carboxylic acid group, or an internal amino acid such as Cys, Lys, Asp or Glu, or an unnatural amino acid containing a similar reactive side chain moiety.

  Various sizes of PEG groups can be used, including but not limited to PEG polymers from about 5 kDa to about 43 kDa. PEG modifications can include a single linear PEG. For example, linear 5, 20 or 30 kDa PEGs linked to maleimide or other bridging groups are available from Nektar and / or NOF (see, eg, Table 2). Modifications may also require branched PEGs containing two or more PEG polymer chains attached to maleimide or other bridging groups but available from Nektar and NOF (see, eg, Table 2) I want to be)

  PEGylation with smaller PEGs (eg linear 5 kDa PEG) may be less likely to reduce the activity of the peptide, while larger PEGs (eg branched 40 kDa PEG) It is possible that it may be more likely to reduce activity. However, larger PEGs can further prolong the plasma half-life such that once weekly injections are possible (Harris et al., Clin. Pharmacokine. 40: 539-551, 2001).

  The linker between the PEG and the peptide bridging group can vary. For example, a commercially available thiol-reactive 40 kDa PEG (mPEG2-MAL) from Nektar (Huntsville, Alab.) Uses a maleimide group for conjugation to Cys and the maleimide group contains Lys Is attached to the PEG via a linker (see, eg, Table 2). As a second example, a commercially available thiol-reactive 43 kDa PEG (GL2-400MA) from NOF uses a maleimide group for conjugation to Cys, and the maleimide group contains a disubstituted alkane linker. To the PEG (see, eg, Table 2). In addition, PEG polymers can be conjugated directly to maleimides as exemplified by the 5 and 20 kDa PEG reagents available from Nektar Therapeutics (Huntsville, Alab.) (See, eg, Table 2).

The invention can include, but is not limited to, the use of mercapto groups as crosslinking sites. Present in amino acids, such as amino groups of N-terminal modified compounds, C-terminal carboxylic acids of either unmodified C-terminal or Z2-modified peptides, and side chains of amino acids such as Lys, Arg, Asp and Glu Other moieties are known to provide reactive groups that provide moieties suitable for covalent modification and attachment to PEG. Numerous examples of suitable cross-linking agents are known to those skilled in the art (eg, Proteins, Structure and
Molecular Properties , 2nd edition, T.M. E. Creighton, W.C. H. (See Freeman and Company, New York, 1993). Such crosslinkers include, but are not limited to, for example, commercially available PEG derivatives containing amines, aldehydes, acetals, maleimides, succinimides and thiols, such as those marketed by Nektar and NOF, Can be conjugated to PEG (eg, Harris et al., Clin. Pharmakinet. 40: 539-551, 2001).

In addition to pegylation, the peptides of the present invention may be modified with fatty acids that improve pharmacodynamic properties. For example, N-terminally modified compounds containing amines can be derivatized with palmitic acid or myristoleic acid or other fatty acids using methods known to those skilled in the art, or alkyl (eg C ₆ -C ₁₈ ) The moiety can be directly included as part of the N-terminal modifying compound.

  The peptides of the present invention have improved stability against proteolysis by DPPIV and in plasma compared to GLP-1. The derivatives of the present invention exhibit prolonged duration of action in vivo, and when derivatized, support less than once dose per day or less than once per week or more.

  Peptides of the present invention (eg Table 1) can be used to treat metabolic disorders such as those resulting from reduced endogenous insulin secretion, especially patients with type 2 diabetes, or impaired glucose tolerance (pre-diabetes with mild changes in insulin secretion). Provide new treatment to patients with (condition). In addition, the peptides of the present invention may include type 1 diabetes, gestational diabetes, juvenile-onset adult type diabetes (MODY), adult autoimmune diabetes (LADA) and related diabetic lipid metabolism disorders, and others Useful in the prevention and / or treatment of diabetes complications, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, fasting hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X and insulin resistance sell.

  The peptides of the present invention (eg Table 1) are used in disorders such as obesity, as well as atherosclerosis, coronary heart disease, coronary artery disease, hyperlipidemia, hypercholesterolemia, low HDL levels, hypertension; It may also be effective in the treatment of cardiovascular diseases, including peripheral diseases; and in the treatment of neurodegenerative diseases (including Parkinson's disease and Alzheimer's disease).

  One aspect of the present invention is a peptide of formula (I), as well as fragments thereof exhibiting at least one biological function that is substantially identical to the peptide of formula (I), including functional equivalents thereof , Derivatives and variants (collectively “peptides of the invention”) (eg, Table 1).

  Antibodies and antibody fragments that selectively bind peptides of the invention (eg, Table 1) are also provided. Such antibodies are useful in detecting the peptides of the present invention and can be identified and generated by procedures known in the art. Polyclonal N-terminal IgG antibodies and monoclonal C-terminal Fab antibodies that recognize the peptides of the present invention have been generated.

  The present invention also includes administering to a mammal a therapeutically effective amount of any of the peptides of the present invention or any GLP-1 active peptide such as the peptide of formula (I) (eg, Table 1). A method for the treatment of diabetes, diabetes-related disorders and / or other diseases or conditions affected by the peptides of the invention, for example achieved by the GLP-1 agonist function of the peptides of the invention in the mammal Also directed to.

  Also disclosed are methods of making the peptides of the present invention.

[Detailed Description of the Invention]
The present invention provides novel modified peptides and fragments, derivatives and variants thereof (peptides of the invention) that exhibit at least one biological function that is substantially identical to the peptide of formula (I) To do. The peptides of the present invention (eg, Table 1) are either in vivo as agonists of GLP-1 or otherwise, both type 1 and type 2 diabetes, gestational diabetes, juvenile-onset adult type diabetes (MODY) (Herman) Diabetes 43:40, 1994); diabetes including adult latent diabetes (LADA) (Zimmet et al., Diabetes Med. 11: 299, 1994); and related diabetic lipid metabolism disorders and other diabetic complications And function in the prevention and / or treatment of diseases or conditions such as hyperglycemia, hyperinsulinemia, impaired glucose tolerance, fasting hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X and insulin resistance To do.

  The peptides of the present invention (eg Table 1) are used in disorders such as obesity, as well as atherosclerosis, coronary heart disease, coronary artery disease, hyperlipidemia, hypercholesterolemia, low HDL levels, hypertension; It may also be effective in the treatment of cardiovascular diseases, including peripheral diseases; and in the treatment of neurodegenerative diseases (including Parkinson's disease and Alzheimer's disease).

  The peptides of the present invention (eg, Table 1) can stimulate insulin release from pancreatic β cells in a glucose-dependent manner. Furthermore, the peptides of the invention are stable in both aqueous and non-aqueous formulations and exhibit a plasma half-life of greater than 1 hour (eg exhibit a plasma half-life of 6 hours or more).

  The peptides of the present invention are GLP-1 agonists (eg Table 1). In addition, these peptides have activity at other relevant receptors including but not limited to VPAC2 receptor activation, GIP receptor activation or glucagon receptor antagonism. sell. Furthermore, the peptides of the invention stimulate insulin release into the plasma in a glucose-dependent manner without inducing a quiescence or increase in plasma glucose levels that is counterproductive for example in the treatment of type 2 diabetes. In addition, the peptides of the present invention can be selective agonists of the GLP-1 receptor, whereby unpleasant or dangerous side effects such as gastrointestinal water retention and / or increased heart rate or It causes the release of insulin into the plasma while being selective for other receptors that are responsible for undesirable cardiovascular effects such as blood pressure.

  The peptides of the invention are also stable in aqueous and non-aqueous formulations. The peptides of the invention can show less than 10% degradation at 37-40 ° C. for 1 week when dissolved in water or non-aqueous organic solvent (pH between 7-8), or The peptides of the present invention can show less than 5% degradation at 37-40 ° C. over a week when dissolved in water (with a pH between 7-8) or non-aqueous organic solvents. Further, the compositions and formulations of the present invention can comprise the peptides of the present invention and from about 2% to about 30% DMSO. In another embodiment of the invention, the compositions and formulations are optionally added such as from about 0.2% to about 3% (w / V) propylene glycol, dimethylformamide, propylene carbonate, polyethylene glycol and triglycerides. Other solvents may be included.

  Finally, derivatized peptides of the invention can exhibit a plasma half-life of, for example, a minimum of 1 hour, a minimum of 3 hours or a minimum of 6 hours in rats after IV infusion. Furthermore, the derivatized peptides can show a significant decrease in plasma glucose AUC after subcutaneous injection in rats, for example at least 24 hours, at least 41 hours or at least 65 hours after injection.

  The peptides of the present invention provide new therapies for patients with reduced endogenous insulin secretion or impaired glucose tolerance, especially type 2 diabetes. That is, the peptides of the present invention are long acting GLP-1 receptor agonists that can be used to maintain, ameliorate, and reverse glucose-stimulated insulin secretion. Furthermore, selective peptide agonists of the GLP-1 receptor can enhance glucose-dependent insulin secretion in the pancreas without causing side effects associated with non-selective activation of other related receptors.

  Certain terms used throughout this specification are defined below and others may be defined as introduced. Single letter abbreviations for specific amino acids, their corresponding amino acids, and three letter abbreviations are as follows. A, alanine (Ala); C, cysteine (Cys); D, aspartic acid (Asp); E, glutamic acid (Glu); F, phenylalanine (Phe); G, glycine (Gly); H, histidine (His) ); I, isoleucine (Ile); K, lysine (Lys); L, leucine (Leu); M, methionine (Met); N, asparagine (Asn); P, proline (Pro); Q, glutamine (Gln) R, arginine (Arg); S, serine (Ser); T, threonine (Thr); V, valine (Val); W, tryptophan (Trp); and Y, tyrosine (Tyr).

  “Functional equivalent” and “substantially identical biological function or activity”, respectively, are indicated by the peptides to which they are compared when the biological activity of each peptide is measured by the same procedure By a biological activity that is within about 30% to about 100% or more of the biological activity is meant.

  As used herein interchangeably, “biological activity”, “activity” or “biological function” refers to a peptide (whether in its native conformation or in a modified conformation). Or an effector function performed directly or indirectly by any fragment, derivative and variant thereof. Biological activity includes, for example, binding to peptides, binding to other proteins or molecules, DNA binding proteins, activity as a transcriptional regulator, ability to bind damaged DNA, and the like.

  The terms “fragment”, “derivative” and “variant” when referring to the peptides of the invention are peptides that retain substantially the same biological function or activity as such peptides, as described further below. Fragments, derivatives and variants of

  A fragment is a portion of a peptide that retains substantially similar functional activity, eg, as described in the in vivo model disclosed herein.

  A derivative is a peptide (eg, a modified N-terminal peptide, a modified C-terminal peptide, or a peptide that substantially preserves the functions disclosed herein and includes additional structures and associated functions, as further described below. PEGylated peptides), all modifications to the fusion peptide that confer additional activity, such as targeting specificity or toxicity to the intended target.

  The peptides of the present invention can be synthetic peptides.

Fragments, derivatives or variants of the peptides of the present invention comprise (i) one or more of the amino acid residues are replaced with conserved or non-conserved amino acid residues, and these substituted amino acid residues are encoded by the genetic code Or (ii) one or more of the amino acid residues include a substituent, or (iii) the mature peptide increases the half-life of the peptide Or (iv) a leader or secretory sequence, or an additional amino acid such as a sequence used to purify the mature peptide Or (v) a peptide having a larger peptide sequence ( Eg to human albumin for increased duration of effect may be one which is fused antibody or Fc) and. Such fragments, derivatives and variants, and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

  The derivatives of the present invention may contain conservative amino acid substitutions (defined further below) made at one or more non-essential amino acid residues. “Non-essential” amino acid residues are those that can be altered from the wild-type sequence of a protein without altering biological activity, whereas “essential” amino acid residues are required for biological activity . A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid having a similar side chain. A family of amino acid residues having similar side chains has been defined in the art. These families include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, Cysteine), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), and aromatic side chains (eg tyrosine) , Phenylalanine, tryptophan, histidine). Fragments, ie biologically active moieties, include peptide fragments suitable for use as pharmaceuticals, to produce antibodies, as research reagents, and the like. A fragment comprises an amino acid sequence sufficiently similar to or derived from the amino acid sequence of a peptide of the invention and represents at least one activity of the peptide, but is disclosed in full length as disclosed herein. Including peptides containing fewer amino acids than these peptides. Typically, the biologically active portion comprises a domain or motif having at least one activity of the peptide. The biologically active portion of the peptide can be, for example, a peptide that is 5 amino acids in length or longer. Such biologically active moieties can be produced synthetically or by recombinant techniques and are disclosed herein and / or described in the art for one or more of the functional activities of the peptides of the invention. It can be evaluated by means known in the art.

  Variants of the peptides of the present invention include peptides having an amino acid sequence sufficiently similar to the amino acid sequence of the peptide of the present invention or its domain. The term “sufficiently similar” refers to a sufficient or minimal number of identity with respect to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and / or a common functional activity. Means the first amino acid sequence containing the amino acid residues of or equivalent thereto. For example, an amino acid sequence containing a common structural domain that is at least about 45% identical and about 75% to 98% identical is defined herein as sufficiently similar. A variant can be sufficiently similar to the amino acid sequence of a peptide of the invention. Such variants generally retain the functional activity of the peptides of the invention.

  Variants include peptides that differ in amino acid sequence due to mutagenesis. Variants that function as GLP-1 receptor agonists can be identified by screening combinatorial libraries of variants of the peptides of the invention, eg, truncated variants, for GLP-1 receptor agonist activity.

The invention also provides chimeric or fusion peptides. The targeting sequence is designed to localize peptide delivery so as to minimize potential side effects. The peptide of the present invention can be composed of amino acids linked to each other by peptide bonds or modified peptide bonds (ie, peptide isosteres), and contains amino acids other than those encoded by 20 genes. sell. Peptides can be modified either by natural processes such as post-translational processing or by chemical modification techniques known in the art. Such modifications are well documented in basic textbooks and in more detailed monographs as well as in a vast research literature. Modifications can be anywhere in the peptide, including the peptide backbone, amino acid side chains, and the amino or carboxyl terminus. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given peptide. Also, a given peptide can contain many types of modifications. The peptides can be branched, for example, as a result of ubiquitination, and they can be cyclic with or without branching. Cyclic, branched and branched cyclic peptides can result from posttranslation natural processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphotidylinositol covalent bond Bond, bridge, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cysteine formation, pyroglutamic acid formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, RNA-mediated addition of amino acids to proteins such as methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitin including reduction (e.g., Proteins, STRU ture and Molecular Properties, 2nd Ed., T.E.Creighton, W.H.Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C.Johnson eds., Academic Press, New York, 1-12 page (1983); Seeifter et al., Meth. Enzymol. 182, 626-646, 1990; see Rattan et al., Ann. NY Acad. Sci., 663, 48-62, 1992).

  The peptides of the present invention include peptides of formula (I) (eg, Table 1), as well as sequences that have very little variation in sequence with them. “Slight variability” substantially reflects at least one biological function of a peptide of the invention, such as GLP-1 receptor agonist activity, and / or an increase in insulin secretion or a decrease in blood glucose as indicated herein. Any sequence addition, substitution, or deletion variants that are maintained in a manner are intended to be included. These functional equivalents include peptides having a minimum of about 90% identity to the peptides of the invention, a minimum of 95% identity to the peptides of the invention, and a minimum of 99% identity to the peptides of the invention. Also included are portions of such peptides that can be made and have substantially the same biological activity. However, any peptide having a slight variation in amino acid sequence with a peptide of the invention that exhibits functional equivalence as further described herein is encompassed by the description of the invention.

  The peptides of the invention can be the product of a chemical synthesis procedure.

  The peptides of the present invention can be conveniently isolated by methods known in the art. The purity of the preparation can also be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis and mass spectrometry and liquid chromatography.

Related peptides within the understanding of those skilled in the art such as chemical mimetics, organic mimetics or peptidomimetics are also provided. As used herein, “mimetic”, “peptidomimetic”, “peptidomimetic”
c) "," organic mimetics "and" chemical mimetics "are peptide derivatives, peptide analogs and compounds having a three-dimensional geometric arrangement of atoms equivalent to that of the peptides of the invention. Is intended to be included. As used herein, the phrase “equivalent to” refers to the same or sufficiently similar arrangement of certain atoms and chemical moieties in the peptide so as to have the biological function of the peptide of the invention. Or understood to encompass peptides having substitution (s) of said atom or chemical moiety having bond length, bond angle and arrangement in a mimetic peptide that results in a geometric configuration Will be done. In a peptidomimetic, the three-dimensional arrangement of chemical components can be structurally and / or functionally equivalent to the three-dimensional arrangement of the peptide backbone and the amino acid side chains of the components in the peptide, substantially Leading to such peptides, organic and chemical mimetics of the peptides of the present invention with unique biological activity. These terms are described, for example, by Fauchere (Adv. Drug Res. 15:29, 1986); Veber and Freidinger (TINS p. 392, 1985); and Evans et al. Chem.Chem.30: 1229, 1987) as used in accordance with the understanding in the art.

  It is understood that a pharmacophore exists for the biological activity of each peptide of the invention. A pharmacophore is understood in the art to comprise an idealized three-dimensional definition of the structural requirements of biological activity. Peptides, organic and chemical mimetics can be designed to adapt each pharmacophore using current computer modeling software (computer assisted drug design). The mimetics can be generated by structure-function analysis based on positional information from substituent atoms in the peptides of the present invention.

  The peptides provided by the present invention are advantageously synthesized by any of the chemical synthesis techniques known in the art, in particular by solid phase synthesis techniques using, for example, commercially available automated peptide synthesizers. Yes. The mimetics of the present invention may be synthesized by solid phase or solution phase methods conventionally used for peptide synthesis (see, eg, Merrifield, J. Amer. Chem. Soc. 85: 2149-54, 1963; Carpino, Acc.Chem.Res.6: 191-98, 1973; Birr, Aspects of the Merrifield Peptide Synthesis, Springer-Verlag: Heidelberg, 1978; The Peptides: Analysis, 3 and 5; Gross and Meinhofer), Academic Press: New York, 1979; Stewart et al., Solid Phase Peptide Synth. sis, 2nd edition, Pierce Chem. Co .: Rockford, IL, 1984; Kent, Ann. Rev. Biochem. 57: 957-89, 1988; and Gregg et al., Int. J. Peptide Protein Res. 55: 161. -214, 1990 (incorporated herein by reference in its entirety).

The peptides of the present invention may be produced by solid phase methodology. Briefly, polyethylene rods coated with N-protected C-terminal amino acid residues with divinylbenzene crosslinked polystyrene, polyacrylamide resin, kieselguhr / polyamide (pepsin K), microporous glass, cellulose, polypropylene membrane, acrylic acid coating, etc. To an insoluble support such as The amino acids are linked from the C-terminus according to the amino acid sequence using successive cycles of deprotection, neutralization and coupling of protected amino acid derivatives. For some synthetic peptides, an FMOC strategy using acid sensitive resins may be used. In this regard, the solid support is chloromethyl resin, hydroxymethyl resin, paraacetamidomethyl resin, benzhydrylamine (BHA) resin, 4-methylbenzhydrylamine (MBHA) resin, oxime resin, 4-alkoxybenzyl alcohol resin. (Wang resin), 4- (2 ′, 4′-dimethoxyphenylaminomethyl) -phenoxymethyl resin, 2,4-dimethoxybenzhydryl-amine resin, and 4- (2 ′, 4′-dimethoxyphenyl-FMOC) It can be a divinylbenzene crosslinked polystyrene resin that is commercially available in a variety of functionalized forms including -amino-methyl) -phenoxyacetamidonorleucyl-MBHA resin (Rink amide MBHA resin). In addition, acid sensitive resins also provide C-terminal acids if desired. The protecting group for α-amino acids is base-labile 9-fluorenylmethoxy-carbonyl (FMOC).

Suitable protecting groups for the side chain functionality of amino acids chemically compatible with BOC (t-butyloxycarbonyl) and FMOC groups are known in the art. When FMOC chemistry is used, the following protected amino acid derivatives: FMOC-Cys (Trt), FMOC-Ser (But), FMOC-Asn (Trt), FMOC-Leu, FMOC-Thr (Trt), FMOC- Val, FMOC-Gly, FMOC-Lys (Boc), FMOC-Gln (Trt), FMOC-Glu (OBut), FMOC-His (Trt), FMOC-Tyr (But), FMOC-Arg (PMC (2, 2 , 5,7,8-pentamethylchroman-6-sulfonyl)), FMOC-Arg (BOC) ₂ , FMOC-Pro and FMOC-Trp (BOC). Amino acid residues are DIC (diisopropyl-carbodiimide), DCC (dicyclohexylcarbodiimide), BOP (benzotriazolyl-N-oxytrisdimethylaminophosphonium hexafluorophosphate), PyBOP (benzotriazol-1-yl hexafluorophosphate). By using various coupling agents and chemistries known in the art such as direct coupling with -oxy-tris-pyrrolidinophosphonium), PyBrOP (bromo-tris-pyrrolidinophosphonium hexafluorophosphate) Through a pre-generated symmetric anhydride; through an active ester such as pentafluorophenyl ester; or through a pre-formed HOBt (1-hydroxybenzotriazole) active ester, or F The use of OC- amino fluorides and chlorides or by using FMOC- amino -N- carboxyanhydride, may couplings. For example, activation can be achieved by HBTU (2- (1H-benzotriazol-1-yl) hexafluorophosphate, 1,1,3,3-tetramethyl in the presence of HOBt or HOAt (7-azahydroxybenzotriazole). (Uronium) or HATU (hexafluorophosphate 2- (1H-7-aza-benzotriazol-1-yl), 1,1,3,3-tetramethyluronium).

  The solid phase method can be performed by automated synthesis on a human or commercially available peptide synthesizer (eg, Applied Biosystems 431A; Applied Biosystems, Foster City, CA). In a typical synthesis, the first (C-terminal) amino acid is loaded onto the chlorotrityl resin. Peptide sequences may be generated using sequential deprotection (using 20% piperidine / NMP (N-methylpyrrolidone)) and coupling cycles according to ABI's FastMoc protocol (Applied Biosystems). Double and triple coupling using coupling with acetic anhydride may also be used.

Synthetic mimetic peptides can be cleaved from the resin and deprotected by treatment with TFA (trifluoroacetic acid) containing an appropriate scavenger. Many such cleavage reagents may be used such as Reagent K (0.75 g crystalline phenol, 0.25 mL ethanedithiol, 0.5 mL thioanisole, 0.5 mL deionized water, 10 mL TFA) and others. . The peptide is separated from the resin by filtration and isolated by ether precipitation. Further purification can be achieved by conventional methods such as gel filtration and reverse phase HPLC (high performance liquid chromatography). The synthetic mimetics of the present invention can be in the form of pharmaceutically acceptable salts, particularly base addition salts, including salts of organic and inorganic bases. Base addition salts of acidic amino acid residues are prepared by treatment of the peptide with an appropriate base or inorganic base according to procedures known to those skilled in the art, or the desired salt is obtained directly by lyophilization of the appropriate base. be able to.

  In general, one of ordinary skill in the art will recognize a variety of peptides as described herein for producing peptides having essentially the same activity as the unmodified peptide, and possibly other desired properties. It will be recognized that it can be modified by chemical techniques. For example, the carboxylic acid group of the peptide can be provided in the form of a pharmaceutically acceptable cationic salt. The amino group in the peptide can be in the form of a pharmaceutically acceptable acid addition salt such as HCl, HBr, acetic acid, benzoic acid, toluenesulfonic acid, maleic acid, tartaric acid and other organic acids, or in the amide. Can be converted. The thiol can be protected with any one of a number of well-recognized protecting groups such as an acetamide group. One skilled in the art will also recognize how to introduce a cyclic structure into the peptides of the invention so that the natural binding configuration can be brought closer. For example, a carboxyl-terminal or amino-terminal cysteine residue can be added to a peptide so that when oxidized, the peptide can contain a disulfide bond and thereby generate a cyclic peptide. Other peptide cyclization methods include the formation of thioethers and carboxyl and amino terminal amides and esters.

In particular, construct peptide derivatives and analogs that have the same or similar desired biological activity as the corresponding peptide but have more favorable activity than the peptide with respect to solubility, stability, and sensitivity to hydrolysis and proteolysis A variety of technologies are available. Such derivatives and analogs include but are not limited to peptides modified with an N-terminal amino group, a C-terminal amide group, and / or a peptide of formula (I) (eg, Table 1) It includes changing one or more amide bonds to non-amide bonds. It is understood that two or more such modifications can be combined in one peptidomimetic structure (eg, modification at the C-terminal amide group and inclusion of a —CH ₂ -carbamate bond between two amino acids in the peptide). Will be done.

Peptidomimetics as understood in the art and provided by the present invention are structurally similar to the peptides of the present invention, but are known in the art and include the following references: Spatola, Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins (Edited by Weinstein), Marcel Dekker: New York, p. 267, 1983; Spatola, Peptide Backbone Modifications 1: 3, 1983; Morley, Trends Pharm. Sci. pp. 463-468, 1980; Hudson et al., Int. J. et al. Pept. Prot. Res. 14: 177-185, 1979; Spatola et al., Life Sci. 38: 1243-1249, 1986; Hann, J. et al. Chem. Soc. Perkin Trans. I 307-314, 1982; Almquist et al. Med. Chem. 23: 1392-1398, 1980; Jennings-White et al., Tetrahedron Lett. 23: 2533, 1982; Szelke et al., EP 045665A; Holladay et al., Tetrahedron Lett. 24: 4401-4404, 1983; and Hruby, Life Sci. 31: 189-199, 1982, each of which is incorporated herein by reference, in accordance with a method such as —CH ₂ NH—, —CH ₂ S—, —CH ₂ CH. ₂ -, - CH = CH- (in conformer of cis and trans _{both), - COCH 2 -, -} CH (OH) CH 2 - and one which is optionally substituted by _-CH 2 SO- Or it has more peptide bonds. Such peptidomimetics are, for example, more economical to manufacture, greater chemical stability or enhanced pharmacological properties (such as half-life, absorption, potency, efficacy), reduced antigenicity And can have significant advantages over peptide embodiments, including and other properties.

  Mimetics analogs of the peptides of the present invention can also be obtained using conventional or rational drug design principles (see, eg, Andrews et al., Proc. Alfred Benz Symp. 28: 145-165, 1990; McPherson , Eur. J. Biochem. 189: 1-24, 1990; Hol et al., Molecular Recognition: Chemical and Biochemical Problems, edited by Roberts; Royal Society of Chemistry; 39: 1016-1018, 1989b; Hol, Agnew Chem. Int. Ed. Engl.25: 767-778, 19 6 See (the disclosures of which are incorporated herein by reference)).

  In accordance with conventional drug design methods, the desired mimetic molecule can be obtained by randomly testing molecules whose structure has attributes in common with the structure of the “natural” peptide. The quantitative contribution resulting from a particular group of changes in binding molecules can be determined by measuring the biological activity of the putative mimetic compared to the activity of the peptide. In one aspect of rational drug design, mimetics are designed to share the attributes of the most stable three-dimensional conformation of the peptide. Thus, for example, mimetics are arranged in a manner sufficient to cause ionic, hydrophobic or van der Waals interactions that are similar to those represented by the peptides of the invention as disclosed herein. It can be designed to have chemical groups that are

One implementation of a rational mimetic design uses a computer system capable of forming a representation of the three-dimensional structure of a peptide, such as those exemplified by Hol, 1989a; Hol, 1989b; and Hol, 1986. . The molecular structures of the peptides, organic and chemical mimetics of the peptides of the present invention can be generated using computer aided design programs commercially available in the art. Examples of such ^{programs, SYBYL 6.5 (R), HQSAR} TM and ^{ALCHEMY 2000 TM (Tripos); GALAXY} TM and ^AM2000 TM (AM Technologies, Inc, San Antonio, TX.); CATALYST ^TM and ^CERIUS TM (Molecular Simulations Inc., San Diego, Calif.); CACHE PRODUCTS ^™ , TSAR ^™ , AMBER ^™ and CHEM-X ^™ (Oxford Molecular Products, Oxford, Calif.) And CHEMBUIDER 3D ^™ , Inc. To do.

Peptides, organic and chemical mimetics produced using the peptides disclosed herein using, for example, art-recognized molecular modeling programs can be used for high-throughput screening, including, for example, combinatorial chemistry methods Can be produced using conventional chemical synthesis techniques designed to accommodate Combinatorial methods useful in the production of peptides, organic and chemical mimetics of the present invention are described, for example, in SIDDCO (Tucson, Arizona); Tripos, Inc. Calbiochem / Novabiochem (San Diego, Calif.); Symyx Technologies, Inc .; (Santa Clara, Calif.); Medichem Research, Inc. (Lemonto, Ill.); Pharm-Eco Laboratories, Inc. (Bethlehem, Pennsylvania); V. Includes phage display arrays, solid phase synthesis and combinatorial chemistry arrays as provided by Organon (Oss, The Netherlands). Methods for combinatorial chemistry production of peptides, organic and chemical mimetics of the present invention are not limited, but include Terrett (Combinatorial Chemistry, Oxford University Press, London, 1998); Gallop et al. Med. Chem. 37: 1233-5
1, 1994; Gordon et al. Med. Chem. 37: 1385-1401, 1994; Look et al., Bioorg. Med. Chem. Lett. 6: 707-12, 1996; Ruhland et al., J. MoI. Amer. Chem. Soc. 118: 253-4, 1996; Gordon et al., Acc. Chem. Res. 29: 144-54, 1996; Thompson and Ellman, Chem. Rev. 96: 555-600, 1996; Fruchtel and Jung, Angew. Chem. Int. Ed. Engl. 35: 17-42, 1996; Pavia, “The Chemical Generation of Molecular Diversity”, Network Science Center, www. netsci. org, 1995; Adnan et al., “Solid Support Combined Chemical Chemistry in Lead Discovery and SAR Optimization,” Id. 1995, Davies and Briant, “Combinatorial Chemistry Library Designing Pharmacophore Diversity,” Id. Pavia, “Chemically Generated Screening Libraries: Present and Future,” Id. And U.S. Pat. Nos. 5,880,972; 5,463,564; 5,331573; and 5,573,905. Can be produced according to methods known in the art.

Newly synthesized peptides can be substantially purified by preparative high performance liquid chromatography (see, eg, Creighton, Proteins: Structure And Molecular Principles , WH Freeman and Co., New York, NY, 1983). The composition of the synthetic peptides of the present invention can be confirmed by amino acid analysis or sequencing by, for example, Edman degradation procedures (Creighton, supra). In addition, any part of the amino acid sequence of the peptide can be altered during direct synthesis and / or combined with sequences from other proteins using chemical methods to produce variant or fusion peptides.

Antibodies and antibody fragments that selectively bind the peptides of the invention are also encompassed by the invention. Any type of antibody known in the art can be generated using methods known in the art. For example, antibodies can be generated that specifically bind to one epitope of a peptide of the invention. As used herein, “antibodies” are intact immunoglobulin molecules and those such as Fab, F (ab ′) ₂ and Fv that are capable of binding one epitope of the peptides of the invention. Of fragments. Typically, a minimum of 6, 8, 10 or 12 consecutive amino acids are required to form one epitope. However, epitopes with non-consecutive amino acids may require more amino acids, for example a minimum of 15, 25 or 50 amino acids.

  Antibodies that specifically bind to one epitope of the peptides of the present invention are therapeutically as well as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitation methods, or other immunochemistry known in the art. It can be used in immunochemical assays such as assays. A variety of immunoassays can be used to identify antibodies with the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are known in the art. Such immunoassays typically require measurement of complex formation between the immunogen and an antibody that specifically binds to the immunogen.

Typically, an antibody that specifically binds to a peptide of the invention provides a detection signal that is higher than that provided by other proteins when used in an immunochemical assay. Antibodies that specifically bind to the peptides of the invention do not detect other proteins in immunochemical assays and can immunoprecipitate the peptides of the invention from solution.

  The peptides of the invention can be used to immunize mammals such as mice, rats, rabbits, guinea pigs, goats, sheep, monkeys or humans to produce polyclonal antibodies. If desired, the peptides of the invention can be conjugated to carrier proteins such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants may be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (eg, aluminum hydroxide), and surface active substances (eg, lysolecithin, pluronic polyhydric alcohols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and Dinitrophenol). Of the adjuvants used in humans, BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are particularly useful.

  Monoclonal antibodies that specifically bind to the peptides of the invention may be produced using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology and EBV hybridoma technology (Kohler et al., Nature 256: 495-97, 1985; Kozbor et al., J. Immunol. Methods. 81: 3142, 1985; Cote et al., Proc. Natl. Acad. Sci. 80: 2026-30, 1983; Cole et al., Mol. Cell Biol. 62: 109-20, 1984).

  In addition, techniques developed for the production of “chimeric antibodies” may be used, ie splicing of mouse antibody genes to human antibody genes to obtain molecules with appropriate antigen specificity and biological activity (Morrison Proc. Natl. Acad. Sci. 81: 6851-55, 1984; Neuberger et al., Nature 312: 604-08, 1984; Takeda et al., Nature 314: 452-54, 1985). Monoclonal and other antibodies may also be “humanized” to prevent a patient from being equipped with an immune response against the antibody when used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require some important residue changes. Sequence differences between rodent antibodies and human sequences can be obtained by substituting residues that differ from those in the human sequence by site-directed mutagenesis of individual residues or grafting of the entire complementarity determining region. Can be minimized. Alternatively, humanized antibodies can be produced using recombinant methods (see, eg, GB 2188638B). Antibodies that specifically bind to peptides of the invention contain an antigen binding site that is either partially or fully humanized, as disclosed in US Pat. No. 5,565,332. Yes.

  Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to the peptides of the invention. Antibodies with related specificity but distinct idiotype composition can be generated by chain shuffling from a random combinatorial immunoglobulin library (Burton, Proc. Natl. Acad. Sci. 88: 11120-23, 1991). .

Single chain antibodies can also be constructed using DNA amplification methods such as PCR using hybridoma cDNA as a template (Thirion et al., Eur. J. Cancer Prev. 5: 507-11, 1996). Single chain antibodies can be mono- or bispecific and can be bivalent or tetravalent. The construction of tetravalent bispecific single chain antibodies is described, for example, by Coloma and Morrison (Nat. Biotechnol. 15: 159-63, 1997).
). The construction of bivalent bispecific single chain antibodies is taught by Mallender and Voss (J. Biol. Chem. 269: 199-206, 1994).

  Nucleotide sequences encoding single chain antibodies are constructed using human or automated nucleotide synthesis, cloned into expression constructs using standard recombinant DNA methods, and introduced into cells as described below. The coding sequence can then be expressed. Alternatively, single chain antibodies use, for example, filamentous phage technology (Verhaar et al., Int. J. Cancer 61: 497-501, 1995; Nichols et al., J. Immunol. Meth. 165: 81-91, 1993). Can be manufactured directly.

  Antibodies that specifically bind to the peptides of the invention can be induced by in vivo production in lymphocyte populations or by immunoglobulin libraries or literature (Orlandi et al., Proc. Natl. Acad. Sci. 86: 38333-37). 1989; Winter et al., Nature 349: 293-99, 1991) can also be produced by screening a panel of highly specific binding reagents.

  Other types of antibodies can be constructed and used therapeutically in the methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins that are derived from immunoglobulins and are multivalent and multispecific, such as “bispecific antibodies” can also be produced (see, eg, WO 94/13804).

Human antibodies with the ability to bind to the peptides of the present invention may also be identified from the MorphoSys of HuCAL ^(R) library where subsequent. The peptides of the present invention were coated onto microtiter plates, and MorphoSys ^{HuCAL (R)} can be incubated with the Fab phage library. Fabs bound to phage that do not bind to the peptides of the invention can be washed off the plate, leaving only phage that bind strongly to the peptides of the invention. Bound phage can be eluted, for example, by a change in pH or by elution with E. coli, and can be amplified by infection of an E. coli host. This panning method can be repeated one or two times to enrich for populations of antibodies that bind tightly to the peptides of the invention. Fabs from the enriched pool are then expressed, purified and screened in an ELISA assay.

  The antibodies of the present invention can be purified by methods known in the art. For example, the antibody can be affinity purified by passage through a column to which a peptide of the invention is bound. The bound antibody can then be eluted from the column using a buffer with a high salt concentration.

Methods of Use As used herein, various terms are defined below.
In the introduction of an element of the invention or preferred embodiments (s) thereof, the articles “a”, “an”, “the”, and “said” include one or more of the elements. Is meant to mean that. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. .

  As used herein, the term “subject” includes mammals (eg, humans and animals).

  The term `` treatment '' is provided with medical assistance for the purpose of improving a subject's condition, directly or indirectly, or delaying the progression of the condition or disorder in the subject, including a human, Includes any process, action, application, treatment, etc.

  The term “combination therapy” or “co-therapy” means the administration of two or more therapeutic agents, for example to treat diabetes. Such administration may involve the co-administration of two or more therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredient, or multiple separate capsules of each inhibitor. Includes administration. In addition, such administration involves the use of each type of therapeutic agent in a sequential manner.

  The phrase “therapeutically effective” is administered to achieve the goal of improving the severity of a diabetic condition or disorder while avoiding or minimizing adverse side effects associated with a given therapeutic treatment It means the amount of each agent.

  The term “pharmaceutically acceptable” means that the subject item is suitable for use in a pharmaceutical product.

  The peptides of formula (I) are expected to be valuable as therapeutic agents (eg Table 1). Accordingly, one aspect of the present invention comprises administering to a patient (including a mammal) a composition containing an amount of a peptide of formula (I) that is effective in the treatment of a target condition, Includes methods for treating various conditions in a patient.

  The peptides of the present invention are of both type 1 and type 2 (non-insulin dependent diabetes) as a result of the ability to stimulate insulin secretion from islet cells in vitro and by causing a decrease in blood glucose in vivo. It can be used in the treatment of diabetes, including diabetes. Such treatment can also delay the onset of diabetes and diabetic complications. The peptide can be used to prevent a subject with impaired glucose tolerance from progressing to develop type 2 diabetes. Other diseases and conditions that can be treated or prevented using the peptides of the invention in the methods of the invention are juvenile-onset adult diabetes (MODY) (Herman et al., Diabetes 43:40, 1994); adult latent diabetes (LADA) (Zimmet et al., Diabetes Med. 11: 299, 1994); Impaired Glucose Tolerance (IGT) (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1); (IFG) (Charles et al., Diabetes 40: 796, 1991); gestational diabetes (Metzger, Diabetes, 40: 197, 1991); and metabolic syndrome X.

  The peptides of the present invention may be used in disorders such as obesity, as well as atherosclerosis, coronary heart disease, coronary artery disease, hyperlipidemia, hypercholesterolemia, low HDL levels, hypertension; cerebrovascular disease; and peripheral It may also be effective in the treatment of cardiovascular diseases, including vascular diseases; and in the treatment of neurodegenerative diseases (including Parkinson's disease and Alzheimer's disease).

  In addition, the peptides of the invention may be used to increase satiety and reduce food intake and in the treatment of obesity (Flint et al., J. Clin. Invest. 101: 515-520, 1998). In addition, peptides of the present invention treat myocardial infarction (Nikolaidis et al., Circ. 109: 962-965, 2004), as well as ameliorate endothelial dysfunction (Nystrom, Am. J. Physiol. Endocrinol. Metab. 287: E1209). -E1215). The peptides of the present invention are also useful for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, as well as other diseases that may benefit from the neuroprotective effects of GLP-1 (During et al., Nat. Med. 9: 1173-1179, 2003).

Peptides of the present invention are, for example, abnormal pancreatic β-cell function, pancreatic β-cell mass, insulin secretion, decreased tissue sensitivity to insulin, liposarcoma cell proliferation, polycystic ovarian disease, chronic anovulation, androgen excess, progesterone Also to treat physiological disorders related to regulation of insulin sensitivity and blood glucose levels such as production, steroidogenesis, cellular redox potential and oxidative stress, plasma triglycerides, HDL, and LDL cholesterol levels, etc. It can also be useful.

  The peptides of the present invention can also be used in the method of treating secondary causes of diabetes of the present invention (Expert Committee on Classification of Diabetes Melitus, Diabetes Care 22 (Supp. 1): S5, 1999). Such secondary causes include glucocorticoid excess, growth hormone excess, pheochromocytoma and drug-induced diabetes. Drugs that can induce diabetes include, but are not limited to, pyriminyl, nicotinic acid, glucocorticoid, phenytoin, thyroid hormone, beta-adrenergic agonist, alpha-interferon, and HIV infections Drugs can be mentioned.

  The peptides of the invention may be used alone or in conjunction with additional therapies and / or compounds known to those skilled in the art for the treatment of diabetes and related disorders. Alternatively, the methods and peptides described herein can be used partially or fully in combination therapy.

  Peptides of the present invention include PPAR ligands (eg agonists, antagonists), insulin secretagogues such as sulfonylurea drugs and nonsulfonylurea secretagogues, α-glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic glucose It can also be administered with other known therapies for the treatment of diabetes, including power reducing compounds, insulin and insulin derivatives, and anti-obesity drugs. Such treatment may be administered before, simultaneously with, or after administration of the peptides of the present invention. Insulin and insulin derivatives include both long and short acting forms of insulin and preparations of insulin. The PPAR ligand can include any agonist and / or antagonist of a PPAR receptor or a combination thereof. For example, PPAR ligands can include PPAR-α, PPAR-g, PPAR-δ, or any combination of two or three receptors of PPAR receptors. PPAR ligands include, for example, rosiglitazone, troglitazone and pioglitazone. Sulfonylurea drugs include, for example, glyburide, glimepiride, chlorpropamide, tolbutamide and glipizide. Alpha-glucosidase inhibitors that may be useful in the treatment of diabetes when administered with the peptides of the present invention include acarbose, miglitol and voglibose. Insulin sensitizers that may be useful in the treatment of diabetes include glitazones (eg, troglitazone, pioglitazone, englitazone, MCC-555, rosiglitazone, and the like) and other PPAR-like compounds other than thiazolidinedione and thiazolidinedione. gamma agonists; biguanides such as metformin and phenformin; protein tyrosine phosphatase-1B (PTP-1B) inhibitors; dipeptidyl peptidase IV (DPPIV) inhibitors; and 11β-HSD inhibitors. Hepatic glucose output reducing compounds that may be useful in the treatment of diabetes when administered with the peptides of the invention include, for example, glucagon antagonists and metformin such as Glucophage and Glucophage XR. Insulin secretagogues that may be useful in the treatment of diabetes when administered with the peptides of the present invention include sulfonylurea and non-sulfonylurea drugs, namely GIP, VIP, PACAP, secretin and their derivatives; nateglinide, meglitinide, repaglinide , Glibenclamide, glimepiride, chlorpropamide and glipizide. In one aspect of the invention, the peptides of the invention are used in combination with an insulin secretagogue to increase the sensitivity of pancreatic beta cells to an insulin secretagogue.

The peptides of the invention may also be used in combination with anti-obesity agents in the methods of the invention. For example, anti-obesity agents include β-3 adrenergic receptor agonists such as CL 316,243; cannabinoid (eg CB-1) antagonists such as Rimonabant; neuropeptide Y receptor antagonists; neuropeptide Y5 inhibitors; B / MTP inhibitor; 11β-hydroxysteroid dehydrogenase-1 inhibitor; peptide YY _3-36 or analog thereof; MCR4 agonist; CCK-A agonist; monoamine reuptake inhibitor; sympathomimetic drug; dopamine agonist; Melanin-concentrating hormone antagonist; leptin; leptin analog; leptin receptor agonist; galanin antagonist; lipase inhibitor; bombesin agonist; thyroid hormone-like agent; Loepiandrosterone or analogs thereof; glucocorticoid receptor antagonist; orexin receptor antagonist; ciliary neurotrophic factor; ghrelin receptor antagonist; histamine-3 receptor antagonist; neuromedin U receptor agonist; Appetite suppressants; and lipase inhibitors such as, for example, Orlistat (Xenical). The peptides of the present invention may also be administered with drug compounds that modulate digestion and / or metabolism, such as drugs that modulate heat production, lipolysis, intestinal motility, fat absorption and satiety.

  The peptides of the present invention may also be used in the methods of the present invention with drugs commonly used to treat lipid disorders. Such drugs include, but are not limited to, HMG-CoA reductase inhibitors, nicotinic acid, compounds that lower fatty acids (eg, acipimox); drugs that lower lipids (eg, stanol esters, sterol glycosides such as chicueside) And azetidinones such as ezetimibe), ACAT inhibitors (such as abashimib), bile acid scavengers, bile acid reuptake inhibitors, microsomal triglyceride transport inhibitors, and fibric acid derivatives. HMG-CoA reductase inhibitors include, for example, lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rivastatin, itavastatin, cerivastatin and ZD-4522. Fibric acid derivatives include, for example, clofibrate, fenofibrate, bezafibrate, ciprofibrate, beclofibrate, etofibrate and gemfibrozil. Capture agents include, for example, cholestyramine, colestipol, and dialkylaminoalkyl derivatives of bridged dextran.

  Furthermore, the peptides of the invention may also be administered in combination with antihypertensive drugs such as, for example, β-blockers and ACE inhibitors. Examples of additional antihypertensive drugs for use with the peptides of the present invention include calcium antagonists (L and T forms; eg diltiazem, verapamil, nifedipine, amlodipine and mibefradil), diuretics (eg chlorothiazide, hydrochlorothiazide, Flumethiazide, hydroflumethiazide, bendroflumethiazide, methyl chlorothiazide, trichloromethiazide, polythiazide, benzthiazide, triclinaphene ethacrylic acid, chlorthalidone, furosemide, musolimine, bumetanide, triamtrenene, amylolide, lactone Agents, ACE inhibitors (eg captopril, zofenopril, fosinopril, enalapril, ceranopril, silazopril, delapril, pentopril, Napril, ramipril, lisinopril), AT-1 receptor antagonists (eg, losartan, irbesartan, valsartan), ET receptor agonists (eg, sitaxsentan, atrusentan, neutral endopeptidase (NEP) inhibitors, vasopepsidase inhibition Agents (dual NEP-ACE inhibitors) (eg omapatrilate and gemopatrilate), and nitrates.

  Such co-therapy may be administered in any combination of two or more drugs (eg, a peptide of the invention in combination with an insulin sensitizer and an anti-obesity agent). Such co-therapy can be administered in the form of a pharmaceutical composition as described above.

Pharmaceutical Compositions Based on known assays used to determine efficacy for treatment of the above-identified conditions in mammals and known pharmaceuticals used to treat these conditions By comparing these results with those of the results, the effective dosage of the peptides of the present invention can be readily determined for the treatment of each desired indication. The amount of active ingredient to be administered in one treatment of these conditions depends on the particular peptide and dosage unit used, the mode of administration, the duration of treatment, the age and sex of the patient being treated, and the condition being treated. It can vary widely according to considerations such as the nature and degree of the.

  The total amount of active ingredient to be administered can generally range, for example, from about 0.0001 mg / kg to about 200 mg / kg, or from about 0.001 mg / kg to about 200 mg / kg body weight per day. A unit dosage can contain, for example, from about 0.05 mg to about 1500 mg of active ingredient, and can be administered one or more times per day. Daily dosages for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques are, for example, from about 0.001 to about 200 mg / kg. It is possible. The daily rectal dosage regimen can be, for example, from about 0.001 to about 200 mg / kg total body weight. The transdermal concentration can be that required to maintain a daily dose of, for example, from about 0.001 to about 200 mg / kg.

  Of course, each patient's specific initial and continuing dosing regimen will depend on the nature and severity of the condition as determined by the attending physician, the activity of the particular peptide used, the age of the patient, the patient's diet, It can vary according to administration time, administration route, drug excretion rate, drug combination, and the like. The desired mode of treatment and number of doses of the peptide of the invention can be ascertained by one skilled in the art using routine treatment tests.

  The peptides of the present invention may be utilized to achieve a desired pharmacological effect by administration to a subject in need thereof with an appropriately formulated pharmaceutical composition. The subject can be a mammal, including, for example, a human in need of treatment for a particular condition or disease. Accordingly, the present invention includes a pharmaceutical composition comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a peptide of the present invention. A pharmaceutically acceptable carrier is relatively non-toxic and harmless to the patient at a concentration consistent with the effective activity of the active ingredient so that any side effects attributed to the carrier do not impair the beneficial effects of the active ingredient. Any carrier that is A pharmaceutically effective amount of peptide is that amount which produces a result or exerts an effect on the particular condition being treated. The peptides of the present invention are pharmaceutically acceptable carriers using oral, parenteral, topical, etc. any effective conventional dosage unit form including, for example, immediate release and sustained release formulations. Can be administered together.

  For oral administration, peptides can be solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions or emulsions. And can be prepared according to methods known in the art of manufacturing pharmaceutical compositions. Solid unit dosage forms can be of the usual hard or soft shell gelatin type containing, for example, surfactants, lubricants, and inert extenders such as lactose, sucrose, calcium phosphate and corn starch. It can be a capsule.

In another embodiment, the peptides of the present invention help to disintegrate and dissolve tablets after administration, such as binders such as gum arabic, corn starch or gelatin; potato starch, alginic acid, corn starch and guar gum. Intended disintegrants; lubricants such as talc, stearic acid, or stearin that are intended to improve the flow of the tablet granulation and prevent sticking of the tablet substance to the surface of the tableting die and punch Of lactose, sucrose and corn starch in combination with magnesium, calcium or zinc; pigments; colorants; and flavorings intended to enhance the aesthetic quality of tablets and make them acceptable to patients Tablets can be compressed with such conventional tablet bases. Suitable excipients for use in oral liquid dosage forms include water and alcohol, either with or without the addition of pharmaceutically acceptable surfactants, suspending agents or emulsifiers, For example, diluents such as ethanol, benzyl alcohol and polyethylene alcohol are included. A variety of other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.

  Dispersible powders and granules are suitable for the production of aqueous suspensions. They provide the active ingredient in admixture with a dispersing aid or wetting agent, suspending agent and one or more preservatives. Suitable dispersing aids or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients may also be present, for example sweetening, flavoring and coloring agents as described above.

  The pharmaceutical composition of the present invention may also exist in the form of an oil-in-water emulsion. The oily phase can be a vegetable oil such as liquid paraffin or a mixture of vegetable oils. Suitable emulsifiers are (1) naturally occurring gums such as gum arabic and gum tragacanth, (2) naturally occurring phosphatides such as soybean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, For example, it may be sorbitan monooleate, and (4) the condensation product of the partial ester with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents.

  Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, as well as a preservative, flavoring and coloring agent.

  The peptides of the present invention may comprise pharmaceutically acceptable surfactants such as soaps or detergents, suspending agents such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, or emulsifiers and other pharmaceuticals. Sterile liquids or mixtures of liquids such as water, saline, aqueous D-glucose and related sugar solutions with or without the addition of auxiliary substances; alcohols such as ethanol, isopropanol or hexadecyl alcohol; propylene glycol or Glycols such as polyethylene glycol; glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as poly (ethylene glycol) 400; oils; fatty acids; fatty acid esters Or as an injectable dosage of peptide in a physiologically acceptable diluent comprising a pharmaceutical carrier, which may be an acetylated fatty acid glyceride, parenterally, ie subcutaneously, intravenously, intramuscularly or intraperitoneally It can also be administered within.

Oils of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petroleum jelly and mineral oil, specifically describe the oils that can be used in the parenteral formulations of the present invention. To do. Suitable fatty acids include oleic acid, stearic acid and isostearic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty alkali metal, ammonium and triethanolamine salts, and suitable detergents are cationic detergents such as dimethyldialkylammonium halides, alkylpyridinium halides and alkylamine acetates; anionic Detergents such as alkyl, aryl and olefin sulfonates, alkyl, olefins, ethers and monoglyceride sulfates, and sulfosuccinates; nonionic detergents such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers; and amphoteric Detergents such as alkyl-β-aminopropionates and 2-alkylimidazoline quaternary ammonium salts, and mixtures are included.

  Parenteral compositions of the present invention may typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may also be used advantageously. In order to minimize or eliminate irritation at the site of injection, such compositions may contain a nonionic surfactant having a hydrophilic lipophilic balance (HLB) of from about 12 to about 17. The amount of surfactant in such formulations ranges from about 5% to about 15% by weight. The surfactant can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB.

  Surfactants used in parenteral formulations are classified as polyethylene sorbitan fatty acid esters, eg sorbitan monooleate, and high molecular weight adducts of ethylene oxide with a hydrophobic base formed by condensation of propylene oxide with propylene glycol Will be described in detail.

  The pharmaceutical composition may be in the form of a sterile injectable aqueous suspension. Such suspending agents include, for example, suitable dispersing or wetting agents and suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; natural such as lecithin Condensation products of alkylene oxides with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain fatty alcohols such as heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate Products of ethylene oxide with various fatty acids and partial esters derived from hexitol, or ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides Condensation products of de, for example using a polyoxyethylene sorbitan monooleate dispersing aid can be a benzoate or wetting agents, may be formulated according to known methods.

  The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent. Diluents and solvents that can be used are, for example, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables.

  The compositions of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions are made by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at the rectal temperature and can therefore melt in the rectum to release the drug. Yes. Such materials are cocoa butter and polyethylene glycols, for example.

  Another formulation used in the methods of the present invention uses a transdermal delivery device (“patch”). Such transdermal patches can be used to provide continuous or non-continuous infusion of controlled amounts of the peptides of the invention. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art (eg, US Pat. No. 5,023,252, incorporated herein by reference). ) Such patches can be constructed for continuous, pulsatile or on demand delivery of pharmaceutical agents.

  Another formulation uses the use of biodegradable microspheres that allow controlled sustained release of the peptides and pegylated peptides of the present invention. Such formulations can be composed of synthetic polymers or copolymers. Such formulations are intended for infusion, inhalation, nasal or oral administration. The construction and use of biodegradable microspheres for the delivery of pharmaceutical agents is known in the art (see, eg, US Pat. No. 6,706,289 (hereby incorporated by reference)). Incorporated)).

  It may be desirable or necessary to introduce the pharmaceutical composition to the patient via a mechanical delivery device. The construction and use of mechanical delivery devices for the delivery of pharmaceutical agents is known in the art. For example, direct techniques for administering drugs directly into the brain typically require the placement of a drug delivery catheter in the patient's ventricular system to bypass the blood brain barrier. One such implantable delivery system used to transport agents to specific anatomical regions of the body is described in US Pat. No. 5,011,472 (incorporated herein by reference). Described).

  The compositions of the present invention may also contain other conventional pharmaceutically acceptable formulation ingredients, commonly referred to as carriers or diluents, as necessary or desired. Any of the compositions of the present invention may be preserved by the addition of an antioxidant such as ascorbic acid or other suitable preservative. Conventional manufacturing procedures for appropriate dosage forms of such compositions may be utilized.

  Commonly used pharmaceutical ingredients that may be used as appropriate to formulate a composition for its intended route of administration include, but are not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid. Acidifying agents that may be mentioned; including but not limited to ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine Includes possible alkalizing agents.

  Peptides identified by the methods described herein can be combined as a single pharmaceutical agent or with one or more other pharmaceutical agents if the combination does not cause unacceptable adverse effects. Can be administered. For example, the peptides of the present invention may be combined with known anti-diabetic drugs, or known anti-obesity drugs, cardiovascular drugs or other indications, etc., and mixtures and combinations thereof.

  Peptides identified by the methods described herein may also be utilized in free base form or compositions, in research and diagnostics, or as reference standards for analysis, and the like. Accordingly, the present invention includes compositions composed of an inert carrier and an effective amount of a peptide of the present invention. An inert carrier is any substance that does not interact with the peptide to be transported and provides support, means of transport, bulk, traceable material, etc. to the peptide to be transported. An effective amount of peptide is that amount that produces a result or exerts an effect on the particular procedure being performed.

  Formulations suitable for subcutaneous, intravenous, intramuscular, etc .; suitable pharmaceutical carriers; and formulation and administration techniques can be prepared by any of the methods known in the art (eg, Remington's Pharmaceutical Sciences, Mack Publishing). Co., Easton, Pa., 20th edition, 2000).

Peptides are known to undergo hydrolysis, amidolysis, oxidation, racemization and isomerization in aqueous and non-aqueous environments. Degradation such as hydrolysis, amide decomposition or oxidation is
It can be easily detected by capillary electrophoresis. Despite enzymatic degradation, peptides with extended plasma half-life or biological time should be stable at least in aqueous solution. For example, a peptide exhibits less than 10% degradation over a day at body temperature, or less than 5% over a day at body temperature. Stability over a week at body temperature (ie, less than a few percent degradation) can allow for less frequent administration. Year-to-digit stability at refrigeration temperatures will allow manufacturers to provide liquid formulations and thus avoid the inconvenience of reconstitution. In addition, stability in organic solvents appears to provide that the peptides are formulated into new dosage forms such as implants.

  It is understood that the structures, materials, compositions, and methods described herein are intended to be representative of the invention, and that the scope of the invention is not limited by the scope of the examples. I will. Those skilled in the art will recognize that the invention can be practiced with modifications to the disclosed structures, materials, compositions and methods, and such modifications are considered within the scope of the invention.

  The following examples are presented to illustrate the invention described herein, but should not be construed to limit the scope of the invention in any way.

EXAMPLES In order that the present invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed to limit the scope of the invention in any way. All publications mentioned in this specification are incorporated by reference in their entirety.

Preparation of N-terminal modifying compounds Air and moisture sensitive liquids and solutions were transferred via syringe or cannula and introduced into the reaction vessel through a rubber septum. Commercial grade reagents and solvents were used without further purification. The term “concentration under reduced pressure” refers to the use of a Buchi rotary evaporator at approximately 15 mm of Hg. All temperatures are reported uncorrected in degrees Celsius (° C). Thin layer chromatography (TLC) was performed on precoated glass supported silica gel 60 A F-250 250 μm plates from EM Science. Column chromatography (flash chromatography) was performed on a Biotage instrument using cartridges pre-filled with 32-63 microns, 60A silica gel. Purification using preparative reverse phase HPLC chromatography was achieved using a Gilson 215 instrument and a YMC Pro-C18 AS-342 (150 × 20 mm ID) column. Typically, the mobile phase used was a mixture of H ₂ O (A) and MeCN (B). Water could or might not be mixed with 0.1% TFA. Typical gradient is:

Met.

Electron impact mass spectra (EI-MS or GC-MS) were obtained from a Hewlett Packard 5890 gas chromatograph equipped with a Hewlett Packard 5890 gas chromatograph with a J & W DB-5 column (0.25 μM coating; 30 m × 0.25 mm).
Obtained on a 5989A mass spectrometer. The ion source was maintained at 250 ° C. and the spectrum was scanned from 50-800 amu at 2 seconds per scan. High performance liquid chromatography-electrospray mass spectrum (LC-MS) is a Finnigan with quadruple pump, variable wavelength detector set at 254 nm, YMC pro C-18 column (2 × 23 mm, 120 A), and electrospray ionization. Obtained using a Hewlett-Packard 1100 HPLC equipped with LCQ ion trap mass spectrometry. The spectrum was scanned from 120 to 1200 amu using a variable ion time according to the number of ions in the ionization source. The eluents were A: 2% acetonitrile in water containing 0.02% TFA and B: 2% water in acetonitrile containing 0.018% TFA. Gradient elution from 10% to 95% B over 3.5 minutes at a flow rate of 1.0 mL / min was used with an initial hold of 0.5 minutes and a final hold at 95% B of 0.5 minutes. Total analysis time was 6.5 minutes. For consistency of characterization data, retention time (RT) is reported in minutes at the peak top as detected by a UV-visible detector set at 254 nm.

Conventional one-dimensional NMR spectroscopy was performed on a 300 or 400 MHz Varian Mercury-plus spectrometer. Samples were dissolved in deuterium solvent obtained from Cambridge Isotopes Labs and transferred to 5 mm ID Wilmad NMR tubes. The spectrum was acquired at 293K. Chemical shifts are recorded on a ppm scale and for ¹ H spectra, DMSO- _d6 2.49 ppm, CD ₃ CN 1.93 ppm, CD ₃ OD 3.30 ppm, CD ₂ Cl ₂ 5.32 ppm, and For 7.26 ppm of CDCl ₃ and the ¹³ C spectrum, 39.5 ppm of DMSO- _d6 , 1.3 ppm of CD ₃ CN, 49.0 ppm of CD ₃ OD, 53.8 ppm of CD ₂ Cl ₂ , and CDCl ₃ Reference was made to an appropriate residual solvent signal, such as 77.0 ppm. General preparation methods are illustrated in the reaction schemes that follow and by specific preparation examples.

Abbreviations and Acronyms As used throughout this disclosure, the following abbreviations have the following meanings.
Celite Corp. of Ac acetyl AcOH acetic acid Boc t-butoxycarbonyl Bu butyl CDCl ₃ deuterated chloroform Celite ^(R) diatomaceous earth Brand climb
Trademark CI Chemical ionization d doublet dd doublet doublet ddd doublet doublet doublet DME dimethoxyethane DMF N, N-dimethylformamide DMSO dimethyl sulfoxide DMSO-d ₆ dimethyl sulfoxide-d ₆
dppf 1,1'-bis (diphenylphosphino) fe
Locene EI Electron impact ionization EI-MS Electron impact-mass spectrometry Et Ethyl EtOH Ethanol EtOAc Ethyl acetate g Gram GC-MS Gas chromatography-Mass spectrometry h Time (s)
¹ H NMR proton nuclear magnetic resonance Hex hexane HPLC high performance liquid chromatography LC-MS liquid chromatography / mass analysis LDA lithium diisopropylamide m multiplet M mol m / z mass to charge ratio Me methyl MeCN acetonitrile mg milligram MHz megahertz min min (1 Or multiple)
mol mol mmol mmol MS mass spectrometry N normality NMR nuclear magnetic resonance NaOAc sodium acetate Pd / C palladium on charcoal PdCl ₂ (dppf). [1,1′-bis (diphenyl) with CH ₂ Cl ₂ dichloromethane
Nylphosphino) ferrocene] dichloroparadi
Um (II) complex (1: 1)
Ph phenyl PPh ₃ triphenylphosphine ppm parts per million Pr propyl q quartet qt quintet quant. Quantitative Rf TLC retention coefficient rt RT RT retention time (HPLC)
s singlet TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TMS tetramethylsilane v / v volume per unit volume vol volume w / w weight per unit weight

Example 1A
Production of (2-mercapto-1H-benzimidazol-1-yl) acetic acid

Stage 1. Production of (2-chloro-1H-benzimidazol-1-yl) methyl acetate

NaH (157.3 mg, 3.93 mmol of 60% dispersant in mineral oil) was suspended in anhydrous DMF (5 mL) and hexane (0.5 mL). 2-Chlorobenzimidazole (500 mg, 3.28 mmol) was added and the resulting solution was allowed to stir at rt for 1 h. Methyl bromoacetate (0.37 mL, 3.93 mmol) was added and the solution was allowed to stir at rt overnight. The reaction mixture was then diluted with water and the resulting tan precipitate was collected by filtration, triturated with ether and dried to yield 385 mg (52%) of crude material. ¹ H NMR (400 MHz, CD ₃ CN) δ 3.78 (s, 3H), 5.03 (s, 2H), 7.28-7.38 (m, 2H), 7.43 (d, 1H), 7.65 (d, 1H).
Stage 2. Preparation of (2-mercapto-1H-benzimidazol-1-yl) methyl acetate

Methyl (2-chloro-1H-benzimidazol-1-yl) acetate (150 mg, 0.67 mmol) and thiourea (101.7 mg, 1.34 mmol) were heated to reflux overnight in ethanol (30 mL). The reaction mixture was concentrated and the residue was triturated with water and dried to yield 142.5 mg (96%) of the crude product. _{^{1 H NMR (400MHz, CD 3}} CN) δ3.78 (s, 3H), 5.03 (s, 2H), 7.20-7.35 (m, 4H), 10.43 (bs, 1H).
Stage 3. Production of (2-mercapto-1H-benzimidazol-1-yl) acetic acid

Methyl (2-mercapto-1H-benzimidazol-1-yl) acetate (143 mg, 0.64 mmol) was dissolved in THF (1 mL), MeOH (1 mL) and water (0.5 mL). The solution was treated with LiOH (17.0 mg, 0.71 mmol) and heated to 80 ° C. for 4 hours. The pH was then adjusted to pH 4 with 1N HCl. The solution was diluted with water and extracted with 5% EtOH / EtOAc. The organics were dried over MgSO ₄ and concentrated in vacuo to yield 75.0 mg (56%) of the desired product. LC / MS m / z 209.1 (M + H) ⁺ ; RT 1.08 min. _{^{1 H NMR (400MHz, CD 3}} CN) δ5.03 (s, 2H), 7.20-7.35 (m, 4H), 10.43 (bs, 1H).

Example 1B
Preparation of lithium 2-{[2- (tritylthio) ethyl] amino} nicotinate

Step 1.2-Preparation of {[2- (tritylthio) ethyl] amino} ethyl nicotinate

Ethyl 2-chloronicotinate (100 mg, 0.54 mmol) was added to [1,1′-bis with 2- (tritylthio) ethanamine (344 mg, 1.08 mmol), cesium carbonate (438 mg, 1.35 mmol), and dichloromethane. Combined with (diphenylphosphino) ferrocene] dichloropalladium (II) complex (1: 1) (110 mg, 0.13 mmol). The solid was dissolved in dioxane (2 mL), water (1 mL) and heated to reflux for 48 hours. The reaction mixture was then diluted with EtOAc and washed with water and brine. The organics were dried over Na ₂ —SO ₄ and concentrated in vacuo. The crude residue was purified by Biotage column chromatography (10% EtOAc / hexanes) to yield 215 mg (85%) of the desired product. ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 1.40 (t, 3H), 2.45 (t, 2H), 3.38 (t, 2H), 4.37 (q, 2H), 7.03 (M, 1H), 7.18-7.45 (m, 16H), 8.18 (d, 1H), 8.45 (d, 1H).
Step 2.2-Preparation of {[2- (tritylthio) ethyl] amino} lithium nicotinate

2-{[2- (Tritylthio) ethyl] amino} ethyl nicotinate (215 mg, 0.46 mmol) was dissolved in THF (1 mL), MeOH (1 mL) and water (0.5 mL). The solution was treated with LiOH (12.1 mg, 0.50 mmol) and heated to 80 ° C. for 4 hours. The crude aqueous mixture was extracted with ether to remove impurities. The solution was then diluted with water and extracted with 5% EtOH / EtOAc. The EtOH / EtOAc extract was dried over MgSO ₄ and concentrated in vacuo to yield 55.0 mg (27%) of the desired product. LC / MS m / z 440.7 (M + H) ⁺ ; RT 2.17 min. ¹ H NMR (400 MHz, CD ₃ CN) δ 2.38 (t, 2H), 3.32 (t, 2H), 7.08 (m, 1H), 7.18-7.45 (m, 16H), 8.19 (d, 1H), 8.45 (d,
1H).

Example 1C
Preparation of lithium 1- [2- (tritylthio) ethyl] -1H-imidazole-2-carboxylate

Step 1.1, Preparation of 1 ', 1 "-{[(2-Bromoethyl) thio] methanetriyl} tribenzene

Triphenylmethyl mercaptan (3.00 g, 10.9 mmol) was dissolved in THF (10 mL) and cooled to 0 ° C. Lithium hexamethyldisilazide (10.85 ml of a 1M solution in THF) was added and the reaction mixture was allowed to stir for 30 minutes. The cooling bath was removed and dibromoethane (1.12 mL, 13.0 mmol) was added. The reaction mixture was allowed to stir at rt for an additional 30 minutes and concentrated in vacuo. The crude residue was dissolved in ethyl acetate and washed with water and brine. The organics were dried over Na ₂ SO ₄ and concentrated to yield 3.44 g of crude material (80% pure by ¹ H NMR integration). This material was used without further purification. _{^{1 H NMR (400MHz, CD 2}} Cl 2) δ2.75 (t, 2H), 2.90 (t, 2H), 7.20-7.45 (m, 18.6H).
Step 2.1—Preparation of [2- (tritylthio) ethyl] -1H-imidazole

NaH (70.5 mg of a 60% suspension in mineral oil, 1.76 mmol) was suspended in anhydrous DMF (3 mL) and hexane (0.5 mL). Imidazole (100 mg, 1.47 mmol) was added and the resulting solution was allowed to stir at rt for 1 h. 1,1 ′, 1 ″-{[(2-Bromoethyl) thio] methanetriyl} tribenzene (845 mg, 1.76 mmol) was added and the solution was allowed to stir overnight at rt. The reaction mixture was diluted with water and Extracted with EtOAc The organics were washed with water and brine and dried over Na ₂ SO _{4. The} crude product was purified by Biotage column chromatography (50% EtOAc / hexanes, 1% Et ₃ N) to give 280 mg. (51%) of the desired product was produced: ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 2.63 (t, 2H), 3.48 (t, 2H), 6.70 (s, 1H) 6.92 (s, 1H), 7.19 (s, 1H), 7.22-7.45 (m, 15H).
Step 3. Preparation of lithium 1- [2- (tritylthio) ethyl] -1H-imidazole-2-carboxylate

1- [2- (Tritylthio) ethyl] -1H-imidazole (140 mg, 0.38 mmol) is dissolved in anhydrous dichloromethane (1.5 mL) and trichloroacetyl chloride (0.06 mL, 0.57 mmol) and N, N -Treated with diisopropylethylamine (0.07 mL, 0.42 mmol). The reaction mixture was allowed to stir at rt overnight. The reaction mixture was then concentrated in vacuo. The crude residue was dissolved in THF (1 mL), MeOH (1 mL) and water (0.5 mL), treated with LiOH (18.1 mg, 0.76 mmol) and allowed to stir at 80 ° C. for 4 hours. The crude reaction mixture was then concentrated in vacuo. Purification by HPLC yielded 70 mg (44%) of the desired product. LC / MS m / z 414.9 (M + H) ^<+> ; RT 2.76 min. ¹ H NMR (400 MHz, CD ₃ OD) δ 2.68 (t, 2H), 4.18 (t, 2H), 6.72 (s, 1H), 6.85 (s, 1H), 7.19- 7.40 (m, 15H).

Example 1D
Preparation of lithium 4-{[2- (tritylthio) ethyl] amino} pyrimidine-5-carboxylate

Step 1.4 Preparation of ethyl 5-hydroxypyrimidine-5-carboxylate

Diethyl malonate (3.14 mL, 20.7 mmol) was combined with N, N ′, N ″ -methylidyne trisformamide (3.00 g, 20.7 mmol) and p-toluenesulfonic acid (356 mg, 2.07 mmol), The reaction mixture was then heated for 4 hours at 180 ° C. The resulting red oil was allowed to cool overnight to rt The crude reaction mixture was dissolved in a minimum amount of water and crystallized overnight, the solid collected by filtration, Washed with water and dried to yield 822 mg (24%) of the desired product: ¹ H NMR (400 MHz, CD ₃ OD) δ 1.38 (t, 3H), 4.32 (q, 2H) , 8.32 (s, 1H), 8.60 (s, 1H).
Step 2. Preparation of ethyl 4-chloropyrimidine-5-carboxylate

Ethyl 4-hydroxypyrimidine-5-carboxylate (380 mg, 2.26 mmol) was dissolved in THF (5 mL) and treated with thionyl chloride (1.65 ml, 22.6 mmol). The solution was heated to reflux for 4 hours and then concentrated in vacuo to yield 417 mg (99%) of the crude product. This material was used without purification or characterization.
Stage 3.4 Preparation of 4-{[2- (tritylthio) ethyl] amino} pyrimidine-5-carboxylate

Ethyl 4-chloropyrimidine-5-carboxylate (200 mg, 1.07 mmol) is dissolved in THF (2 mL) and 2- (tritylthio) ethanamine (514 mg, 1.61 mmol) and N, N-diisopropylethylamine (0. 56 ml, 3.22 mmol) was added. The reaction mixture was heated to reflux overnight and then concentrated in vacuo. The crude residue was purified by Biotage column chromatography to yield 230 mg (46%) of product. The product was confirmed by HNMR. ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 1.42 (t, 3H), 2.48 (bs, 2H), 3.40 (bs, 2H), 4.40 (q, 2H), 7.18 -7.50 (m, 16H), 8.85 (s, 1H), 8.96 (s, 1H).
Step 4.4 Preparation of {{2- (tritylthio) ethyl] amino} pyrimidine-5-carboxylate

Ethyl 4-{[2- (tritylthio) ethyl] amino} pyrimidine-5-carboxylate (230 mg, 0.49 mmol) was dissolved in THF (1 mL), MeOH (1 mL) and water (0.5 mL). The solution was treated with LiOH (23.5 mg, 0.98 mmol) and allowed to stir at rt for 2 days. The crude reaction mixture was then diluted with water and extracted with EtOAc. The organic extract was dried over Na ₂ SO ₄ and concentrated in vacuo to yield 180 mg (82%) of the desired product. LC / MS m / z 441.6 (M + H) ^<+> ; RT 2.54 min. ¹ H NMR (400 MHz, CD ₃ OD) δ 2.43 (t, 2H), 3.32 (m below solvent peak), 7.10-7.42 (m, 1H), 8.70 (d, 2H ).

Example 1E
Preparation of lithium 1- [2- (tritylthio) ethyl] -1H-benzimidazole-2-carboxylate

Step 1.1 Preparation of ethyl H-benzimidazole-2-carboxylate

1H-benzimidazole-2-carboxylic acid (500 mg, 3.08 mmol) was suspended in EtOH (5 mL), treated with thionyl chloride (1.12 mL, 15.4 mmol) and heated to reflux overnight. The reaction mixture was concentrated in vacuo to yield 644 mg (99%) of the crude product. ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 1.32 (t, 3H), 4.38 (q, 2H), 7.30 (m, 2H), 7.63 (m, 2H).
Step 2.1—Preparation of ethyl 2- (2-bromoethyl) -1H-benzimidazole-2-carboxylate

NaH (504 mg of a 60% suspension in mineral oil, 1.07 mmol) was suspended in anhydrous DMF (1.5 mL). Ethyl 1H-benzimidazole-2-carboxylate (170 mg, 0.89 mmol) was added and the solution was allowed to stir at rt for 1 h. 1,2-Dibromoethane (0.23 mL, 2.7 mmol) was added and the solution was heated to 50 ° C. overnight. The reaction mixture was diluted with water and extracted with EtOAc. The organic extract was dried over Na ₂ SO ₄ , concentrated in vacuo and purified by Biotage column chromatography (15% EtOAc / hexanes) to yield 100 mg (38%) of the desired product. ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 1.48 (t, 3H), 3.80 (t, 2H), 4.50 (q, 2H), 5.02 (t, 2H), 7.39 (T, 1H), 7.45 (t, 1H), 7.53 (d, 1H), 7.87 (d, 1H).
Step 3. Preparation of ethyl 1- [2- (tritylthio) ethyl] -1H-benzimidazole-2-carboxylate

Ethyl 1- (2-bromoethyl) -1H-benzimidazole-2-carboxylate (100 mg, 0.34 mmol), triphenylmethyl mercaptan (112 mg, 0.40 mmol) and N, N-diisopropylethylamine in THF (1 ml) A solution of (0.07 ml, 0.40 mmol) was allowed to stir at rt for 2 hours. In a separate flask, a solution of triphenylmethyl mercaptan (112 mg, 0.40 mmol) in THF (1 ml) was treated with lithium hexamethyldisilazide (0.40 mL of a 1M solution in THF) and 10 at rt. Stir for minutes. This solution was added to the original reaction mixture resulting in a red solution that was allowed to stir overnight at rt. The reaction mixture was concentrated in vacuo and the crude residue was purified by Biotage column chromatography to yield 80 mg (48%) of the desired product. ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 1.44 (t, 3H), 2.78 (t, 2H), 4.40-4.52 (m, 4H), 7.03 (m, 1H) 7.20-7.40 (m, 15H), 7.42 (m, 1H), 7.82 (m, 1H).
Step 4.1 Preparation of 1- [2- (tritylthio) ethyl] -1H-benzimidazole-2-carboxylate lithium

Ethyl 1- [2- (tritylthio) ethyl] -1H-benzimidazole-2-carboxylate (75 mg, 0.15 mmol) was dissolved in THF (1 mL), MeOH (1 mL) and water (0.5 mL). The solution was treated with LiOH (7.3 mg, 0.30 mmol) and allowed to stir at rt for 2 days. The crude reaction mixture was then diluted with water and extracted with EtOAc. The organic extract was dried over Na ₂ SO ₄ and concentrated in vacuo to yield 74 mg (99%) of the desired product. LC / MS m / z 464.9 (M + H) ^<+> ; RT 3.16 min. ¹ H NMR (400 MHz, CD ₃ OD) δ2.72 (t, 2H), 4.58 (t, 2H), 6.98 (m, 1H), 7.15-7.30 (m, 16H), 7.38 (d, 1
H), 7.63 (m, 1H).

Example 1F
Production of (2-mercapto-1H-imidazol-1-yl) acetic acid

Stage 1. Production of ethyl N- (2,2-dimethoxyethyl) glycinate hydrochloride

Bromoacetaldehyde dimethyl acetal (0.85 mL, 7.2 mmol) was added glycine ethyl ester hydrochloride (500 mg, 3.58 mmol) and N, N-diisopropylethylamine (1.37 mL, in THF (4 mL) and EtOH (1 mL). 7.88 mmol) solution. The reaction mixture was heated to 70 ° C. and allowed to stir overnight. The solution was diluted with water and extracted with dichloromethane. The organic extract was dried over Na ₂ SO ₄ and concentrated in vacuo. The crude residue was treated with 1N HCl in ether and the resulting precipitate was collected by filtration and dried to yield 371 mg (23%) of the desired product. ¹ H NMR (400 MHz, CD ₃ CN) δ 1.30 (t, 3H), 3.18 (bs, 2H), 3.42 (s, 6H), 3.85 (bs, 2H), 4.27 ( q, 2H), 4.92 (t, 1H), 9.40 (bs, 2H).
Stage 2. Preparation of (2-mercapto-1H-imidazol-1-yl) ethyl acetate

N- (2,2-dimethoxyethyl) ethyl glycinate hydrochloride (370 mg, 1.63 mmol) was dissolved in EtOH (2 mL) and a solution of potassium thiocyanate (237 mg, 2.44 mmol) in EtOH (8 mL). Was processed. The pink suspension was heated to reflux overnight. Concentrated HCl (0.136 mL, 1.63 mmol) was added and the solution was refluxed for 3 hours. The reaction mixture was concentrated in vacuo and the resulting solid was recrystallized from EtOAc to yield 130 mg (43%) of the desired product. ¹ H NMR (400 MHz, C
D ₃ CN) δ 1.33 (t, 3H), 4.20 (q, 2H), 4.77 (s, 2H), 6.77 (s, 1H), 6.83 (s, 1H), 9 .92 (bs, 1H).
Stage 3. Production of (2-mercapto-1H-imidazol-1-yl) acetic acid

(2-Mercapto-1H-imidazol-1-yl) ethyl acetate (130 mg, 0.70 mmol) was dissolved in THF (1 mL), MeOH (1 mL) and water (0.5 mL). The solution was treated with LiOH (33.4 mg, 1.40 mmol) and allowed to stir at rt for 2 days. The crude reaction mixture was acidified to pH 2 with 2N HCl, diluted with water and extracted with 5: 1 EtOAc / EtOH. The organic extract was dried over Na ₂ SO ₄ and concentrated in vacuo to yield 70 mg (63%) of the desired product. LC / MS m / z 159.1 (M + H) ⁺ ; RT 1.05 min. ¹ H NMR (400 MHz, CD ₃ OD) δ 4.82 (s, 2H), 6.82 (s, 1H), 6.99 (s, 1H).

Example 1G
Production of ({1- [2- (tritylthio) ethyl] -1H-imidazol-2-yl} thio) lithium acetate

Stage 1. Production of methyl (1H-imidazol-2-ylthio) acetate

2-thioimidazole (300 mg, 3.00 mmol) was dissolved in THF (3 mL) and treated with N, N-diisopropylethylamine (0.63 mL, 3.59 mmol) and methyl bromoacetate (0.31 mL, 3.3 mmol). And allowed to stir at rt for 1 hour. The solid precipitate was filtered off and the filtrate was diluted with EtOAc. The organics were washed with water, dried over Na ₂ SO ₄ and concentrated in vacuo to yield 385 mg (75%) of the desired product. _{^{1 H NMR (400MHz, CD 3}} CN) δ3.68 (s, 3H), 3.82 (s, 2H), 7.05 (s, 2H).
Stage 2. Preparation of ({1- [2- (tritylthio) ethyl] -1H-imidazol-2-yl} thio) methyl acetate

NaH (55.7 mg of a 60% suspension in mineral oil, 1.39 mmol) was suspended in anhydrous DMF (1.5 mL). Methyl (1H-imidazol-2-ylthio) acetate (200 mg, 1.16 mmol) was added and the resulting solution was allowed to stir at rt for 1 h. 1,1 ′, 1 ″-{[(2-Bromoethyl) thio] methanetriyl} tribenzene (668 mg, 1.39 mmol) was added and the reaction mixture was heated to 50 ° C. overnight. The reaction mixture was diluted with water. The organic extract was washed with water and brine, dried over MgSO ₄ and concentrated in vacuo The crude residue was purified by Biotage column chromatography (20% EtOAc / hexanes) to give 180 mg ( 33%) of the desired product: ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 2.60 (t, 2H), 3.65 (m, 5H), 3.78 (s, 2H), 6.69 (s, 1H), 6.96 (s, 1H), 7.20-7.40 (m, 15H).
Stage 3. Production of ({1- [2- (tritylthio) ethyl] -1H-imidazol-2-yl} thio) lithium acetate

({1- [2- (Tritylthio) ethyl] -1H-imidazol-2-yl} thio) methyl acetate (180 mg, 0.38 mmol) in THF (1 mL), MeOH (1 mL) and water (0.5 mL). Dissolved. The solution was treated with LiOH (18.2 mg, 0.76 mmol) and allowed to stir at rt overnight. The reaction mixture was diluted with water and extracted with EtOAc. The organic extract was dried over Na ₂ SO ₄ and concentrated in vacuo. Purification of the crude material by HPLC yielded 61 mg (34%) of the desired product. LC / MS m / z 460.9 (M +
H) ⁺ ; RT 2.81 minutes. ¹ H NMR (400 MHz, CD ₃ OD) δ 2.59 (t, 2H), 3.60 (s, 2H), 3.92 (t, 2H), 6.89 (s, 1H), 6.92 ( s, 1H), 7.19-7.35 (m, 15H).

Example 1H
Preparation of lithium 3- (2-tritylsulfanylethylamino) pyrazine-2-carboxylate

Step 1.2-Preparation of Tritylsulfanylethylamine

A suspension of cysteamine hydrochloride (1.14 g, 9.80 mmol) and triethylamine (3.0 mL, 21.6 mmol) in dichloromethane (20 mL) was stirred at rt for 10 min. N, O-bis (trimethylsilyl) acetamide (1.99 g, 9.80 mmol) was then added and the reaction was allowed to stir under nitrogen for 30 minutes. The reaction mixture was cooled to 0 ° C. and trityl chloride (2.46 g, 8.82 mmol) was added in one portion. The suspension was warmed to rt and allowed to stir for 16 hours. The reaction mixture was quenched with water (15 mL). The mixture was washed sequentially with 1N HCl (2 × 10 mL), water (2 × 10 mL), 15% ammonia solution (4 mL) and water (5 × 20 mL). The organics were dried over Na ₂ SO ₄ and concentrated in vacuo to yield 1.9 g (61%) of a light brown oil. _{^{1 H NMR (400MHz, CDCl 3}} ) δ7.70-7.18 (m, 15H), 2.71 (t, 2H), 2.4 (b, 2H).
Step 2. Preparation of 3-bromopyrazine-2-carboxylic acid methyl ester

Bromine (3.91 g, 24.46 mmol) was added to a stirred mixture of 3-aminopyrazine-2-carboxylic acid methyl ester (1.27 g, 8.29 mmol) and hydrobromic acid (4.70 mL, 41.5 mmol). Added dropwise at 0 ° C. A solution of sodium nitrite (1.44 g, 20.9 mmol) in water (6 mL) was then added dropwise. The reaction mixture was stirred for 15 minutes, brought to pH 8 with NaHCO ₃ (saturated, aqueous) and extracted with ethyl acetate (80 mL) and chloroform (50 mL). The combined organics were dried over MgSO ₄ and concentrated in vacuo to yield 1.13 g (63%) of an orange oil that solidified on standing. LC-MS m / z 217 (M + H ^< + ^> ); RT 1.15 min.
Step 3. Preparation of 3- (2-tritylsulfanylethylamino) pyrazine-2-carboxylic acid methyl ester

3-Bromopyrazine-2-carboxylic acid methyl ester (0.10 g, 0.45 mmol), 2-tritylsulfanylethylamine (0.29 g, 0.90 mmol) and triethylamine (0.06 mL, 0.005 mmol) in acetonitrile (5 mL). 44 mmol) was heated to reflux under argon for 18 hours. The mixture was concentrated under reduced pressure and purified by column chromatography (3: 1 Hex: EtOAc) to give 0.058 g (28%) of the desired product. ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 7.95 (s, 1H), 8.44 (s, 1H), 8.31 (s, 1H), 7.57.18 (m, 15H), 4 .20 (s, 3H), 3.36 (t, 2H), 2.50 (t, 2H).
Step 4. Preparation of lithium 3- (2-tritylsulfanylethylamino) pyrazine-2-carboxylate

3- (2-Tritylsulfanylethylamino) pyrazine-2-carboxylic acid methyl ester (0.12 g, 0.27 mmol) and LiOH (0. 0) in THF (5 mL), methanol (5 mL) and water (2.5 mL). 030 g, 1.3 mmol) was stirred at rt for 18 h. The reaction mixture was concentrated and purified by HPLC to yield 0.075 g (63%) of the desired product. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.26 (s, 1H), 8.15 (s, 1H), 7.42-7 (m, 15H), 3.30 (t, 2H), 2. 40 (t, 2H).

Example 1I
Preparation of 4-mercaptothiazole-5-carboxylic acid

Step 1.4 Preparation of 2- (2-methoxycarbonylethylsulfanyl) thiazole-5-carboxylic acid ethyl ester

A solution of ethyl isocyanoacetate (0.92 g, 7.7 mmol) in THF (8 mL) was added to a suspension of potassium tert-butoxide (1.0 g, 8.5 mmol) in THF (6 mL) at −40 ° C. One drop was added. The mixture was cooled to −60 ° C. and a solution of carbon disulfide (0.59 g, 7.7 mmol) in THF (8 mL) was added dropwise, keeping the temperature below −50 ° C. The mixture was warmed to 10 ° C. and methyl 3-bromopropionate (1.33 g, 7.70 mmol) was added. The mixture was allowed to stir for 2 hours and concentrated in vacuo. The product is recrystallized from dichloromethane / hexane to give 1.28 g (60%)
Of the desired product as a white solid. LC-MS m / z 276 (M + H ^< + ^> ); RT 1.65 min.
Step 2. Preparation of 4-mercaptothiazole-5-carboxylic acid ethyl ester

Sodium hydroxide (0.14 g, 3.5 mmol) was added to 4- (2-methoxycarbonylethylsulfanyl) thiazole-5-carboxylic acid ethyl ester (0.96 g, 3.5 mmol) in methanol (13.6 mL). Added to the solution. The mixture was refluxed for 1 hour and then concentrated in vacuo. The residue was dissolved in ethyl acetate / water and the pH was adjusted to 2 with 2N HCl. The organic layer was isolated and concentrated in vacuo to yield 0.66 g (100%) of the desired product, which was used without further purification. LC-MS m / z 189 (M + H ^< + ^> ); RT 1.47 min.
Step 3. Preparation of 4-mercaptothiazole-5-carboxylic acid

Sodium hydroxide (0.25 g, 6.3 mmol) was added to a solution of 4-mercaptothiazole-5-carboxylic acid ethyl ester (0.60 g, 3.2 mmol) in methanol (5 mL) and water (5 mL). And the mixture was heated to 80 ° C. for 3 hours. Upon cooling to rt, the reaction mixture was concentrated in vacuo. The residue was acidified with 2N HCl and extracted with dichloromethane. The organic layer was dried over MgSO ₄ , concentrated in vacuo, and purified by HPLC to yield 0.059 g (12%) of the desired product. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.95 (s, 1H); LC-MS m / z 162 (M + H ⁺ ); RT 1.01 min.

Example 1J
Production of 2-mercaptothiazole-5-carboxylic acid

Step 1.2-Preparation of mercaptothiazole-5-carboxylic acid methyl ester

A mixture of 2-bromothiazole-5-carboxylic acid methyl ester (0.40 g, 1.8 mmol) and thiourea (0.16 g, 2.1 mmol) in ethanol (6 mL) was heated to reflux for 2 hours. The mixture was allowed to cool to rt and the resulting suspension was filtered to give 0.19 g (61%) of the desired product as a yellow solid. LC-MS m / z 176.1 (M + H ^< + ^> ); RT 1.17 min.
Step 2.2-Preparation of mercaptothiazole-5-carboxylic acid

Sodium hydroxide (0.08 g, 1.9 mmol) was added to a solution of 2-mercaptothiazole-5-carboxylic acid methyl ester (0.17 g, 0.97 mmol) in methanol (5 mL) and water (5 mL). . The reaction mixture was stirred at rt for 3 h and concentrated in vacuo. The residue was acidified with 2N HCl and the resulting suspension was filtered to yield 0.061 g (39%) of the desired product as an off-white solid. ¹ H NMR (400 MHz, CD ₃ OD) δ 7.80 (s, 1H); LC-MS m / z 161 (M + H ⁺ ); RT 1.02 min.

Example 1K
Preparation of lithium 2- (2-tritylsulfanylethylamino) thiazole-5-carboxylate

Step 1.2 Preparation of 2- (2-tritylsulfanyl-ethylamino) -thiazole-5-carboxylic acid methyl ester

2-Bromothiazole-5-carboxylic acid methyl ester (0.20 g, 0.88 mmol), 2-tritylsulfanylethylamine (0.42 g, 1.3 mmol) and triethylamine (0.12 mL, 0.005 mmol) in acetonitrile (5 mL). 88 mmol) was heated to reflux under argon for 18 hours. The mixture was concentrated in vacuo and purified by column chromatography (3: 1 Hex: EtOAc) to give 0.12 g (29%) of the desired product. ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 8.15 (s, 1H), 7.60-7.15 (m, 15H), 4.20 (s, 3H), 3.45 (t, 2H) 2.50 (br, 2H).
Step 2.2—Preparation of lithium (2-tritylsulfanylethylamino) -thiazole-5-carboxylate

2- (2-Tritylsulfanylethylamino) -thiazole-5-carboxylic acid methyl ester (0.12 g, 0.26 mmol) and LiOH (0) in THF (5 mL), methanol (5 mL) and water (2.5 mL). (0.03 g, 1.3 mmol) was stirred at rt for 18 h. The reaction mixture was concentrated in vacuo and purified by HPLC to yield 0.087 g (75%) of the desired product. ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 7.90 (s, 1H), 7.40-7.10 (m, 15H), 3.30 (s, 3H), 3.45 (t, 2H) 2.40 (br, 2H).

Example 1L
Preparation of 2-mercapto-6-methylpyrimidine-4-carboxylic acid

Step 1.2 Preparation of Carbamimidoylsulfanyl-6-methylpyrimidine-4-carboxylic acid methyl ester hydrobromide

A mixture of 2-chloro-6-methylpyrimidine-4-carboxylic acid methyl ester (0.50 g, 2.7 mmol) and thiourea (0.41 g, 5.4 mmol) in ethanol (8 mL) was heated to reflux for 16 hours. And then concentrated in vacuo. Attempts to purify the product by recrystallization (methanol / ether) yielded 0.229 g (38%) of an oil which was identified as the desired product. LC-MS m / z 226.9 (M + H ^< + ^> ); RT 1.07 min.
Step 2.2-Preparation of mercapto-6-methylpyrimidine-4-carboxylic acid

Mixture of 1N sodium hydroxide (6.98 mL, 6.98 mmol) and 2-carbamimidoylsulfanyl-6-methylpyrimidine-4-carboxylic acid methyl ethyl ester hydrobromide (0.37 g, 1.4 mmol) Was heated to reflux for 2 hours. Upon cooling to rt, the reaction mixture was concentrated in vacuo. The residue was dissolved in water and MP-TsOH (Argonaut technologies) was added. The mixture was stirred for 18 hours until a pH of 4 was achieved. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was dissolved in methanol, filtered, and the filtrate was concentrated in vacuo to give 0.139 g (59%) of the desired product as a brownish solid. ¹ H NMR (400 MHz, CD ₃ OD) δ 7.10 (s, 1H), 3.40 (s, 3H); LC-MS m / z 171 (M + H ⁺ ); RT 1.15 min.

Example 1M
Production of 5-mercaptonicotinic acid

Step 1.5—Preparation of tert-butylsulfanylnicotinonitrile

tert-Butylthiol (0.43 g, 4.8 mmol) was added to a suspension of NaH (0.19 g, 4.8 mmol) in DMF (15 mL) and the reaction mixture was heated at 50 ° C. for 1 h. 5-Bromonicotinonitrile (0.60 g, 3.2 mmol) was added to the resulting suspension and the reaction mixture was heated at 120 ° C. for 5 hours. Upon cooling to rt, the mixture was concentrated in vacuo and purified by HPLC to yield 0.329 g (54%) of the desired product as an off-white solid. LC-MS m / z 193 (M + H ^< + ^> ); RT 2.90 min.
Step 2.5-Preparation of tert-butylsulfanylnicotinic acid

A mixture of 5-tert-butylsulfanylnicotinonitrile (0.43 g, 2.2 mmol) and sodium hydroxide (0.89 g, 22 mmol) in ethanol (5 mL) and water (5 mL) was heated to reflux for 1 hour. Upon cooling to rt, the reaction mixture was diluted with water and extracted with ether. The aqueous layer was acidified with 2N HCl and extracted with dichloromethane. The dichloromethane extract was dried over MgSO ₄ and concentrated to yield 0.433 g (79%) of the desired product as a white solid. LC-MS m / z 212 (M + H ^< + ^> ); RT 2.36 min.
Step 3.5-Production of mercaptonicotinic acid

A solution of 5-tert-butylsulfanylnicotinic acid (0.30 g, 1.2 mmol) in 2N HCl (9 mL, 18 mmol) was heated to reflux for 32 hours. Upon cooling to rt, the reaction mixture was concentrated in vacuo to yield 0.054 g (28%) of the desired product. ¹ H NMR (400 MHz, CD ₃ OD) δ 9.00-8.80 (m, 2H), 8.40 (d, 1H); LC-MS m / z 156 (M + H ⁺ ); RT 2.37 min.

Example 1N
Preparation of 5-isopropyl-2-mercaptothiazole-4-carboxylic acid

Step 1.2 Preparation of 2-chloro-4-methyl-3-oxopentanoic acid ethyl ester

A solution of sulfuryl chloride (1.63 mL, 19.7 mmol) in toluene (5 mL) was added to a solution of 4-methyl-3-oxopentanoic acid ethyl ester (3.28 g, 19.7 mmol) in toluene (25 mL). Added dropwise over 10 minutes. The resulting mixture was stirred at rt for 18 h and then slowly quenched with water and NaHCO ₃ (saturated, aqueous). The mixture was extracted with ethyl acetate and the combined organics were dried over MgSO ₄ and concentrated in vacuo to yield 3.4 g (70%) of the desired product, which was used without further purification. LC-MS m / z 194.1 (M + H ^< + ^> ); RT 2.69 min.
Step 2. Preparation of 2-amino-5-isopropylthiazole-4-carboxylic acid ethyl ester

A mixture of 2-chloro-4-methyl-3-oxopentanoic acid ethyl ester (2.0 g, 7.3 mmol) and thiourea (0.43 g, 5.6 mmol) in ethanol (8 mL) was refluxed for 18 hours, Then it was concentrated in vacuo. The residue was treated with aqueous ammonia and the resulting yellow solid was dissolved in water and extracted with dichloromethane. The combined organics were dried over Na ₂ SO ₄ and concentrated in vacuo. The solid was dissolved in a small amount of dichloromethane and filtered to yield 1.02 g (85%) of the desired product as a cream colored solid. LC-MS m / z 215.1 (M + H ^< + ^> ); RT 1.96 min.
Step 3. Preparation of 2-bromo-5-isopropylthiazole-4-carboxylic acid ethyl ester

To a dark brown solution of copper (II) bromide (2.47 g, 11.1 mmol) in acetonitrile (10 mL) in a two-necked flask equipped with a condenser was added tert-butylnitrile (0.63 g, 5.5 mmol). Was slowly added at rt. The mixture was heated to 60 ° C. and a suspension of 2-amino-5-isopropylthiazole-4-carboxylic acid ethyl ester (0.79 g, 3.7 mmol) in acetonitrile (14 mL) was added dropwise. The mixture was then heated at 60 ° C. for 3 hours. Upon cooling to rt, the reaction mixture was poured into 1N NaOH (40 mL) and extracted with ethyl acetate. The combined organics were dried over Na ₂ SO ₄ , concentrated in vacuo and purified by column chromatography (2: 1 EtOAc / hexanes) to give 0.88 g (86%) of the desired product as a yellow oil. As a result. LC-MS m / z 280 (M + H ^< + ^> ); RT 3.65 min.
Step 4.5 Preparation of Isopropyl-2-mercaptothiazole-4-carboxylic acid ethyl ester

A mixture of 2-bromo-5-isopropylthiazole-4-carboxylic acid ethyl ester (0.20 g, 0.72 mmol) and thiourea (0.07 g, 0.86 mmol) in ethanol (6 mL) was heated to reflux for 2 hours. did. Upon cooling to rt, the resulting suspension was filtered to yield 0.11 g (66%) of the desired product as a yellow solid. LC-MS
m / z 232.1 (M + H ⁺ ); RT 2.72 min.
Step 5. Preparation of 5-isopropyl-2-mercaptothiazole-4-carboxylic acid

NaOH (1N, 0.78 mL, 0.78 mmol) was added to 5-isopropyl-2-mercaptothiazole-4-carboxylic acid ethyl ester (0.09 g, 0.39 mmol) in methanol (3 mL) and water (2 mL). To the solution was added and the mixture was stirred at rt for 3 h. The reaction mixture was concentrated in vacuo and the residue was acidified with 2N HCl. The resulting suspension was filtered to yield 0.045 g (57%) of the desired product as an off-white solid. ¹ H NMR (400 MHz, CD ₃ OD) δ 3.85-4.10 (m, 1H), 1.22 (d, 6H); LC-MS m / z 204.2 (M + H ⁺ ); RT 1.65 Minutes.

Example 1O
Preparation of (1-hexadecyl-1H-benzimidazol-2-ylsulfanyl) acetic acid

Stage 1. Preparation of (1H-benzimidazol-2-ylsulfanyl) acetic acid ethyl ester

A mixture of 2-mercaptobenzimidazole (0.3 g, 2 mmol), ethyl bromoacetate (0.50 g, 2.9 mmol) and potassium carbonate (0.14 g, 0.98 mmol) in ethanol (3.1 mL) was brought to reflux. Heated for 8 hours and then concentrated in vacuo. Purification by HPLC yielded 0.22 g (46%) of the desired product. LC-MS
m / z 237.2 (M + H ^< + ^> ); RT 1.41 min.
Stage 2. Preparation of (1-hexadecyl-1H-benzimidazol-2-ylsulfanyl) acetic acid methyl ester

Sodium hydride (0.03 g, 0.8 mmol) was added to a solution of (1H-benzimidazol-2-ylsulfanyl) acetic acid ethyl ester (0.21 g, 0.79 mmol) in DMF (10 mL) at 0 ° C. And the mixture was stirred at rt for 1 h. Hexadecyl bromide (0.22 g, 0.71 mmol) was added and the mixture was stirred at rt for 18 h. The reaction mixture was diluted with water and methanol and concentrated in vacuo. Purification by column chromatography (20% ethyl acetate in hexane) yielded 0.24 g (67%) of the desired product. LC-MS m / z 447.4 (M + H ^< + ^> ); RT 5.03 min.
Stage 3. Preparation of (1-hexadecyl-1H-benzimidazol-2-ylsulfanyl) acetic acid

A mixture of lithium hydroxide (0.060 g, 2.4 mmol) and (1-hexadecyl-1H-benzimidazol-2-ylsulfanyl) acetic acid methyl ester (0.21 g, 0.47 mmol) was heated to reflux for 2 hours. Rt, cooled to rt and concentrated in vacuo. The residue was dissolved in water and the suspension was acidified with 1N HCl and filtered. The solid was collected and dried to yield 0.16 g (79%) of the desired product. ¹ H NMR (400 MHz, CD ₃ OD) δ 7.70-7.20 (m, 4H), 4.20 (t, 2H), 3.80 (s, 2H), 1.85 (m, 2H), 1.21-1.50 (m, 28H), 0.90 (t, 3H); LC-MS m / z 433.2 (M + H ⁺ ); RT 4.72 min.

Example 1P
Production of lithium 6- (2-tritylsulfanylethylamino) nicotinate

Step 1.6 Preparation of 2- (2-tritylsulfanylethylamino) nicotinic acid methyl ester.

With methyl 6-chloronicotinate (0.20 g, 1.17 mmol), 2-tritylsulfanylethylamine (0.56 g, 1.75 mmol), cesium carbonate (0.95 g, 2.91 mmol), and dichloromethane [1, 1′-bis (diphenylphosphino) ferrocene] dichloropalladium (II) complex (1: 1) (0.24 g, 0.29 mmol) was added to 120 ° C. overnight in 1,4-dioxane (4.0 mL) and water. Heated. Upon cooling to rt, the reaction mixture was filtered through Celite ^(R), concentrated in vacuo, and purified by Biotage column chromatography (5% EtOAc / hexanes). This yielded 0.3442 g of a white solid. The material was recrystallized from 10% EtOAc / hexanes to yield 0.272 g (51%) of the desired product as a white solid. Rf = 0.42 (20% EtOAc / hexane). ¹ H NMR (400 MHz, CD ₂ Cl ₂ ) δ 2.0 (bs, 1H), 2.45 (t, 2H), 3.40 (t, 2H), 3.92 (s, 3H), 7.15 -7.30 (m, 10H), 7.42-7.50 (m, 6H), 8.02 (d, 1H), 8.90 (s, 1H).
Step 2. Preparation of 6- (2-tritylsulfanylethylamino) lithium nicotinate

6- (2-Tritylsulfanylethylamino) nicotinic acid methyl ester (270 mg, 0.59 mmol) was suspended in THF (2 mL), MeOH (2 mL) and water (1 mL). The reaction mixture was treated with LiOH (20 mg, 0.65 mmol) and heated to 50 ° C. for 2 hours. Upon cooling to rt, the reaction mixture was concentrated in vacuo. Recrystallization from EtOAc / hexane (3: 1) yielded 236 mg (89%) of the desired product as a white solid. ¹ H NMR (400 MHz, DMSO- _d6 ) δ 2.25 (t, 2H), 3.00 (bt, 1H), 3.28 (t, 2H), 7.00-7.40 (m, 16H), 7.90 (d, 1H), 8.72 (s, 1H).

Peptide synthesis Peptides are synthesized on an Applied Biosystems 430A peptide synthesizer using FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied Biosystems protocol is used. The peptide is cleaved with 84.6% TFA, 4.4% phenol, 4.4% water, 4.4% thioanisole and 2.2% ethanedithiol. The peptide is precipitated from the cleavage cocktail using cold tert butyl methyl ether. The precipitate is washed with cold ether and dried under argon. The peptide is purified by reverse phase C ₁₈ HPLC using a linear water / acetonitrile gradient containing 0.1% TFA. Peptide identity is confirmed by MALDI and electrospray mass spectrometry and amino acid analysis.

Methods for adding N-terminal modified compounds Peptides are synthesized on an Applied Biosystems 430A peptide synthesizer using FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied Biosystems protocol is used. The N-terminal modifying compound is attached to the peptide according to natural amino acid coupling during FMOC chemistry. N-terminal modifying compounds are either commercially available or synthesized as described in Example 1. In the case of N-terminal modified compounds containing amine and mercapto, the amine and mercapto groups are protected with Fmoc or trityl, respectively, during peptide coupling. The peptide is cleaved with 84.6% TFA, 4.4% phenol, 4.4% water, 4.4% thioanisole and 2.2% ethanedithiol. The peptide is precipitated from the cleavage cocktail using cold tert butyl methyl ether. The precipitate is washed with cold ether and dried under argon. The peptide is purified by reverse phase C18 HPLC using a linear water / acetonitrile gradient containing 0.1% TFA. Peptide identity is confirmed by MALDI and electrospray mass spectrometry and amino acid analysis.

Methods for Adding C-Terminal Modifying Compounds Peptides are synthesized on an Applied Biosystems 433A peptide synthesizer using FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied Biosystems protocol is used. HBTU activated C-terminal modified compounds are subjected to coupling of natural amino acids during FMOC chemistry, such as Wang resin to produce peptides with C-terminal modified compounds containing free carboxylic acids, or Rink Amide) for producing amide variants. The peptides are then synthesized by stepwise addition of amino acids using standard FOMC protocols. The peptide is cleaved with 84.6% TFA, 4.4% phenol, 4.4% water, 4.4% thioanisole and 2.2% ethanedithiol. The peptide is precipitated from the cleavage cocktail using cold tert butyl methyl ether. The precipitate is washed with cold ether and dried under argon. The peptide is purified by reverse phase C18 HPLC using a linear water / acetonitrile gradient containing 0.1% TFA. Peptide identity is confirmed by MALDI and electrospray mass spectrometry and amino acid analysis.

Preparation of Pegylated Peptides Pegylated derivatives are prepared by incubating methoxypolyethylene glycol derivatized with maleimide to attach to the mercapto moiety of the N-terminal modifying group. Use mPEG-MAL or mPEG2-MAL products supplied by Nektar Therapeutics (Huntsville, Alabama, USA), or GLE-200MA or GLE-400MA products supplied by NOF (Tokyo, Japan). The coupling reaction is performed by incubating the peptide and a 2-fold molar excess of maleimide-PEG in 50 mM Tris, pH 7 for 2-12 hours. The peptide concentration can be 1 mg / ml or less. Underivatized peptide and PEG are purified from PEGylated peptide by cation exchange chromatography and dialysis or by reverse phase C ₁₈ HPLC. The purified PEG-peptide conjugate is then lyophilized.

Preparation of Fatty Acid Derivatized Peptides Fatty acid (palmitic acid) derivatives of N-terminal modified compounds containing amines are selectively treated with 0.1% TFA for the Fmoc protecting group of the amine portion of the N-terminal modified group prior to deprotection and cleavage Prepared as the N-terminal modified peptide as described in Example 3 except that it was removed and derivatized with palmitic acid using the same conditions as for normal amino acid coupling.

  Fatty acid derivatives were synthesized as described in Example 1, using 1-hexadecyl-1H-benzimidazol-2-ylsulfanyl) acetic acid as the N-terminal modifying group as described in Example 3. Can also be manufactured.

Pharmaceutical Compositions-IV and SC Formulations Sterile IV injectable formulations are prepared using 4 mg of the peptide of formula (I) or 4 mg peptide content equivalent using any manufacturing method known in the art. The product is prepared using a derivatized peptide having a product and 1 L sterilized physiological saline. Higher concentrations of peptides can be used in SC formulations. For the peptide of formula (I) or derivatized peptide, 4 mg is dissolved in 100 mL saline or DMSO and a sterile vial after sterile filtration is filled with the composition.

Peptide Mass Spectrometry Dilute 40 pmol / 2 μl aliquots of peptide to 10 μl with water. HEPES buffer is removed by application of 50% (20 pmol / 5 μl) of sample to conditioned Millipore C18 ZipTip according to manufacturer's instructions. Samples are eluted directly from ZipTip onto MALDI plates with matrix (10 mg / ml α-cyanohydroxycinnamic acid in 50% ACN, 0.1% TFA). Applied Biosystems Voyager running sample in reflector ion mode
Analyze with DE-PRO MALDI. Data was collected in the 500-4000 Da range and the resulting mass was compared to that expected by human calculations.

Edman analysis of peptides Peptide samples are supplied for Edman degradation at 1 nmol / 10 μl in 10 mM HEPES, pH 7.4, 5% TFA. Prior to Edman analysis, HEPES buffer salt was
Remove the Applied Biosystems ProSorb sample cartridge by using according to the manufacturer's instructions. Briefly, the sample is applied to a PVDF membrane and washed with 0.1% TFA, after which the membrane is removed and inserted into a protein sequencer for Edman degradation. Edman degradation is performed on an Applied Biosystems Procedure 494HT protein sequencer using the pulsed liquid method according to the manufacturer's instructions. Read the array manually.

Peptide stability The formulation described in Example 4 is placed in a stationary stability chamber. The peptides are also analyzed for stability to degradation of DPPIV in solution and plasma. Samples are periodically removed for analysis by methods of detection of peptide degradation sensitivity, capillary electrophoresis, mass spectrometry, Edman degradation, ELISA and protein activity assays. The areas of the various peaks are summed, and the area of the parent peptide peak is divided by the total peak area. The quotient is% purity. Due to the presence of impurities present in the fresh peptide, the purity change is normalized by dividing the purity at various times by the initial purity. For stability against DPPIV and plasma, 20 pmol / μl peptide was incubated at 37 ° C. in the presence of 300 pM DPPIV in 100 mM HEPES, pH 7.4. At various time points, the reaction (2 μl aliquot) is terminated by adding 1 μM DPPIV inhibitor and freezing. For MALDI mass spectrometry, time points of T = 0, 1, 5, and 24 hours are evaluated. The results are plotted as a percentage of intact peptide or peptide derivative compared to the degradation product.

Binding of peptides to GLP-1 receptor RINm5F cell membranes are prepared according to the following procedure. RINm5F cell flasks are washed with PBS, scraped in 20 mM Hepes / 1 mM EDTA / 250 mM sucrose (HES) buffer containing protease inhibitors and homogenized with a dounce homogenizer, then repeated through a 23 gauge needle. And resuspend. Unbroken cells and nuclei are removed by centrifugation at 500 × g for 5 minutes. The pellet is resuspended in HES buffer using a 23 gauge needle and the centrifugation is repeated. Combine the supernatants from the two spins and centrifuge at 40,000 xg for 20 minutes. The resulting plasma membrane pellet is resuspended in HES buffer using a 23 gauge needle and then a 25 gauge needle. The membrane is stored at −80 ° C. until use.

IC ₅₀ values for competitive binding of peptides of the invention and peptide fragments, variants and analogs to the GLP-1 receptor in RINm5F membranes typically range from a minimum of about 0.01 nM to about 20 nM (ie 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nM). The IC ₅₀ values of peptide derivatives of the invention typically have a minimum of 0.01 nM to about 500 nM (ie 0.01, 0.1, 1, 10, ₅₀ , 100, 150, 200, 250, 300, 350, 400 , 450 or 500 nM).

Competitive binding of peptides using the RINm5F cell plasma membrane is measured as follows. 96 well GF / C filtration plates (Millipore, Bedford, Mass.) Were blocked with 0.3% PEI for a minimum of 1 hour and 20 mM Tris, 2 mM
Wash twice with binding buffer consisting of EDTA, pH 7.5, 1 mg / ml BSA and 1 mg / ml bacitracin. Five micrograms of RINm5F cell plasma membrane diluted in binding buffer was added to 0.05 mCi of ¹²⁵ I-labeled GLP-1 and peptide concentrations ranging from 1 × 10 ⁻¹² to 1 × 10 ⁻⁵ M. Apply to each well together. rt
After 60 minutes of incubation, the plates were washed 3 times with ice-cold PBS containing 1 mg / ml BSA. Plates are dried, scintillant is added to each well, and cpm per well is measured using a Wallac Microbeta counter.

The number of ¹²⁵ I counts bound to the membrane at each concentration of peptide is plotted and analyzed by non-linear regression using Prizm software to determine IC ₅₀ . The disclosed peptides bind to the GLP-1 receptor present in the plasma membrane isolated from RINm5F cells with IC ₅₀ values between 1.4 nM and 248 nM (determined from a minimum of 3 trials) did. IC ₅₀ is the concentration of peptide at which the maximum binding of labeled GLP-1 (7-36) is reduced by 50%.

Elevation of cAMP in response to peptide Peptide signaling of the GLP-1 receptor is measured using a cAMP scintillation approximation assay. RINm5F cells were plated at 1.5 × 10 ⁵ cells / well into 96 well plates (Costar) and grown in RPMI 1640, 5% FBS, antibiotic / antimycotic (Gibco BRL) for 24 hours at 37 ° C. Let The medium is removed and the cells are washed twice with PBS. Cells are incubated for 15 minutes at 37 ° C. with peptide concentrations ranging from 1 × 10 ⁻¹² to 1 × 10 ⁻⁵ M in Hepes-PBS containing 1% BSA and 100 μM IBMX. For assays of peptides conjugated with fatty acids, BSA was omitted from the incubation buffer. Incubation buffer is removed and cells are lysed with lysis reagent provided with cAMP scintillation approximation assay (SPA) direct screening assay system (Amersham Pharmacia Biotech Inc, Piscataway, NJ). The amount (pmol) of cAMP present in the lysate is measured according to the instructions provided with this kit. The amount of cAMP produced (pmol) at each concentration of peptide is plotted and analyzed by non-linear regression using Prizm software to determine the EC ₅₀ for each peptide.

Insulin secretion from dispersed rat islet cells Insulin secretion in dispersed rat islets mediated by a number of peptides of the invention is measured as follows. Islets of Langerhans isolated from SD rats (200-250 g) are digested using collagenase. Dispersed islet cells are treated with trypsin, seeded in 96V bottom plates and pelleted. The cells are then cultured overnight in media with or without the peptides of the invention. The medium is aspirated and cells are preincubated with Krebs-Ringer-HEPES buffer containing 3 mM glucose for 30 minutes at 37 ° C. The preincubation buffer is removed and the cells are incubated at 37 ° C. for an appropriate time with Krebs-Ringer-HEPES buffer containing the appropriate glucose concentration (eg, 8 mM) with or without peptide. A portion of the supernatant is removed and its insulin content is measured by SPA. The results are expressed as “fold over control” (FOC).

Generation of peptide-specific antibodies and measurement of peptides by ELISA Polyclonal antibodies specific for the peptides of the invention are generated by synthesizing specific fragments of the peptides of the invention using an ABI 433A peptide synthesizer. The peptide is then cleaved from the resin and purified with Beckman System Gold analysis and preparative HPLC equipment. The correct product is identified using a Perspective MALDI mass spectrometer. The peptide is dried using a lyophilizer. The peptide (2 mg) is then conjugated to keyhole limpet hemocyanin (KLH) via the free sulfhydryl group of Cys.

  Female New Zealand white rabbits are immunized on days 0, 14, 35, 56 and 77. On day 0, each rabbit is injected subcutaneously with 250 μg of peptide and Freund's complete adjuvant. Subsequent immunization utilizes 125 μg of peptide per rabbit. Blood collection begins on day 21 and continues at 21 day intervals thereafter. Purification of the anti-peptide antibody is performed by passing the crude serum through a specific peptide affinity purification column. Antibody titer is measured by ELISA.

A 96-well Immulon 4HBX plate is coated with a C-terminal F (ab) antibody specific for the peptide of the invention and allowed to incubate overnight at 4 ° C. The plate is then blocked to prevent nonspecific binding. The peptide standard (2500 ng / mL to 160 pg / mL) is then diluted with 33% plasma and the sample is diluted 3-fold with buffer and then incubated at rt for 1.5 hours. After washing, a polyclonal N-terminal antibody specific for the peptide of the invention is incubated on the plate for 1 hour. This is followed by the addition of horseradish peroxidase (HRP) -donkey anti-rabbit antibody and the samples and standards are incubated for another 1 hour. Detection is assessed after incubation with 3,3 ′, 5,5′-tetramethylbenzidine (TMB) solution and the plate is read at OD ₄₅₀ .

Alternatively, 96-well Immulon 4HBX plates are coated with a polyclonal N-terminal antibody specific for the peptides of the invention and allowed to incubate overnight at 4 ° C. The plate is then blocked to prevent nonspecific binding. The peptide standard (2500 ng / mL to 160 pg / mL) is then diluted with 50% plasma and the sample is diluted 2-fold with buffer and then incubated at rt for 1.5 hours. After washing, a monoclonal anti-PEG antibody specific for the peptide of the invention is incubated on the plate for 1 hour. This is followed by the addition of horseradish peroxidase (HRP) -anti-mouse antibody and the samples and standards are incubated for another hour. Detection is assessed after incubation with 3,3 ′, 5,5′-tetramethylbenzidine (TMB) solution and the plate is read at OD ₄₅₀ .

Peptide Pharmacokinetics After IV and Subcutaneous Administration Transfer the plasma sample to a microcentrifuge tube and add an equal volume of acetonitrile to the sample (50% final concentration). The sample is vortexed vigorously for about 5 minutes and allowed to stand on ice for 10 minutes. The sample is again vortexed for about 1 minute and then centrifuged at maximum (about 15,000 × g) for 30 minutes in a microcentrifuge (4 ° C.).

  After centrifugation, the aqueous layer is carefully transferred to a clean centrifuge tube and the sample is centrifuged at maximum speed (about 15,000 × g) for 5 minutes in a microcentrifuge (4 ° C.). The extracted sample is dried under vacuum until dried using a Speed Vac SC110 (Savant) with moderate heating settings. The sample is resuspended in an appropriate volume of sterile water and maintained at 4 ° C. Samples are then sonicated for 10 minutes at rt in an ultrasonic bath prior to analysis.

Effect of peptides on intraperitoneal glucose tolerance in rats The in vivo activity of peptides of the invention when administered subcutaneously is tested in rats. Rats fasted overnight are given a subcutaneous injection of control or peptide (1-100 μg / kg). Basal blood glucose is measured after 3 hours and rats are given 2 g / kg glucose intraperitoneally. Blood glucose is measured again after 15, 30 and 60 minutes.

Demonstration of the activity of the peptides of the present invention is well known in the art, in vitro, ex
It can be achieved by in vivo and in vivo assays. For example, diabetes and related disorders such as syndrome X, impaired glucose tolerance, fasting hyperglycemia and antiinsulinemia; atherosclerotic diseases, and related disorders such as hypertriglyceridemia and hypercholesterolemia; and obesity The following assays may be used to demonstrate the effectiveness of pharmaceutical agents for the treatment of

Blood glucose measurement methods db / db mice (obtained from Jackson Laboratories, Bar Harbor, Maine) are bled (either in the eye or tail vein) and grouped according to equivalent mean blood glucose levels. They are administered the test peptide for 14 days. At this point, the animals were bled again by eye or tail vein and blood glucose levels were measured. In each case, the glucose concentration is measured with Glucometer Elite XL (Bayer Corporation, Elkhart, Ind.).

Methods for Measuring Effects on Cardiovascular Parameters Cardiovascular parameters (eg heart rate and blood pressure) are also evaluated. SHR rats are administered vehicle or test peptide for 2 weeks. Blood pressure and heart rate are measured using the tail cuff method as described by Grinsell et al. (Am. J. Hypertens. 13: 370-375, 2000). In monkeys, blood pressure and heart rate are monitored as described by Shen et al. (J. Pharmacol. Exp. Therap. 278: 1435-1443, 1996).

Methods for Measuring Triglyceride Levels hApoA1 mice (obtained from Jackson Laboratories, Bar Harbor, Maine) are bled (in either the eye or tail vein) and grouped according to equivalent mean serum triglyceride levels. They are administered the test peptide for 8 days. The animals are then bled again by eye or tail vein and serum triglyceride levels are measured. In each case, triglyceride levels are measured using a Technicon Axon automated analyzer (Bayer Corporation, Tarrytown, NY).

Methods for Measuring HDL Cholesterol Levels To measure plasma HDL cholesterol levels, hApoP1 mice are bled and grouped with equivalent mean plasma HDL cholesterol levels. Mice are administered vehicle or test peptide for 7 days and then blood is collected on day 8. Plasma is analyzed for HDL cholesterol using a Synchron clinical device (CX4) (Beckman Coulter, Fullerton, Calif.).

Methods for Measuring Total Cholesterol, HDL Cholesterol, Triglyceride and Glucose Levels In another in vivo assay, obese monkeys are bled, followed by vehicle or test peptide for 4 weeks, and then bled again. Serum, total cholesterol, H
DL cholesterol, triglycerides and glucose are analyzed using a Syncron clinical device (CX4) (Beckman Coulter, Fullerton, Calif.). Lipoprotein subclass analysis is performed by NMR spectroscopy as described by Oliver et al. (Proc. Natl. Acad. Sci. USA 98: 5306-5311, 2001).

  All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described with reference to certain preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in biochemistry or related fields are intended to be within the scope of the following claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (51)

Formula (I)
Z1-A1-A2-A3-Gly-A5-Phe-Thr-A8-Asp-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-Phe-A23-A24- A25-A26-A27-A28-A29-A30-A31-A32-A33-A34-A35-A36-A37-A38-A39-A40-Z2 (SEQ ID NO: 1)
Where
A1 is His, Phe, Tyr, d-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N ^α -acetylhistidine, α-fluoromethyl-histidine, α-methyl-histidine , 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
A2 is Ser, Gly, Ala, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1 -Aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid or (1-aminocyclooctyl) carboxylic acid;
A3 is Glu, Gln or Ala;
A5 is Thr or Ala;
A8 is Ser or Ala;
A10 is Val, Leu, Tyr or Ala;
A11 is Ser, Ala, Arg, Gly, Lys or Thr;
A12 is Lys, Ser, Arg, Ala, Asn, His, or Gln;
A13 is Tyr or Gln;
A14 is Leu, Met or Ala;
A15 is Asp or Glu;
A16 is Gly, Glu, Ala, Ser, Phe, Trp, Thr, His, Lys, Arg, Val, Ile, Leu, Met, Asn, Gln or Tyr;
A17 is Gln, Glu, Arg, Ala, Lys, Leu, Met, Val, Phe, Ile, Trp, Asn, Asp, His, Ser, Thr, Gly or Tyr;
A18 is Ala, Arg, Lys, Phe, Ile, Leu, Tyr, Val, Met, Gly or His;
A19 is Ala or Val;
A20 is Lys, Arg, Ala, Gln or Cys;
A21 is Glu, Leu, Ala or Asp;
A23 is Ile or Val;
A24 is Ala, Glu, Lys, Gln, Asn or Lys-X;
A25 is Trp or Ala;
A26 is Leu or Ala;
A27 is Val, Lys, Ala or Met;
A28 is Lys, Asn, Arg, Ala, Gln or Cys;
A29 is Gly, Ala, Lys, Thr or Cys;
A30 is Arg, Gly, Ala, Cys or has been deleted;
A31 is Gly Pro, Lys, Cys, Lys-X or has been deleted;
A32 is Ser, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A33 is Ser, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A34 is Gly, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A35 is Ala, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A36 is Pro, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A37 is Pro, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A38 is Pro, Lys, Cys, Lys-X, Cys-PEG or has been deleted;
A39 is Ser, Lys, Cys, Lys-X, Cys-PEG or deleted; and A40 is Lys-X, Cys-PEG, or deleted;
Lys-X is Lys modified with fatty acid at N ^ε ,
Z1 is

Selected from
And
Z2 is

Selected from the
Peptides.   2. The peptide of claim 1 selected from SEQ ID NOs: 1-56.   The peptide according to claim 2, which is selected from SEQ ID NOs: 1-30.   The peptide according to claim 2, which is selected from SEQ ID NOs: 31-56.   The peptide according to claim 2, selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.   The peptide according to claim 2, selected from SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.   The peptide according to claim 2, which is selected from SEQ ID NOs: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45.   The peptide according to claim 2, selected from SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 56.   The peptide according to any one of claims 1 to 8, wherein the peptide is PEGylated.   The peptide according to any one of claims 1 to 8, wherein the peptide is PEGylated at the C-terminus. PEG

The peptide according to claim 9 or 10, which is selected from:   The peptide according to any one of claims 1 to 11, wherein the peptide is acetylated. Z1 is

The peptide according to any one of claims 1 to 8, which is selected from: Z1 is

The peptide according to any one of claims 1 to 8, which is selected from: PEG

The peptide according to claim 13 or 14, which is selected from:   A polynucleotide encoding the peptide of SEQ ID NO: 1-56 or a degenerate variant thereof.   A vector comprising the polynucleotide according to claim 16.   A host cell comprising the vector of claim 17. 19. A method for producing a peptide comprising a) culturing the host cell of claim 18 under conditions suitable for expression of the polypeptide; and b) recovering the peptide from the host cell culture.   A purified antibody that specifically binds to the peptide of any one of claims 1-15.   16. A pharmaceutical composition comprising an effective amount of the peptide of any one of claims 1-15 in combination with a pharmaceutically acceptable carrier.   A pharmaceutical preparation comprising a therapeutically effective amount of a peptide according to any one of claims 1 to 15 in combination with a pharmaceutically acceptable carrier and one or more pharmaceutical agents. Composition.   Pharmaceutical agents include PPAR ligands, insulin secretagogues, sulfonylurea drugs, α-glucosidase inhibitors, insulin sensitizers, hepatic glucose output lowering compounds, insulin and insulin derivatives, biguanides, protein tyrosine phosphatase 1B, dipeptidyl Peptidase IV, 11β-HSD inhibitor, anti-obesity agent, HMG-CoA reductase inhibitor, nicotinic acid, lipid lowering agent, ACAT inhibitor, bile acid scavenger, bile acid uptake inhibitor, microsomal triglyceride transport inhibitor, fibrin The group consisting of acid derivatives, β-blockers, ACE inhibitors, calcium antagonists, diuretics, renin inhibitors, AT-1 receptor antagonists, ET receptor antagonists, neutral endopeptidase inhibitors, vasopepsidase inhibitors and nitrates Selected from Is the pharmaceutical composition according to claim 22.   Administering to a subject in need thereof a therapeutically effective amount of a peptide according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 21, 22 or 23. A method for treating diabetes, comprising:   25. The method of claim 24, wherein the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, juvenile onset diabetes, adult latent diabetes and gestational diabetes.   Administering to a subject in need thereof a therapeutically effective amount of a peptide according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 21, 22 or 23. A method of treating syndrome X, comprising:   Administering to a subject in need thereof a therapeutically effective amount of a peptide according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 21, 22 or 23. A method of treating a diabetes-related disorder comprising.   28. The method of claim 27, wherein the diabetes related disorder is selected from the group consisting of hyperglycemia, hyperinsulinemia, impaired glucose tolerance, fasting hyperglycemia, dyslipidemia, hypertriglyceridemia, and insulin resistance. .   Administering to a subject in need thereof a therapeutically effective amount of a peptide according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 21, 22 or 23. A method of treating or preventing a secondary cause of diabetes, comprising:   30. The method of claim 29, wherein the secondary cause is selected from the group consisting of glucocorticoid excess, growth hormone excess, pheochromocytoma and drug-induced diabetes. A therapeutically effective amount of a combination of one or more pharmaceutical agents with one or more pharmaceutical agents.
A method for treating diabetes comprising the step of administering the peptide according to any one of 5 to a subject in need thereof.   The pharmaceutical agent comprises a PPAR agonist, a sulfonylurea drug, a non-sulfonylurea secretagogue, an α-glucosidase inhibitor, an insulin sensitizer, an insulin secretagogue, a hepatic glucose output lowering compound, insulin and an antiobesity agent 32. The method of claim 31, wherein the method is selected from the group.   33. The method of claim 32, wherein the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, juvenile onset diabetes, adult latent diabetes, and gestational diabetes.   Administering to a subject in need thereof a therapeutically effective amount of a peptide according to any one of claims 1-15 in combination with one or more pharmaceutical agents. A method for treating syndrome X.   The pharmaceutical agent comprises a PPAR agonist, a sulfonylurea drug, a non-sulfonylurea secretagogue, an α-glucosidase inhibitor, an insulin sensitizer, an insulin secretagogue, a hepatic glucose output lowering compound, insulin and an antiobesity agent 35. The method of claim 34, selected from the group.   Administering to a subject in need thereof a therapeutically effective amount of a peptide according to any one of claims 1-15 in combination with one or more pharmaceutical agents. A method for treating a diabetes-related disorder.   37. The method of claim 36, wherein the diabetes related disorder is selected from the group consisting of hyperglycemia, hyperinsulinemia, impaired glucose tolerance, fasting hyperglycemia, dyslipidemia, hypertriglyceridemia and insulin resistance. .   The pharmaceutical agent comprises a PPAR agonist, a sulfonylurea drug, a non-sulfonylurea secretagogue, an α-glucosidase inhibitor, an insulin sensitizer, an insulin secretagogue, a hepatic glucose output lowering compound, insulin and an antiobesity agent 38. The method of claim 37, selected from the group.   Administering to a subject in need thereof a therapeutically effective amount of a peptide according to any one of claims 1-15 in combination with one or more pharmaceutical agents. A method for treating or preventing a secondary cause of diabetes.   The pharmaceutical agent comprises a PPAR agonist, a sulfonylurea drug, a non-sulfonylurea secretagogue, an α-glucosidase inhibitor, an insulin sensitizer, an insulin secretagogue, a hepatic glucose output lowering compound, insulin and an antiobesity agent 40. The method of claim 39, selected from the group.   HMG-CoA reductase inhibitor, nicotinic acid, lipid lowering drug, ACAT inhibitor, bile acid scavenger, bile acid uptake inhibitor, microsomal triglyceride transport inhibitor, fibric acid derivative, β-blocker, ACE inhibitor, calcium Combination with one or more agents selected from the group consisting of antagonists, diuretics, renin inhibitors, AT-1 receptor antagonists, ET receptor antagonists, neutral endopeptidase inhibitors, vasopepsidase inhibitors and nitrates Administering a therapeutically effective amount of the peptide of any one of claims 1 to 15 to a subject in need thereof, diabetes, syndrome X, diabetes-related disorder, or diabetes To treat secondary causes of   42. The method of claim 41, wherein the diabetes related disorder is selected from the group consisting of hyperglycemia, hyperinsulinemia, impaired glucose tolerance, fasting hyperglycemia, dyslipidemia, hypertriglyceridemia, and insulin resistance. .   43. The peptide of any one of claims 31 to 42, wherein the peptide of any one of claims 1 to 15 and one or more pharmaceutical agents are administered as a single pharmaceutical dosage formulation. The method described in 1.   Administering to a subject in need thereof a therapeutically effective amount of a peptide according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 21, 22 or 23. A method of treating a cardiovascular disease, comprising:   45. The method of claim 44, wherein the cardiovascular disease is selected from atherosclerosis, coronary heart disease, coronary artery disease and hypertension.   Administering to a subject in need thereof a therapeutically effective amount of a peptide according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 21, 22 or 23. A method of treating obesity comprising.   Insulin secretion in the subject by administering to the subject in need thereof a peptide according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 21, 22 or 23. Stimulation method.   16. A peptide according to any one of claims 1 to 15 for the treatment and / or prevention of diabetes and diabetes related disorders.   16. A medicament comprising at least one peptide according to any one of claims 1 to 15 in combination with at least one pharmaceutically acceptable pharmaceutically safe carrier or excipient.   Use of a peptide according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment and / or prevention of diabetes and diabetes related disorders.   50. A pharmaceutical product according to claim 49 for the treatment and / or prevention of diabetes.