WO2006121904A1 - Glucose-dependent insulinotropic polypeptide (gip) receptor agonists and their pharmacological methods of use - Google Patents

Glucose-dependent insulinotropic polypeptide (gip) receptor agonists and their pharmacological methods of use Download PDF

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Publication number
WO2006121904A1
WO2006121904A1 PCT/US2006/017494 US2006017494W WO2006121904A1 WO 2006121904 A1 WO2006121904 A1 WO 2006121904A1 US 2006017494 W US2006017494 W US 2006017494W WO 2006121904 A1 WO2006121904 A1 WO 2006121904A1
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lys
peptide
cys
diabetes
deleted
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PCT/US2006/017494
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French (fr)
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Kevin Clairmont
Kevin Lumb
James Whelan
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Bayer Pharmaceuticals Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel modifications that provide suitable derivatization sites to improve the pharmacokinetic properties of the peptides.
  • Such N-terminal modifications at the mainchain amino group of the first amino acid residue or C-terminal modifications at the mainchain carboxylate group of the last amino acid residue may include aliphatics, C3 to C7 cycloalkyl, aryl, or mono- or bi-cyclic heteroaromatics containing one or more nitrogen, oxygen, and/or sulfur heteroatoms.
  • the N-terminal or C-terminal modifications may provide suitable derivatization sites (exemplified, but not limited to, amino and thiol groups).
  • modified peptides of the present invention are useful in stimulating the release of insulin from pancreatic ⁇ -cells in a glucose-dependent manner, thereby providing a treatment option for those individuals afflicted with metabolic disorders such as diabetes or impaired glucose tolerance, a prediabetic state.
  • Diabetes is characterized by impaired glucose metabolism manifesting itself, among other things, by an elevated blood glucose level in the diabetic patient. Underlying defects lead to a classification of diabetes into two major groups: type 1 diabetes, or insulin dependent diabetes mellitus (IDDM), which arises when patients lack ⁇ -cells producing insulin in their pancreatic islets of Langerhans; and type 2 diabetes, or non-insulin dependent diabetes mellitus (NIDDM), which occurs in patients with an impaired ⁇ -cell function and alterations in insulin action.
  • IDDM insulin dependent diabetes mellitus
  • NIDDM non-insulin dependent diabetes mellitus
  • Type 1 diabetic patients are currently treated with insulin, while the majority of type 2 diabetic patients are treated with agents that stimulate ⁇ -cell function or with agents that enhance the tissue sensitivity of the patients towards insulin. Over time almost one-half of type 2 diabetic subjects lose their response to these agents and then must be placed on insulin therapy.
  • the drugs presently used to treat type 2 diabetes are described below.
  • Alpha-glucosidase inhibitors e.g., Precose®, VogliboseTM, and Miglitol®
  • Precose® e.g., Precose®, VogliboseTM, and Miglitol®
  • Insulin sensitizers are drugs that enhance the body's response to insulin.
  • Thiozolidinediones such as AvandiaTM (rosiglitazone) and ActosTM (pioglitazone) activate the peroxisome proliferator-activated receptor (PPAR) gamma subtype and modulate the activity of a set of genes that have not been well described.
  • RezulinTM troglitazone
  • AvandiaTM and ActosTM seem to be associated with fluid retention and edema. Another potential side effect is weight gain. AvandiaTM is not indicated for use with insulin because of concern about congestive heart failure.
  • Insulin secretagogues e.g., sulfonylureas (SFUs) and other agents that act by the ATP- dependent K+ channel
  • SFUs are standard therapy for type 2 diabetics that have mild to moderate fasting hyperglycemia.
  • the SFUs have limitations that include a potential for inducing hypoglycemia, weight gain, and high primary and secondary failure rates. Ten to 20% of initially treated patients fail to show a significant treatment effect (primary failure). Secondary failure is demonstrated by an additional 20-30% loss of treatment effect after six months on an SFU. Insulin treatment is required in 50% of the SFU responders after 5-7 years of therapy (Scheen, et al., Diabetes Res. Clin. Pract. 6:533-543, 1989).
  • GlucophageTM (metformin HCI) is a biguanide that lowers blood glucose by decreasing hepatic glucose output and increasing peripheral glucose uptake and utilization. The drug is effective at lowering blood glucose in mildly and moderately affected subjects and does not have the side effects of weight gain or the potential to induce hypoglycemia.
  • GlucophageTM has a number of side effects including gastrointestinal disturbances and the potential for lactic acidosis. GlucophageTM is contraindicated in diabetics over the age of 70 and in subjects with impairment in renal or liver function.
  • GlucophageTM has primary and secondary failure rates similar to the SFUs.
  • Insulin treatment is instituted after diet, exercise, and oral medications have failed to adequately control blood glucose. This treatment has the drawbacks that it is an injectable, that it can produce hypoglycemia, and that it causes weight gain.
  • new therapies to treat type 2 diabetes are needed.
  • new treatments to retain normal (glucose-dependent) insulin secretion are needed.
  • Such new drugs should have the following characteristics: dependent on glucose for promoting insulin secretion (i.e., produce insulin secretion only in the presence of elevated blood glucose); low primary and secondary failure rates; and preservation of islet cell function.
  • the strategy to develop the new therapy disclosed herein is based on the cyclic adenosine monophosphate (cAMP) signaling mechanism and its effects on insulin secretion.
  • cAMP cyclic adenosine monophosphate
  • Cyclic AMP is a major regulator of the insulin secretion process. Elevation of this signaling molecule promotes the closure of the K+ channels following the activation of protein kinase A pathway. Closure of the K+ channels causes cell depolarization and subsequent opening of Ca ++ channels, which in turn leads to exocytosis of insulin granules. Little if any effects on insulin secretion occurs in the absence of low glucose concentrations (Weinhaus, et al., Diabetes 47:1426-1435, 1998).
  • Secretagogues like PACAP pituitary adenylate cyclase activating peptide
  • VIP vasoactive intestinal peptide
  • GIP glucose-dependent insulinotropic polypeptide
  • GLP-1 glucagon-like peptide 1
  • Insulin secretagogues working through the elevation of cAMP such as GLP-1 , VIP, GIP, and PACAP are also able to enhance insulin synthesis in addition to insulin release (Skoglund, et al., Diabetes 49:1156-1164, 2000; Borboni, et al., Endocrinology 140:5530-5537, 1999).
  • GLP-1 is released from the intestinal L-cell after a meal and functions as an incretin hormone (i.e., it potentiates glucose-induced insulin release from the pancreatic ⁇ -cell). It is a 37- amino acid peptide that is differentially expressed by the glucagon gene, depending upon tissue type. The clinical data that support the beneficial effect of raising cAMP levels in ⁇ -cells have been collected with GLP-1. Infusions of GLP-1 in poorly controlled type 2 diabetics normalized their fasting blood glucose levels (Gutniak, et al., New Eng. J. Med.
  • Amyli ⁇ Pharmaceuticals is conducting Phase III trials with Exendin-4 (AC2993), a 39 amino acid peptide originally identified in GiIa Monster. Amylin has reported that clinical studies demonstrated improved glycemic control in type 2 diabetic patients treated with Exendin-4. However, the incidence of nausea and vomiting was significant.
  • PACAP is a potent stimulator of glucose-dependent insulin secretion from pancreatic ⁇ - cells.
  • Three different PACAP receptor types (PAC1, VPAC1 , and VPAC2) have been described (Harmar, et al., Pharmacol. Reviews 50:265-270, 1998; Vaudry, et al., Pharmacol. Reviews 52:269-324, 2000).
  • PACAP displays no receptor selectivity, having comparable activities and potencies at all three receptors.
  • PAC1 is located predominately in the CNS, whereas VPAC1 and VPAC2 are more widely distributed.
  • VPAC1 is located in the CNS as well as in liver, lungs, and intestine.
  • VPAC2 is located in the CNS, pancreas, skeletal muscle, heart, kidney, adipose tissue, testis, and stomach. Recent work argues that VPAC2 is responsible for the insulin secretion from ⁇ -cells (Inagaki, et al., Proc. Natl. Acad. Sci. USA 91:2679-2683, 1994; Tsutsumi, et al., Diabetes 51:1453-1460, 2002). This insulinotropic action of PACAP is mediated by the GTP binding protein Gs. Accumulation of intracellular cAMP in turn activates the nonselective cation channels in ⁇ - cells increasing [Ca++], and promotes exocytosis of insulin-containing secretory granules.
  • Glucose-dependent insulinotropic polypeptide previously known as gastric inhibitory polypeptide (GIP) is a 42-residue gut peptide secreted by enteroendocrine K cells that is involved in the regulation of fat and glucose metabolism, with the insulinotropic function localized to residues 1-30. GIP is degraded by DPPIV proteolysis and eliminated by renal clearance, resulting in short-lived efficacy in vivo that limits the therapeutic value of the unmodified peptide. Limited clinical data have been collected with GIP. Intravenous (IV) administration or continuous in type 2 diabetics caused an acute increase in plasma insulin levels (Kindmark, et al., J. Clin. Endocrinol. Metab. 86:2015-2019, 2001 ; Meier, et al., Diabetes 53 (Suppl 3):S220-S224, 2004). These effects, however, are short-lived because of the short half-life of the peptide.
  • the incretin GIP is a 42-residue peptide involved in the regulation of fat and glucose metabolism (Kieffer, Ann. Endocrinol. 65:13-21 , 2004; Meier and Nauck, Horm. Metab. Res. 36:859-66, 2004; Vilsboll and Hoist, Diabetologia 47:357-66, 2004).
  • GIP not only enhances insulin secretion acutely, but stimulates insulin production through enhancement of proinsulin transcription and translation (Fehmann and Goke, Peptides 16:1149-1152, 1995; Wang, et al., MoI. Cell. Endocrinol.
  • GIP insulin target tissues directly to lower plasma glucose: enhancement of glucose uptake in adipose (Eckel, et al., Diabetes 28:1141-1142, 1979) and muscle (O'Harte, et al., J. Endocrinol.
  • GIP or GIP variants hold promise as a peptide therapeutic for conditions such as diabetes and obesity (Gault, et al., Neuropeptides 37:253-63, 2003; Green, et al., Curr. Pharm. Des. 10:3651-62, 2004).
  • modified GIP peptides improve glucose tolerance in rodents (Green, et al., Curr. Pharm. Des. 10:3651-62, 2004; Meier and Nauck, Horm. Metab. Res.
  • Vasoactive intestinal peptide is a 28 amino acid peptide that was first isolated from hog upper small intestine (Said and Mutt, Science 169:1217-1218, 1970; U.S. Patent No. 3,879,371). This peptide belongs to a family of structurally-related, small polypeptides that includes helodermin, secretin, the somatostatins, and glucagon. The biological effects of VIP are mediated by the 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 displays comparable activities and potencies at VPAC1 and VPAC2.
  • Modifications at the N-terminal tyrosine or at alanine 2 maintain functional activity, while modifications reported at positions 3 and 13 have reduced with biological effectiveness of the peptides to generate cyclic AMP and to enhance insulin secretion.
  • modified GIP molecules may act as functional antagonists.
  • the present invention provides novel modifications that provide suitable derivatization sites to improve the pharmacokinetic properties of the peptides.
  • Such N-terminal modifications at the amino group of the first peptide residue may include aliphatics, C3 to C7 cycloalkyl, aryl, or mono- or bi-cyclic heteroaromatics containing one or more nitrogen, oxygen, and/or sulfur heteroatoms.
  • the N-terminal modifications may provide suitable derivatization sites (exemplified, but not limited to, amino and thiol groups).
  • N-terminal modifications include, but are not limited to, 2-amino benzoic acid, 3-amino benzoic acid, 4-amino benzoic 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-aminocyclohexane carboxylic acid, D-pipecolinic acid, 4-amino-1-methyl-1 H-imidazole-2-carboxylic acid, 4-methythiobenzoic acid, 2- methythiobenzoic acid, 2-methythionicotinic acid, proline, 6-aminohexanoic acid, benzoic acid, (S)- tetrahydroisoquinoline acetic acid, indoline-2-carboxylic acid, cis-3-aminocyclohexane carboxylic acid,
  • the invention relates to a peptide of Formula (I)
  • A1 is any naturally occurring amino acid, any d-amino acid, or deleted;
  • A2 is any naturally occurring amino acid, any d-amino acid, or deleted;
  • A3 is any naturally occurring amino acid, any d-amino acid, or deleted;
  • A7 is lle or Thr
  • A10 is VaI or Tyr
  • A12 is lie, Ala, or Ser
  • A13 is Ala, lie, or VaI
  • A14 is Leu or Met
  • A15 is Asp, GIu, Asn, or Lys
  • A16 is Lys or GIy
  • A17 is lie or GIn
  • A18 is His, Ala, or Arg
  • A19 is GIn, Ala, or GIu;
  • A20 is GIn, Lys, or GIu;
  • A21 is GIu, Asp, or Ala
  • A23 is He or VaI
  • A24 is Ala, Asn, or Asp
  • A26 is Leu or Ala
  • A28 is Ala or Ser
  • A29 is GIn, VaI, or GIu;
  • A30 is Lys, Arg, Asp, GIu, Asn, GIn, or Cys;
  • A31 is GIy, Lys, Cys, GIn, Lys-X, Cys-PEG, or deleted;
  • A32 is Lys, Cys, GIy, Lys-X, Cys-PEG or deleted;
  • A33 is Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A34 is Asn, Ser, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A35 is Asp, Asn, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A36 is Trp, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A37 is Lys, lie, Cys, Lys-X, Cys-PEG or deleted;
  • A38 is His, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A39 is As
  • Lys-X is Lys modified at N ⁇ with a fatty acid exemplified by CH 3 (CH 2 ) n COOH where n ranges from 0 to about 24.
  • Z2 may be a hydroxyl group such that the peptide has an unmodified carboxylate C- terminus or Z2 may be a modification of the C-terminal carboxylate group. Z2 may be a modification such as amidation, or Z2 may also be an unnatural amino acid or amide thereof. Z2 is exemplified by, but not limited to,
  • the invention relates to a peptide of Formula (II)
  • A3 is GIu, d-Glu, Lys, Tyr, Pro, Trp, Phe, Asp, or deleted;
  • A7 is lie or Thr
  • A10 is VaI or Tyr
  • A12 is lie, Ala, or Ser
  • A13 is Ala, lie, or VaI
  • A14 is Leu or Met
  • A15 is Asp, Asn, Lys, or GIu;
  • A16 is Lys or GIy
  • A17 is lie or GIn
  • A18 is His, Ala, or Arg
  • A19 is GIn, GIu, or Ala
  • A20 is GIn, GIu, or Lys
  • A21 is GIu, Ala, or Asp
  • A23 is lie or VaI
  • A24 is Ala, Asp, or Asn
  • A26 is Leu or Ala
  • A31 is GIy, GIn, Lys, Cys, Lys-X, Cys-PEG, or deleted;
  • A32 is GIy, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A33 is Lys, Cys, Lys-X, Cys-PEG or deleted
  • A34 is Asn, Ser, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A35 is Asp, Asn, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A36 is Trp, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A37 is Lys, He, Cys, Lys-X, Cys-PEG or deleted;
  • A38 is His, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A39 is Asn, Asp, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A40 is lie, Leu, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A41 is Thr, Ser, lie, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A42 is GIn, GIu, Lys, Cys, Lys-X, Cys-PEG or deleted;
  • A43 is Lys-X, Cys-PEG or deleted. [026] The definitions for Z1 , Z2, and Lys-X are as defined above for Formula (I).
  • the N-terminal modifications are attached via an amide bond to the alpha-amino group of the first amino acid of said peptide.
  • the C-terminal modifications are attached via an amide bond to the main chain carboxylate group of the last amino acid of said peptide.
  • Examples of peptides of Formula (I) or Formula (II) may be found in, but are not limited to, the peptides described in Table 1 (e.g., SEQ ID NO: 1-82).
  • Derivatives of the present invention may include peptides that have been fused with another compound, such as a compound to increase the half-life of the peptide and/or to reduce potential immunogenicity of the peptide (e.g., polyethylene glycol, "PEG").
  • PEG polyethylene glycol
  • PEGylated peptides typically have greater half-life in vivo (Greenwald, Adv. Drug. Del. Rev. 55:217-250, 2003).
  • the fusion of the peptide to PEG may be accomplished by any means known to one skilled in the art.
  • PEGylation may be accomplished by first introducing a cysteine mutation into the peptide to provide a linker upon which to attach the PEG, followed by site-specific derivatization with PEG-maleimide.
  • the N-terminal modification may incorporate a reactive moiety for coupling to PEG, as exemplified by the amine group, the mercapto group, or the carboxylate group of the N-terminal modifying compounds disclosed above.
  • PEGylation may be accomplished by first introducing a mercapto moiety into the peptide via the N-terminal modifying group to provide a linker upon which to attach the PEG, followed by site-specific derivatization with methoxy-PEG-maleimide reagents supplied by, for example, either Nektar Therapeutics (San Carlos, CA, USA) and/or NOF (Tokyo, Japan).
  • methoxy-PEG-maleimide reagents supplied by, for example, either Nektar Therapeutics (San Carlos, CA, USA) and/or NOF (Tokyo, Japan).
  • Nektar Therapeutics Ses, CA, USA
  • NOF Tokyo, Japan
  • numerous Cys reactive groups are known to those skilled in the art of protein cross-linking, such as the use of alkyl halides and vinyl sulfones (see, e.g., Proteins, Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H.
  • the PEG could be introduced by direct attachment to the C-terminal carboxylate group, or to an internal amino acid such as Cys, Lys, Asp, or GIu or to unnatural amino acids that contain similar reactive sidechain moieties.
  • PEG polymers can be used, as exemplified but not limited to, PEG polymers of from about 5 kDa to about 43 kDa.
  • the PEG modification may include a single, linear PEG.
  • linear 5, 20, or 30 kDa PEGs that are attached to maleidmide or other cross-linking groups are available from Nektar and/or NOF (see, e.g., Table 2).
  • the modification may involve branched PEGs that contain two or more PEG polymer chains that are attached to maleimide or other cross-linking groups are available from Nektar and NOF (see, e.g., Table 2).
  • the commercially available thiol-reactive 40 kDa PEG (mPEG2-MAL) from Nektar (Huntsville, Al) employs a maleimide group for conjugation to Cys, and the maleimide group is attached to the PEG via a linker that contains a Lys (see, e.g., Table 2).
  • the commercially available thiol-reactive 43 kDa PEG (GL2-400MA) from NOF employs a maleimide group for conjugation to Cys, and the maleimide group is attached to the PEG via a bi-substituted alkane linker (see, e.g., Table 2).
  • the PEG polymer can be attached directly to the maleimide, as exemplified by PEG reagents of molecular weight 5 and 20 kDa available form Nektar Therapeutics (Huntsville, Al) (see, e.g., Table 2).
  • the present invention exemplifies, but is not limited to, the use of a mercapto group as a cross-linking site.
  • a mercapto group As a cross-linking site.
  • other moieties present in amino acids such as the amino group of the N-terminal modifying compound, the C-terminal carboxylate of either an unmodified C-terminus or a peptide modified with Z2, and the side chains of amino acids such as Lys, Arg, Asp, and GIu provide reactive groups that provide moieties suitable for covalent modification and attachment to PEG.
  • suitable cross-linking agents are known to those skilled in the art (see, e.g., Proteins, Structure and Molecular Properties, 2nd ed., T. E. Creighton, W.H.
  • cross-linking agents can be linked to PEG as exemplified by, but not limited to, commercially available PEG derivatives containing amines, aldehydes, acetals, maleimide, succinimides, and thiols that are marketed, for example, by Nektar and NOF (e.g., Harris, et al., Clin. Pharmokinet. 40:539-551, 2001).
  • Nektar and NOF e.g., Harris, et al., Clin. Pharmokinet. 40:539-551, 2001.
  • the peptides of the present invention may be modified with fatty acids that improve pharmacodynamic properties.
  • the amine containing N-terminal modifying compounds can be derivatized with palmitate or myristolate or other fatty acids using methods known to those skilled in the art or an alkyl (e.g., C 6 -Ci 8 ) moiety can be included directly as part of the N-terminal modifying compound.
  • the peptides of the present invention have improved stability to proteolysis by DPPIV and in plasma as compared to PACAP or VIP. While both VIP and PACAP27 have been reported to be resistant to cleavage by DPPIV (Zhu, et al., J. Biol. Chem 278: 22418-22423, 2003), the inventors have demonstrated that these peptides are cleaved at longer time points.
  • the derivatives of the present invention demonstrate an extended duration of action in vivo, supporting a dosing interval of less than once per day or once per week or greater, when derivatized.
  • the peptides of the present invention provide a new therapy for patients with, for example, metabolic disorders such as those resulting from decreased endogenous insulin secretion, in particular type 2 diabetics, or for patients with impaired glucose tolerance, a prediabetic state that has a mild alteration in insulin secretion.
  • the peptides of the present invention may be useful in the prevention and/or treatment of type 1 diabetes, gestational diabetes, maturity-onset diabetes of the young (MODY), latent autoimmune diabetes adult (LADA), and associated diabetic dyslipidemia and other diabetic complications, as well as hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, Syndrome X, and insulin resistance.
  • the peptides of the present invention may also be utilized in the prevention and/or treatment of obesity (e.g., regulation of appetite and food intake); disorders of energy homeostasis; disorders of lipid and carbohydrate metabolism; cardiovascular disease, including atherosclerosis, coronary heart disease, coronary artery disease, hyperlipidemia, hypercholesteremia, low HDL levels and hypertension; cerebrovascular disease and peripheral vessel disease; polycystic ovary syndrome; carcinogenesis, and hyperplasia; ulcers; growth disorders, and other conditions identified herein, or function otherwise as described later herein.
  • One aspect of the invention is a peptide of Formula (I) and Formula (II), and fragments, derivatives, and variants thereof that demonstrate at least one biological function that is substantially the same as the peptides of Formula (I) and Formula (II) (collectively, “peptides of this invention"), including functional equivalents thereof (e.g., Table 1).
  • Antibodies and antibody fragments that selectively bind the peptides of this invention are also provided. Such antibodies are useful in detecting the peptides of this invention, and can be identified and made by procedures well known in the art.
  • a polyclonal N-terminal IgG antibody and a monoclonal C-terminal Fab antibody have been generated which recognize peptides of this invention.
  • the invention is also directed to a method of treating diabetes, diabetes-related disorders, and/or other diseases or conditions affected by the peptides of this invention, for example, effected by the GIP agonist function of the peptides of this invention, in a mammal, comprising administering a therapeutically effective amount of any of the peptides of the present invention or any peptide active at GIP such as a peptide of Formula (I) and Formula (II) to said mammal (e.g., Table 1).
  • This invention provides novel modified peptides, and fragments, derivatives, and variants thereof that demonstrate at least one biological function that is substantially the same as the peptides of Formula (I) and Formula (II) (peptides of this invention).
  • the peptides of this invention e.g., Table 1
  • the peptides of the present invention may also be utilized in the prevention and/or treatment of obesity (e.g., regulation of appetite and food intake); disorders of energy homeostasis; disorders of lipid and carbohydrate metabolism; cardiovascular disease, including atherosclerosis, coronary heart disease, coronary artery disease, hyperlipidemia, hypercholesteremia, low HDL levels and hypertension; cerebrovascular disease and peripheral vessel disease; polycystic ovary syndrome; carcinogenesis, and hyperplasia; ulcers; growth disorders, and other conditions identified herein, or function otherwise as described later herein.
  • the peptides of this invention (e.g., Table 1) will stimulate insulin release from pancreatic ⁇ -cells in a glucose-dependent fashion. Furthermore, the peptides of this invention are stable in both aqueous and non-aqueous formulations and exhibit a plasma half-life of greater than one hour, for example, demonstrating a plasma half-life greater than 6 hours.
  • the peptides of this invention are GIP agonists.
  • these peptides may possess activities at other related receptors including, but not limited to, VPAC2 receptor agonism, GLP receptor agonism, or glucagon receptor antagonism.
  • the peptides of this invention stimulate insulin release into plasma in a glucose-dependent fashion without inducing a stasis or increase in the level of plasma glucose that is counterproductive to the treatment of, for example, type 2 diabetes.
  • the peptides of this invention may be selective agonists of the GIP receptor, thereby causing, for example, an increase in insulin release into plasma, while being selective against other receptors that are responsible for such disagreeable or dangerous side effects as gastrointestinal water retention, and/or unwanted cardiovascular effects such as increased heart rate or blood pressure.
  • compositions and formulations of the present invention may comprise peptides of the present invention and about 2% to about 30% DMSO.
  • compositions and formulations may optionally include about 0.2% to about 3% (w/v) of additional solvents such as propylene glycol, dimethyl formamide, propylene carbonate, polyethylene glycol, and triglycerides.
  • additional solvents such as propylene glycol, dimethyl formamide, propylene carbonate, polyethylene glycol, and triglycerides.
  • the derivatized peptides of this invention may exhibit a plasma half-life of, for example, at least 1 hour in rats after IV injection, at least 3 hours, or at least 6 hours. Furthermore, the derivatized peptide may demonstrate a significant lowering of the plasma glucose AUC following subcutaneous injection in rats, for example, at least 24 hours, at least 41 hours, or at least 65 hours following injection.
  • the peptides of this invention provide a new therapy for patients with decreased endogenous insulin secretion or impaired glucose tolerance, in particular, type 2 diabetes. That is, the peptides of the present invention are long-acting GIP receptor agonists that may be used to maintain, improve, and restore glucose-stimulated insulin secretion. Furthermore, a selective peptide agonist of the GIP receptor will enhance glucose-dependent insulin secretion in the pancreas without causing the side effects associated with non-selective activation of the other related receptors.
  • “Functional equivalent” and “substantially the same biological function or activity” each means that degree of biological activity that is within about 30% to about 100% or more of that biological activity demonstrated by the peptide to which it is being compared when the biological activity of each peptide is determined by the same procedure.
  • Bioactivity means an effector function that is directly or indirectly performed by a peptide (whether in its native or denatured conformation), or by any fragments, derivatives, and variants thereof.
  • Biological activities include, for example, binding to peptides, binding to other proteins or molecules, activity as a DNA binding protein, as a transcription regulator, ability to bind damaged DNA, etc.
  • fragment when referring to the peptides of the present invention, means fragments, derivatives, and variants of the peptides which retain substantially the same biological function or activity as such peptides, as described further below.
  • a fragment is a portion of the peptide which retains substantially similar functional activity, for example, as described in the in vivo models disclosed herein.
  • a derivative includes all modifications to the peptide which substantially preserve the functions disclosed herein and include additional structure and attendant function (e.g., modified N-terminus peptides, modified C-terminus peptides, or PEGylated peptides), fusion peptides which confer targeting specificity or an additional activity such as toxicity to an intended target, as described further below.
  • additional structure and attendant function e.g., modified N-terminus peptides, modified C-terminus peptides, or PEGylated peptides
  • fusion peptides which confer targeting specificity or an additional activity such as toxicity to an intended target, as described further below.
  • the peptides of the present invention may be synthetic peptides.
  • the fragment, derivative, or variant of the peptides of the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature peptide is fused with another compound, such as a compound to increase the half-life of the peptide (e.g., polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature peptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature peptide, or (v) one in which the peptide sequence is fused with a larger peptide (e.g., human albumin, an antibody or Fc, for increased duration of effect).
  • a larger peptide e.g., human albumin, an
  • the derivatives of the present invention may contain conservative amino acid substitutions (defined further below) made at one or more nonessential amino acid residues.
  • a "nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an "essential” amino acid residue is required for biological activity.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • Fragments, or biologically active portions include peptide fragments suitable for use as a medicament, to generate antibodies, as a research reagent, and the like. Fragments include peptides comprising amino acid sequences sufficiently similar to or derived from the amino acid sequences of a peptide of this invention and exhibiting at least one activity of that peptide, but which include fewer amino acids than the full-length peptides disclosed herein.
  • biologically active portions comprise a domain or motif with at least one activity of the peptide.
  • a biologically active portion of a peptide can be a peptide which is, for example, five or more amino acids in length. Such biologically active portions can be prepared synthetically or by recombinant techniques and can be evaluated for one or more of the functional activities of a peptide of this invention by means disclosed herein and/or well known in the art.
  • Variants of the peptides of this invention include peptides having an amino acid sequence sufficiently similar to the amino acid sequence of the peptides of this invention or a domain thereof.
  • the term "sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity.
  • amino acid sequences that contain a common structural domain that is at least about 45%, about 75% through 98%, identical are defined herein as sufficiently similar.
  • Variants will be sufficiently similar to the amino acid sequence of the peptides of this invention. Such variants generally retain the functional activity of the peptides of this invention.
  • Variants include peptides that differ in amino acid sequence due to mutagenesis. Variants that function as GIP receptor agonists may be identified by screening combinatorial libraries of mutants, for example truncation mutants, of the peptides of this invention for GIP receptor agonist activity.
  • the invention also provides chimeric or fusion peptides.
  • the targeting sequence is designed to localize the delivery of the peptide to minimize potential side effects.
  • the peptides of this invention may be composed of amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres), and may contain amino acids other than the 20 gene- encoded amino acids.
  • the peptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • Modifications may occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. 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 may contain many types of modifications. Peptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic peptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross- linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, e.g., Protein
  • the peptides of the present invention include the peptides of Formula (I) and Formula (II) (e.g., Table 1), as well as those sequences having insubstantial variations in sequence from them.
  • An "insubstantial variation” would include any sequence addition, substitution, or deletion variant that maintains substantially at least one biological function of the peptides of this invention, for example, GIP receptor agonist activity, and/or enhancement of insulin secretion or lowering of blood glucose demonstrated herein.
  • These functional equivalents may include peptides which have at least about 90% identity to the peptides of the present invention, at least 95% identity to the peptides of the present invention, and at least 99% identity to the peptides of the present invention, and also include portions of such peptides having substantially the same biological activity.
  • any peptide having insubstantial variation in amino acid sequence from the peptides of the present invention that demonstrates functional equivalency as described further herein is included in the description of the present invention.
  • the peptides of this invention may be a product of chemical synthetic procedures.
  • the peptides of this invention may be conveniently isolated by methods that are well known in the art. Purity of the preparations may also be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis and mass spectroscopy and liquid chromatography.
  • peptide mimetic peptide mimetic
  • peptidomimetic peptidomimetic
  • organomimetic organic mimetic
  • chemical mimetic are intended to encompass peptide derivatives, peptide analogs, and chemical compounds having an arrangement of atoms in a three-dimensional orientation that is equivalent to that of a peptide of the present invention.
  • the phrase "equivalent to” as used herein is intended to encompass peptides having substitution(s) of certain atoms, or chemical moieties in said peptide, having bond lengths, bond angles, and arrangements in the mimetic peptide that produce the same or sufficiently similar arrangement or orientation of said atoms and moieties to have the biological function of the peptides of the invention.
  • the three- dimensional arrangement of the chemical constituents may be structurally and/or functionally equivalent to the three-dimensional arrangement of the peptide backbone and component amino acid sidechains in the peptide, resulting in such peptide-, organo-, and chemical mimetics of the peptides of the invention having substantial biological activity.
  • a pharmacophore exists for the biological activity of each peptide of the invention.
  • a pharmacophore is understood in the art as comprising an idealized, three- dimensional definition of the structural requirements for biological activity.
  • Peptido-, organo-, and chemical mimetics may be designed to fit each pharmacophore with current computer modeling software (computer aided drug design). Said mimetics may be produced by structure-function analysis, based on the positional information from the substituent atoms in the peptides of the invention.
  • Peptides as provided by the invention may be advantageously synthesized by any of the chemical synthesis techniques known in the art, particularly solid-phase synthesis techniques, for example, using commercially-available automated peptide synthesizers.
  • the mimetics of the present invention may be synthesized by solid phase or solution phase methods conventionally used for the synthesis of peptides (see, e.g., Merrifield, J. Amer. Chem. Soc. 85:2149-54, 1963; Carpino, Ace. Chem. Res. 6:191-98, 1973; Birr, Aspects of the Merrifield Peptide Synthesis, Springer- Verlag: Heidelberg, 1978; The Peptides: Analysis, Synthesis, Biology, VoIs.
  • Peptides of the present invention may be prepared by solid phase methodology. Briefly, an N-protected C-terminal amino acid residue is linked to an insoluble support such as divinylbenzene cross-linked polystyrene, polyacrylamide resin, Kieselguhr/polyamide (pepsyn K), controlled pore glass, cellulose, polypropylene membranes, acrylic acid-coated polyethylene rods, or the like. Cycles of deprotection, neutralization, and coupling of successive protected amino acid derivatives are used to link the amino acids from the C-terminus according to the amino acid sequence. For some synthetic peptides, an FMOC strategy using an acid-sensitive resin may be used.
  • an insoluble support such as divinylbenzene cross-linked polystyrene, polyacrylamide resin, Kieselguhr/polyamide (pepsyn K), controlled pore glass, cellulose, polypropylene membranes, acrylic acid-coated polyethylene rods, or the like. Cycles of deprotection, neutralization, and coupling of
  • Solid supports in this regard may be divinylbenzene cross-linked polystyrene resins, which are commercially available in a variety of functionalized forms, including chloromethyl resin, hydroxymethyl resin, paraacetamidomethyl resin, benzhydrylamine (BHA) resin, A- methylbenzhydrylamine (MBHA) resin, oxime resins, 4-alkoxybenzyl alcohol resin (Wang resin), 4- (2',4'-dimethoxyphenylaminomethyl)-phenoxymethyl resin, 2,4-dimethoxybenzhydryl-amine resin, and 4-(2',4'-dimethoxyphenyl-FMOC-amino-methyl)-phenoxyacetamidonorleucyl-MBHA resin (Rink amide MBHA resin).
  • acid-sensitive resins also provide C-terminal acids, if desired.
  • a protecting group for alpha amino acids is base-labile 9-fluorenylmethoxy-carbonyl (FMOC).
  • Suitable protecting groups for the side chain functionalities of amino acids chemically compatible with BOC (t-butyloxycarbonyl) and FMOC groups are well known in the art.
  • FMOC chemistry the following protected amino acid derivatives may be utilized: FMOC- Cys(Trt), FMOC-Ser(But), FMOC-Asn(Trt), FMOC-Leu, FMOC-Th r(Trt), FMOC-VaI, FMOC-GIy, 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).
  • the amino acid residues may be coupled by using a variety of coupling agents and chemistries known in the art, such as direct coupling with DIC (diisopropyl-carbodiimide), DCC (dicyclohexylcarbodiimide), BOP (benzotriazolyl-N-oxytrisdimethylaminophosphonium hexa- fluorophosphate), PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluoro- phosphate), PyBrOP (bromo-tris-pyrrolidinophosphonium hexafluorophosphate); via performed symmetrical anhydrides; via active esters such as pentafluorophenyl esters; or via performed HOBt (1-hydroxybenzotriazole) active esters or by using FMOC-amino acid fluoride and chlorides or by using FMOC-amino acid-N-carboxy anhydrides.
  • activation may be performed with HBTU (2-(1H-benzotriazole-1-yl),1 ,1 ,3 ! 3-tetramethyluronium hexafluorophosphate) or HATU (2-(1H-7-aza-benzotriazole-1-yl),1 ,1 ,3,3-tetramethyluronium hexafluoro-phosphate) in the presence of HOBt or HOAt (7-azahydroxybenztriazole).
  • the solid phase method may be carried out manually, or by automated synthesis on a commercially available peptide synthesizer (e.g., Applied Biosystems 431 A or the like; Applied Biosystems, Foster City, CA).
  • the first (C-terminal) amino acid is loaded on the chlorotrityl resin.
  • Successive deprotection with 20% piperidine/NMP (N-methylpyrrolidone)
  • coupling cycles according to ABI FastMoc protocols (Applied Biosystems) may be used to generate the peptide sequence.
  • Double and triple coupling, with capping by acetic anhydride, may also be used.
  • the synthetic mimetic peptide may be cleaved from the resin and deprotected by treatment with TFA (trifluoroacetic acid) containing appropriate scavengers.
  • TFA trifluoroacetic acid
  • Many such cleavage reagents 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, may be used.
  • Reagent K 0.75 g crystalline phenol, 0.25 ml_ ethanedithiol, 0.5 ml_ thioanisole, 0.5 mL deionized water, 10 ml_ TFA
  • the peptide is separated from the resin by filtration and isolated by ether precipitation. Further purification may be achieved by conventional methods, such as gel filtration and reverse phase HPLC (high performance liquid chromatography).
  • Synthetic mimetics according to the present invention may be in the form of pharmaceutically acceptable salts, especially base-addition salts including salts of organic bases and inorganic bases.
  • the base-addition salts of the acidic amino acid residues are prepared by treatment of the peptide with the appropriate base or inorganic base, according to procedures well known to those skilled in the art, or the desired salt may be obtained directly by lyophilization of the appropriate base.
  • peptides as described herein may be modified by a variety of chemical techniques to produce peptides having essentially the same activity as the unmodified peptide, and optionally having other desirable properties.
  • carboxylic acid groups of the peptide may be provided in the form of a salt of a pharmaceutically- acceptable cation.
  • Amino groups within the peptide may be in the form of a pharmaceutically- acceptable acid addition salt, such as the HCI, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric, and other organic salts, or may be converted to an amide.
  • Thiols may be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • protecting groups such as acetamide groups.
  • Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this invention so that the native binding configuration will be more nearly approximated.
  • a carboxyl terminal or amino terminal cysteine residue may be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, thereby generating a cyclic peptide.
  • Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino- terminal amides and esters.
  • peptide derivatives and analogs with the same or similar desired biological activity as the corresponding peptide but with more favorable activity than the peptide with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis.
  • Such derivatives and analogs include peptides modified at the N- terminal amino group, as exemplified by, but not limited to, the peptides of Formula (I) and Formula (II) (e.g., Table 1), the C-terminal amide group, and/or changing one or more of the amido linkages in the peptide to a non-amido linkage.
  • two or more such modifications may be coupled in one peptide mimetic structure (e.g., modification at the G-terminal amide group and inclusion of a -CH 2 - carbamate linkage between two amino acids in the peptide).
  • Such peptide mimetics may have significant advantages over peptide embodiments, including, for example, more economical to produce, having greater chemical stability or enhanced pharmacological properties (such as half-life, absorption, potency, efficacy, etc.), reduced antigenicity, and other properties.
  • Mimetic analogs of the peptides of the invention may also be obtained using the principles of conventional or rational drug design (see, e.g., Andrews, et al., Proc. Alfred Benzon Symp. 28:145-165, 1990; McPherson, Eur. J. Biochem. 189:1-24, 1990; HoI, et al., in Molecular Recognition: Chemical and Biochemical Problems, (Roberts, ed.); Royal Society of Chemistry; pp. 84-93, 1989a; HoI, Arzneim-Forsch. 39:1016-1018, 1989b; HoI, Agnew Chem. Int. Ed. Engl. 25:767-778, 1986; the disclosures of which are herein incorporated by reference).
  • the desired mimetic molecules may be obtained by randomly testing molecules whose structures have an attribute in common with the structure of a "native" peptide.
  • the quantitative contribution that results from a change in a particular group of a binding molecule may be determined by measuring the biological activity of the putative mimetic in comparison with the activity of the peptide.
  • the mimetic is designed to share an attribute of the most stable three- dimensional conformation of the peptide.
  • the mimetic may be designed to possess chemical groups that are oriented in a way sufficient to cause ionic, hydrophobic, or van der Waals interactions that are similar to those exhibited by the peptides of the invention, as disclosed herein.
  • One method for performing rational mimetic design employs a computer system capable of forming a representation of the three-dimensional structure of the peptide, such as those exemplified by HoI, 1989a; HoI, 1989b; and HoI, 1986.
  • Molecular structures of the peptido-, organo-, and chemical mimetics of the peptides of the invention may be produced using computer- assisted design programs commercially available in the art.
  • Examples of such programs include SYBYL 6.5®, HQSARTM, and ALCHEMY 2000TM (Tripos); GALAXYTM and AM2000TM (AM Technologies, Inc., San Antonio, TX); CATALYSTTM and CERIUSTM (Molecular Simulations, Inc., San Diego, CA); CACHE PRODUCTSTM, TSARTM, AMBERTM, and CHEM-XTM (Oxford Molecular Products, Oxford, CA) and CHEMBUILDER3DTM (Interactive Simulations, Inc., San Diego, CA).
  • the peptido-, organo-, and chemical mimetics produced using the peptides disclosed herein using, for example, art-recognized molecular modeling programs may be produced using conventional chemical synthetic techniques, for example, designed to accommodate high throughput screening, including combinatorial chemistry methods.
  • Combinatorial methods useful in the production of the peptido-, organo-, and chemical mimetics of the invention include phage display arrays, solid-phase synthesis, and combinatorial chemistry arrays, as provided, for example, by SIDDCO (Tuscon, Arizona); Tripos, Inc.; Calbiochem/Novabiochem (San Diego, CA); Symyx Technologies, Inc. (Santa Clara, CA); Medichem Research, Inc.
  • Combinatorial chemistry production of the peptido-, organo-, and chemical mimetics of the invention may be produced according to methods known in the art, including, but not limited to, techniques disclosed in Terrett, (Combinatorial Chemistry, Oxford University Press, London, 1998); Gallop, et al., J. Med. Chem. 37:1233-51, 1994; Gordon, et al., J. Med. Chem. 37:1385-1401, 1994; Look, et al., Bioorg. Med. Chem. Lett. 6:707-12, 1996; Ruhland, et al., J.
  • the newly synthesized peptides may be substantially purified by preparative high performance liquid chromatography (see, e.g., Creighton, Proteins: Structures And Molecular Principles, WH Freeman and Co., New York, N.Y., 1983).
  • the composition of a synthetic peptide of the present invention may be confirmed by amino acid analysis or sequencing by, for example, the Edman degradation procedure (Creighton, supra). Additionally, any portion of the amino acid sequence of the peptide may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant peptide or a fusion peptide.
  • antibodies and antibody fragments that selectively bind the peptides of this invention. Any type of antibody known in the art may be generated using methods well known in the art. For example, an antibody may be generated to bind specifically to an epitope of a peptide of this invention.
  • "Antibody” as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of a peptide of this invention.
  • Fab fragment antigen binding fragments of a peptide of this invention.
  • Fv fragment antigen binding fragments of a peptide of this invention.
  • at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope.
  • epitopes which involve non-contiguous amino acids may require more amino acids, for example, at least 15, 25, or 50 amino acids.
  • An antibody which specifically binds to an epitope of a peptide of this invention may be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays may be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.
  • an antibody which specifically binds to a peptide of this invention provides a detection signal higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • Antibodies which specifically bind to a peptide of this invention do not detect other proteins in immunochemical assays and can immunoprecipitate a peptide of this invention from solution.
  • Peptides of this invention may be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, goat, sheep, monkey, or human, to produce polyclonal antibodies.
  • a peptide of this invention may be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants may be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • mineral gels e.g., aluminum hydroxide
  • surface active substances e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies which specifically bind to a peptide of this invention may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma technique (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., MoI. Cell Biol. 62:109-20, 1984).
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, may be used (Morrison, et al., 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 also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues.
  • rodent antibodies and human sequences may be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grafting of entire complementarity determining regions.
  • humanized antibodies may be produced using recombinant methods (see, e.g., GB2188638B).
  • Antibodies which specifically bind to a peptide of this invention may contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Patent No. 5,565,332.
  • single chain antibodies may be adapted using methods known in the art to produce single chain antibodies which specifically bind to a peptide of this invention.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88:11120-23, 1991).
  • Single-chain antibodies also may be constructed using a DNA amplification method, 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. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison (Nat. Biotechnol. 15:159-63, 1997). Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss (J. Biol. Chem. 269:199-206, 1994).
  • a nucleotide sequence encoding a single-chain antibody may be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar, et al., Int. J. Cancer 61 :497-501 , 1995; Nicholls, et al., J. Immunol. Meth. 165:81-91 , 1993).
  • Antibodies which specifically bind to a peptide of this invention may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, et al., Proc. Natl. Acad. Sci. 86:38333-37, 1989; Winter, et al., Nature 349:293-99, 1991).
  • chimeric antibodies may be constructed as disclosed in WO 93/03151.
  • Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" also can be prepared (see, e.g., WO 94/13804,).
  • Human antibodies with the ability to bind to the peptides of this invention may also be identified from the MorphoSys HuCAL ® library as follows.
  • a peptide of this invention may be coated on a microtiter plate and incubated with the MorphoSys HuCAL ® Fab phage library.
  • Those phage-linked Fabs not binding to the peptide of this invention can be washed away from the plate, leaving only phage which tightly bind to the peptide of this invention.
  • the bound phage can be eluted, for example, by a change in pH or by elution with E. coli and amplified by infection of E. coli hosts. This panning process can be repeated once or twice to enrich for a population of antibodies that tightly bind to the peptide of this invention.
  • the Fabs from the enriched pool are then expressed, purified, and screened in an ELISA assay.
  • Antibodies according to the invention may be purified by methods well known in the art. For example, antibodies may be affinity purified by passage over a column to which a peptide of this invention is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • subject includes mammals (e.g., humans and animals).
  • treatment includes any process, action, application, therapy, or the like, wherein a subject, including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject.
  • combination therapy means the administration of two or more therapeutic agents to treat, for example, diabetes.
  • administration encompasses coadministration of two or more therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each inhibitor agent.
  • administration encompasses use of each type of therapeutic agent in a sequential manner.
  • terapéuticaally effective means the amount of each agent administered that will achieve the goal of improvement in a diabetic condition or disorder severity, while avoiding or minimizing adverse side effects associated with the given therapeutic treatment.
  • pharmaceutically acceptable means that the subject item is appropriate for use in a pharmaceutical product.
  • an embodiment of this invention includes a method of treating the various conditions in a patient (including mammals) which comprises administering to said patient a composition containing an amount of the peptide of Formula (I) and Formula (II), that is effective in treating the target condition.
  • the peptides of the present invention may be employed in treatment diabetes, including both type 1 and type 2 diabetes (non-insulin dependent diabetes mellitus). Such treatment may also delay the onset of diabetes and diabetic complications.
  • treatment diabetes including both type 1 and type 2 diabetes (non-insulin dependent diabetes mellitus).
  • non-insulin dependent diabetes mellitus non-insulin dependent diabetes mellitus
  • the peptides may be used to prevent subjects with impaired glucose tolerance from proceeding to develop type 2 diabetes.
  • Other diseases and conditions that may be treated or prevented using peptides of the invention in methods of the invention include: Maturity-Onset Diabetes of the Young (MODY) (Herman, et al., Diabetes 43:40, 1994); Latent Autoimmune Diabetes Adult (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):S5, 1999); impaired fasting glucose (IFG) (Charles, et al., Diabetes 40:796, 1991); gestational diabetes (Metzger, Diabetes, 40:197, 1991); and metabolic syndrome X.
  • MODY Maturity-Onset Diabetes of the Young
  • LADA Latent Autoimmune Diabetes Adult
  • IGT impaired glucose tolerance
  • IGF impaired fasting glucose
  • the peptides of the present invention may also be utilized in the prevention and/or treatment of obesity (e.g., regulation of appetite and food intake); disorders of energy homeostasis; disorders of lipid and carbohydrate metabolism; cardiovascular disease, including atherosclerosis, coronary heart disease, coronary artery disease, hyperlipidemia, hypercholesteremia, low HDL levels and hypertension; cerebrovascular disease and peripheral vessel disease; polycystic ovary syndrome; carcinogenesis, and hyperplasia; ulcers; growth disorders, and other conditions identified herein, or function otherwise as described later herein.
  • obesity e.g., regulation of appetite and food intake
  • disorders of energy homeostasis e.g., obesity, regulation of appetite and food intake
  • disorders of energy homeostasis e.g., obesity and food intake
  • disorders of energy homeostasis e.g., obesity, regulation of appetite and food intake
  • disorders of energy homeostasis e.g., obesity, regulation of appetite and food intake
  • the peptides of the present invention may also be useful for treating physiological disorders related to, for example, cell differentiation to produce lipid accumulating cells, regulation of insulin sensitivity and blood glucose levels, which are involved in, for example, abnormal pancreatic ⁇ -cell function, carcinogenesis, hyperplasia, reduction in the pancreatic ⁇ -cell mass, insulin secretion, tissue sensitivity to insulin, cell growth, polycystic ovarian disease, chronic anovulation, hyperandrogenism, progesterone production, steroidogenesis, plasma triglycerides, HDL, and LDL cholesterol levels, and the like.
  • Peptides of the invention may also be used in methods of the invention to treat secondary causes of diabetes (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1):S5, 1999).
  • Such secondary causes include glucocorticoid excess, growth hormone excess, pheochromocytoma, and drug-induced diabetes.
  • Drugs that may induce diabetes include, but are not limited to, pyriminil, nicotinic acid, glucocorticoids, phenytoin, thyroid hormone, ⁇ - adrenergic agents, ⁇ -interferon and drugs used to treat HIV infection.
  • the peptides of the present invention may be used alone or in combination with additional therapies and/or compounds known to those skilled in the art in the treatment of diabetes and related disorders. Alternatively, the methods and peptides described herein may be used, partially or completely, in combination therapy.
  • the peptides of the invention may also be administered in combination with other known therapies for the treatment of diabetes, including PPAR ligands (e.g., agonists, antagonists), insulin secretagogues, for example, sulfonylurea drugs and non-sulfonylurea secretagogues, ⁇ - glucosidase inhibitors, insulin sensitizers, hepatic glucose output lowering compounds, insulin and insulin derivatives, and anti-obesity drugs.
  • Such therapies may be administered prior to, concurrently with, or following administration of the peptides of the invention.
  • Insulin and insulin derivatives include both long and short acting forms and formulations of insulin.
  • PPAR ligands may include agonists and/or antagonists of any of the PPAR receptors or combinations thereof.
  • PPAR ligands may include ligands of PPAR- ⁇ , PPAR- ⁇ , PPAR- ⁇ or any combination of two or three of the receptors of PPAR.
  • PPAR ligands include, for example, rosiglitazone, troglitazone, and pioglitazone.
  • Sulfonylurea drugs include, for example, glyburide, glimepiride, chlorpropamide, tolbutamide, and glipizide, - ⁇ -glucosidase inhibitors that may be useful in treating diabetes when administered with a peptide of the invention include acarbose, miglitol, and voglibose.
  • PPAR- ⁇ agonists such as the glitazones (e.g., troglitazone, pioglitazone, englitazone, MCC-555, rosiglitazone, and the like) and other thiazolidinedione and non-thiazolidinedione compounds
  • biguanides such as metform
  • Hepatic glucose output lowering compounds that may be useful in treating diabetes when administered with a peptide of the invention include, for example, glucagon anatgonists and metformin, such as Glucophage and Glucophage XR.
  • Insulin secretagogues that may be useful in treating diabetes when administered with a peptide of the invention include sulfonylurea and non-sulfonylurea drugs: GLP-1 , VIP, PACAP, secretin, and derivatives thereof; nateglinide, meglitinide, repaglinide, glibenclamide, glimepiride, chlorpropamide, and glipizide.
  • GLP-1 includes derivatives of GLP-1 with longer half-lives than native GLP-1 , such as, for example, fatty-acid derivatized GLP-1 and exendin.
  • anti-obesity drugs include ⁇ -3 adrenergic receptor agonists such as CL 316,243; cannabinoid (e.g., CB-1) antagonists such as Rimonabant; neuropeptide-Y receptor antagonists; neuropeptide Y5 inhibitors; apo-B/MTP inhibitors; 11 ⁇ -hydroxy steroid dehydrogenase-1 inhibitors; peptide YY 3-36 or analogs thereof; MCR4 agonists; CCK-A agonists; monoamine reuptake inhibitors; sympathomimetic agents; dopamine agonists; melanocyte- stimulating hormone receptor analogs; melanin concentrating hormone antagonists; leptin; leptin analogs; leptin receptor agonists; galanin antagonists; lipase inhibitors; bombesin agonists; thyromimetic agents; dehydro
  • Peptides of the invention may also be used in methods of the invention in combination with drugs commonly used to treat lipid disorders.
  • drugs include, but are not limited to, HMG-CoA reductase inhibitors, nicotinic acid, fatty acid lowering compounds (e.g., acipimox); lipid lowering drugs (e.g., stanol esters, sterol glycosides such as tiqueside, and azetidinones such as ezetimibe), ACAT inhibitors (such as avasimibe), bile acid sequestrants, bile acid reuptake inhibitors, microsomal triglyceride transport inhibitors, and fibric acid derivatives.
  • HMG-CoA reductase inhibitors e.g., nicotinic acid, fatty acid lowering compounds (e.g., acipimox); lipid lowering drugs (e.g., stanol esters, sterol glycosides such as tiqueside
  • 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.
  • Sequestrants include,. for example, cholestyramine, colestipol, and dialkylaminoalkyl derivatives of a cross-linked dextran.
  • peptides of the invention may also be administered in combination with antihypertensive drugs, such as, for example, ⁇ -blockers and ACE inhibitors.
  • antihypertensive drugs such as, for example, ⁇ -blockers and ACE inhibitors.
  • additional anti-hypertensive agents for use in combination with the peptides of the present invention include calcium channel blockers (L-type and T-type; e.g., diltiazem, verapamil, nifedipine, amlodipine and mybefradil), diuretics (e.g., chlorothiazide, hydrochlorothiazide, flumethiazide, hydroflumethiazide, bendroflumethiazide, methylchlorothiazide, trichloromethiazide, polythiazide, benzthiazide, ethacrynic acid tricrynafen, chlorthalidone, furosemide, mus
  • ET receptor antagonists e.g., sitaxsentan, atrsentan, neutral endopeptidase (NEP) inhibitors, vasopepsidase inhibitors (dual NEP-ACE inhibitors) (e.g., omapatrilat and gemopatrilat), and nitrates.
  • Such co-therapies may be administered in any combination of two or more drugs ⁇ e.g., a peptide of the invention in combination with an insulin sensitizer and an anti-obesity drug).
  • Such co-therapies may be administered in the form of pharmaceutical compositions, as described above.
  • the effective dosage of the peptides of this invention can readily be determined for treatment of each desired indication.
  • the amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular peptide and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.
  • the total amount of the active ingredient to be administered may generally range from, for example, 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 may contain from, for example, about 0.05 mg to about 1500 mg of active ingredient, and may be administered one or more times per day.
  • the daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous, and parenteral injections, and use of infusion techniques may be from, for example, about 0.001 to about 200 mg/kg.
  • the daily rectal dosage regimen may be from, for example, about 0.001 to about 200 mg/kg of total body weight.
  • the transdermal concentration may be that required to maintain a daily dose of from, for example, about 0.001 to about 200 mg/kg.
  • the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific peptide employed, the age of the patient, the diet of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like.
  • the desired mode of treatment and number of doses of a peptide of the present invention may be ascertained by those skilled in the art using conventional treatment tests.
  • the peptides of this invention may be utilized to achieve the desired pharmacological effect by administration to a subject in need thereof in an appropriately formulated pharmaceutical composition.
  • a subject for example, may be a mammal, including a human, in need of treatment for a particular condition or disease. Therefore, the present invention includes pharmaceutical compositions which are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a peptide of the present invention.
  • a pharmaceutically acceptable carrier is any carrier which is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient.
  • a pharmaceutically effective amount of a peptide is that amount which produces a result or exerts an influence on the particular condition being treated.
  • the peptides of the present invention may be administered with a pharmaceutically-acceptable carrier using any effective conventional dosage unit forms, including, for example, immediate and timed release preparations, orally, parenterally, topically, or the like.
  • the peptides may be formulated into solid or liquid preparations such as, for example, capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions, and may be prepared according to methods known to the art for the manufacture of pharmaceutical compositions.
  • the solid unit dosage forms may be a capsule which can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.
  • the peptides of this invention may be tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin; disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum; lubricants intended to improve the flow of tablet granulation and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example, talc, stearic acid, or magnesium, calcium or zinc stearate; dyes; coloring agents; and flavoring agents intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient.
  • conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin
  • disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch
  • Suitable excipients for use in oral liquid dosage forms include diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • Various 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 preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, those sweetening, flavoring and coloring agents described above, may also be present.
  • the pharmaceutical compositions of this invention may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil such as liquid paraffin or a mixture of vegetable oils.
  • Suitable emulsifying agents may be (1) naturally occurring gums such as gum acacia and gum tragacanth, (2) naturally occurring phosphatides such as soy bean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and (4) condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents such as, for example, glycerol, propylene glycol, sorbitol, or sucrose. Such formulations may also contain a demulcent, and preservative, flavoring and coloring agents.
  • sweetening agents such as, for example, glycerol, propylene glycol, sorbitol, or sucrose.
  • Such formulations may also contain a demulcent, and preservative, flavoring and coloring agents.
  • the peptides of this invention may also be administered parenterally, that is, subcutaneously, intravenously, intramuscularly, or interperitoneally, as injectable dosages of the peptide in a physiologically acceptable diluent with a pharmaceutical carrier which may be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related sugar solutions; an alcohol such as ethanol, isopropanol, or hexadecyl alcohol; glycols such as propylene glycol or polyethylene glycol; glycerol ketals such as 2,2-dimethyl-1,1-dioxo!ane-4-methanol, ethers such as poly(ethyleneglycol) 400; an oil; a fatty acid; a fatty acid ester or glyceride; or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending
  • oils which can be used in the parenteral formulations of this invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil.
  • 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 include cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates; anionic detergents, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; nonionic detergents, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers; and amphoteric detergents, for example, alkyl- beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, as well as mixtures.
  • suitable detergents include cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl
  • compositions of this 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 non-ionic surfactant having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulation 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 the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • the pharmaceutical compositions may be in the form of sterile injectable aqueous suspensions.
  • Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents which may be a naturally occurring phosphatide such as lecithin, a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate, a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol, a condensation product of ethylene oxide with a partial ester derived form a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product of an ethylene oxide with a partial ester derived from
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
  • Diluents and solvents that may be employed are, for example, water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile fixed oils are conventionally employed as solvents or suspending media.
  • any bland, fixed oil may be employed including synthetic mono or diglycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • a composition of the invention may also be administered in the form of suppositories for rectal administration of the drug.
  • These compositions may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are, for example, cocoa butter and polyethylene glycol.
  • transdermal delivery devices Such transdermal patches may be used to provide continuous or discontinuous infusion of the peptides of the present invention in controlled amounts.
  • the construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art (see, e.g., U.S. Patent No! 5,023,252, incorporated herein by reference).
  • patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
  • Another formulation employs the use of biodegradable microspheres that allow controlled, sustained release of the peptides and PEGylated peptides of this invention.
  • Such formulations can be comprised of synthetic polymers or copolymers.
  • Such formulations allow for injection, inhalation, nasal or oral administration.
  • the construction and use of biodegradable microspheres for the delivery of pharmaceutical agents is well known in the art (e.g., US Patent No. 6, 706,289, incorporated herein by reference).
  • compositions of the invention may also contain other conventional pharmaceutically acceptable compounding ingredients, generally referred to as carriers or diluents, as necessary or desired. Any of the compositions of this invention may be preserved by the addition of an antioxidant such as ascorbic acid or by other suitable preservatives. Conventional procedures for preparing such compositions in appropriate dosage forms can be utilized.
  • compositions for its intended route of administration include: acidifying agents, for example, but are not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid; and alkalinizing agents such as, but are not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine.
  • acidifying agents for example, but are not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid
  • alkalinizing agents such as, but are not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine.
  • the peptides identified by the methods described herein may be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutical agents where the combination causes no unacceptable adverse effects.
  • the peptides of this invention can be combined with known anti-diabetic, or with known anti-obesity, cardiovascular or other indication agents, and the like, as well as with admixtures and combinations thereof.
  • the peptides identified by the methods described herein may also be utilized, in free base form or in compositions, in research and diagnostics, or as analytical reference standards, and the like. Therefore, the present invention includes compositions which are comprised of an inert carrier and an effective amount of a peptide of the present invention.
  • An inert carrier is any material which does not interact with the peptide to be carried and which lends support, means of conveyance, bulk, traceable material, and the like to the peptide to be carried.
  • An effective amount of peptide is that amount which produces a result or exerts an influence on the particular procedure being performed.
  • Formulations suitable for subcutaneous, intravenous, intramuscular, and the like; suitable pharmaceutical carriers; and techniques for formulation and administration may be prepared by any of the methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20 th edition, 2000)
  • Peptides are known to undergo hydrolysis, deamidation, oxidation, racemization and isomerization in aqueous and non-aqueous environment.
  • Degradation such as hydrolysis, deamidation or oxidation can readily detected by capillary electrophoresis.
  • Enzymatic degradation notwithstanding, peptides having a prolonged plasma half-life, or biological resident time, should, at minimum, be stable in aqueous solution.
  • a peptide exhibits less than 10% degradation over a period of one day at body temperature or less than 5% degradation over a period of one day at body temperature.
  • Stability i.e., less than a few percent of degradation
  • Stability in the magnitude of years at refrigeration temperature will allow the manufacturer to present a liquid formulation, thus avoid the inconvenience of reconstitution.
  • stability in organic solvent would provide peptide be formulated into novel dosage forms such as implant.
  • Electron impact mass spectra (EI-MS or GC-MS) were obtained with a Hewlett Packard 5989A mass spectrometer equipped with a Hewlett Packard 5890 Gas Chromatograph with a J & W DB-5 column (0.25 uM coating; 30 m x 0.25 mm). The ion source was maintained at 250 0 C and spectra were scanned from 50-800 amu at 2 sec per scan.
  • High pressure liquid chromatography- electrospray mass spectra were obtained using a Hewlett-Packard 1100 HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (2 x 23 mm, 120A), and a Finnigan LCQ ion trap mass spectrometer with electrospray ionization. Spectra were scanned from 120-1200 amu using a variable ion time according to the number of ions in the source. The eluents were A: 2% acetonitrile in water with 0.02% TFA and B: 2% water in acetonitrile with 0.018% TFA.
  • Ethyl 2- ⁇ [2-(tritylthio)ethyl]amino ⁇ 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 0 C for 4 h. 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 extracts were dried over MgSO 4 and concentrated in vacuo, yielding 55.0 mg (27%) of the desired product.
  • Triphenylmethylmercaptan (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 min. 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 min and was concentrated in vacuo. The crude residue was dissolved in ethyl acetate and washed with water and brine.
  • Ethyl N-(2,2-dimethoxyethyl)glycinate hydrochloride (370 mg, 1.63 mmol) was dissolved in EtOH (2 mL) and treated with a solution of potassium thiocyanate (237 mg, 2.44 mmol) in EtOH (8 mL). The pink suspension was heated to reflux overnight. Concentrated HCI (0.136 mL, 1.63 mmol) was added, and the solution was allowed to reflux for 3 h. The reaction mixture was concentrated in vacuo, and the resulting solid was recrystalized from EtOAc, yielding 130 mg (43%) of the desired product.
  • Peptides are synthesized with an Applied Biosystems 430A peptide synthesizer using FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied Biosystems protocols are used. The peptides are cleaved with 84.6% TFA, 4.4% phenol, 4.4% water, 4.4% thioanisol, and 2.2% ethanedithiol. Peptides are precipitated from the cleavage cocktail using cold tertbutylmethyl ether. The precipitate is washed with the cold ether and dried under argon. Peptides are purified with by reversed phase C 18 HPLC with linear water/acetonitrile gradients containing 0.1% TFA. Peptide identity is confirmed with MALDI and electrospray mass spectrometry and with amino acid analysis. Example 3. Methods for Adding N-Terminal Modifying Compound
  • N-terminal modifying compounds are synthesized with an Applied Biosystems 430A peptide synthesizer using FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied Biosystems protocols are used.
  • the N-terminal modifying compounds are coupled to the peptide as per a 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 amine and mercapto containing N-terminal modifying compounds, the amine and mercapto groups are protected with FMOC or trityl, respectively, during coupling to the peptide.
  • the peptides are cleaved with 84.6% TFA, 4.4% phenol, 4.4% water, 4.4% thioanisol, and 2.2% ethanedithiol.
  • Peptides are precipitated from the cleavage cocktail using cold tertbutylmethyl ether. The precipitate is washed with the cold ether and dried under argon.
  • Peptides are purified with by reversed phase C18 HPLC with linear water/acetonitrile gradients containing 0.1% TFA. Peptide identity is confirmed with MALDI and electrospray mass spectrometry and with amino acid analysis.
  • Peptides are synthesized with an Applied Biosystems 433A peptide synthesizer using FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied Biosystems protocols are used.
  • the HBTU-activated C-terminal modifying compounds are coupled to the resin (e.g., Wang resin for producing peptides with a C-terminal modifying compound containing the free carboxylate or Rink Amide for producing amide variants) as per a natural amino acid coupling during FMOC chemistry.
  • the peptides are then synthesized by the stepwise addition of amino acids using standard FMOC protocols.
  • the peptides are cleaved with 84.6% TFA 1 4.4% phenol, 4.4% water, 4.4% thioanisol, and 2.2% ethanedithiol.
  • Peptides are precipitated from the cleavage cocktail using cold tertbutylmethyl ether. The precipitate is washed with the cold ether and dried under argon.
  • Peptides are purified with by reversed phase C18 HPLC with linear water/acetonitrile gradients containing 0.1% TFA. Peptide identity is confirmed with MALDI and electrospray mass spectrometry and with amino acid analysis.
  • PEG derivatives are prepared by incubating methoxypolyethlene glycols derivatized with maledimide for coupling to the mercapto moiety of the N-terminal modifying group.
  • mPEG-MAL or mPEG2-MAL products supplied by Nektar Therapeutics (Huntsville, Al, USA) or GLE-200MA or GLE-400MA products supplied by NOF (Toyko, Japan) are used.
  • Coupling reactions are performed by incubating the peptide and a two-fold molar excess of maleimide-PEG in 50 mM Tris, pH 7 at rt for 2-12 h.
  • the peptide concentration may be 1 mg/ml or less.
  • the fatty acid (palmitate) derivatives of amine containing N-terminal modifying compounds are prepared as N-terminal modified peptides as described in Example 3 except that prior to deprotection and cleavage the FMOC protecting group of the amine moiety of the N-terminal modifying group was selectively removed with 0.1 % TFA and derivativized with palmitic acid using the same conditions as for a normal amino acid coupling.
  • the fatty acid derivative can also be prepared as described in Example 3 using 1- hexadecyl-1 H-benzoimidazol-2-ylsulfanyl)acetic acid as the N-terminal modifying group, which was synthesized as described in Example 1.
  • a sterile IV injectable formulation is prepared with 4 mg of a peptide of Formula (I) or Formula (II), or a derivatized polypetide having equivalent of 4 mg peptide content, and 1 L sterile saline, using any manufacturing process well known in the art. Higher concentrations of peptide may be used for SC formulation.
  • 4 mg is dissolved in 100 mL saline or DMSO and sterile vials after aseptic filtration, are filled with the composition.
  • Peptide samples are supplied for Edman degradation at 1 nmol/10 ⁇ l in 10 mM HEPES, pH 7.4, 5% TFA.
  • HEPES buffer salt Prior to Edman analysis, the HEPES buffer salt is removed by using an Applied Biosystems ProSorb sample cartridge as per manufacturers instructions. Briefly, the sample is applied to a PVDF membrane and washed with 0.1% TFA, then the membrane is removed and inserted into the protein sequencer for Edman degradation. Edman degradation is carried out on an Applied Biosystems Procise 494HT protein sequencing system using the pulsed-liquid method according to manufacturer instructions. Sequences are read manually.
  • Example 4 The formulations described in Example 4 are placed in constant stability chamber. Peptides are also analyzed for stability to degradation in solutions of DPPIV and in plasma. Samples are removed periodically for analysis by capillary electrophoresis, mass spectrometry, Edman degradation, ELISA, and assays of peptide activity, which are sensitive methods to detect degradation of peptide. The area of various peaks is summed and the area for peak of the parent peptide is divided by the total peak area. The quotient is the % purity. Since there are impurities present in the fresh peptide, the purity change is normalized by dividing the purity at different time point by the initial purity.
  • peptides at 20 pmol/ ⁇ l were incubated at 37 0 C in the presence of 300 pM DPPIV in 100 mM HEPES, pH 7.4. At various timepoints, the reaction (2 ⁇ l aliquot) is terminated by addition of 1 ⁇ M DPPIV inhibitor and freezing.
  • T 0 hr, 1 hr, 5 hr, and 24 hr timepoints are evaluated. The results are plotted as percent intact peptide or peptide derivative as compared to degradation products.
  • CHO cells transfected with GIP receptor are Dounce homogenized in 20 mM Tris-HCI, pH 7.4, 5 mM MgCI 2 , 3 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (Sigma A8456), and 1 ⁇ g/ml aprotinin (Sigma A1153).
  • the homogenate is centrifuged at 20k rpm for 20 min at 4°C.
  • the pellet is suspended in 20 mM Tris-HCI, pH 7.4, 1 mM EDTA and 1 ⁇ g/ml aprotinin. An aliquot is removed for protein concentration determination by the Bradford assay (BioRad, 500-0006).
  • the suspension is brought to a final concentration of 3-10 mg protein/ml and stored in aliquots at -80 0 C.
  • GF/C filtration 96-well plates (Millipore MAFCNOB) are pretreated with 0.1 ml/well 0.3% polyethyleneamine (Sigma P3143) for 1-2 hours at rt.
  • the plates are washed (2 x 0.1 ml) with ice- cold 20 mM Tris-HCI, pH 7.4, 1 mM EDTA, 0.1% (w/v) BSA (Sigma 8806) and 0.1 % (w/v) bacitracin (Sigma 5150).
  • binding buffer (20 mM Tris-HCI, pH 7.4, 1 mM EDTA, 0.5% BSA and 0.1% bacitracin) containing 15-25 ⁇ g CHO membrane protein and peptide.
  • 125 l-labeled GIP (Amersham IM303, 2000 Ci/mmol) is added to a final concentration of 50 pM and the plates incubated for 2 h at rt. The bound from free 125 l-labeled GIP is separated with a Milllipore vacuum apparatus. The plates are washed (3 x 0.2 ml) with ice cold PBS containing 0.1% (w/v) BSA. The plates are dried at rt, 0.030 ml Ultima gold (Packard 6013329) is added, and radioactive content isdetermined with a Wallac Microbeta counter.
  • CHO cells transfected with the GIP receptor are plated on clear bottom white 96-well plates and washed (2 x 0.2 ml) with phosphate buffered saline (PBS).
  • Assay buffer (90 ⁇ l: 10 mM HEPES, pH 7.4,150 mM NaCI, 5 mM KCI, 2.5 mM CaCI 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 25 mM NaHCO 3 , 0.1 mM 3-isobutyl-1-methylxanthine, and 1%(w/v) BSA) are added to each well followed by 10 ⁇ l peptide (0.1-100 nM final).
  • the plates are incubated for 15 min at 37°C under 5% CO 2 . Buffer is removed, the cells lysed, and assayed for cAMP content using an SPA kit (Amersham RPA559). Radioactivity is determined on a Wallac Microbeta counter. Data are fit to a single-site binding model with Prism 3.03 (GraphPad Software, San Diego, California, USA).
  • Insulin secretion of dispersed rat islets mediated by a number of peptides of the present invention is measured as follows. Islets of Langerhans, isolated from SD rats (200-250 g), are digested using collagenase. The dispersed islet cells are treated with trypsin, seeded into 96 V- bottom plates, and pelleted. The cells are then cultured overnight in media with or without peptides of this invention. The media is aspirated, and the cells are pre-incubated with Krebs- Ringer-HEPES buffer containing 3 mM glucose for 30 minutes at 37°C.
  • the pre-incubation buffer is removed, and the cells are incubated at 37°C with Krebs-Ringer-HEPES buffer containing the appropriate glucose concentration (e.g., 8 mM) with or without peptides for an appropriate time. A portion of the supernatant is removed and its insulin content is measured by SPA. The results are expressed as "fold over control” (FOC).
  • FOC fold over control
  • Polyclonal antibodies specific to the peptides of the present invention are generated by synthesizing a specific fragment of a peptide of this invention using an ABI 433A peptide synthesizer. The peptide is then cleaved from the resin, and purified on a Beckman System Gold Analytical and Preparative HPLC system. A Perspective MALDI mass spectrophotometer system is used to identify the correct product. The peptide is dried using a lyophilizer. The peptide (2 mg) is then conjugated to keyhole limpet hemocyanin (KLH) via the free sulphydryl group on the Cys.
  • KLH keyhole limpet hemocyanin
  • a 96-well lmmulon 4HBX plate is coated with a C-terminal F(ab) antibody, specific to the peptides of the present invention, and allowed to incubate ovenight at 4 0 C. The plate is then blocked to prevent non-specific binding. Then, peptide standards (2500 ng/mL-160 pg/mL) are diluted in 33% plasma and the samples are diluted 1 :3 in buffer followed by incubation for 1.5 h at rt. After washing, a polyclonal N-terminal antibody specific to the peptides of this invention is incubated on the plate for 1 h.
  • HRP horseradish peroxidase
  • TMB 3,3',5,5'-tetramethylbenzidine
  • the plate is read at OD 450 .
  • the 96-well lmmulon 4HBX plate is coated with a polyclonal N-terminal antibody, specific to the peptides of the present invention, and allowed to incubate overnight at 4 0 C. The plate is then blocked to prevent non-specific binding.
  • peptide standards (2500 ng/mL-160 pg/mL) are diluted in 50% plasma and the samples are diluted 1 :2 in buffer followed by incubation for 1.5 h at rt. After washing, a monoclonal anti-PEG antibody specific to the peptides of this invention is incubated on the plate for one hour. This was followed by the addition of horseradish peroxidase (HRR)- anti-mouse antibody and the samples and standards are incubated for another hour. Detection is assessed following incubation with 3,3',5,5'-tetramethylbenzidine (TMB) solution, and the plate is read at OD 450 .
  • HRR horseradish peroxidase
  • Plasma samples are transferred to a microcentrifuge tube and an equal volume of acetonitrile is added to the sample (a 50% final concentration).
  • the sample is vigorously vortexed for about 5 min and allowed to sit on ice for 10 min.
  • the sample is again vortexed for about 1 min, and then centrifuged for 30 min in a microcentrifuge (4 0 C) at maximum (about 15,000 x g).
  • the aqueous phase is carefully transferred to a clean centrifuge tube, and the sample is centrifuged for 5 min in a microcentrifuge (4 0 C) at maximum speed (about 15,000 x g).
  • the extracted sample is dried under vacuum using a Speed Vac SC110 (Savant) with a medium heat setting until dry.
  • the sample is resuspended in an appropriate volume of sterile water and is maintained at 4 0 C.
  • the sample is then sonicated in a sonibath for 10 min at rt prior to analysis.
  • Demonstration of the activity of the peptides of the present invention may be accomplished through in vitro, ex vivo, and in vivo assays that are well known in the art.
  • in vitro, ex vivo, and in vivo assays that are well known in the art.
  • the following assays may be used.
  • Example 17 Method for Measuring Blood Glucose Levels
  • db/db mice obtained from Jackson Laboratories, Bar Harbor, ME are bled (by either eye or tail vein) and grouped according to equivalent mean blood glucose levels. They are dosed with the test peptide for 14 days. At this point, the animals are bled again by eye or tail vein and blood glucose levels were determined. In each case, glucose levels are measured with a Glucometer Elite XL (Bayer Corporation, Elkhart, IN).
  • Cardiovascular parameters e.g., heart rate and blood pressure
  • SHR rats are dosed with vehicle or test peptide for 2 weeks.
  • Blood pressure and heart rate are determined using atail-c ⁇ ff method as described by Grinsell, et al., (Am. J. Hypertens. 13:370- 375, 2000).
  • blood pressure and heart rate are monitored as described by Shen, et al., (J. Pharmacol. Exp. Therap. 278:1435-1443, 1996).
  • hApoAI mice obtained from Jackson Laboratories, Bar Harbor, ME are bled (by either eye or tail vein) and grouped according to equivalent mean serum triglyceride levels. They are dosed with the test peptide for 8 days. The animals are then bled again by eye or tail vein, and serum triglyceride levels are determined. In each case, triglyceride levels are measured using a Technicon Axon Autoanalyzer (Bayer Corporation, Tarrytown, NY).
  • hApoAI mice are bled and grouped with equivalent mean plasma HDL-cholesterol levels. The mice are dosed with vehicle or test peptide for 7 days, and then bled again on day 8. Plasma is analyzed for HDL-cholesterol using the Synchron Clinical System (CX4) (Beckman Coulter, Fullerton, CA).
  • CX4 Synchron Clinical System
  • Example 21 Method for Measuring Total Cholesterol, HDL-Cholesterol, Triglycerides, and Glucose Levels

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Abstract

This invention provides peptides with novel modifications that provide suitable derivatization sites to improve the pharmacokinetic properties of the peptides. These modified peptides function in vivo as agonists of the GIP receptor. The peptides of the present invention provide a new therapy for patients with decreased endogenous insulin secretion, for example, type 2 diabetics.

Description

GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE (GIP) RECEPTOR AGONISTS AND THEIR PHARMACOLOGICAL METHODS OF USE
[001] This application claims benefit of U.S. Provisional Application Serial No. 60/678,722; filed on May 6, 2005, the contents of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[002] This invention relates to novel modifications that provide suitable derivatization sites to improve the pharmacokinetic properties of the peptides. Such N-terminal modifications at the mainchain amino group of the first amino acid residue or C-terminal modifications at the mainchain carboxylate group of the last amino acid residue may include aliphatics, C3 to C7 cycloalkyl, aryl, or mono- or bi-cyclic heteroaromatics containing one or more nitrogen, oxygen, and/or sulfur heteroatoms. In addition, the N-terminal or C-terminal modifications may provide suitable derivatization sites (exemplified, 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 β-cells in a glucose-dependent manner, thereby providing a treatment option for those individuals afflicted with metabolic disorders such as diabetes or impaired glucose tolerance, a prediabetic state.
BACKGROUND OF THE INVENTION
[003] Diabetes is characterized by impaired glucose metabolism manifesting itself, among other things, by an elevated blood glucose level in the diabetic patient. Underlying defects lead to a classification of diabetes into two major groups: type 1 diabetes, or insulin dependent diabetes mellitus (IDDM), which arises when patients lack β-cells producing insulin in their pancreatic islets of Langerhans; and type 2 diabetes, or non-insulin dependent diabetes mellitus (NIDDM), which occurs in patients with an impaired β-cell function and alterations in insulin action.
[004] Type 1 diabetic patients are currently treated with insulin, while the majority of type 2 diabetic patients are treated with agents that stimulate β-cell function or with agents that enhance the tissue sensitivity of the patients towards insulin. Over time almost one-half of type 2 diabetic subjects lose their response to these agents and then must be placed on insulin therapy. The drugs presently used to treat type 2 diabetes are described below.
[005] Alpha-glucosidase inhibitors (e.g., Precose®, Voglibose™, and Miglitol®) reduce the excursion of postprandial glucose by delaying the absorption of glucose from the gut. These drugs are safe and provide treatment for mild to moderately affected diabetic subjects. However, gastrointestinal side effects have been reported in the literature.
[006] Insulin sensitizers are drugs that enhance the body's response to insulin. Thiozolidinediones such as Avandia™ (rosiglitazone) and Actos™ (pioglitazone) activate the peroxisome proliferator-activated receptor (PPAR) gamma subtype and modulate the activity of a set of genes that have not been well described. Rezulin™ (troglitazone), the first drug in this class, was withdrawn because elevated liver enzyme levels and drug induced hepatotoxicity. These hepatic effects do not appear to be a significant problem in patients using Avandia™ and Actos™. Even so, liver enzyme testing is recommended every 2 months in the first year of therapy and periodically thereafter. Avandia™ and Actos™ seem to be associated with fluid retention and edema. Another potential side effect is weight gain. Avandia™ is not indicated for use with insulin because of concern about congestive heart failure.
[007] Insulin secretagogues (e.g., sulfonylureas (SFUs) and other agents that act by the ATP- dependent K+ channel) are another drug type presently used to treat type 2 diabetes. SFUs are standard therapy for type 2 diabetics that have mild to moderate fasting hyperglycemia. The SFUs have limitations that include a potential for inducing hypoglycemia, weight gain, and high primary and secondary failure rates. Ten to 20% of initially treated patients fail to show a significant treatment effect (primary failure). Secondary failure is demonstrated by an additional 20-30% loss of treatment effect after six months on an SFU. Insulin treatment is required in 50% of the SFU responders after 5-7 years of therapy (Scheen, et al., Diabetes Res. Clin. Pract. 6:533-543, 1989).
[008] Glucophage™ (metformin HCI) is a biguanide that lowers blood glucose by decreasing hepatic glucose output and increasing peripheral glucose uptake and utilization. The drug is effective at lowering blood glucose in mildly and moderately affected subjects and does not have the side effects of weight gain or the potential to induce hypoglycemia. However, Glucophage™ has a number of side effects including gastrointestinal disturbances and the potential for lactic acidosis. Glucophage™ is contraindicated in diabetics over the age of 70 and in subjects with impairment in renal or liver function. Finally, Glucophage™ has primary and secondary failure rates similar to the SFUs.
[009] Insulin treatment is instituted after diet, exercise, and oral medications have failed to adequately control blood glucose. This treatment has the drawbacks that it is an injectable, that it can produce hypoglycemia, and that it causes weight gain.
[010] Because of the problems with current treatments, new therapies to treat type 2 diabetes are needed. In particular, new treatments to retain normal (glucose-dependent) insulin secretion are needed. Such new drugs should have the following characteristics: dependent on glucose for promoting insulin secretion (i.e., produce insulin secretion only in the presence of elevated blood glucose); low primary and secondary failure rates; and preservation of islet cell function. The strategy to develop the new therapy disclosed herein is based on the cyclic adenosine monophosphate (cAMP) signaling mechanism and its effects on insulin secretion.
[011] Cyclic AMP is a major regulator of the insulin secretion process. Elevation of this signaling molecule promotes the closure of the K+ channels following the activation of protein kinase A pathway. Closure of the K+ channels causes cell depolarization and subsequent opening of Ca++ channels, which in turn leads to exocytosis of insulin granules. Little if any effects on insulin secretion occurs in the absence of low glucose concentrations (Weinhaus, et al., Diabetes 47:1426-1435, 1998). Secretagogues like PACAP (pituitary adenylate cyclase activating peptide), VIP (vasoactive intestinal peptide), GIP (glucose-dependent insulinotropic polypeptide), and GLP-1 (glucagon-like peptide 1) use the cAMP system to regulate insulin secretion in a glucose- dependent fashion (Komatsu, et al., Diabetes 46:1928-1938, 1997; Filipsson, et al., Diabetes 50:1959-1969, 2001 ; Drucker, Endocrinology 142:521-527, 2001). Insulin secretagogues working through the elevation of cAMP such as GLP-1 , VIP, GIP, and PACAP are also able to enhance insulin synthesis in addition to insulin release (Skoglund, et al., Diabetes 49:1156-1164, 2000; Borboni, et al., Endocrinology 140:5530-5537, 1999).
[012] GLP-1 is released from the intestinal L-cell after a meal and functions as an incretin hormone (i.e., it potentiates glucose-induced insulin release from the pancreatic β-cell). It is a 37- amino acid peptide that is differentially expressed by the glucagon gene, depending upon tissue type. The clinical data that support the beneficial effect of raising cAMP levels in β-cells have been collected with GLP-1. Infusions of GLP-1 in poorly controlled type 2 diabetics normalized their fasting blood glucose levels (Gutniak, et al., New Eng. J. Med. 326:1316-1322, 1992) and with longer infusions improved the β-cell function to those of normal subjects (Rachman, et al., Diabetes 45:1524-1530, 1996). A recent report has shown that GLP-1 improves the ability of β- cells to respond to glucose in subjects with impaired glucose tolerance (Byrne, et al., Diabetes 47:1259-1265, 1998). All of these effects, however, are short-lived because of the short half-life of the peptide.
[013] Amyliπ Pharmaceuticals is conducting Phase III trials with Exendin-4 (AC2993), a 39 amino acid peptide originally identified in GiIa Monster. Amylin has reported that clinical studies demonstrated improved glycemic control in type 2 diabetic patients treated with Exendin-4. However, the incidence of nausea and vomiting was significant.
[014] 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 (Harmar, et al., Pharmacol. Reviews 50:265-270, 1998; Vaudry, et al., Pharmacol. Reviews 52:269-324, 2000). PACAP displays no receptor selectivity, having comparable activities and potencies at all three receptors. PAC1 is located predominately in the CNS, whereas VPAC1 and VPAC2 are more widely distributed. VPAC1 is located in the CNS as well as in liver, lungs, and intestine. VPAC2 is located in the CNS, pancreas, skeletal muscle, heart, kidney, adipose tissue, testis, and stomach. Recent work argues that VPAC2 is responsible for the insulin secretion from β-cells (Inagaki, et al., Proc. Natl. Acad. Sci. USA 91:2679-2683, 1994; Tsutsumi, et al., Diabetes 51:1453-1460, 2002). This insulinotropic action of PACAP is mediated by the GTP binding protein Gs. Accumulation of intracellular cAMP in turn activates the nonselective cation channels in β- cells increasing [Ca++], and promotes exocytosis of insulin-containing secretory granules.
[015] Glucose-dependent insulinotropic polypeptide, previously known as gastric inhibitory polypeptide (GIP) is a 42-residue gut peptide secreted by enteroendocrine K cells that is involved in the regulation of fat and glucose metabolism, with the insulinotropic function localized to residues 1-30. GIP is degraded by DPPIV proteolysis and eliminated by renal clearance, resulting in short-lived efficacy in vivo that limits the therapeutic value of the unmodified peptide. Limited clinical data have been collected with GIP. Intravenous (IV) administration or continuous in type 2 diabetics caused an acute increase in plasma insulin levels (Kindmark, et al., J. Clin. Endocrinol. Metab. 86:2015-2019, 2001 ; Meier, et al., Diabetes 53 (Suppl 3):S220-S224, 2004). These effects, however, are short-lived because of the short half-life of the peptide.
[016] The incretin GIP is a 42-residue peptide involved in the regulation of fat and glucose metabolism (Kieffer, Ann. Endocrinol. 65:13-21 , 2004; Meier and Nauck, Horm. Metab. Res. 36:859-66, 2004; Vilsboll and Hoist, Diabetologia 47:357-66, 2004). In pancreatic islets, GIP not only enhances insulin secretion acutely, but stimulates insulin production through enhancement of proinsulin transcription and translation (Fehmann and Goke, Peptides 16:1149-1152, 1995; Wang, et al., MoI. Cell. Endocrinol. 116:81-87, 1996), enhances the growth and survival of pancreatic β- cells (Trumper, et al., MoI. Endocrinol. 15:1559-1570, 2001 ; Trumper, et al., J. Endocrinol. 174:233-245, 2002; Pospisilik, et al., Diabetes 52:741-750, 2003). In addition to effects on the pancreas to enhance insulin secretion, GIP also has effects insulin target tissues directly to lower plasma glucose: enhancement of glucose uptake in adipose (Eckel, et al., Diabetes 28:1141-1142, 1979) and muscle (O'Harte, et al., J. Endocrinol. 156:237-243, 1998), and inhibition of hepatic glucose output (Elahi, et al., Can. J. Physiol. Pharmacol. 65:18, 1986). The use of GIP or GIP variants holds promise as a peptide therapeutic for conditions such as diabetes and obesity (Gault, et al., Neuropeptides 37:253-63, 2003; Green, et al., Curr. Pharm. Des. 10:3651-62, 2004). For example, modified GIP peptides improve glucose tolerance in rodents (Green, et al., Curr. Pharm. Des. 10:3651-62, 2004; Meier and Nauck, Horm. Metab. Res. 36:859-66, 2004) and intravenous administration and continuous infusion of GIP causes an acute stimulation of insulin secretion in type 2 diabetic patients (Kindmark, et al., J. Clin. Endocrinol. Metab. 86:2015-9, 2001 ; Meier, et al., Diabetes 53(Suppl 3):S220-4, 2003). The use of unmodified GIP as a therapeutic, however, is limited by the short in vivo lifetime of the native peptide (Meier and Nauck, Horm. Metab. Res. 36:859-66, 2004).
[017] Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide that was first isolated from hog upper small intestine (Said and Mutt, Science 169:1217-1218, 1970; U.S. Patent No. 3,879,371). This peptide belongs to a family of structurally-related, small polypeptides that includes helodermin, secretin, the somatostatins, and glucagon. The biological effects of VIP are mediated by the 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 displays comparable activities and potencies at VPAC1 and VPAC2.
[018] Improving the stability of GIP to proteolysis not only maintains the activity of GIP at its receptor but, more importantly, prevents the production of GIP fragments, some of which act as GIP receptor antagonists (Gault, et al., J. Endocrinol. 175:525-533, 2002). Reported modifications have included protection of the N-terminus of GIP from proteolysis by DPPIV through modification of the N-terminal tyrosine (O'Harte, et al., Biochim. Biophys. Acta 1425:319-327, 1998; O'Harte, et al., Diabetes 48:758-765, 1999; O'Harte, et al., J. Endocrinol. 165:639-648, 2000; O'Harte, et al., Diabetologia 45:1281-1291 , 2002; Gault, et al., Biochem. Biophys. Res. Commun. 290:1420-1426, 2002), mutation of the alanine at position 2 (Hinke, et al., Diabetes 51 :656-661 , 2002; Gault, et al., J. Endocrinol. 176:133-141 , 2003; Gault, et al., Metabolism 52:679-687, 2003), mutation of glutamic acid at position 3 (Gault, et al., Biochem. Biophys. Res. Commun. 308:207-213, 2003), and alanine at position 13 (Gault, et al., Cell Biol. International 27:41-46, 2003). Modifications at the N-terminal tyrosine or at alanine 2 maintain functional activity, while modifications reported at positions 3 and 13 have reduced with biological effectiveness of the peptides to generate cyclic AMP and to enhance insulin secretion. Such modified GIP molecules may act as functional antagonists.
[019] There exists a need for improved peptides that have the glucose-dependent insulin secretagogue activity of PACAP, VIP, GIP, GLP-1 , or Exendin-4, but with fewer side-effects, and preferably which are stable in formulation and have long plasma half-lives in vivo. Such improved in vivo half-life results from peptides with both decreased clearance and decreased susceptibility to proteolysis. Furthermore, tighter control of plasma glucose levels may prevent long-term diabetic complications. Thus, new diabetic drugs should provide an improved quality of life for patients.
SUMMARY OF THE INVENTION
[020] The present invention provides novel modifications that provide suitable derivatization sites to improve the pharmacokinetic properties of the peptides. Such N-terminal modifications at the amino group of the first peptide residue may include aliphatics, C3 to C7 cycloalkyl, aryl, or mono- or bi-cyclic heteroaromatics containing one or more nitrogen, oxygen, and/or sulfur heteroatoms. In addition, the N-terminal modifications may provide suitable derivatization sites (exemplified, but not limited to, amino and thiol groups). Several examples of such N-terminal modifications include, but are not limited to, 2-amino benzoic acid, 3-amino benzoic acid, 4-amino benzoic 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-aminocyclohexane carboxylic acid, D-pipecolinic acid, 4-amino-1-methyl-1 H-imidazole-2-carboxylic acid, 4-methythiobenzoic acid, 2- methythiobenzoic acid, 2-methythionicotinic acid, proline, 6-aminohexanoic acid, benzoic acid, (S)- tetrahydroisoquinoline acetic acid, indoline-2-carboxylic acid, cis-3-aminocyclohexane carboxylic acid, L-pipecolinic acid, 9-gluorenylmethoxycarbonyl, 2-thio-polyethylene glycol benzoic acid, 2- thio-polyethylene glycol nicotinic acid, 4-amino-1-methyl-1 H-imidazole-2-carboxylic acid, 1-amino- cyclopentane-3-carboxylic acid, 4-amino-1-methyl-1 H-imidazole-2-carboxylic acid, 1-amino- cyclopentane-3-carboxylic acid, 1-amino-cyclopentane-3-carboxylic acid, (2-mercapto-1 H- benzimidazol-1-yl)acetic acid, 2-(tritylthio)ethyl]amino}nicotinate, 2-{[2-(tritylthio)ethyl] aminojnicotinate, 1 -[2-(tritylthio)ethyl]-1 H-imidazole-2-carboxylate, 4-{[2-(tritylthio)ethyl]amino} pyrimidine-5-carboxylate, 1 -[2-(tritylthio)ethyl]-1 H-benzimidazole-2-carboxylate, 2-mercapto-1 H- imidazol-1-yl)acetic acid, ({1-[2-(tritylthio)ethyl]-1 H-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-carboxylae, 2- mercapto-e-methylpyrimidine-^carboxylic acid, 5-mercaptonicotinic acid, 5-isopropyl-2- mercaptothiazole-4-carboxylic acid, i-hexadecyl-I H-benzoimidazol^-ylsulfanylJacetic acid, and 2- (2-tert-butoxycarbonylaminoethylamino)thiazoIe-5-carboxylic acid.
[021] The invention relates to a peptide of Formula (I)
Z1 -A1 -A2-A3-Gly-Thr-Phe-A7-Ser-Asp-A10-Ser-A12-A13-A14-A15-A16-A17-A18-A19-A20- A21-Phe-A23-A24-Tφ-A26-Leu-A28-A29-A30-A31-A32-A33-A34-A35-A36-A37-A38-A39- A40-A41-A42-A43-Z2 (SEQ ID NO: 1) wherein
A1 is any naturally occurring amino acid, any d-amino acid, or deleted;
A2 is any naturally occurring amino acid, any d-amino acid, or deleted;
A3 is any naturally occurring amino acid, any d-amino acid, or deleted;
A7 is lle or Thr;
A10 is VaI or Tyr;
A12 is lie, Ala, or Ser;
A13 is Ala, lie, or VaI;
A14 is Leu or Met;
A15 is Asp, GIu, Asn, or Lys;
A16 is Lys or GIy;
A17 is lie or GIn;
A18 is His, Ala, or Arg;
A19 is GIn, Ala, or GIu;
A20 is GIn, Lys, or GIu;
A21 is GIu, Asp, or Ala;
A23 is He or VaI;
A24 is Ala, Asn, or Asp;
A26 is Leu or Ala;
A28 is Ala or Ser;
A29 is GIn, VaI, or GIu;
A30 is Lys, Arg, Asp, GIu, Asn, GIn, or Cys; A31 is GIy, Lys, Cys, GIn, Lys-X, Cys-PEG, or deleted; A32 is Lys, Cys, GIy, Lys-X, Cys-PEG or deleted; A33 is Lys, Cys, Lys-X, Cys-PEG or deleted; A34 is Asn, Ser, Lys, Cys, Lys-X, Cys-PEG or deleted; A35 is Asp, Asn, Lys, Cys, Lys-X, Cys-PEG or deleted; A36 is Trp, Lys, Cys, Lys-X, Cys-PEG or deleted; A37 is Lys, lie, Cys, Lys-X, Cys-PEG or deleted; A38 is His, Lys, Cys, Lys-X, Cys-PEG or deleted; A39 is Asn, Asp, Lys, Cys, Lys-X, Cys-PEG or deleted; A40 is lie, Leu, Lys, Cys, Lys-X, Cys-PEG or deleted; A41 is Thr, Ser, lie, Lys, Cys, Lys-X, Cys-PEG or deleted; A42 is GIn, GIu, Lys, Cys, Lys-X, Cys-PEG or deleted; and A43 is Lys-X, Cys-PEG or deleted.
[022] Lys-X is Lys modified at Nε with a fatty acid exemplified by CH3(CH2)nCOOH where n ranges from 0 to about 24.
[023] Z1 is selected from
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000009_0001
Figure imgf000009_0002
Figure imgf000010_0001
Figure imgf000010_0002
Figure imgf000010_0003
Figure imgf000011_0001
Figure imgf000011_0002
Figure imgf000012_0001
Figure imgf000012_0002
[024] Z2 may be a hydroxyl group such that the peptide has an unmodified carboxylate C- terminus or Z2 may be a modification of the C-terminal carboxylate group. Z2 may be a modification such as amidation, or Z2 may also be an unnatural amino acid or amide thereof. Z2 is exemplified by, but not limited to,
Figure imgf000013_0001
Figure imgf000013_0002
Figure imgf000013_0003
[025] The invention relates to a peptide of Formula (II)
Z1 -A1 -A2-A3-Gly-Thr-Phe-A7-Ser-Asp-A10-Ser-A12-A13-A14-A15-A16-A17-A18-A19-A20- A21-Phe-A23-A24-Trp-A26-Leu-Ala-Gln-Lys-A31-A32-A33-A34-A35-A36-A37-A38-A39- A40-A41-A42-A43-Z2 (SEQ ID NO: 2) wherein A1 is Tyr, His, Ala, or deleted; A2 is Ala, d-Ala, Tyr, Ser, GIy, VaI, or deleted;
A3 is GIu, d-Glu, Lys, Tyr, Pro, Trp, Phe, Asp, or deleted;
A7 is lie or Thr;
A10 is VaI or Tyr;
A12 is lie, Ala, or Ser;
A13 is Ala, lie, or VaI;
A14 is Leu or Met;
A15 is Asp, Asn, Lys, or GIu;
A16 is Lys or GIy;
A17 is lie or GIn;
A18 is His, Ala, or Arg;
A19 is GIn, GIu, or Ala;
A20 is GIn, GIu, or Lys;
A21 is GIu, Ala, or Asp;
A23 is lie or VaI;
A24 is Ala, Asp, or Asn;
A26 is Leu or Ala;
A31 is GIy, GIn, Lys, Cys, Lys-X, Cys-PEG, or deleted;
A32 is GIy, Lys, Cys, Lys-X, Cys-PEG or deleted;
A33 is Lys, Cys, Lys-X, Cys-PEG or deleted;
A34 is Asn, Ser, Lys, Cys, Lys-X, Cys-PEG or deleted;
A35 is Asp, Asn, Lys, Cys, Lys-X, Cys-PEG or deleted;
A36 is Trp, Lys, Cys, Lys-X, Cys-PEG or deleted;
A37 is Lys, He, Cys, Lys-X, Cys-PEG or deleted;
A38 is His, Lys, Cys, Lys-X, Cys-PEG or deleted;
A39 is Asn, Asp, Lys, Cys, Lys-X, Cys-PEG or deleted;
A40 is lie, Leu, Lys, Cys, Lys-X, Cys-PEG or deleted;
A41 is Thr, Ser, lie, Lys, Cys, Lys-X, Cys-PEG or deleted;
A42 is GIn, GIu, Lys, Cys, Lys-X, Cys-PEG or deleted; and
A43 is Lys-X, Cys-PEG or deleted. [026] The definitions for Z1 , Z2, and Lys-X are as defined above for Formula (I).
[027] For the peptide of Formula (I) or Formula (II), the N-terminal modifications are attached via an amide bond to the alpha-amino group of the first amino acid of said peptide. The C-terminal modifications are attached via an amide bond to the main chain carboxylate group of the last amino acid of said peptide. Examples of peptides of Formula (I) or Formula (II) may be found in, but are not limited to, the peptides described in Table 1 (e.g., SEQ ID NO: 1-82).
[028] Derivatives of the present invention may include peptides that have been fused with another compound, such as a compound to increase the half-life of the peptide and/or to reduce potential immunogenicity of the peptide (e.g., polyethylene glycol, "PEG"). For example, PEGylated peptides typically have greater half-life in vivo (Greenwald, Adv. Drug. Del. Rev. 55:217-250, 2003).
[029] In the case of PEGylation, the fusion of the peptide to PEG may be accomplished by any means known to one skilled in the art. For example, PEGylation may be accomplished by first introducing a cysteine mutation into the peptide to provide a linker upon which to attach the PEG, followed by site-specific derivatization with PEG-maleimide. Alternatively, the N-terminal modification may incorporate a reactive moiety for coupling to PEG, as exemplified by the amine group, the mercapto group, or the carboxylate group of the N-terminal modifying compounds disclosed above. For example, PEGylation may be accomplished by first introducing a mercapto moiety into the peptide via the N-terminal modifying group to provide a linker upon which to attach the PEG, followed by site-specific derivatization with methoxy-PEG-maleimide reagents supplied by, for example, either Nektar Therapeutics (San Carlos, CA, USA) and/or NOF (Tokyo, Japan). In addition to maleimide, numerous Cys reactive groups are known to those skilled in the art of protein cross-linking, such as the use of alkyl halides and vinyl sulfones (see, e.g., Proteins, Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993). In addition, the PEG could be introduced by direct attachment to the C-terminal carboxylate group, or to an internal amino acid such as Cys, Lys, Asp, or GIu or to unnatural amino acids that contain similar reactive sidechain moieties.
[030] Various size PEG groups can be used, as exemplified but not limited to, PEG polymers of from about 5 kDa to about 43 kDa. The PEG modification may include a single, linear PEG. For example, linear 5, 20, or 30 kDa PEGs that are attached to maleidmide or other cross-linking groups are available from Nektar and/or NOF (see, e.g., Table 2). Also, the modification may involve branched PEGs that contain two or more PEG polymer chains that are attached to maleimide or other cross-linking groups are available from Nektar and NOF (see, e.g., Table 2).
[031] It is possible that PEGylation with a smaller PEG (e.g., a linear 5 kDa PEG) will less likely reduce activity of the peptide, whereas a larger PEG (e.g., a branched 40 kDa PEG) will more likely reduce activity. However, a larger PEG will increase plasma half-life further so that once a week injection may be possible (Harris, et al., Clin. Pharmacokinet. 40:539-551, 2001). [032] The linker between the PEG and the peptide cross-linking group can be varied. For example, the commercially available thiol-reactive 40 kDa PEG (mPEG2-MAL) from Nektar (Huntsville, Al) employs a maleimide group for conjugation to Cys, and the maleimide group is attached to the PEG via a linker that contains a Lys (see, e.g., Table 2). As a second example, the commercially available thiol-reactive 43 kDa PEG (GL2-400MA) from NOF employs a maleimide group for conjugation to Cys, and the maleimide group is attached to the PEG via a bi-substituted alkane linker (see, e.g., Table 2). In addition, the PEG polymer can be attached directly to the maleimide, as exemplified by PEG reagents of molecular weight 5 and 20 kDa available form Nektar Therapeutics (Huntsville, Al) (see, e.g., Table 2).
[033] The present invention exemplifies, but is not limited to, the use of a mercapto group as a cross-linking site. It is well known that other moieties present in amino acids such as the amino group of the N-terminal modifying compound, the C-terminal carboxylate of either an unmodified C-terminus or a peptide modified with Z2, and the side chains of amino acids such as Lys, Arg, Asp, and GIu 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 (see, e.g., Proteins, Structure and Molecular Properties, 2nd ed., T. E. Creighton, W.H. Freeman and Company, New York, 1993). Such cross-linking agents can be linked to PEG as exemplified by, but not limited to, commercially available PEG derivatives containing amines, aldehydes, acetals, maleimide, succinimides, and thiols that are marketed, for example, by Nektar and NOF (e.g., Harris, et al., Clin. Pharmokinet. 40:539-551, 2001).
[034] In addition to PEGylation, the peptides of the present invention may be modified with fatty acids that improve pharmacodynamic properties. For example, the amine containing N-terminal modifying compounds can be derivatized with palmitate or myristolate or other fatty acids using methods known to those skilled in the art or an alkyl (e.g., C6-Ci8) moiety can be included directly as part of the N-terminal modifying compound.
[035] The peptides of the present invention have improved stability to proteolysis by DPPIV and in plasma as compared to PACAP or VIP. While both VIP and PACAP27 have been reported to be resistant to cleavage by DPPIV (Zhu, et al., J. Biol. Chem 278: 22418-22423, 2003), the inventors have demonstrated that these peptides are cleaved at longer time points. The derivatives of the present invention demonstrate an extended duration of action in vivo, supporting a dosing interval of less than once per day or once per week or greater, when derivatized.
[036] The peptides of the present invention (e.g., Table 1 ) provide a new therapy for patients with, for example, metabolic disorders such as those resulting from decreased endogenous insulin secretion, in particular type 2 diabetics, or for patients with impaired glucose tolerance, a prediabetic state that has a mild alteration in insulin secretion. In addition, the peptides of the present invention may be useful in the prevention and/or treatment of type 1 diabetes, gestational diabetes, maturity-onset diabetes of the young (MODY), latent autoimmune diabetes adult (LADA), and associated diabetic dyslipidemia and other diabetic complications, as well as hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, Syndrome X, and insulin resistance.
[037] The peptides of the present invention (e.g., Table 1) may also be utilized in the prevention and/or treatment of obesity (e.g., regulation of appetite and food intake); disorders of energy homeostasis; disorders of lipid and carbohydrate metabolism; cardiovascular disease, including atherosclerosis, coronary heart disease, coronary artery disease, hyperlipidemia, hypercholesteremia, low HDL levels and hypertension; cerebrovascular disease and peripheral vessel disease; polycystic ovary syndrome; carcinogenesis, and hyperplasia; ulcers; growth disorders, and other conditions identified herein, or function otherwise as described later herein.
[038] One aspect of the invention is a peptide of Formula (I) and Formula (II), and fragments, derivatives, and variants thereof that demonstrate at least one biological function that is substantially the same as the peptides of Formula (I) and Formula (II) (collectively, "peptides of this invention"), including functional equivalents thereof (e.g., Table 1).
[039] Antibodies and antibody fragments that selectively bind the peptides of this invention (e.g., Table 1) are also provided. Such antibodies are useful in detecting the peptides of this invention, and can be identified and made by procedures well known in the art. A polyclonal N-terminal IgG antibody and a monoclonal C-terminal Fab antibody have been generated which recognize peptides of this invention.
[040] The invention is also directed to a method of treating diabetes, diabetes-related disorders, and/or other diseases or conditions affected by the peptides of this invention, for example, effected by the GIP agonist function of the peptides of this invention, in a mammal, comprising administering a therapeutically effective amount of any of the peptides of the present invention or any peptide active at GIP such as a peptide of Formula (I) and Formula (II) to said mammal (e.g., Table 1).
[041] Also disclosed are methods of making the peptides of this invention.
DETAILED DESCRIPTION OF THE INVENTION
[042] This invention provides novel modified peptides, and fragments, derivatives, and variants thereof that demonstrate at least one biological function that is substantially the same as the peptides of Formula (I) and Formula (II) (peptides of this invention). The peptides of this invention (e.g., Table 1) function in vivo as GlP agonists or otherwise in the prevention and/or treatment of such diseases or conditions as diabetes including both type 1 and type 2 diabetes, gestational diabetes, maturity-onset diabetes of the young (MODY) (Herman, et al., Diabetes 43:40, 1994); latent autoimmune diabetes adult (LADA) (Zimmet, et al., Diabetes Med. 11:299, 1994); and associated diabetic dyslipidemia and other diabetic complications, as well as hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, Syndrome X, and insulin resistance. [043] The peptides of the present invention (e.g., Table 1) may also be utilized in the prevention and/or treatment of obesity (e.g., regulation of appetite and food intake); disorders of energy homeostasis; disorders of lipid and carbohydrate metabolism; cardiovascular disease, including atherosclerosis, coronary heart disease, coronary artery disease, hyperlipidemia, hypercholesteremia, low HDL levels and hypertension; cerebrovascular disease and peripheral vessel disease; polycystic ovary syndrome; carcinogenesis, and hyperplasia; ulcers; growth disorders, and other conditions identified herein, or function otherwise as described later herein.
[044] The peptides of this invention (e.g., Table 1) will stimulate insulin release from pancreatic β-cells in a glucose-dependent fashion. Furthermore, the peptides of this invention are stable in both aqueous and non-aqueous formulations and exhibit a plasma half-life of greater than one hour, for example, demonstrating a plasma half-life greater than 6 hours.
[045] The peptides of this invention (e.g., Table 1) are GIP agonists. In addition, these peptides may possess activities at other related receptors including, but not limited to, VPAC2 receptor agonism, GLP receptor agonism, or glucagon receptor antagonism. Furthermore, the peptides of this invention stimulate insulin release into plasma in a glucose-dependent fashion without inducing a stasis or increase in the level of plasma glucose that is counterproductive to the treatment of, for example, type 2 diabetes. Additionally, the peptides of this invention may be selective agonists of the GIP receptor, thereby causing, for example, an increase in insulin release into plasma, while being selective against other receptors that are responsible for such disagreeable or dangerous side effects as gastrointestinal water retention, and/or unwanted cardiovascular effects such as increased heart rate or blood pressure.
[046] The peptides of this invention are also stable in aqueous and non-aqueous formulations. The peptides of this invention will exhibit less than 10% degradation at 37-4O0C over a period of one week, when dissolved in water (at pH between 7-8) or non-aqueous organic solvent, or the peptides of this invention will exhibit less than 5% degradation at 37-400C over a period of one week, when dissolved in water (at pH between 7-8) or non-aqueous organic solvent. Furthermore, compositions and formulations of the present invention may comprise peptides of the present invention and about 2% to about 30% DMSO. In another embodiment of the present invention, the compositions and formulations may optionally include about 0.2% to about 3% (w/v) of additional solvents such as propylene glycol, dimethyl formamide, propylene carbonate, polyethylene glycol, and triglycerides.
[047] Finally, the derivatized peptides of this invention may exhibit a plasma half-life of, for example, at least 1 hour in rats after IV injection, at least 3 hours, or at least 6 hours. Furthermore, the derivatized peptide may demonstrate a significant lowering of the plasma glucose AUC following subcutaneous injection in rats, for example, at least 24 hours, at least 41 hours, or at least 65 hours following injection.
[048] The peptides of this invention provide a new therapy for patients with decreased endogenous insulin secretion or impaired glucose tolerance, in particular, type 2 diabetes. That is, the peptides of the present invention are long-acting GIP receptor agonists that may be used to maintain, improve, and restore glucose-stimulated insulin secretion. Furthermore, a selective peptide agonist of the GIP receptor will enhance glucose-dependent insulin secretion in the pancreas without causing the side effects associated with non-selective activation of the other related receptors.
[049] Certain terms used throughout this specification are defined below, and others will be defined as introduced. The single letter abbreviation for a particular amino acid, its corresponding amino acid, and three letter abbreviation are as follows: A, alanine (Ala); C, cysteine (Cys); D, aspartic acid (Asp); E, glutamic acid (GIu); F, phenylalanine (Phe); G, glycine (GIy); H, histidine (His); I, isoleucine (lie); K, lysine (Lys); L, leucine (Leu); M, methionine (Met); N, asparagine (Asn); P, proline (Pro); Q, glutamine (GIn); R, arginine (Arg); S, serine (Ser); T, threonine (Thr); V, valine (VaI); W, tryptophan (Trp); and Y, tyrosine (Tyr).
[050] "Functional equivalent" and "substantially the same biological function or activity" each means that degree of biological activity that is within about 30% to about 100% or more of that biological activity demonstrated by the peptide to which it is being compared when the biological activity of each peptide is determined by the same procedure.
[051] "Biological activity," "activity," or "biological function," which are used interchangeably, herein mean an effector function that is directly or indirectly performed by a peptide (whether in its native or denatured conformation), or by any fragments, derivatives, and variants thereof. Biological activities include, for example, binding to peptides, binding to other proteins or molecules, activity as a DNA binding protein, as a transcription regulator, ability to bind damaged DNA, etc.
[052] The terms "fragment," "derivative," and "variant," when referring to the peptides of the present invention, means fragments, derivatives, and variants of the peptides which retain substantially the same biological function or activity as such peptides, as described further below.
[053] A fragment is a portion of the peptide which retains substantially similar functional activity, for example, as described in the in vivo models disclosed herein.
[054] A derivative includes all modifications to the peptide which substantially preserve the functions disclosed herein and include additional structure and attendant function (e.g., modified N-terminus peptides, modified C-terminus peptides, or PEGylated peptides), fusion peptides which confer targeting specificity or an additional activity such as toxicity to an intended target, as described further below.
[055] The peptides of the present invention may be synthetic peptides.
[056] The fragment, derivative, or variant of the peptides of the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature peptide is fused with another compound, such as a compound to increase the half-life of the peptide (e.g., polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature peptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature peptide, or (v) one in which the peptide sequence is fused with a larger peptide (e.g., human albumin, an antibody or Fc, for increased duration of effect). Such fragments, derivatives, and variants and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
[057] The derivatives of the present invention may contain conservative amino acid substitutions (defined further below) made at one or more nonessential amino acid residues. A "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Fragments, or biologically active portions include peptide fragments suitable for use as a medicament, to generate antibodies, as a research reagent, and the like. Fragments include peptides comprising amino acid sequences sufficiently similar to or derived from the amino acid sequences of a peptide of this invention and exhibiting at least one activity of that peptide, but which include fewer amino acids than the full-length peptides disclosed herein. Typically, biologically active portions comprise a domain or motif with at least one activity of the peptide. A biologically active portion of a peptide can be a peptide which is, for example, five or more amino acids in length. Such biologically active portions can be prepared synthetically or by recombinant techniques and can be evaluated for one or more of the functional activities of a peptide of this invention by means disclosed herein and/or well known in the art.
[058] Variants of the peptides of this invention include peptides having an amino acid sequence sufficiently similar to the amino acid sequence of the peptides of this invention or a domain thereof. The term "sufficiently similar" means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain that is at least about 45%, about 75% through 98%, identical are defined herein as sufficiently similar. Variants will be sufficiently similar to the amino acid sequence of the peptides of this invention. Such variants generally retain the functional activity of the peptides of this invention. [059] Variants include peptides that differ in amino acid sequence due to mutagenesis. Variants that function as GIP receptor agonists may be identified by screening combinatorial libraries of mutants, for example truncation mutants, of the peptides of this invention for GIP receptor agonist activity.
[060] The invention also provides chimeric or fusion peptides. The targeting sequence is designed to localize the delivery of the peptide to minimize potential side effects. The peptides of this invention may be composed of amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres), and may contain amino acids other than the 20 gene- encoded amino acids. The peptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. 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 may contain many types of modifications. Peptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic peptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross- linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, e.g., Proteins. Structure and Molecular Properties. 2nd ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, ed., Academic Press, New York, pgs. 1-12 (1983); Seifter, et al., Meth. Enzymol. 182:626-646, 1990; Rattan, et al., Ann. N.Y. Acad. Sci. 663:48-62, 1992).
[061] The peptides of the present invention include the peptides of Formula (I) and Formula (II) (e.g., Table 1), as well as those sequences having insubstantial variations in sequence from them. An "insubstantial variation" would include any sequence addition, substitution, or deletion variant that maintains substantially at least one biological function of the peptides of this invention, for example, GIP receptor agonist activity, and/or enhancement of insulin secretion or lowering of blood glucose demonstrated herein. These functional equivalents may include peptides which have at least about 90% identity to the peptides of the present invention, at least 95% identity to the peptides of the present invention, and at least 99% identity to the peptides of the present invention, and also include portions of such peptides having substantially the same biological activity. However, any peptide having insubstantial variation in amino acid sequence from the peptides of the present invention that demonstrates functional equivalency as described further herein is included in the description of the present invention.
[062] The peptides of this invention may be a product of chemical synthetic procedures.
[063] The peptides of this invention may be conveniently isolated by methods that are well known in the art. Purity of the preparations may also be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis and mass spectroscopy and liquid chromatography.
[064] Also provided are related peptides within the understanding of those with skill in the art, such as chemical mimetics, organomimetics, or peptidomimetics. As used herein, the terms "mimetic," "peptide mimetic," "peptidomimetic," "organomimetic," and "chemical mimetic" are intended to encompass peptide derivatives, peptide analogs, and chemical compounds having an arrangement of atoms in a three-dimensional orientation that is equivalent to that of a peptide of the present invention. It will be understood that the phrase "equivalent to" as used herein is intended to encompass peptides having substitution(s) of certain atoms, or chemical moieties in said peptide, having bond lengths, bond angles, and arrangements in the mimetic peptide that produce the same or sufficiently similar arrangement or orientation of said atoms and moieties to have the biological function of the peptides of the invention. In peptide mimetics, the three- dimensional arrangement of the chemical constituents may be structurally and/or functionally equivalent to the three-dimensional arrangement of the peptide backbone and component amino acid sidechains in the peptide, resulting in such peptide-, organo-, and chemical mimetics of the peptides of the invention having substantial biological activity. These terms are used according to the understanding in the art, as illustrated, for example, by Fauchere, (Adv. Drug Res. 15:29, 1986); Veber & Freidinger, (TINS p.392, 1985); and Evans, et al., (J. Med. Chem. 30:1229, 1987), incorporated herein by reference.
[065] It is understood that a pharmacophore exists for the biological activity of each peptide of the invention. A pharmacophore is understood in the art as comprising an idealized, three- dimensional definition of the structural requirements for biological activity. Peptido-, organo-, and chemical mimetics may be designed to fit each pharmacophore with current computer modeling software (computer aided drug design). Said mimetics may be produced by structure-function analysis, based on the positional information from the substituent atoms in the peptides of the invention.
[066] Peptides as provided by the invention may be advantageously synthesized by any of the chemical synthesis techniques known in the art, particularly solid-phase synthesis techniques, for example, using commercially-available automated peptide synthesizers. The mimetics of the present invention may be synthesized by solid phase or solution phase methods conventionally used for the synthesis of peptides (see, e.g., Merrifield, J. Amer. Chem. Soc. 85:2149-54, 1963; Carpino, Ace. Chem. Res. 6:191-98, 1973; Birr, Aspects of the Merrifield Peptide Synthesis, Springer- Verlag: Heidelberg, 1978; The Peptides: Analysis, Synthesis, Biology, VoIs. 1 , 2, 3, and 5, (Gross & Meinhofer, eds.), Academic Press: New York, 1979; Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, III., 1984; Kent, Ann. Rev. Biochem. 57:957-89, 1988; and Gregg, et al., Int. J. Peptide Protein Res. 55:161-214, 1990, which are incorporated herein by reference in their entirety.)
[067] Peptides of the present invention may be prepared by solid phase methodology. Briefly, an N-protected C-terminal amino acid residue is linked to an insoluble support such as divinylbenzene cross-linked polystyrene, polyacrylamide resin, Kieselguhr/polyamide (pepsyn K), controlled pore glass, cellulose, polypropylene membranes, acrylic acid-coated polyethylene rods, or the like. Cycles of deprotection, neutralization, and coupling of successive protected amino acid derivatives are used to link the amino acids from the C-terminus according to the amino acid sequence. For some synthetic peptides, an FMOC strategy using an acid-sensitive resin may be used. Solid supports in this regard may be divinylbenzene cross-linked polystyrene resins, which are commercially available in a variety of functionalized forms, including chloromethyl resin, hydroxymethyl resin, paraacetamidomethyl resin, benzhydrylamine (BHA) resin, A- methylbenzhydrylamine (MBHA) resin, oxime resins, 4-alkoxybenzyl alcohol resin (Wang resin), 4- (2',4'-dimethoxyphenylaminomethyl)-phenoxymethyl resin, 2,4-dimethoxybenzhydryl-amine resin, and 4-(2',4'-dimethoxyphenyl-FMOC-amino-methyl)-phenoxyacetamidonorleucyl-MBHA resin (Rink amide MBHA resin). In addition, acid-sensitive resins also provide C-terminal acids, if desired. A protecting group for alpha amino acids is base-labile 9-fluorenylmethoxy-carbonyl (FMOC).
[068] Suitable protecting groups for the side chain functionalities of amino acids chemically compatible with BOC (t-butyloxycarbonyl) and FMOC groups are well known in the art. When using FMOC chemistry, the following protected amino acid derivatives may be utilized: FMOC- Cys(Trt), FMOC-Ser(But), FMOC-Asn(Trt), FMOC-Leu, FMOC-Th r(Trt), FMOC-VaI, FMOC-GIy, 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). The amino acid residues may be coupled by using a variety of coupling agents and chemistries known in the art, such as direct coupling with DIC (diisopropyl-carbodiimide), DCC (dicyclohexylcarbodiimide), BOP (benzotriazolyl-N-oxytrisdimethylaminophosphonium hexa- fluorophosphate), PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluoro- phosphate), PyBrOP (bromo-tris-pyrrolidinophosphonium hexafluorophosphate); via performed symmetrical anhydrides; via active esters such as pentafluorophenyl esters; or via performed HOBt (1-hydroxybenzotriazole) active esters or by using FMOC-amino acid fluoride and chlorides or by using FMOC-amino acid-N-carboxy anhydrides. For example, activation may be performed with HBTU (2-(1H-benzotriazole-1-yl),1 ,1 ,3!3-tetramethyluronium hexafluorophosphate) or HATU (2-(1H-7-aza-benzotriazole-1-yl),1 ,1 ,3,3-tetramethyluronium hexafluoro-phosphate) in the presence of HOBt or HOAt (7-azahydroxybenztriazole). [069] The solid phase method may be carried out manually, or by automated synthesis on a commercially available peptide synthesizer (e.g., Applied Biosystems 431 A or the like; Applied Biosystems, Foster City, CA). In a typical synthesis, the first (C-terminal) amino acid is loaded on the chlorotrityl resin. Successive deprotection (with 20% piperidine/NMP (N-methylpyrrolidone)) and coupling cycles according to ABI FastMoc protocols (Applied Biosystems) may be used to generate the peptide sequence. Double and triple coupling, with capping by acetic anhydride, may also be used.
[070] The synthetic mimetic peptide may be cleaved from the resin and deprotected by treatment with TFA (trifluoroacetic acid) containing appropriate scavengers. Many such cleavage reagents, 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, may be used. The peptide is separated from the resin by filtration and isolated by ether precipitation. Further purification may be achieved by conventional methods, such as gel filtration and reverse phase HPLC (high performance liquid chromatography). Synthetic mimetics according to the present invention may be in the form of pharmaceutically acceptable salts, especially base-addition salts including salts of organic bases and inorganic bases. The base-addition salts of the acidic amino acid residues are prepared by treatment of the peptide with the appropriate base or inorganic base, according to procedures well known to those skilled in the art, or the desired salt may be obtained directly by lyophilization of the appropriate base.
[071] Generally, those skilled in the art will recognize that peptides as described herein may be modified by a variety of chemical techniques to produce peptides having essentially the same activity as the unmodified peptide, and optionally having other desirable properties. For example, carboxylic acid groups of the peptide may be provided in the form of a salt of a pharmaceutically- acceptable cation. Amino groups within the peptide may be in the form of a pharmaceutically- acceptable acid addition salt, such as the HCI, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric, and other organic salts, or may be converted to an amide. Thiols may be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this invention so that the native binding configuration will be more nearly approximated. For example, a carboxyl terminal or amino terminal cysteine residue may be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, thereby generating a cyclic peptide. Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino- terminal amides and esters.
[072] Specifically, a variety of techniques are available for constructing peptide derivatives and analogs with the same or similar desired biological activity as the corresponding peptide but with more favorable activity than the peptide with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis. Such derivatives and analogs include peptides modified at the N- terminal amino group, as exemplified by, but not limited to, the peptides of Formula (I) and Formula (II) (e.g., Table 1), the C-terminal amide group, and/or changing one or more of the amido linkages in the peptide to a non-amido linkage. It will be understood that two or more such modifications may be coupled in one peptide mimetic structure (e.g., modification at the G-terminal amide group and inclusion of a -CH2- carbamate linkage between two amino acids in the peptide).
[073] Peptide mimetics as understood in the art and provided by the invention are structurally similar to the peptides of the invention, but have one or more peptide linkages optionally replaced by a linkage, for example, -CH2NH-, -CH2S-, -CH2CH2-, --CH=CH- (in both cis and trans conformers), -CDCH2-, -CH(OH)CH2 --, and -CH2SO-, by methods known in the art and further described in the following references: Spatola, Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, (Weinstein, ed.), 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. Pept. Prot. Res. 14:177-185, 1979; Spatola, et al., Life Sci. 38:1243-1249, 1986; Hann, J. Chem. Soc. Perkin Trans. I 307-314, 1982; Almquist, et al., J. Med. Chem. 23:1392-1398, 1980; Jennings-White, et al., Tetrahedron Lett. 23:2533, 1982; Szelke, et al., EP045665A; 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. Such peptide mimetics may have significant advantages over peptide embodiments, including, for example, more economical to produce, having greater chemical stability or enhanced pharmacological properties (such as half-life, absorption, potency, efficacy, etc.), reduced antigenicity, and other properties.
[074] Mimetic analogs of the peptides of the invention may also be obtained using the principles of conventional or rational drug design (see, e.g., Andrews, et al., Proc. Alfred Benzon Symp. 28:145-165, 1990; McPherson, Eur. J. Biochem. 189:1-24, 1990; HoI, et al., in Molecular Recognition: Chemical and Biochemical Problems, (Roberts, ed.); Royal Society of Chemistry; pp. 84-93, 1989a; HoI, Arzneim-Forsch. 39:1016-1018, 1989b; HoI, Agnew Chem. Int. Ed. Engl. 25:767-778, 1986; the disclosures of which are herein incorporated by reference).
[075] In accordance with the methods of conventional drug design, the desired mimetic molecules may be obtained by randomly testing molecules whose structures have an attribute in common with the structure of a "native" peptide. The quantitative contribution that results from a change in a particular group of a binding molecule may be determined by measuring the biological activity of the putative mimetic in comparison with the activity of the peptide. In one embodiment of rational drug design, the mimetic is designed to share an attribute of the most stable three- dimensional conformation of the peptide. Thus, for example, the mimetic may be designed to possess chemical groups that are oriented in a way sufficient to cause ionic, hydrophobic, or van der Waals interactions that are similar to those exhibited by the peptides of the invention, as disclosed herein.
[076] One method for performing rational mimetic design employs a computer system capable of forming a representation of the three-dimensional structure of the peptide, such as those exemplified by HoI, 1989a; HoI, 1989b; and HoI, 1986. Molecular structures of the peptido-, organo-, and chemical mimetics of the peptides of the invention may be produced using computer- assisted design programs commercially available in the art. Examples of such programs include SYBYL 6.5®, HQSAR™, and ALCHEMY 2000™ (Tripos); GALAXY™ and AM2000™ (AM Technologies, Inc., San Antonio, TX); CATALYST™ and CERIUS™ (Molecular Simulations, Inc., San Diego, CA); CACHE PRODUCTS™, TSAR™, AMBER™, and CHEM-X™ (Oxford Molecular Products, Oxford, CA) and CHEMBUILDER3D™ (Interactive Simulations, Inc., San Diego, CA).
[077] The peptido-, organo-, and chemical mimetics produced using the peptides disclosed herein using, for example, art-recognized molecular modeling programs may be produced using conventional chemical synthetic techniques, for example, designed to accommodate high throughput screening, including combinatorial chemistry methods. Combinatorial methods useful in the production of the peptido-, organo-, and chemical mimetics of the invention include phage display arrays, solid-phase synthesis, and combinatorial chemistry arrays, as provided, for example, by SIDDCO (Tuscon, Arizona); Tripos, Inc.; Calbiochem/Novabiochem (San Diego, CA); Symyx Technologies, Inc. (Santa Clara, CA); Medichem Research, Inc. (Lemont, IL); Pharm-Eco Laboratories, Inc. (Bethlehem, PA); or N. V. Organon (Oss, Netherlands). Combinatorial chemistry production of the peptido-, organo-, and chemical mimetics of the invention may be produced according to methods known in the art, including, but not limited to, techniques disclosed in Terrett, (Combinatorial Chemistry, Oxford University Press, London, 1998); Gallop, et al., J. Med. Chem. 37:1233-51, 1994; Gordon, et al., J. Med. Chem. 37:1385-1401, 1994; Look, et al., Bioorg. Med. Chem. Lett. 6:707-12, 1996; Ruhland, et al., J. Amer. Chem. Soc. 118: 253-4, 1996; Gordon, et al., Ace. Chem. Res. 29:144-54, 1996; Thompson & Ellman, Chem. Rev. 96:555-600, 1996; Fruchtel & 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 Combinatorial Chemistry in Lead Discovery and SAR Optimization," Id., 1995; Davies and Briant, "Combinatorial Chemistry Library Design using Pharmacophore Diversity," Id., 1995; Pavia, "Chemically Generated Screening Libraries: Present and Future," Id., 1996; and U.S. Patents, Nos. 5,880,972; 5,463,564; 5,331573; and 5,573,905.
[078] The newly synthesized peptides may be substantially purified by preparative high performance liquid chromatography (see, e.g., Creighton, Proteins: Structures And Molecular Principles, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic peptide of the present invention may be confirmed by amino acid analysis or sequencing by, for example, the Edman degradation procedure (Creighton, supra). Additionally, any portion of the amino acid sequence of the peptide may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant peptide or a fusion peptide.
[079] Also included in this invention are antibodies and antibody fragments that selectively bind the peptides of this invention. Any type of antibody known in the art may be generated using methods well known in the art. For example, an antibody may be generated to bind specifically to an epitope of a peptide of this invention. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')2, and Fv, which are capable of binding an epitope of a peptide of this invention. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more amino acids, for example, at least 15, 25, or 50 amino acids.
[080] An antibody which specifically binds to an epitope of a peptide of this invention may be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays may be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.
[081] Typically, an antibody which specifically binds to a peptide of this invention provides a detection signal higher than a detection signal provided with other proteins when used in an immunochemical assay. Antibodies which specifically bind to a peptide of this invention do not detect other proteins in immunochemical assays and can immunoprecipitate a peptide of this invention from solution.
[082] Peptides of this invention may be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, goat, sheep, monkey, or human, to produce polyclonal antibodies. If desired, a peptide of this invention may be conjugated to a carrier protein, 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 (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially useful.
[083] Monoclonal antibodies which specifically bind to a peptide of this invention may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma technique (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., MoI. Cell Biol. 62:109-20, 1984).
[084] In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, may be used (Morrison, et al., 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 also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences may be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grafting of entire complementarity determining regions. Alternatively, humanized antibodies may be produced using recombinant methods (see, e.g., GB2188638B). Antibodies which specifically bind to a peptide of this invention may contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Patent No. 5,565,332.
[085] Alternatively, techniques described for the production of single chain antibodies may be adapted using methods known in the art to produce single chain antibodies which specifically bind to a peptide of this invention. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88:11120-23, 1991).
[086] Single-chain antibodies also may be constructed using a DNA amplification method, 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. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison (Nat. Biotechnol. 15:159-63, 1997). Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss (J. Biol. Chem. 269:199-206, 1994).
[087] A nucleotide sequence encoding a single-chain antibody may be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar, et al., Int. J. Cancer 61 :497-501 , 1995; Nicholls, et al., J. Immunol. Meth. 165:81-91 , 1993).
[088] Antibodies which specifically bind to a peptide of this invention may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, et al., Proc. Natl. Acad. Sci. 86:38333-37, 1989; Winter, et al., Nature 349:293-99, 1991).
[089] Other types of antibodies may be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies may be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" also can be prepared (see, e.g., WO 94/13804,).
[090] Human antibodies with the ability to bind to the peptides of this invention may also be identified from the MorphoSys HuCAL® library as follows. A peptide of this invention may be coated on a microtiter plate and incubated with the MorphoSys HuCAL® Fab phage library. Those phage-linked Fabs not binding to the peptide of this invention can be washed away from the plate, leaving only phage which tightly bind to the peptide of this invention. The bound phage can be eluted, for example, by a change in pH or by elution with E. coli and amplified by infection of E. coli hosts. This panning process can be repeated once or twice to enrich for a population of antibodies that tightly bind to the peptide of this invention. The Fabs from the enriched pool are then expressed, purified, and screened in an ELISA assay.
[091] Antibodies according to the invention may be purified by methods well known in the art. For example, antibodies may be affinity purified by passage over a column to which a peptide of this invention is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
Methods of Use
[092] As used herein, various terms are defined below.
[093] When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.
[094] The term "subject" as used herein includes mammals (e.g., humans and animals).
[095] The term "treatment" includes any process, action, application, therapy, or the like, wherein a subject, including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject.
[096] The term "combination therapy" or "co-therapy" means the administration of two or more therapeutic agents to treat, for example, diabetes. Such administration encompasses coadministration of two or more therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each inhibitor agent. In addition, such administration encompasses use of each type of therapeutic agent in a sequential manner.
[097] The phrase "therapeutically effective" means the amount of each agent administered that will achieve the goal of improvement in a diabetic condition or disorder severity, while avoiding or minimizing adverse side effects associated with the given therapeutic treatment.
[098] The term "pharmaceutically acceptable" means that the subject item is appropriate for use in a pharmaceutical product.
[099] The peptides of Formula (I) and Formula (II) (e.g., Table 1) are expected to be valuable as therapeutic agents. Accordingly, an embodiment of this invention includes a method of treating the various conditions in a patient (including mammals) which comprises administering to said patient a composition containing an amount of the peptide of Formula (I) and Formula (II), that is effective in treating the target condition.
[100] The peptides of the present invention, as a result of the ability to stimulate insulin secretion from pancreatic islet cells in vitro, and by causing a decrease in blood glucose in vivo, may be employed in treatment diabetes, including both type 1 and type 2 diabetes (non-insulin dependent diabetes mellitus). Such treatment may also delay the onset of diabetes and diabetic complications. The peptides may be used to prevent subjects with impaired glucose tolerance from proceeding to develop type 2 diabetes. Other diseases and conditions that may be treated or prevented using peptides of the invention in methods of the invention include: Maturity-Onset Diabetes of the Young (MODY) (Herman, et al., Diabetes 43:40, 1994); Latent Autoimmune Diabetes Adult (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):S5, 1999); impaired fasting glucose (IFG) (Charles, et al., Diabetes 40:796, 1991); gestational diabetes (Metzger, Diabetes, 40:197, 1991); and metabolic syndrome X.
[101] The peptides of the present invention may also be utilized in the prevention and/or treatment of obesity (e.g., regulation of appetite and food intake); disorders of energy homeostasis; disorders of lipid and carbohydrate metabolism; cardiovascular disease, including atherosclerosis, coronary heart disease, coronary artery disease, hyperlipidemia, hypercholesteremia, low HDL levels and hypertension; cerebrovascular disease and peripheral vessel disease; polycystic ovary syndrome; carcinogenesis, and hyperplasia; ulcers; growth disorders, and other conditions identified herein, or function otherwise as described later herein.
[102] The peptides of the present invention may also be useful for treating physiological disorders related to, for example, cell differentiation to produce lipid accumulating cells, regulation of insulin sensitivity and blood glucose levels, which are involved in, for example, abnormal pancreatic β-cell function, carcinogenesis, hyperplasia, reduction in the pancreatic β-cell mass, insulin secretion, tissue sensitivity to insulin, cell growth, polycystic ovarian disease, chronic anovulation, hyperandrogenism, progesterone production, steroidogenesis, plasma triglycerides, HDL, and LDL cholesterol levels, and the like.
[103] Peptides of the invention may also be used in methods of the invention to treat secondary causes of diabetes (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1):S5, 1999). Such secondary causes include glucocorticoid excess, growth hormone excess, pheochromocytoma, and drug-induced diabetes. Drugs that may induce diabetes include, but are not limited to, pyriminil, nicotinic acid, glucocorticoids, phenytoin, thyroid hormone, β- adrenergic agents, α-interferon and drugs used to treat HIV infection.
[104] The peptides of the present invention may be used alone or in combination with additional therapies and/or compounds known to those skilled in the art in the treatment of diabetes and related disorders. Alternatively, the methods and peptides described herein may be used, partially or completely, in combination therapy.
[105] The peptides of the invention may also be administered in combination with other known therapies for the treatment of diabetes, including PPAR ligands (e.g., agonists, antagonists), insulin secretagogues, for example, sulfonylurea drugs and non-sulfonylurea secretagogues, α- glucosidase inhibitors, insulin sensitizers, hepatic glucose output lowering compounds, insulin and insulin derivatives, and anti-obesity drugs. Such therapies may be administered prior to, concurrently with, or following administration of the peptides of the invention. Insulin and insulin derivatives include both long and short acting forms and formulations of insulin. PPAR ligands may include agonists and/or antagonists of any of the PPAR receptors or combinations thereof. For example, PPAR ligands may include ligands of PPAR-α, PPAR-γ, PPAR-δ or any combination of two or three of the receptors of PPAR. PPAR ligands include, for example, rosiglitazone, troglitazone, and pioglitazone. Sulfonylurea drugs include, for example, glyburide, glimepiride, chlorpropamide, tolbutamide, and glipizide, -α-glucosidase inhibitors that may be useful in treating diabetes when administered with a peptide of the invention include acarbose, miglitol, and voglibose. Insulin sensitizers that may be useful in treating diabetes include PPAR-γ agonists such as the glitazones (e.g., troglitazone, pioglitazone, englitazone, MCC-555, rosiglitazone, and the like) and other thiazolidinedione and non-thiazolidinedione compounds; biguanides such as metformin and phenformin; protein tyrosine phosphatase-1 B (PTP-1B) inhibitors; dipeptidyl peptidase IV (DPPIV) inhibitors; and 11beta-HSD inhibitors. Hepatic glucose output lowering compounds that may be useful in treating diabetes when administered with a peptide of the invention include, for example, glucagon anatgonists and metformin, such as Glucophage and Glucophage XR. Insulin secretagogues that may be useful in treating diabetes when administered with a peptide of the invention include sulfonylurea and non-sulfonylurea drugs: GLP-1 , VIP, PACAP, secretin, and derivatives thereof; nateglinide, meglitinide, repaglinide, glibenclamide, glimepiride, chlorpropamide, and glipizide. For example, GLP-1 includes derivatives of GLP-1 with longer half-lives than native GLP-1 , such as, for example, fatty-acid derivatized GLP-1 and exendin.
[106] Peptides of the invention may also be used in methods of the invention in combination with anti-obesity drugs. For example, anti-obesity drugs include β-3 adrenergic receptor agonists such as CL 316,243; cannabinoid (e.g., CB-1) antagonists such as Rimonabant; neuropeptide-Y receptor antagonists; neuropeptide Y5 inhibitors; apo-B/MTP inhibitors; 11 β-hydroxy steroid dehydrogenase-1 inhibitors; peptide YY3-36 or analogs thereof; MCR4 agonists; CCK-A agonists; monoamine reuptake inhibitors; sympathomimetic agents; dopamine agonists; melanocyte- stimulating hormone receptor analogs; melanin concentrating hormone antagonists; leptin; leptin analogs; leptin receptor agonists; galanin antagonists; lipase inhibitors; bombesin agonists; thyromimetic agents; dehydroepiandrosterone or analogs thereof; glucocorticoid receptor antagonists; orexin receptor antagonists; ciliary neurotrophic factor; ghrelin receptor antagonists; histamine-3 receptor antagonists; neuromedin U receptor agonists; appetite suppressants, such as, for example, sibutramine (Meridia); and lipase inhibitors, such as, for example, orlistat (Xenical). The peptides of the present invention may also be administered in combination with a drug compound that modulates digestion and/or metabolism such as drugs that modulate thermogenesis, lipolysis, gut motility, fat absorption, and satiety.
[107] Peptides of the invention may also be used in methods of the invention in combination with drugs commonly used to treat lipid disorders. Such drugs include, but are not limited to, HMG-CoA reductase inhibitors, nicotinic acid, fatty acid lowering compounds (e.g., acipimox); lipid lowering drugs (e.g., stanol esters, sterol glycosides such as tiqueside, and azetidinones such as ezetimibe), ACAT inhibitors (such as avasimibe), bile acid sequestrants, 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. Sequestrants include,. for example, cholestyramine, colestipol, and dialkylaminoalkyl derivatives of a cross-linked dextran.
[108] Furthermore, 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 anti-hypertensive agents for use in combination with the peptides of the present invention include calcium channel blockers (L-type and T-type; e.g., diltiazem, verapamil, nifedipine, amlodipine and mybefradil), diuretics (e.g., chlorothiazide, hydrochlorothiazide, flumethiazide, hydroflumethiazide, bendroflumethiazide, methylchlorothiazide, trichloromethiazide, polythiazide, benzthiazide, ethacrynic acid tricrynafen, chlorthalidone, furosemide, musolimine, bumetanide, triamtrenene, amiloride, spironolactone), renin inhibitors, ACE inhibitors (e.g., captopril, zofenopril, fosinopril, enalapril, ceranopril, cilazopril, delapril, pentopril, quinapril, ramipril, lisinopril), AT-1 receptor antagonists (e. g., losartan, irbesartan, valsartan), ET receptor antagonists (e.g., sitaxsentan, atrsentan, neutral endopeptidase (NEP) inhibitors, vasopepsidase inhibitors (dual NEP-ACE inhibitors) (e.g., omapatrilat and gemopatrilat), and nitrates.
[109] Such co-therapies may be administered in any combination of two or more drugs {e.g., a peptide of the invention in combination with an insulin sensitizer and an anti-obesity drug). Such co-therapies may be administered in the form of pharmaceutical compositions, as described above.
Pharmaceutical Compositions
[110] Based on well known assays used to determine the efficacy for treatment of conditions identified above in mammals, and by comparison of these results with the results of known medicaments that are used to treat these conditions, the effective dosage of the peptides of this invention can readily be determined for treatment of each desired indication. The amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular peptide and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.
[111] The total amount of the active ingredient to be administered may generally range from, for example, 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 may contain from, for example, about 0.05 mg to about 1500 mg of active ingredient, and may be administered one or more times per day. The daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous, and parenteral injections, and use of infusion techniques may be from, for example, about 0.001 to about 200 mg/kg. The daily rectal dosage regimen may be from, for example, about 0.001 to about 200 mg/kg of total body weight. The transdermal concentration may be that required to maintain a daily dose of from, for example, about 0.001 to about 200 mg/kg.
[112] Of course, the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific peptide employed, the age of the patient, the diet of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a peptide of the present invention may be ascertained by those skilled in the art using conventional treatment tests.
[113] The peptides of this invention may be utilized to achieve the desired pharmacological effect by administration to a subject in need thereof in an appropriately formulated pharmaceutical composition. A subject, for example, may be a mammal, including a human, in need of treatment for a particular condition or disease. Therefore, the present invention includes pharmaceutical compositions which are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a peptide of the present invention. A pharmaceutically acceptable carrier is any carrier which is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. A pharmaceutically effective amount of a peptide is that amount which produces a result or exerts an influence on the particular condition being treated. The peptides of the present invention may be administered with a pharmaceutically-acceptable carrier using any effective conventional dosage unit forms, including, for example, immediate and timed release preparations, orally, parenterally, topically, or the like.
[114] For oral administration, the peptides may be formulated into solid or liquid preparations such as, for example, capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions, and may be prepared according to methods known to the art for the manufacture of pharmaceutical compositions. The solid unit dosage forms may be a capsule which can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.
[115] In another embodiment, the peptides of this invention may be tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin; disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum; lubricants intended to improve the flow of tablet granulation and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example, talc, stearic acid, or magnesium, calcium or zinc stearate; dyes; coloring agents; and flavoring agents intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient. Suitable excipients for use in oral liquid dosage forms include diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Various 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.
[116] Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, those sweetening, flavoring and coloring agents described above, may also be present.
[117] The pharmaceutical compositions of this invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as liquid paraffin or a mixture of vegetable oils. Suitable emulsifying agents may be (1) naturally occurring gums such as gum acacia and gum tragacanth, (2) naturally occurring phosphatides such as soy bean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, and (4) condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
[118] Syrups and elixirs may be formulated with sweetening agents such as, for example, glycerol, propylene glycol, sorbitol, or sucrose. Such formulations may also contain a demulcent, and preservative, flavoring and coloring agents.
[119] The peptides of this invention may also be administered parenterally, that is, subcutaneously, intravenously, intramuscularly, or interperitoneally, as injectable dosages of the peptide in a physiologically acceptable diluent with a pharmaceutical carrier which may be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related sugar solutions; an alcohol such as ethanol, isopropanol, or hexadecyl alcohol; glycols such as propylene glycol or polyethylene glycol; glycerol ketals such as 2,2-dimethyl-1,1-dioxo!ane-4-methanol, ethers such as poly(ethyleneglycol) 400; an oil; a fatty acid; a fatty acid ester or glyceride; or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pectin, carbomers, methycellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agent and other pharmaceutical adjuvants.
[120] Illustrative of oils which can be used in the parenteral formulations of this invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil. 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 include cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates; anionic detergents, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; nonionic detergents, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers; and amphoteric detergents, for example, alkyl- beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, as well as mixtures.
[121] The parenteral compositions of this 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 non-ionic surfactant having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulation 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.
[122] Illustrative of surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
[123] The pharmaceutical compositions may be in the form of sterile injectable aqueous suspensions. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents which may be a naturally occurring phosphatide such as lecithin, a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate, a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol, a condensation product of ethylene oxide with a partial ester derived form a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product of an ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, for example polyoxyethylene sorbitan monooleate.
[124] The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Diluents and solvents that may be employed are, for example, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as solvents or suspending media. 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.
[125] A composition of the invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are, for example, cocoa butter and polyethylene glycol.
[126] Another formulation employed in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the peptides of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art (see, e.g., U.S. Patent No! 5,023,252, incorporated herein by reference). Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
[127] Another formulation employs the use of biodegradable microspheres that allow controlled, sustained release of the peptides and PEGylated peptides of this invention. Such formulations can be comprised of synthetic polymers or copolymers. Such formulations allow for injection, inhalation, nasal or oral administration. The construction and use of biodegradable microspheres for the delivery of pharmaceutical agents is well known in the art (e.g., US Patent No. 6, 706,289, incorporated herein by reference).
[128] 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 well known in the art. For example, direct techniques for administering a drug directly to the brain usually involve placement of a drug delivery catheter into the patient's ventricular system to bypass the blood-brain barrier. One such implantable delivery system, used for the transport of agents to specific anatomical regions of the body, is described in U.S. Patent No. 5,011 ,472, incorporated herein by reference.
[129] The compositions of the invention may also contain other conventional pharmaceutically acceptable compounding ingredients, generally referred to as carriers or diluents, as necessary or desired. Any of the compositions of this invention may be preserved by the addition of an antioxidant such as ascorbic acid or by other suitable preservatives. Conventional procedures for preparing such compositions in appropriate dosage forms can be utilized.
[130] Commonly used pharmaceutical ingredients which may be used as appropriate to formulate the composition for its intended route of administration include: acidifying agents, for example, but are not limited to, acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid; and alkalinizing agents such as, but are not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine.
[131] The peptides identified by the methods described herein may be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutical agents where the combination causes no unacceptable adverse effects. For example, the peptides of this invention can be combined with known anti-diabetic, or with known anti-obesity, cardiovascular or other indication agents, and the like, as well as with admixtures and combinations thereof. [132] The peptides identified by the methods described herein may also be utilized, in free base form or in compositions, in research and diagnostics, or as analytical reference standards, and the like. Therefore, the present invention includes compositions which are comprised of an inert carrier and an effective amount of a peptide of the present invention. An inert carrier is any material which does not interact with the peptide to be carried and which lends support, means of conveyance, bulk, traceable material, and the like to the peptide to be carried. An effective amount of peptide is that amount which produces a result or exerts an influence on the particular procedure being performed.
[133] Formulations suitable for subcutaneous, intravenous, intramuscular, and the like; suitable pharmaceutical carriers; and techniques for formulation and administration may be prepared by any of the methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20th edition, 2000)
[134] Peptides are known to undergo hydrolysis, deamidation, oxidation, racemization and isomerization in aqueous and non-aqueous environment. Degradation such as hydrolysis, deamidation or oxidation can readily detected by capillary electrophoresis. Enzymatic degradation notwithstanding, peptides having a prolonged plasma half-life, or biological resident time, should, at minimum, be stable in aqueous solution. For example, a peptide exhibits less than 10% degradation over a period of one day at body temperature or less than 5% degradation over a period of one day at body temperature. Stability (i.e., less than a few percent of degradation) over a period of weeks at body temperature will allow less frequent dosing. Stability in the magnitude of years at refrigeration temperature will allow the manufacturer to present a liquid formulation, thus avoid the inconvenience of reconstitution. Additionally, stability in organic solvent would provide peptide be formulated into novel dosage forms such as implant.
[135] The structures, materials, compositions, and methods described herein are intended to be representative examples of the invention, and it will be understood that the scope of the invention is not limited by the scope of the examples. Those skilled in the art will recognize that the invention may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the invention.
[136] The following examples are presented to illustrate the invention described herein, but should not be construed as limiting the scope of the invention in any way.
EXAMPLES
[137] In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner. All publications mentioned herein are incorporated by reference in their entirety. Example 1. Preparation of N-terminal Modifying Compounds
[138] Air and moisture sensitive liquids and solutions were transferred via syringe or cannula, and introduced into reaction vessels through rubber septa. Commercial grade reagents and solvents were used without further purification. The term "concentration under reduced pressure" refers to use of a Buchi rotary evaporator at approximately 15 mm of Hg. All temperatures are reported uncorrected in degrees Celsius (0C). Thin layer chromatography (TLC) was performed on EM Science pre-coated glass-backed silica gel 60 A F-254 250 μm plates. Column chromatography (flash chromatography) was performed on a Biotage system using 32-63 micron, 60 A, silica gel pre-packed cartridges. Purification using preparative reversed-phase HPLC chromatography were accomplished using a Gilson 215 system and a YMC Pro-C18 AS-342 (150 x 20 mm LD.) column. Typically, the mobile phase used was a mixture of H2O (A) and MeCN (B). The water could be mixed or not with 0.1 % TFA. A typical gradient was:
Figure imgf000038_0001
[139] Electron impact mass spectra (EI-MS or GC-MS) were obtained with a Hewlett Packard 5989A mass spectrometer equipped with a Hewlett Packard 5890 Gas Chromatograph with a J & W DB-5 column (0.25 uM coating; 30 m x 0.25 mm). The ion source was maintained at 2500C and spectra were scanned from 50-800 amu at 2 sec per scan. High pressure liquid chromatography- electrospray mass spectra (LC-MS) were obtained using a Hewlett-Packard 1100 HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (2 x 23 mm, 120A), and a Finnigan LCQ ion trap mass spectrometer with electrospray ionization. Spectra were scanned from 120-1200 amu using a variable ion time according to the number of ions in the source. The eluents were A: 2% acetonitrile in water with 0.02% TFA and B: 2% water in acetonitrile with 0.018% TFA. Gradient elution from 10% to 95% B over 3.5 minutes at a flowrate 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 run time was 6.5 minutes. For consistency in characterization data, the retention time (RT) is reported in minutes at the apex of the peak as detected by the UV-Vis detector set at 254 nm.
[140] Routine one-dimensional NMR spectroscopy was performed on 300 or 400 MHz Varian Mercury-plus spectrometers. The samples were dissolved in deuterated solvents obtained from Cambridge Isotope Labs, and transferred to 5 mm ID Wilmad NMR tubes. The spectra were acquired at 293 K. The chemical shifts were recorded on the ppm scale and were referenced to the appropriate residual solvent signals, such as 2.49 ppm for DMSO-d6, 1 -93 ppm for CD3CN, 3.30 ppm for CD3OD, 5.32 ppm for CD2CI2, and 7.26 ppm for CDCI3 for 1H spectra, and 39.5 ppm for DMS0-d6, 1 -3 ppm for CD3CN, 49.0 ppm for CD3OD, 53.8 ppm for CD2CI2, and 77.0 ppm for CDCI3 for 13C spectra. General methods of preparation are illustrated in the reaction schemes, and by the specific preparative examples that follow.
Abbreviations and Acronyms
[141] When the following abbreviations are used throughout the disclosure, they have the following meaning:
Ac acetyl
AcOH acetic acid
Boc f-butoxycarbonyl
Bu butyl
CDCI3 deuterochloroform
Celite® registered trademark of Celite Corp. brand of diatomaceous earth
Cl chemical ionization d doublet dd doublet of doublet ddd doublet of doublet of doublet
DME dimethoxyethane
DMF Λ/,/V-dimethyl formamide
DMSO dimethylsulfoxide
DMS0-d6 dimethylsulfoxide-c/6 dppf 1 ,1 '-bis(diphenylphosphino)ferrocene
El 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 hour(s)
1H NMR proton nuclear magnetic resonance
Hex hexanes
HPLC high performance liquid chromatography
LC-MS liquid chromatography/mass spectroscopy
LDA lithium diisopropylamide m multiplet
M molar m/z mass over charge
Me methyl
MeCN acetonitrile mg milligram
MHz megahertz min minute(s) mol mole mmol millimole
MS mass spectrometry
N normal
NMR nuclear magnetic resonance
NaOAc sodium acetate
Pd/C palladium on carbon
PdCI2(dppf). CH2CI2 [1 ,1 '-bis(diphenylphosphino)ferrocene] dichloropalladium (II) complex with dichloromethane (1 :1)
Ph phenyl
PPh3 triphenylphosphine ppm parts per million
Pr propyl q quartet qt quintet quant. quantitative
Rf TLC retention factor rt room temperature
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 1 A
[142] Preparation of (2-Mercapto-1H-benzimidazol-1-yI)acetic acid
Figure imgf000041_0001
[143] Step 1. Preparation of Methyl (2-chloro-1 H-benzimidazoI- 1-yl)acetatθ
Figure imgf000041_0002
[144] NaH (157.3 mg of a 60% dispersion in mineral oil, 3.93 mmol) was suspended in dry DMF (5 mL) and hexanes (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 overnight at rt. The reaction mixture was then diluted with water, and the resulting tan precipitate was collected by filtration, triturated with ether, and dried, yielding 385 mg (52%) of crude material. 1H NMR (400 MHz, CD3CN) δ 3.78 (s, 3H), 5.03 (s, 2H), 7.28-7.38 (m, 2H), 7.43 (d, 1 H), 7.65 (d, 1 H).
[145] Step 2. Preparation of Methyl (2-mercapto-1H-benzimidazol-1-yl)acetate
Figure imgf000041_0003
[146] 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 in ethanol (30 mL) overnight. The reaction mixture was concentrated, and the residue was triturated with water and dried, yielding 142.5 mg (96%) of crude product. 1H NMR (400 MHz, CD3CN) δ 3.78 (s, 3H), 5.03 (s, 2H), 7.20-7.35 (m, 4H), 10.43 (bs, 1 H).
[147] Step 3. Preparation of (2-Mercapto- 1 H-benzimidazol- 1-yl)acetic acid
Figure imgf000041_0004
[148] Methyl (2-mercapto-1H-benzimidazol-1-yl)acetate (143 mg, 0.64 mmol) was dissolved in THF (1mL), 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 h. The pH was then adjusted to pH 4 with 1 N HCI. The solution was diluted with water and extracted with 5% EtOH/EtOAc. The organics were dried over MgSO4 and concentrated in vacuo, yielding 75.0 mg (56%) of the desired product. LC/MS m/z 209.1 (M+H)+; RT 1.08 min. 1H NMR (400 MHz, CD3CN) δ 5.03 (s, 2H), 7.20-7.35 (m, 4H), 10.43 (bs, 1H).
EXAMPLE 1 B [149] Preparation of Lithium 2-{[2-(tritylthio)ethyl]amino}nicotinate
Figure imgf000042_0001
[150] Step 1. Preparation of Ethyl 2-{[2-(tritylthio)ethyl]amino}nicotinate
Figure imgf000042_0002
[151] Ethyl 2-chloronicotinate (100 mg, 0.54 mmol) was combined with 2-(tritylthio)ethanamine (344 mg, 1.08 mmol), cesium carbonate (438 mg, 1.35 mmol), and [1 ,1'-bis(diphenylphosphino) ferrocene]dichloropalladium(ll) complex with dichloromethane (1 :1) (110 mg, 0.13 mmol). The solids were dissolved in dioxane (2 mL), water (1 mL), and heated to reflux over 48 h. The reaction mixture was then diluted with EtOAc and washed with water and brine. The organics were dried over Na2SO4 and concentrated in vacuo. The crude residue was purified by Biotage column chromatography (10% EtOAc/hexane), yielding 215 mg (85%) of the desired product. 1H NMR (400 MHz, CD2CI2) δ 1.40 (t, 3H), 2.45 (t, 2H), 3.38 (t, 2H), 4.37 (q, 2H), 7.03 (m, 1 H), 7.18- 7.45 (m, 16H), 8.18 (d, 1 H), 8.45 (d, 1H). [152] Step 2. Preparation of Lithium 2-{[2-(tritylthio)θthyl]amino}nicotinate
Figure imgf000043_0001
[153] Ethyl 2-{[2-(tritylthio)ethyl]amino}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 800C for 4 h. 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 extracts were dried over MgSO4 and concentrated in vacuo, yielding 55.0 mg (27%) of the desired product. LC/MS m/z 440.7 (M+H)+; RT 2.17 min. 1H NMR (400 MHz, CD3CN) 52.38 (t, 2H), 3.32 (t, 2H), 7.08 (m, 1H), 7.18-7.45 (m, 16H), 8.19 (d, 1 H), 8.45 (d, 1 H).
EXAMPLE 1C
[154] Preparation of Lithium 1-[2-(tritylthio)ethyl]-1H-imidazole-2-carboxylate
Figure imgf000043_0002
[155] Step 1. Preparation of 1, 1 ', 1 "-{[(2-bromoethyl)thio]methanetriyl}tribenzene
Figure imgf000043_0003
[156] Triphenylmethylmercaptan (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 min. 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 min and was concentrated in vacuo. The crude residue was dissolved in ethyl acetate and washed with water and brine. The organics were dried over Na2SO4 and concentrated, yielding 3.44 g crude material (80% pure by 1H NMR integration). This material was used without further purification. 1H NMR (400 MHz, CD2CI2) δ 2.75 (t, 2H), 2.90 (t, 2H), 7.20-7.45 (m, 18.6H).
[157] Step 2. Preparation of 1-[2-(Tritylthio)ethyl]-1H-imidazole
Figure imgf000044_0001
[158] NaH (70.5 mg of a 60% suspension in mineral oil, 1.76 mmol) was suspended in dry DMF (3 mL) and hexanes (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 at rt overnight. The reaction mixture was diluted with water and extracted with EtOAc. The organics were washed with water and brine and dried over Na2SO4. The crude product was purified by Biotage column chromatography (50% EtOAc/hexane, 1% Et3N), yielding 280 mg (51%) of the desired product. 1H NMR (400 MHz, CD2CI2) 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).
[159] Step 3. Preparation of Lithium 1-[2-(tritylthio)ethyl]-1H-imidazole-2-carboxylate
Figure imgf000044_0002
[160] 1 -[2-(Tritylthio)ethyl]-1 H-imidazole (140 mg, 0.38 mmol) was dissolved in dry dichloromethane (1.5 mL) and treated with trichloroacetyl chloride (0.06 mL, 0.57 mmol) and N1N- diisopropylethylamine (0.07 mL, 0.42 mmol). The reaction mixture was allowed to stir overnight at rt. 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 h. The crude reaction mixture was then concentrated in vacuo. Purification by HPLC gave 70 mg (44%) of the desired product. LC/MS m/z414.9 (M+H)+; RT 2.76 min. 1H NMR (400 MHz, CD3OD) .δ 2.68 (t, 2H), 4.18 (t, 2H), 6.72 (s, 1 H), 6.85 (s, 1H), 7.19-7.40 (m, 15H). EXAMPLE 1 D
[161] Preparation of Lithium 4-{[2-(tritylthio)ethyl]amino}pyrimidine-5-carboxylate
[162]
Figure imgf000045_0001
[163] Diethyl malonate (3.14 mL, 20.7 mmol) was combined with N,N',N"- methylidynetrisformamide (3.00 g, 20.7 mmol), and p-toluenesulfonic acid (356 tng, 2.07 mmol), and the reaction mixture was heated to 180°C for 4 h. The resulting red oil was allowed to cool to rt overnight. The crude reaction mixture was dissolved in a minimum amount of water and allowed to crystallize overnight. The solids were collected by filtration, washed with water, and dried, yielding 822 mg (24%) of the desired product. 1H NMR (400 MHz, CD3OD) δ 1.38 (t, 3H), 4.32 (q, 2H), 8.32 (s, 1 H), 8.60 (s, 1 H).
[164] Step 2. Preparation of ethyl 4-chloropyrimidine-5-carboxylate
N ~ .CO2B
SA.
[165] 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 h and then concentrated in vacuo, yielding 417 mg (99%) of the crude product. This material was used without purification or characterization.
[166] Step 3. Preparation of ethyl 4-{[2-(tritylthio)ethyl]amino}pyrimidine-5-carboxylate
Figure imgf000045_0002
[167] Ethyl 4-chloropyrimidine-5-carboxylate (200 mg, 1.07 mmol) was dissolved in THF (2 ml_), and 2-(tritylthio)ethanamine (514 mg, 1.61 mmol) and N,N-diisopropylethylamine (0.56 ml, 3.22 mmol) were added. The reaction mixture was heated to reflux overnight and then concentrated in vacuo. The crude residue was purified by Biotage column chromatography, yielding 230 mg (46%) product. Product confirmed by HNMR. 1H NMR (400 MHz, CD2CI2) § 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, 1 H).
[168] Step 4. Preparation of Lithium 4-{[2-(tritylthio)ethyl]amino}pyrimidine-5-carboxylate
Figure imgf000046_0001
[169] 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 mmo!) and allowed to stir at rt for two days. The crude reaction mixture was then diluted with water and extracted with EtOAc. The organic extracts were dried over Na2SO4 and concentrated in vacuo, yielding 180 mg (82%) of the desired product. LC/MS m/z441.6 (M+H)+; RT 2.54 min. 1H NMR (400 MHz, CD3OD) δ 2.43 (t, 2H)1 3.32 (m beneath solvent peak), 7.10-7.42 (m, 1H), 8.70 (d,2H).
EXAMPLE 1 E [170] Preparation of Lithium 1-[2-(tritylthio)ethyl]-1H-benzimidazole-2-carboxylate
Figure imgf000046_0002
[171] Step 1. Preparation of ethyl 1 H-benzimidazole-2-carboxylate
Figure imgf000046_0003
[172] 1 H-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, yielding 644 mg (99%) of the crude product. 1H NMR (400 MHz, CD2CI2) δ 1.32 (t, 3H), 4.38 (q, 2H), 7.30 (m, 2H), 7.63 (m, 2H).
[173] Step 2. Preparation of ethyl 1 -(2-bromoethyl)- 1 H-benzimidazole-2-carboxylate
Figure imgf000047_0001
[174] NaH (504 mg of a 60% suspension in mineral oil, 1.07 mmol) was suspended in dry DMF (1.5 mL). Ethyl 1 H-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 extracts were dried over Na2SO4, concentrated in vacuo, and purified by Biotage column chromatography (15% EtOAc/hexanes), yielding 100 mg (38%) of the desired product. 1H NMR (400 MHz, CD2CI2) δ 1.48 (t, 3H), 3.80 (t, 2H), 4.50 (q, 2H), 5.02 (t, 2H), 7.39 (t, 1 H), 7.45 (t, 1H), 7.53 (d, 1 H)1 7.87 (d, 1 H).
[175] Step 3. Preparation of ethyl 1 -[2-(tritylthio)βthyl]-1 H-benzimidazole-2-carboxylate
Figure imgf000047_0002
[176] A solution of ethyl 1-(2-bromoethyl)-1 H-benzimidazole-2-carboxylate (100 mg, 0.34 mmol), triphenylmethylmercaptan (112 mg, 0.40 mmol), and N,N-diisopropylethylamine (0.07 ml, 0.40 mmol) in THF (1 mL) were allowed to stir for 2 h at rt. In a separate flask, a solution of triphenylmethylmercaptan (112 mg, 0.40 mmol) in THF (1 mL) was treated with lithium hexamethyldisilazide (0.40 mL of a 1M solution in THF) and allowed to stir for 10 min at rt. This solution was added to the original reaction mixture, immediately resulting in a red solution that was allowed to stir at rt overnight. The reaction mixture was concentrated in vacuo, and the crude residue was purified by Biotage column chromatography, yielding 80 mg (48%) of the desired product. 1H NMR (400 MHz, CD2CI2) § 1.44 (t, 3H), 2.78 (t, 2H), 4.40-4.52 (m, 4H), 7.03 (m, 1 H), 7.20-7.40 (m, 15H), 7.42 (m, 1H), 7.82 (m, 1 H). [177] Step 4. Preparation of Lithium 1-[2-(tritylthio)ethyl]-1H-benzimidazole-2-carboxylate
Figure imgf000048_0001
[178] Ethyl 1 -[2-(tritylthio)ethyl]-1 H-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 extracts were dried over Na2SO4 and concentrated in vacuo, yielding 74 mg (99%) of the desired product. LC/MS m/z 464.9 (Mn-H)+; RT 3.16 min. 1H NMR (400 MHz, CD3OD) δ 2.72 (t, 2H), 4.58 (t, 2H), 6.98 (m, 1 H), 7.15-7.30 (m, 16H), 7.38 (d, 1 H), 7.63 (m, 1H).
EXAMPLE 1 F [179] Preparation of (2-Mercapto-1H-imidazol-1-yl)acetic Acid
Figure imgf000048_0002
[180] Step 1. Preparation of ethyl N-(2,2-dimethoxyethyl)glycinate hydrochloride
Figure imgf000048_0003
HCI
[181] Bromoacetaldehyde dimethylacetal (0.85 mL, 7.2 mmol) was added to a solution of glycine ethyl ester hydrochloride (500 mg, 3.58 mmol) and N,N-diisopropylethyIamine (1.37 mL, 7.88 mmol) in THF (4mL) and EtOH (1 mL). 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 extracts were dried over Na2SO4 and concentrated in vacuo. The crude residue was treated with 1 N HCI in ether, and the resulting precipitate was collected by filtration and dried, yielding 371 mg (23%) of the desired product. 1H NMR (400 MHz, CD3CN) 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). [182] Step 2. Preparation of ethyl (2-mercapto- 1 H-imidazol- 1 -yl)acetatβ
Figure imgf000049_0001
[183] Ethyl N-(2,2-dimethoxyethyl)glycinate hydrochloride (370 mg, 1.63 mmol) was dissolved in EtOH (2 mL) and treated with a solution of potassium thiocyanate (237 mg, 2.44 mmol) in EtOH (8 mL). The pink suspension was heated to reflux overnight. Concentrated HCI (0.136 mL, 1.63 mmol) was added, and the solution was allowed to reflux for 3 h. The reaction mixture was concentrated in vacuo, and the resulting solid was recrystalized from EtOAc, yielding 130 mg (43%) of the desired product. 1H NMR (400 MHz, CD3CN) δ 1.33 (t, 3H), 4.20 (q, 2H), 4.77 (s, 2H), 6.77 (s, 1H), 6.83 (s, 1 H), 9.92 (bs, 1H).
[184] Step 3. Preparation of (2-Mercapto- 1 H-imidazol- 1 -yl)acetic acid
Figure imgf000049_0002
[185] Ethyl (2-mercapto-1 H-imidazol-1-yl)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 HCI, diluted with water, and extracted with 5:1 EtOAc/EtOH. The organic extracts were dried over Na2SO4 and concentrated in vacuo, yielding 70 mg (63%) of the desired product. LC/MS m/z 159.1 (M+H)+; RT 1.05 min. 1H NMR (400 MHz, CD3OD) δ 4.82 (s, 2H), 6.82 (s, 1H), 6.99 (s,
1H).
EXAMPLE 1G
[186] Preparation of Lithium ({1-[2-(tritylthio)ethyl]-1H-imidazol-2-yl}thio)acetate
Figure imgf000049_0003
[187] Step 1. Preparation of methyl (1H-imidazol-2-ylthio)acetate
Figure imgf000050_0001
[188] 2-Thioimidazole (300 mg, 3.00 mmol) was dissolved in THF (3 mL), treated with N1N- diisopropylethylamine (0.63 mL, 3.59 mmol) and methyl bromoacetate (0.31 mL, 3.3 mmol), and allowed to stir at rt for 1 h. The solid precipitate was filtered off, and the filtrate was diluted with EtOAc. The organics were washed with water, dried over Na2SO4 and concentrated in vacuo, yielding 385 mg (75%) of the desired product. 1H NMR (400 MHz, CD3CN) δ 3.68 (s, 3H), 3.82 (s, 2H), 7.05 (s, 2H).
[189] Step 2. Preparation of Methyl ({1-[2-(tritylthio)ethyl]-1H-imidazol-2-yl}thio)acetate
Figure imgf000050_0002
[190] NaH (55.7 mg of a 60% suspension in mineral oil, 1.39 mmol) was suspended in dry DMF (1.5 mL). Methyl (1 H-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 and extracted with EtOAc. The organic extracts were washed with water and brine, dried over MgSO4, and concentrated in vacuo. The crude residue was purified by Biotage column chromatography (20% EtOAc/hexanes), yielding 180 mg (33%) of the desired product. 1H NMR (400 MHz, CD2CI2) δ 2.60 (t, 2H), 3.65 (m, 5H), 3.78 (s, 2H), 6.69 (s, 1 H), 6.96 (s, 1H), 7.20-7.40 (m, 15H).
[191] Step 3. Preparation of Lithium ({1-[2-(tritylthio)ethyl]-1H-imidazol-2-yl}thio)acetate
Figure imgf000050_0003
[192] Methyl ({1-[2-(tritylthio)ethyl]-1H-imidazol-2-yl}thio)acetate (180 mg, 0.38 mmol) was dissolved in THF (1 mL), MeOH (1 ml_), and water (0.5 mL). 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 extracts were dried over Na2SO4 and concentrated in vacuo. Purification of the crude material by HPLC gave 61 mg (34%) of the desired product. LC/MS m/z 460.9 (M+H)+; RT 2.81 min. 1H NMR (400 MHz, CD3OD) δ 2.59 (t, 2H), 3.60 (s, 2H), 3.92 (t, 2H)-, 6.89 (s, 1 H), 6.92 (s, 1H), 7.19-7.35 (m, 15H).
EXAMPLE 1 H
[193] Preparation of Lithium 3-(2-tritylsulfanylethylamino)pyrazine-2-carboxylate
Figure imgf000051_0001
[194] Step 1. Preparation of 2-tritylsulfanylethylamine
Figure imgf000051_0002
[195] 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 for 30 min under nitrogen. 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 allowed to warm to rt and stir for 16 h. The reaction mixture was quenched with water (15 mL). The mixture was washed successively with 1 N HCI (2 x 10 mL), water (2 x 10 mL), 15 % ammonia solution (4 mL), and water (5 x 20 mL). The organics were dried over Na2SO4 and concentrated in vacuo to give 1.9 g (61%) of a light brown oil. 1H NMR (400 MHz, CDCI3) 87.70-7.18 (m, 15H), 2.71 (t, 2H), 2.4 (b, 2H).
[196] Step 2. Preparation of 3-bromopyrazine-2-carboxylic acid methyl ester
Figure imgf000051_0003
[197] Bromine (3.91 g, 24.46 mmol) was added dropwise 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) at 00C. 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 min, brought to pH 8 with NaHCO3 (saturated, aqueous), and extracted with ethyl acetate (80 mL) and chloroform (50 mL). The combined organics were dried over MgSO4 and concentrated in vacuo to give 1.13 g (63%) of an orange oil which solidified on standing. LC-MS- m/z 217 (M+H+); RT 1.15 min.
[198] Step 3. Preparation of 3-(2-tritylsulfanylethylamino)pyrazine-2-carboxylic acid methyl ester
Figure imgf000052_0001
[199] A solution of 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, 0.44 mmol) in acetonitrile (5 mL) was heated to reflux for 18 h under argon. The mixture was concentrated under reduced pressure and purified by column chromatography (3:1 Hex:EtOAc), yielding 0.058 g (28%) of the desired product. 1H NMR (400 MHz, CD2CI2) δ 7.95 (s, 1 H), 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).
[200] Step 4. Preparation of lithium 3-(2-tritylsulfanylethylamino)pyrazine-2-carboxylate
Figure imgf000052_0002
[201] A mixture of 3-(2-tritylsulfanylethylamino)pyrazine-2-carboxylic acid methyl ester (0.12 g, 0.27 mmol) and LiOH (0.030 g, 1.3 mmol) in THF (5 mL), methanol (5 mL), and water (2.5 mL) was stirred at rt for 18 h. The reaction mixture was concentrated and purified by HPLC, yielding 0.075 g (63%) of the desired product. 1H NMR (400 MHz, CD3OD) δ 8.26 (s, 1 H), 8.15 (s, 1 H), 7.42-7 (m, 15H), 3.30 (t, 2H), 2.40 (t, 2H). EXAMPLE 11 [202] Preparation of Λ-mercaptothiazole-δ-carboxylic acid
Figure imgf000053_0001
[203] Step 1. Preparation of 4-(2-methoxycarhonylethylsulfanyl)thiazole-5-carboxylic acid ethyl ester
Figure imgf000053_0002
[204] A solution of ethyl isocyanoacetate (0.92 g, 7.7 mmol) in THF (8 ml_) was added dropwise to a suspension of potassium tert-butoxide (1.0 g, 8.5 mmol) in THF (6 ml.) at -40°C. The mixture was cooled to -600C1 and a solution of carbon disulfide (0.59 g, 7.7 mmol) in THF (8 mL) was added dropwise while keeping the temperature below -500C. The mixture was warmed to 100C and methyl 3-bromopropionate (1.33 g, 7.70 mmol) was added. The mixture was allowed to stir for 2 h and was concentrated in vacuo. The product was recrystalized from dichloromethane/hexanes to give 1.28 g (60%) of the desired product as a white solid. LC-MS m/z276 (M+H+); RT 1.65 min.
[205] Step 2. Preparation of ^mercaptothiazole-δ-carboxylic acid ethyl ester
Oγθv^
Figure imgf000053_0003
[206] Sodium hydroxide (0.14 g, 3.5 mmol) was added to a solution of 4-(2- methoxycarbonylethylsulfanyOthiazole-δ-carboxylic acid ethyl ester (0.96 g, 3.5 mmol) in methanol (13.6 mL). The mixture was refluxed for 1 h and then concentrated in vacuo. The residue was taken up in ethyl acetate/water and the pH adjusted to 2 with 2 N HCI. The organic layer was isolated and concentrated in vacuo, yielding 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.
[207] Step 3. Preparation of4-mercaptothiazole-δ-carboxylic acid
Figure imgf000053_0004
[208] 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 800C for 3 h. Upon cooling to rt, the reaction mixture was concentrated in vacuo. The residue was acidified with 2 N HCI and extracted with dichloromethane. The organic layer was dried over MgSO4, concentrated in vacuo, and purified by HPLC to give 0.059 g (12%) of the desired product. 1H NMR (400 MHz, CD3OD) δ 8.95 (s, 1 H); LC-MS m/z 162 (M+H+); RT 1.01 min.
EXAMPLE U
[209] Preparation of2-mercaptothiazole-5-carboxylic acid
Figure imgf000054_0001
[210] Step 1. Preparation of2-mercaptothiazole-5-carhoxylic acid methyl ester
Figure imgf000054_0002
[211] 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 h. 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.
[212] Step 2. Preparation of2-mercapiothiazole-5-carboxylic acid
Figure imgf000054_0003
[213] 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 2 N HCI and the resulting suspension was filtered to give 0.061 g (39%) of the desired product as an off-white solid. 1H NMR (400 MHz, CD3OD) 57.80 (s, 1 H); LC-MS mfc 161 (M+H+); RT 1.02 min.
EXAMPLE 1 K
[214] Preparation of Lithium 2-(2-tritylsulfanylethylamino)thiazole-5-carboxylate
Figure imgf000054_0004
[215] Step 1. Preparation of 2-(2-tritylsulfanyl-ethylamino)-thiazole-5-carboxylic acid methyl ester
Figure imgf000055_0001
[216] A solution of 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, 0.88 mmol) in acetonitrile (5 mL) was heated to reflux for 18 h under argon. 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. 1H NMR (400 MHz, CD2CI2) δ 8.15 (s, 1H), 7.60-7.15 (m, 15H), 4.20 (s, 3H), 3.45(t, 2H), 2.50 (br, 2H).
[217] Step 2. Preparation of lithium 2-(2-tritylsulfanylethylamino)-thiazole-5-carboxylate
Figure imgf000055_0002
[218] A mixture of 2-(2-tritylsulfanylethylamino)-thiazole-5-carboxylic acid methyl ester (0.12 g, 0.26 mmol) and LiOH (0.03 g, 1.3 mmol) in THF (5 mL), methanol (5 mL) and water (2.5 mL) was stirred at rt for 18 h. The reaction mixture was concentrated in vacuo and purified by HPLC to give 0.087 g (75%) of the desired product. 1H NMR (400 MHz, CD2CI2) 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
[219] Preparation of2-mercapto-6-methylpyrimidine-4-carboxylic acid
Figure imgf000055_0003
[220] Step 1. Preparation of2-carbamimidoylsulfanyl-6-methylpyrimidine-4-carboxylic acid methyl ester hydrobromide
Figure imgf000056_0001
[221] 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 h and then concentrated in vacuo. Attempt to purify the product by recrystalization (methanol/ether) gave 0.229 g (38%) of an oil which was confirmed as the desired product. LC-MS m/z 226.9 (M+H+); RT 1.07 min.
[222] Step 2. Preparation of2-mercapto-6-methylpyrimidine-4-carboxylic acid
Figure imgf000056_0002
[223] A mixture of 1 N sodium hydroxide (6.98 mL, 6.98 mmol) and 2-carbamimidoylsulfanyl-6- methylpyrimidine-4-carboxylic acid methyl ester hydrobromide (0.37 g, 1.4 mmol) was heated to reflux for 2 h. Upon cooling to rt, the reaction mixture was concentrated in vacuo. The residue was taken up in water and MP-TsOH (Argonaut technologies) was added. The mixture was stirred for 18 h until a pH of 4 was achieved. The mixture was filtered, and the filtrate was concentrated in vacuo. The residue was taken up in methanol, filtered, and the filtrate was concentrated in vacuo to give 0.139 g (59%) of the desired product as a brownish solid. 1H NMR (400 MHz, CD3OD) δ 7.10 (s, 1 H), 3.40 (s, 3H); LC-MS m/z 171 (M+H+); RT 1.15 min.
EXAMPLE 1 M [224] Preparation of 5-mercaptonicotinic acid
Figure imgf000056_0003
[225] Step 1. Preparation of δ-tert-butylsulfanylnicotinonitrile
Figure imgf000056_0004
[226] 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 5O0C for 1 h. 5-Bromonicotinonitrile (0.60 g, 3.2 mmol) was added to the resulting suspension, and the reaction mixture was heated at 12O0C for 5 h. Upon cooling to rt, the mixture was concentrated in vacuo and purified by HPLC, yielding 0.329 g (54%) of the desired product as an off-white solid. LC-MS m/z 193 (M+H+); RT 2.90 min.
[227] Step 2. Preparation of 5-tert-butylsulfanylnicotinic acid
Figure imgf000057_0001
[228] 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 h. Upon cooling to rt, the reaction mixture was diluted with water and was extracted with ether. The aqueous layer was acidified with 2 N HCl and was extracted with dichloromethane. The dichloromethane extracts were dried over MgSO4 and concentrated to give 0.433 g (79%) of the desired product as a white solid. LC-MS m/z 212 (M+H+); RT 2.36 min.
[229] Step 3. Preparation 5-mercaptonicotinic acid
Figure imgf000057_0002
[230] A solution of 5-tert-butylsulfanylnicotinic acid (0.30 g, 1.2 mmol) in 2 N HCI (9 mL, 18 mmol) was heated to reflux for 32 h. Upon cooling to rt, the reaction mixture was concentrated in vacuo to give 0.054 g (28%) of the desired product. 1H NMR (400 MHz, CD3OD) δ 9.00-8.80 (m, 2H), 8.40 (d, 1 H); LC-MS m/z 156 (M+H+); RT 2.37 min.
EXAMPLE 1 N
[231 ] Preparation of 5-\sopropy\-2-mercaptothiazoie-4-carboxyiic acid
Figure imgf000057_0003
[232] Step 1. Preparation of2-chloro-4-methyl-3-oxopentanoic acid ethyl ester
Figure imgf000057_0004
[233] A solution of sulfuryl chloride (1.63 mL, 19.7 mmol) in toluene (S mL) was added dropwise to a solution of 4-methyl-3-oxopentanoic acid ethyl ester (3.28 g, 19.7 mmol) in toluene (25 mL) over 10 min. The resulting mixture was stirred at rt for 18 h and then slowly quenched with water and NaHCO3 (saturated, aqueous). The mixture was extracted with ethyl acetate, and the combined organics were dried over MgSO4 and concentrated in vacuo to give 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.
[234] Step 2. Preparation of 2-amino-5-isopropylthiazole-4-carboxylic acid ethyl ester
Figure imgf000058_0001
[235] 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 h and then concentrated in vacuo. The residue was treated with aqueous ammonia, and the resultant yellow solid was taken up in water and extracted with dichloromethane. The combined organics were dried over Na2SO4 and concentrated in vacuo. The solid was taken up in a small amount of dichloromethane and filtered to give 1.02 g (85%) of the desired product as a cream colored solid. LC-MS m/z215.1 (M+H+); RT 1.96 min.
[236] Step 3. Preparation of2-bromo-5-isopropylthia∑ole-4-carboxylic acid ethyl ester
Figure imgf000058_0002
[237] 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-butylnitrite (0.63 g, 5.5 mmol) slowly at rt. The mixture was heated to 600C, 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 h. Upon cooling to rt, the reaction mixture was poured into 1 N NaOH (40 mL) and extracted with ethyl acetate. The combined organics were dried over Na2SO4, 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. LC-MS m/z 280 (M+H+); RT 3.65 min.
[238] Step 4. Preparation of δ-isopropyl-Σ-mercaptothiazole^-carboxylic acid ethyl ester
Figure imgf000058_0003
[239] 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 h. Upon cooling to rt, the resulting suspension was filtered to give 0.11 g (66%) of the desired product as a yellow solid. LC-MS m/z 232.1 (M+H+); RT 2.72 min.
[240] Step 5. Preparation of 5-isopropy\-2-mercaptoMazo\e-4-carboxylic acid
Figure imgf000059_0001
[241] NaOH (1 N, 0.78 mL, 0.78 mmol) was added to a solution of 5-isopropyl-2- mercaptothiazole-4-carboxylic acid ethyl ester (0.09 g, 0.39 mmol) in methanol (3 mL) and water (2 mL), and the mixture was stirred at rt for 3 h. The reaction mixture was concentrated in vacuo, and the residue was acidified with 2 N HCI. The resulting suspension was filtered to give 0.045 g (57%) of the desired product as an off-white solid. 1H NMR (400 MHz, CD3OD) δ 3.85-4.10 (m, 1 H), 1.22 (d, 6H); LC-MS m/z 204.2 (M+H+); RT 1.65 min.
EXAMPLE 10
[242] Preparation of (1-hexadecyl- 1H-benzoimidazol-2~ylsulfanyl)acetic acid
Figure imgf000059_0002
[243] Step 1. Preparation of (1H-benzoimidazol-2-ylsulfanyl)acetic acid ethyl ester
Figure imgf000059_0003
[244] 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 heated to reflux for 8 h 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. [245] Step 2. Preparation of (1-hexadecyl- 1H-henzoimidazol-2-ylsulfanyl)acetic acid methyl ester
Figure imgf000060_0001
[246] Sodium hydride (0.03 g, 0.8 mmol) was added to a solution of (1 H-benzoimidazol-2- ylsulfanyl) acetic acid ethyl ester (0.21 g, 0.79 mmol) in DMF (10 ml.) at O0C, and the mixture was stirred at rt for 1 h. Hexadecylbromide (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 hexanes) gave 0.24 g (67%) of the desired product. LC-MS m/z 447.4 (M+H+); RT 5.03 min.
[247] Step 3. Preparation of (i-hexadecyl-IH-benzoimidazol-Σ-ylsulfanyfyacetic acid
Figure imgf000060_0002
[248] A mixture of lithium hydroxide (0.060 g, 2.4 mmoi) and (1-hexadecyl-1 H-benzoimidazol-2- ylsulfanyl) acetic acid methyl ester (0.21 g, 0.47 mmoi) was heated to reflux for 2 h, cooled to rt, and concentrated in vacuo. The residue was taken up in water, and the suspension was acidified with 1 N HCI and filtered. The solid was collected and dried to give 0.16 g (79%) of the desired product. 1H NMR (400 MHz, CD3OD) 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 1 P
[249] Preparation of Lithium 6-(2-tritylsulfanylethylamino)nicotinate
Figure imgf000060_0003
[250] Step 1. Preparation of 6-(2-tritylsulfanylβthylamino)nicotinic acid methyl ester
Figure imgf000061_0001
[251] Methyl 6-chloronicotiriate (0.20 g, 1.17 mmtil), 2-tritylsulfanylethylamine (0.56 g, 1.75 mmol), cesium carbonate (0.95 g, 2.91 mmol), and [1 ,1'-bis(diphenylphosphino)ferrocene] dichloropalladium(ll) complex with dichloromethane (1 :1) (0.24 g, 0.29 mmol) were heated to 1200C overnight in 1 ,4-dioxane (4.0 ml_) and water. Upon cooling to rt, the reaction mixture was filtered through Celite®, 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, yielding 0.272 g (51%) of the desired product as a white solid. Rf = 0.42 (20% EtOAc/Hexanes). 1H NMR (400 MHz, CD2CI2) δ 2.0 (bs, 1H)1 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, 1 H), 8.90 (s, 1 H).
[252] Step 2. Preparation of Lithium 6-(2-tritylsulfanylethylamino)nicotinate
Figure imgf000061_0002
[253] 6-(2-Tritylsulfany!ethylamino)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/Hexanes (3:1) yielded 236 mg (89%) of the desired product as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 2.25 (t, 2H), 3.00 (bt, 1 H), 3.28 (t, 2H), 7.00-7.40 (m, 16H), 7.90 (d, 1H), 8.72 (s, 1H).
Example 2. Peptide Synthesis
[254] Peptides are synthesized with an Applied Biosystems 430A peptide synthesizer using FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied Biosystems protocols are used. The peptides are cleaved with 84.6% TFA, 4.4% phenol, 4.4% water, 4.4% thioanisol, and 2.2% ethanedithiol. Peptides are precipitated from the cleavage cocktail using cold tertbutylmethyl ether. The precipitate is washed with the cold ether and dried under argon. Peptides are purified with by reversed phase C18 HPLC with linear water/acetonitrile gradients containing 0.1% TFA. Peptide identity is confirmed with MALDI and electrospray mass spectrometry and with amino acid analysis. Example 3. Methods for Adding N-Terminal Modifying Compound
[255] Peptides are synthesized with an Applied Biosystems 430A peptide synthesizer using FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied Biosystems protocols are used. The N-terminal modifying compounds are coupled to the peptide as per a 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 amine and mercapto containing N-terminal modifying compounds, the amine and mercapto groups are protected with FMOC or trityl, respectively, during coupling to the peptide. The peptides are cleaved with 84.6% TFA, 4.4% phenol, 4.4% water, 4.4% thioanisol, and 2.2% ethanedithiol. Peptides are precipitated from the cleavage cocktail using cold tertbutylmethyl ether. The precipitate is washed with the cold ether and dried under argon. Peptides are purified with by reversed phase C18 HPLC with linear water/acetonitrile gradients containing 0.1% TFA. Peptide identity is confirmed with MALDI and electrospray mass spectrometry and with amino acid analysis.
Example 4. Methods for Adding C-terminal Modifying Compound
[256] Peptides are synthesized with an Applied Biosystems 433A peptide synthesizer using FMOC chemistry with HBTU activation on Rink amide resin. Standard Applied Biosystems protocols are used. The HBTU-activated C-terminal modifying compounds are coupled to the resin (e.g., Wang resin for producing peptides with a C-terminal modifying compound containing the free carboxylate or Rink Amide for producing amide variants) as per a natural amino acid coupling during FMOC chemistry. The peptides are then synthesized by the stepwise addition of amino acids using standard FMOC protocols. The peptides are cleaved with 84.6% TFA1 4.4% phenol, 4.4% water, 4.4% thioanisol, and 2.2% ethanedithiol. Peptides are precipitated from the cleavage cocktail using cold tertbutylmethyl ether. The precipitate is washed with the cold ether and dried under argon. Peptides are purified with by reversed phase C18 HPLC with linear water/acetonitrile gradients containing 0.1% TFA. Peptide identity is confirmed with MALDI and electrospray mass spectrometry and with amino acid analysis.
Example 5. Preparation of PEGylated Peptides
[257] PEG derivatives are prepared by incubating methoxypolyethlene glycols derivatized with maledimide for coupling to the mercapto moiety of the N-terminal modifying group. mPEG-MAL or mPEG2-MAL products supplied by Nektar Therapeutics (Huntsville, Al, USA) or GLE-200MA or GLE-400MA products supplied by NOF (Toyko, Japan) are used. Coupling reactions are performed by incubating the peptide and a two-fold molar excess of maleimide-PEG in 50 mM Tris, pH 7 at rt for 2-12 h. The peptide concentration may be 1 mg/ml or less. Underivatized peptides and PEG are purified from the PEGylated peptide with cation exchange chromatography and dialysis or by reversed phase Ci8 HPLC. The purified PEG-peptide conjugate is then freeze dried. Example 6. Preparation of Fatty-acid Derivatized Peptides
[258] The fatty acid (palmitate) derivatives of amine containing N-terminal modifying compounds are prepared as N-terminal modified peptides as described in Example 3 except that prior to deprotection and cleavage the FMOC protecting group of the amine moiety of the N-terminal modifying group was selectively removed with 0.1 % TFA and derivativized with palmitic acid using the same conditions as for a normal amino acid coupling.
[259] The fatty acid derivative can also be prepared as described in Example 3 using 1- hexadecyl-1 H-benzoimidazol-2-ylsulfanyl)acetic acid as the N-terminal modifying group, which was synthesized as described in Example 1.
Example 7. Pharmaceutical Composition - IV and SC Formulations
[260] A sterile IV injectable formulation is prepared with 4 mg of a peptide of Formula (I) or Formula (II), or a derivatized polypetide having equivalent of 4 mg peptide content, and 1 L sterile saline, using any manufacturing process well known in the art. Higher concentrations of peptide may be used for SC formulation. In the case of the peptide of Formula (I) or Formula (II), or a derivatized peptide, 4 mg is dissolved in 100 mL saline or DMSO and sterile vials after aseptic filtration, are filled with the composition.
Example 8. Mass Spectrometr Analysis of Peptides
[261] Forty pmol/2 μl aliquots of peptides are diluted up to 10 μl with water. The HEPES buffer is removed by application of 50% of the sample (20 pmol/5 μl) to a conditioned Millipore C18 ZipTip, as per manufacturers instructions. Samples are eluted from the ZipTip with matrix (10 mg/ml alpha-cyanohydroxycinnamic acid in 50% ACN, 0.1% TFA) directly onto the MALDl plate. Samples are analyzed on an Applied Biosystems Voyager DE-PRO MALDI operated in the reflector ion mode. Data is collected in the 500 - 4000 Da range and resulting masses were compared to those expected by manual calculation.
Example 9. Edman Analysis of Peptides
[262] 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, the HEPES buffer salt is removed by using an Applied Biosystems ProSorb sample cartridge as per manufacturers instructions. Briefly, the sample is applied to a PVDF membrane and washed with 0.1% TFA, then the membrane is removed and inserted into the protein sequencer for Edman degradation. Edman degradation is carried out on an Applied Biosystems Procise 494HT protein sequencing system using the pulsed-liquid method according to manufacturer instructions. Sequences are read manually. Example 10. Stability of Peptides
[263] The formulations described in Example 4 are placed in constant stability chamber. Peptides are also analyzed for stability to degradation in solutions of DPPIV and in plasma. Samples are removed periodically for analysis by capillary electrophoresis, mass spectrometry, Edman degradation, ELISA, and assays of peptide activity, which are sensitive methods to detect degradation of peptide. The area of various peaks is summed and the area for peak of the parent peptide is divided by the total peak area. The quotient is the % purity. Since there are impurities present in the fresh peptide, the purity change is normalized by dividing the purity at different time point by the initial purity. For stability to DPPIV and plasma, peptides at 20 pmol/μl were incubated at 370C in the presence of 300 pM DPPIV in 100 mM HEPES, pH 7.4. At various timepoints, the reaction (2 μl aliquot) is terminated by addition of 1 μM DPPIV inhibitor and freezing. For MALDI mass spectrometric analysis, T = 0 hr, 1 hr, 5 hr, and 24 hr timepoints are evaluated. The results are plotted as percent intact peptide or peptide derivative as compared to degradation products.
Example 11. Binding of Peptides to the GIP Receptor
[264] CHO cells transfected with GIP receptor are Dounce homogenized in 20 mM Tris-HCI, pH 7.4, 5 mM MgCI2, 3 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (Sigma A8456), and 1 μg/ml aprotinin (Sigma A1153). The homogenate is centrifuged at 20k rpm for 20 min at 4°C. The pellet is suspended in 20 mM Tris-HCI, pH 7.4, 1 mM EDTA and 1 μg/ml aprotinin. An aliquot is removed for protein concentration determination by the Bradford assay (BioRad, 500-0006). The suspension is brought to a final concentration of 3-10 mg protein/ml and stored in aliquots at -800C. GF/C filtration 96-well plates (Millipore MAFCNOB) are pretreated with 0.1 ml/well 0.3% polyethyleneamine (Sigma P3143) for 1-2 hours at rt. The plates are washed (2 x 0.1 ml) with ice- cold 20 mM Tris-HCI, pH 7.4, 1 mM EDTA, 0.1% (w/v) BSA (Sigma 8806) and 0.1 % (w/v) bacitracin (Sigma 5150). To each well is added 150 μl binding buffer (20 mM Tris-HCI, pH 7.4, 1 mM EDTA, 0.5% BSA and 0.1% bacitracin) containing 15-25 μg CHO membrane protein and peptide. 125l-labeled GIP (Amersham IM303, 2000 Ci/mmol) is added to a final concentration of 50 pM and the plates incubated for 2 h at rt. The bound from free 125l-labeled GIP is separated with a Milllipore vacuum apparatus. The plates are washed (3 x 0.2 ml) with ice cold PBS containing 0.1% (w/v) BSA. The plates are dried at rt, 0.030 ml Ultima gold (Packard 6013329) is added, and radioactive content isdetermined with a Wallac Microbeta counter.
Example 12. Elevation ofcAMP in Response to Peptides
[265] CHO cells transfected with the GIP receptor are plated on clear bottom white 96-well plates and washed (2 x 0.2 ml) with phosphate buffered saline (PBS). Assay buffer (90 μl: 10 mM HEPES, pH 7.4,150 mM NaCI, 5 mM KCI, 2.5 mM CaCI2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 0.1 mM 3-isobutyl-1-methylxanthine, and 1%(w/v) BSA) are added to each well followed by 10 μl peptide (0.1-100 nM final). The plates are incubated for 15 min at 37°C under 5% CO2. Buffer is removed, the cells lysed, and assayed for cAMP content using an SPA kit (Amersham RPA559). Radioactivity is determined on a Wallac Microbeta counter. Data are fit to a single-site binding model with Prism 3.03 (GraphPad Software, San Diego, California, USA).
Example 13. Insulin Secretion from Dispersed Rat Islet Cells
[266] Insulin secretion of dispersed rat islets mediated by a number of peptides of the present invention is measured as follows. Islets of Langerhans, isolated from SD rats (200-250 g), are digested using collagenase. The dispersed islet cells are treated with trypsin, seeded into 96 V- bottom plates, and pelleted. The cells are then cultured overnight in media with or without peptides of this invention. The media is aspirated, and the cells are pre-incubated with Krebs- Ringer-HEPES buffer containing 3 mM glucose for 30 minutes at 37°C. The pre-incubation buffer is removed, and the cells are incubated at 37°C with Krebs-Ringer-HEPES buffer containing the appropriate glucose concentration (e.g., 8 mM) with or without peptides for an appropriate time. A portion of the supernatant is removed and its insulin content is measured by SPA. The results are expressed as "fold over control" (FOC).
Example 14. Generation of Peptide Specific Antibodies and Peptide Measurement by ELISA
[267] Polyclonal antibodies specific to the peptides of the present invention are generated by synthesizing a specific fragment of a peptide of this invention using an ABI 433A peptide synthesizer. The peptide is then cleaved from the resin, and purified on a Beckman System Gold Analytical and Preparative HPLC system. A Perspective MALDI mass spectrophotometer system is used to identify the correct product. The peptide is dried using a lyophilizer. The peptide (2 mg) is then conjugated to keyhole limpet hemocyanin (KLH) via the free sulphydryl group on the Cys.
[268] Female New Zealand White rabbits are immunized on Day 0, 14, 35, 56, and 77. On Day 0, each rabbit is injected subcutaneous with 250 μg peptide and complete Freund's adjuvant. Subsequent immunizations utilize 125 μg peptide per rabbit. Bleeds are started on Day 21 and continued at 21 -day intervals thereafter. Purification of anti-peptide antibodies is performed by passing the crude serum over a specific peptide affinity purification column. The antibody titer is determined by ELISA.
[269] A 96-well lmmulon 4HBX plate is coated with a C-terminal F(ab) antibody, specific to the peptides of the present invention, and allowed to incubate ovenight at 40C. The plate is then blocked to prevent non-specific binding. Then, peptide standards (2500 ng/mL-160 pg/mL) are diluted in 33% plasma and the samples are diluted 1 :3 in buffer followed by incubation for 1.5 h at rt. After washing, a polyclonal N-terminal antibody specific to the peptides of this invention is incubated on the plate for 1 h. This is followed by the addition of horseradish peroxidase (HRP)- donkey-anti-rabbit antibody and the samples and standards are incubated for another hour. Detection is assessed following incubation with 3,3',5,5'-tetramethylbenzidine (TMB) solution, and the plate is read at OD450 . [270] Alternatively, the 96-well lmmulon 4HBX plate is coated with a polyclonal N-terminal antibody, specific to the peptides of the present invention, and allowed to incubate overnight at 40C. The plate is then blocked to prevent non-specific binding. Then, peptide standards (2500 ng/mL-160 pg/mL) are diluted in 50% plasma and the samples are diluted 1 :2 in buffer followed by incubation for 1.5 h at rt. After washing, a monoclonal anti-PEG antibody specific to the peptides of this invention is incubated on the plate for one hour. This was followed by the addition of horseradish peroxidase (HRR)- anti-mouse antibody and the samples and standards are incubated for another hour. Detection is assessed following incubation with 3,3',5,5'-tetramethylbenzidine (TMB) solution, and the plate is read at OD450.
Example 15. Phamacokinetics of Peptides Following IV and Subcutaneous Dosing
[271] Plasma samples are transferred to a microcentrifuge tube and an equal volume of acetonitrile is added to the sample (a 50% final concentration). The sample is vigorously vortexed for about 5 min and allowed to sit on ice for 10 min. The sample is again vortexed for about 1 min, and then centrifuged for 30 min in a microcentrifuge (40C) at maximum (about 15,000 x g).
[272] Following centrifugation, the aqueous phase is carefully transferred to a clean centrifuge tube, and the sample is centrifuged for 5 min in a microcentrifuge (40C) at maximum speed (about 15,000 x g). The extracted sample is dried under vacuum using a Speed Vac SC110 (Savant) with a medium heat setting until dry. The sample is resuspended in an appropriate volume of sterile water and is maintained at 40C. The sample is then sonicated in a sonibath for 10 min at rt prior to analysis.
Example 16. Effect of Peptides on Intraperitoneal Glucose Tolerance in Rats
[273] The in vivo activity of the peptides of this invention when administered subcutaneously is examined in rats. Rats fasted overnight are given a subcutaneous injection of control or peptide (1-100 μg/kg). Three hours later, basal blood glucose is measured, and the rats are given 2 g/kg of glucose intraperitoneal^. Blood glucose is measured again after 15, 30, and 60 min.
[274] Demonstration of the activity of the peptides of the present invention may be accomplished through in vitro, ex vivo, and in vivo assays that are well known in the art. For example, to demonstrate the efficacy of a pharmaceutical agent for the treatment of diabetes and related disorders such as Syndrome X, impaired glucose tolerance, impaired fasting glucose, and hyperinsulinemia; atherosclerotic disease and related disorders such as hypertriglyceridemia and hypercholesteremia; and obesity, the following assays may be used.
Example 17. Method for Measuring Blood Glucose Levels
[275] db/db mice (obtained from Jackson Laboratories, Bar Harbor, ME) are bled (by either eye or tail vein) and grouped according to equivalent mean blood glucose levels. They are dosed with the test peptide for 14 days. At this point, the animals are bled again by eye or tail vein and blood glucose levels were determined. In each case, glucose levels are measured with a Glucometer Elite XL (Bayer Corporation, Elkhart, IN).
Example 18. Method for Measuring an Effect on Cardiovascular Parameters
[276] Cardiovascular parameters (e.g., heart rate and blood pressure) are also evaluated. SHR rats are dosed with vehicle or test peptide for 2 weeks. Blood pressure and heart rate are determined using atail-cύff 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).
Example 19. Method for Measuring Triglyceride Levels
[277] hApoAI mice (obtained from Jackson Laboratories, Bar Harbor, ME) are bled (by either eye or tail vein) and grouped according to equivalent mean serum triglyceride levels. They are dosed with the test peptide for 8 days. The animals are then bled again by eye or tail vein, and serum triglyceride levels are determined. In each case, triglyceride levels are measured using a Technicon Axon Autoanalyzer (Bayer Corporation, Tarrytown, NY).
Example 20. Method for Measuring HDL-Cholesterol Levels
[278] To determine plasma HDL-cholesterol levels, hApoAI mice are bled and grouped with equivalent mean plasma HDL-cholesterol levels. The mice are dosed with vehicle or test peptide for 7 days, and then bled again on day 8. Plasma is analyzed for HDL-cholesterol using the Synchron Clinical System (CX4) (Beckman Coulter, Fullerton, CA).
Example 21. Method for Measuring Total Cholesterol, HDL-Cholesterol, Triglycerides, and Glucose Levels
[279] In another in vivo assay, obese monkeys are bled, then dosed with vehicle or test peptide for 4 weeks, and then bled again. Serum is analyzed for total cholesterol, HDL-cholesterol, triglycerides, and glucose using the Synchron Clinical System (CX4) (Beckman Coulter, Fullerton, CA). Lipoprotein subclass analysis is performed by NMR spectroscopy as described by Oliver, et al., (Proc. Natl. Acad. Sci. USA 98:5306-5311 , 2001).
[280] All publications and patents mentioned in the above specification are incorporated herein 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 in connection with specific preferred embodiments, it should 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 the field of 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.
TABLE 1
Figure imgf000069_0001
TABLE 1 (cont'd)
Figure imgf000070_0001
TABLE 1 (cont'd)
Figure imgf000071_0001
TABLE 2
Figure imgf000072_0001

Claims

Claims:
1. A peptide of Formula (I)
Z1 -A1 -A2-A3-Gly-Thr-Phe-A7-Ser-Asp-A10-Ser-A12-A13-A14-A15-A16-A17-A18-A19-A20- A21-Phe-A23-A24-Trp-A26-Leu-A28-A29-A30-A31-A32-A33-A34-A35-A36-A37-A38-A39- A40-A41-A42-A43-Z2 (SEQ ID NO: 1) wherein
A1 is any naturally occurring amino acid, any d-amino acid, or deleted;
A2 is any naturally occurring amino acid, any d-amino acid, or deleted;
A3 is any naturally occurring amino acid, any d-amino acid, or deleted;
A7 is lie or Thr;
A10 is Val or Tyr;
A12 is He, Ala, or Ser;
A13 is Ala, lie, or VaI;
A14 is Leu or Met;
A15 is Asp, GIu, Asn, or Lys;
A16 is Lys or GIy;
A17 is He or GIn;
A18 is His, Ala, or Arg;
A19 is GIn, Ala, or GIu;
A20 is GIn, Lys, or GIu;
A21 is GIu, Asp, or Ala;
A23 is lie or VaI;
A24 is Ala, Asn, or Asp;
A26 is Leu or Ala;
A28 is Ala or Ser;
A29 is GIn, VaI, or GIu;
A30 is Lys, Arg, Asp, GIu, Asn, GIn, or Cys;
A31 is GIy, Lys, Cys, GIn, Lys-X, Cys-PEG, or deleted;
A32 is Lys, Cys, GIy, Lys-X, Cys-PEG or deleted;
A33 is Lys, Cys, Lys-X, Cys-PEG or deleted; A34 is Asn, Ser, Lys, Cys, Lys-X, Cys-PEG or deleted; A35 is Asp, Asn, Lys, Cys, Lys-X, Cys-PEG or deleted; A36 is Trp, Lys, Cys, Lys-X, Cys-PEG or deleted; A37 is Lys, lie, Cys, Lys-X, Cys-PEG or deleted; A38 is His, Lys, Cys, Lys-X, Cys-PEG or deleted; A39 is Asn, Asp, Lys, Cys, Lys-X, Cys-PEG or deleted; A40 is lie, Leu, Lys, Cys, Lys-X, Cys-PEG or deleted; A41 is Thr, Ser, lie, Lys, Cys, Lys-X, Cys-PEG or deleted; A42 is GIn, GIu, Lys, Cys, Lys-X, Cys-PEG or deleted; and A43 is Lys-X, Cys-PEG or deleted;
Lys-X is Lys modified at Nε with a fatty acid 21 is selected from
Figure imgf000074_0001
Figure imgf000074_0002
Figure imgf000074_0003
Figure imgf000075_0001
Figure imgf000075_0002
Figure imgf000075_0003
Figure imgf000076_0001
Figure imgf000076_0002
Figure imgf000076_0003
Figure imgf000076_0004
Figure imgf000077_0001
Figure imgf000077_0002
Figure imgf000077_0003
Figure imgf000078_0001
Figure imgf000078_0002
and Z2 is selected from
Figure imgf000079_0001
Figure imgf000079_0002
Figure imgf000079_0003
2. A peptide of Formula (II)
Z1 -A1 -A2-A3-Gly-Thr-Phe-A7-Ser-Asp-A10-Ser-A12-A13-A14-A15-A16-A17-A18-A19-A20- A21-Phe-A23-A24-Trp-A26-Leu-Ala-Gln-Lys-A31-A32-A33-A34-A35-A36-A37-A38-A39- A40-A41-A42-A43-Z2 (SEQ ID NO: 2) wherein A1 is Tyr, His, Ala, or deleted;
A2 is Ala, d-Ala, Tyr, Ser, GIy, VaI, or deleted;
A3 is GIu, d-Glu, Lys, Tyr, Pro, Trp, Phe, Asp, or deleted;
A7 is He or Thr;
A10 is VaI or Tyr;
A12 is He, Ala, or Ser;
A13 is Ala, lie, or VaI;
A14 is Leu or Met;
A15 is Asp, Asn, Lys, or GIu;
A16 is Lys or GIy;
A17 is lie or GIn;
A18 is His, Ala, or Arg;
A19 is GIn, GIu, or Ala;
A20 is GIn, GIu, or Lys;
A21 is GIu, Ala, or Asp;
A23 is He or VaI;
A24 is Ala, Asp, or Asn;
A26 is Leu or Ala;
A31 is GIy, GIn, Lys, Cys, Lys-X, Cys-PEG, or deleted;
A32 is GIy, Lys, Cys, Lys-X, Cys-PEG or deleted;
A33 is Lys, Cys, Lys-X, Cys-PEG or deleted;
A34 is Asn, Ser, Lys, Cys, Lys-X, Cys-PEG or deleted;
A35 is Asp, Asn, Lys, Cys, Lys-X, Cys-PEG or deleted;
A36 is Trp, Lys, Cys, Lys-X, Cys-PEG or deleted;
A37 is Lys, He, Cys, Lys-X, Cys-PEG or deleted;
A38 is His, Lys, Cys, Lys-X, Cys-PEG or deleted;
A39 is Asn, Asp, Lys, Cys, Lys-X, Cys-PEG or deleted;
A40 is lie, Leu, Lys, Cys, Lys-X, Cys-PEG or deleted;
A41 is Thr, Ser, He, Lys, Cys, Lys-X, Cys-PEG or deleted;
A42 is GIn, GIu, Lys, Cys, Lys-X, Cys-PEG or deleted; and A43 is Lys-X, Cys-PEG or deleted; wherein
Z1 , Z2, and Lys-X are as defined in claim 1.
3. The peptide of claim 1 or 2, wherein said peptide is selected from SEQ ID NO: 3-82.
4. The peptide of claim 3, wherein said peptide is selected from SEQ ID NO: 3-45.
5. The peptide of claim 3, wherein said peptide is selected from SEQ ID NO: 46-82.
6. The peptide of claim 3, wherein said peptide is selected from SEQ ID NO: 3-25.
7. The peptide of claim 3, wherein said peptide is selected from SEQ ID NO: 26-45.
8. The peptide of claim 3, wherein said peptide is selected from SEQ ID NO: 46-65.
9. The peptide of claim 3, wherein said peptide is selected from SEQ ID NO: 66-82.
10. The peptide of any one of claims 1-9, wherein said peptide is PEGylated.
11. The peptide of any one of claims 1-9, wherein said peptide is PEGylated at the C- terminus.
12. The peptide of claim 10 or 11 , wherein PEG is selected from
Figure imgf000081_0001
Figure imgf000081_0002
Figure imgf000081_0003
Figure imgf000082_0001
and
Figure imgf000082_0002
13. The peptide of any one of claims 1 -12, wherein said peptide is acetylated.
14. The peptide of any one of claims 1-9, wherein Z1 is selected from
Figure imgf000082_0003
Figure imgf000082_0004
Figure imgf000083_0001
Figure imgf000083_0002
Figure imgf000083_0003
Figure imgf000083_0004
Figure imgf000083_0005
Figure imgf000084_0001
Figure imgf000084_0002
5. The peptide of any one of claims 1-9, wherein Z1 is selected from
Figure imgf000085_0001
Figure imgf000085_0002
Figure imgf000085_0003
and
6. The peptide of claim 14 or 15, wherein PEG is selected from
Figure imgf000086_0001
Figure imgf000086_0002
Figure imgf000086_0003
Figure imgf000086_0004
Figure imgf000086_0005
17. A polynucleotide encoding a peptide of SEQ ID NO: 1-42, or a degenerate variant thereof.
18. A vector comprising a polynucleotide of claim 17.
19. A host cell comprising a vector of claim 18.
20. A method for producing a peptide comprising: a) culturing the host cell of claim 19 under conditions suitable for the expression of said polypeptide; and b) recovering the peptide from the host cell culture.
21. A purified antibody which binds specifically to the peptide of any one of claims 1-16.
22. A pharmaceutical composition comprising an effective amount of a peptide of any one of claims 1-16, in combination with a pharmaceutically acceptable carrier.
23. A pharmaceutical composition comprising a therapeutically effective amount of a peptide of any one of claims 1-16, in combination with a pharmaceutically acceptable carrier and one or more pharmaceutical agents.
24. The pharmaceutical composition of claim 23, wherein said pharmaceutical agent is selected from the group consisting of PPAR ligands, insulin secretagogues, sulfonylurea drugs, α-glucosidase inhibitors, insulin sensitizers, hepatic glucose output lowering compounds, insulin and insulin derivatives, biguanides, protein tyrosine phosphatase-1 B, dipeptidyl peptidase IV, 11 beta-HSD inhibitors, anti-obesity drugs, HMG-CoA reductase inhibitors, nicotinic acid, lipid lowering drugs, ACAT inhibitors, bile acid sequestrants, bile acid reuptake inhibitors, microsomal triglyceride transport inhibitors, fibric acid derivatives, β-blockers, ACE inhibitors, calcium channel blockers, diuretics, renin inhibitors, AT-1 receptor antagonists, ET receptor antagonists, neutral endopeptidase inhibitors, vasopepsidase inhibitors, and nitrates.
25. A method of treating diabetes comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 or a pharmaceutical composition of claims 22, 23, or 24.
26. The method of claim 25, wherein said diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, maturity-onset diabetes of the young, latent autoimmune diabetes adult, and gestational diabetes.
27. A method of treating Syndrome X comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 or a pharmaceutical composition of claims 22, 23, or 24.
28. A method of treating diabetes-related disorders comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 or a pharmaceutical composition of claims 22, 23, or 24.
29. The method of claim 28, wherein said diabetes-related disorder is selected from the group consisting of hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, and insulin resistance.
30. A method of treating or preventing secondary causes of diabetes comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 or a pharmaceutical composition of claims 22, 23, or 24.
31. The method of claim 30, wherein said secondary cause is selected from the group consisting of glucocorticoid excess, growth hormone excess, pheochromocytoma, and drug-induced diabetes.
32. A method of treating diabetes comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 in combination with one or more pharmaceutical agents.
33. The method of claim 32, wherein said pharmaceutical agent is selected from the group consisting of PPAR agonists, sulfonylurea drugs, non-sulfonylurea secretagogues, α- glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic glucose output lowering compounds, insulin, and anti-obesity agents.
34. The method of claim 33, wherein said diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, maturity-onset diabetes of the young, latent autoimmune diabetes adult, and gestational diabetes.
35. A method of treating Syndrome X comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 in combination with one or more pharmaceutical agents.
36. The method of claim 35, wherein said pharmaceutical agent is selected from the group consisting of PPAR agonists, sulfonylurea drugs, non-sulfonylurea secretagogues, α- glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic glucose output lowering compounds, insulin, and anti-obesity agents.
37. A method of treating diabetes-related disorders comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 in combination with one or more pharmaceutical agents.
38. The method of claim 37, wherein said diabetes-related disorder is selected from the group consisting of hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, and insulin resistance.
39. The method of claim 38, wherein said pharmaceutical agent is selected from the group consisting of PPAR agonists, sulfonylurea drugs, non-sulfonylurea secretagogues, α- glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic glucose output lowering compounds, insulin, and anti-obesity agents.
40. A method of treating or preventing secondary causes of diabetes comprising the step of administering a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 in combination with one or more pharmaceutical agents.
41. The method of claim 40, wherein said pharmaceutical agent is selected from the group consisting of PPAR agonists, sulfonylurea drugs, non-sulfonylurea secretagogues, α- glucosidase inhibitors, insulin sensitizers, insulin secretagogues, hepatic glucose output lowering compounds, insulin, and anti-obesity agents
42. A method of treating diabetes, Syndrome X, diabetes-related disorders or secondary causes of diabetes comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-15 in combination with one or more agents selected from the group consisting of HMG-CoA reductase inhibitors, nicotinic acid, lipid lowering drugs, ACAT inhibitors, bile acid sequestrants, bile acid reuptake inhibitors, microsomal triglyceride transport inhibitors, fibric acid derivatives, β- blockers, ACE inhibitors, calcium channel blockers, diuretics, renin inhibitors, AT-1 receptor antagonists, ET receptor antagonists, neutral endopeptidase inhibitors, vasopepsidase inhibitors, and nitrates.
43. The method of claim 42, wherein said diabetes-related disorder is selected from the group consisting of hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, hypertriglyceridemia, and insulin resistance.
44. The method of any one of claims 32 to 43, wherein the peptide of any one of claims 1 -16 and one or more pharmaceutical agents are administered as a single pharmaceutical dosage formulation.
45. A method of treating cardiovascular disease comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 or a pharmaceutical composition of claims 22, 23, or 24.
46. The method of claim 45, wherein said cardiovascular disease is selected from atherosclerosis, coronary heart disease, coronary artery disease, and hypertension.
47. A method of treating obesity comprising the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of claims 1-16 or a pharmaceutical composition of claims 22, 23, or 24.
48. A method of stimulating insulin secretion in a subject in need thereof by administering to said subject a peptide of any one of claims 1-16 or a pharmaceutical composition of claims 22, 23, or 24.
49. Peptides according to any one of claims 1-16 for the treatment and/or prophylaxis of diabetes and diabetes-related disorders.
50. Medicament containing at least one peptide according to any one of claims 1-16 in combination with at least one pharmaceutically acceptable, pharmaceutically safe carrier or excipient.
51. Use of peptides according to any one of claims 1 -16 for manufacturing a medicament for the treatment and/or prophylaxis of diabetes and diabetes-related disorders.
52. Medicaments according to claim 50 for the treatment and/or prophylaxis of diabetes.
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