Source: https://patents.google.com/patent/US8445647B2/en
Timestamp: 2019-07-16 03:09:30
Document Index: 55063651

Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 2', 'Application No. 2', 'Application No. 00928', 'Application No. 00928685', 'Application No. 00', 'Application No. 00928685', 'Application No. 00']

US8445647B2 - Modified exendins and exendin agonists - Google Patents
US8445647B2
US8445647B2 US13/296,120 US201113296120A US8445647B2 US 8445647 B2 US8445647 B2 US 8445647B2 US 201113296120 A US201113296120 A US 201113296120A US 8445647 B2 US8445647 B2 US 8445647B2
US13/296,120
US20120135922A1 (en
2011-11-14 Application filed by Amylin Pharmaceuticals LLC, AstraZeneca Pharmaceuticals LP filed Critical Amylin Pharmaceuticals LLC
2011-11-14 Priority to US13/296,120 priority patent/US8445647B2/en
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2012-05-31 Publication of US20120135922A1 publication Critical patent/US20120135922A1/en
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2013-05-21 Publication of US8445647B2 publication Critical patent/US8445647B2/en
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2015-12-16 PTAB case IPR2016-00354 filed (Settlement) litigation https://portal.unifiedpatents.com/ptab/case/IPR2016-00354 Petitioner: Institution date: 2016-06-28 Termination date: 2016-10-28 "Unified Patents PTAB Data" by Unified Patents is licensed under a Creative Commons Attribution 4.0 International License.
This application is a continuation of U.S. patent application Ser. No. 11/174,089, filed Jun. 30, 2005 now U.S. Pat. No. 8,057,822, which is a continuation of U.S. patent application Ser. No. 09/561,226, filed Apr. 28, 2000 now U.S. Pat. No. 6,924,264, which claims priority to, and the benefit of, U.S. provisional patent application Ser. No. 60/132,018, filed Apr. 30, 1999, each of which is hereby incorporated by reference in its entirety.
The exendins are peptides that are found in the salivary secretions of the Gila monster and the Mexican Bearded Lizard, reptiles that are endogenous to Arizona and Northern Mexico. Exendin-3 [SEQ ID NO: 1] is present in the salivary secretions of Heloderma horridum (Mexican Beaded Lizard), and exendin-4 [SEQ. ID. NO. 2] is present in the salivary secretions of Heloderma suspectum (Gila monster) (Eng, J., et al., J. Biol. Chem., 265:20259-62, 1990; Eng, J., et al., J. Biol. Them., 267:7402-05, 1992). The amino acid sequence of exendin-3 is shown in FIG. 1. The amino acid sequence of exendin-4 is shown in FIG. 2. Exendin-4 was first thought to be a (potentially toxic) component of the venom. It now appears that exendin-4 is devoid of toxicity, and that it instead is made in salivary glands in the Gila monster.
The exendins have some sequence similarity to several members of the glucagon-like peptide family, with the highest homology, 53%, being to GLP-1 [7-36]NH2 [SEQ ID NO: 3] (Goke, et al., J. Biol. Chem., 268:19650-55, 1993). GLP-1 [7-36]NH2, also sometimes referred to as proglucagon[78-107] or simply “GLP-1”, has an insulinotropic effect, stimulating insulin secretion from pancreatic beta-cells; GLP-1 has also been reported to inhibit glucagon secretion from pancreatic alpha-cells (Ørsov, et al., Diabetes, 42:658-61, 1993; D'Alessio, et al., J. Clin. Invest., 97:133-38, 1996). GLP-1 has been reported to inhibit gastric emptying (Willms B, et al., J. Clin. Endocrinol. Metab. 81 (1): 327-32, 1996; Wettergren A, et al., Dig. Dis. Sci. 38 (4): 665-73, 1993), and gastric acid secretion (Schjoldager B T, et al., Dig. Dis. Sci. 34 (5): 703-8, 1989; O'Halloran D J, et al., J. Endocrinol. 126 (1): 169-73, 1990; Wettergren A, et al., Dig. Dis. Sci. 38 (4): 665-73, 1993)). GLP-1 [7-37], which has an additional glycine residue at its carboxy terminus, is reported to stimulate insulin secretion in humans (Ørsov, et al., Diabetes, 42:658-61, 1993). Other reports relate to the inhibition of glucagon secretion (Creutzfeldt WOC, et al., Glucagonostatic actions and reduction of fasting hyperglycemia by exogenous glucagon-like peptide I (7-36) amide in Type 1 diabetic patients, Diabetes Care 1996; 19(6):580-6), and a purported role in appetite control (Turton M D, et al., A role for glucagon-like peptide-1 in the central regulation of feeding, Nature 1996 January; 379(6560):69-72). A transmembrane G-protein adenylate-cyclase-coupled receptor, said to be responsible at least in part for the insulinotropic effect of GLP-1, has reportedly been cloned from a beta-cell line (Thorens, Proc. Natl. Acad. Sci. USA 89:8641-45, 1992). GLP-1 has been the focus of significant investigation in recent years due to its reported action on the amplification of stimulated insulin production (Byrne M M, Goke B. Lessons from human studies with glucagon-like peptide-1: Potential of the gut hormone for clinical use. In: Fehmann H C, Goke B. Insulinotropic Gut Hormone Glucagon-Like Peptide 1. Basel, Switzerland: Karger, 1997:219-33).
Pharmacological studies have led to reports that exendin-4 can act at GLP-1 receptors in vitro on certain insulin-secreting cells, at dispersed acinar cells from guinea pig pancreas, and at parietal cells from stomach; the peptide is also reported to stimulate somatostatin release and inhibit gastrin release in isolated stomachs (coke, et al., J. Biol. Chem. 268:19650-55, 1993; Schepp, et al., Eur. J. Pharmacol., 69:183-91, 1994; Eissele, et al., Life Sci., 55:629-34, 1994). Exendin-3 and exendin-4 were reportedly found to stimulate cAMP production in, and amylase release from, pancreatic acinar cells (Malhotra, R., et al., Regulatory Peptides, 41:149-56, 1992; Raufman, et al., J. Biol. Chem. 267:21432-37, 1992; Singh, et al., Regul. Pept. 53:47-59, 1994). Exendin-4 has a significantly longer duration of action than GLP-1. For example, in one experiment, glucose lowering by exendin-4 in diabetic mice was reported to persist for several hours, and, depending on dose, for up to 24 hours (Eng, J. Prolonged effect of exendin-4 on hyperglycemia of db/db mice, Diabetes 1996 May; 45(Suppl 2):152A (abstract 554)). Based on their insulinotropic activities, the use of exendin-3 and exendin-4 for the treatment of diabetes mellitus and the prevention of hyperglycemia has been proposed (Eng, U.S. Pat. No. 5,424,286).
The present invention relates to novel modified exendins and exendin agonists having an exendin or exendin agonist linked to one or more molecular weight increasing compounds, of which polyethylene glycol polymers (or other molecular weight increasing agents), and related products and methods. Such products and methods that are useful for many applications, including, for example, in the treatment of diabetes, including Type 1 and 2 diabetes, gestational diabetes (see U.S. patent application Ser. No. 09/323,867, entitled, “Use of Exendins and Agonists Thereof For The Treatment of Gestational Diabetes Mellitus,” filed Jun. 1, 1999), in the treatment of disorders which would be benefited by agents which modulate plasma glucose levels, in the treatment of disorders which would be benefited by the administration of agents useful in modulating the rate of gastric emptying or food intake, including obesity, eating disorders, and insulin-resistance syndrome, and to modulate triglyceride levels and to treat subjects suffering from dyslipidemia (i.e., increased LDL cholesterol, increased VLDL cholesterol, and/or decreased HDL cholesterol) (see U.S. provisional patent application Ser. No. 60/175,365, entitled, “Use of Exendins and Agonists Thereof for Modulation of Triglyceride Levels and Treatment of Dyslipidemia,” filed Jan. 10, 2000). The methods are also useful for lowering plasma lipid levels, reducing cardiac risk, reducing the appetite, and reducing the weight of subjects. Still other embodiments concern methods for suppressing glucagon secretion (see U.S. provisional patent application Ser. No. 60/132,017, entitled, “Methods for Glucagon Suppression,” filed Apr. 30, 1999, which is commonly owned). Pharmaceutical compositions for use in the methods of the invention are also disclosed.
By “exendin agonist” is meant a compound which mimics the effects of exendins, e.g., on gastric motility and gastric emptying (namely, a compound which effectively binds to the receptor at which exendins exert their action on gastric motility and gastric emptying, preferably an analog or derivative of an exendin) or a compound, e.g., that mimics the effects of exendin on the reduction of food intake by binding to the receptor or receptors where exendin causes this effect. Preferred exendin agonist compounds include those described in U.S. Patent Application Ser. No. 90/003,869, entitled, “Use of Exendin And Agonists Thereof For The Reduction of Food Intake”, filed Jan. 7, 1998, (and the priority applications thereto) which enjoys common ownership with the present application and which is incorporated by this reference into the present application as though fully set forth herein. Effects of exendins or exendin agonists on reducing food intake can be identified, evaluated, or screened for, using the methods described herein, or other methods known in the art for determining exendin effects, e.g., on food intake or appetite.
In another aspect, a therapeutically effective amount of an amylin agonist is also administered to the subject. In a preferred aspect, the amylin agonist is an amylin or an amylin agonist analog such as 25,23,29Pro-human-amylin. The use of amylin agonists to treat post-prandial hyperglycemia, as well as to beneficially regulate gastrointestinal motility, is described in International Application No. PCT/US94/10225, published Mar. 16, 1995 which has been incorporated by reference herein.
exendin-4 (1-30) [SEQ ID NO: 3: His Gly Glu Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly Gly];
exendin-4 (1-30) amide [SEQ ID NO: 4: His Gly Glu
Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly-NH2];
exendin-4 (1-28) amide [SEQ ID NO: 5: His Gly Glu
14Leu, 25Phe exendin-4 amide [SEQ ID NO: 6: His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln
Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe
Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro
Pro Ser-NH2];
14Leu, 25Phe exendin-4 (1-28) amide [SEQ ID NO: 7:
Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Lys Asn-NH2];
14Leu, 22Ala, 25Phe exendin-4 (1-28) amide [SEQ ID
NO: 8: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Ala Ile
Glu Phe Leu Lys Asn-NH2].
The term “amino acid” refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers if their structure allow such stereoisomeric forms. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Aan), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), typtophan (Trp), tyrosine (Tyr) and valine (Val). Unnatural amino acids include, but are not limited to azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine, pentylglycine, pipecolic acid and thioproline. Amino acid analogs include the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or modified on their N-terminal amino group or their side chain groups, as for example, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone.
FIG. 3 depicts the amino acid sequences for certain exendin agonist compounds useful in the present invention [SEQ ID NOs: 9 TO 25].
FIG. 4 depicts the amino acid sequences for certain compounds of the present invention, Compounds 1-174 [SEQ ID NOs: 26-199].
Exendin-4 dose dependently slowed gastric emptying in HSD rats and was −90-fold more potent than GLP-1 for this action. Exendin-4 has also been shown to reduce food intake in NIH/Sw (Swiss) mice following peripheral administration, and was at least 1000 times more potent than GLP-1 for this action. Exendin-4 reduced plasma glucagon concentrations by approximately 40% in anesthetized ZDF rats during hyperinsulinemic, hyperglycemic clamp conditions, but did not affect plasma glucagon concentrations during euglycemic conditions in normal rats. Exendin-4 has been shown to dose-dependently reduce body weight in obese ZDF rats, while in lean ZDF rats, the observed decrease in body weight appears to be transient.
Exendin agonist compounds also include those described in U.S. Provisional Application No. 60/065,442, including compounds of the formula (I)
[SEQ ID NO: 200]:
Xaa27 Xaa28-Z1;
[SEQ ID NO: 201]
Gly Gly-Z2,
Gly Gly Xaa31-Z2,
Gly Gly Xaa31 Ser-Z2,
Gly Gly Xaa31 Ser Ser-Z2,
Gly Gly Xaa31 Ser Ser Gly-Z2,
Gly Gly Xaa31 Ser Ser Gly ASD-149564.1Gly Xaa31
Ser Ser Gly Ala Xaa36-Z2,
Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2
Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-
N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and
According to an especially preferred aspect, especially preferred compounds include those of formula (I) wherein: Xaa1 is His or Arg; Xaa2 is Gly or Ala; Xaa3 is Asp or Glu; Xaas is Ala or Thr; Xaa6 is Ala, Phe or nephthylalaine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser or Thr; Xaa9 is Asp or Glu; Xaa10 is Ala, Leu or pentylglycine; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln; Xaa14 is Ala, Leu or pentylglycine; Xaa15 is Ala or Glu; Xaa16 is Ala or Glu; Xaa17 is Ala or Glu; Xaa19 is Ala or Val; Xaa20 is Ala or Arg; Xaa21 is Ala or Leu; Xaa22 is Phe or naphthylalanine; Xaa23 is Ile, Val or tert-butylglycine; Xaa24 is Ala, Glu or Asp; Xaa25 is Ala, Trp or Phe; Xaa26 is Ala or Leu; Xaa27 is Ala or Lys; Xaan is Ala or Asn; Z1 is —OH, —NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2; Xaa31, Xaa36, Xaa37 and Xaa38 being independently Pro homoproline, thioproline or N-methylalanine; and Z2 being —OH or —NH2; provided that no more than three of Xaa3, Xaa5, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala. Especially preferred compounds include those set forth in PCT application Serial No. PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds” identified therein as compounds 2-23.
Exendin agonist compounds also include those described in U.S. Provisional Application No. 60/066,029, including compounds of the formula (II)
[SEQ ID NO: 202]:
[SEQ ID NO: 203]
Gly Gly Xaa31 Ser Ser Gly Ala-Z2,
Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2,
Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2,
Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38
According to an especially preferred aspect, especially preferred compounds include those of formula (II) wherein: Xaa1 is His or Ala; Xaa2 is Gly or Ala; Xaa3 is Ala, Asp or Glu; Xaa4 is Ala or Gly; Xaa6 is Ala or Thr; Xaa6 is Phe or naphthylalanine; Xaa7 is Thr or Ser; Xaa8 is Ala, Ser or Thr; Xaa9 is Ala, Asp or Glu; Xaa10 is Ala, Leu or pentylglycine; Xaa11 is Ala or Ser; Xaa12 is Ala or Lys; Xaa13 is Ala or Gln; Xaa14 is Ala, Leu, Met or pentylglycine; Xaa15 is Ala or Glu; Xaa16 is Ala or Glu; Xaa17 is Ala or Glu; Xaa19 is Ala or Val; Xaa20 is Ala or Arg; Xaa21 is Ala or Leu; Xaa22 is Phe or naphthylalanine; Xaa23 is Ile, Val or tert-butylglycine; Xaa24 is Ala, Glu or Asp; Xaa25 is Ala, Trp or Phe; Xaa26 is Ala or Leu; Xaa27 is Ala or Lys; Xaa28 is Ala or Asn; Z1 is —OH, —NH2, Gly-Z2, Gly Gly-Z2, Gly Gly Xaa31-Z2, Gly Gly Xaa31 Ser-Z2, Gly Gly Xaa31 Ser Ser-Z2, Gly Gly Xaa31 Ser Ser Gly-Z2, Gly Gly Xaa31 Ser Ser Gly Ala-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37-Z2, Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2 or Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38 Xaa39-Z2; Xaa31, Xaa36, Xaa3, and Xaa38 being independently Pro homoproline, thioproline or N-methylalanine; and Z2 being —OH or —NH2; provided that no more than three of Xaa3, Xaa5, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Xaa13, Xaa14, Xaa15, Xaa16, Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, Xaa26, Xaa27 and Xaa28 are Ala; and provided also that, if Xaa1 is His, Arg or Tyr, then at least one of Xaa3, Xaa4 and Xaa9 is Ala. Especially preferred compounds of formula (II) include those described in application Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds” as having the amino acid sequence of SEQ ID NOs: 5-93 therein.
Also within the scope of the present invention are narrower genera of compounds having peptides of various lengths, for example genera of compounds which do not include peptides having a length of 28, 29 or 30 amino acid residues, respectively. Additionally, the present invention includes narrower genera of compounds described in PCT application Ser. No. PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds” and having particular amino acid sequences, for example, compounds of the formula (III)
[SEQ ID NO: 204]:
Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25
Xaa26 Xaa27 Xaa28-Z1;
Gly Gly -Z2,
Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2;
Additionally, the present invention includes narrower genera of peptide compounds described in PCT Application Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds” as having particular amino acid sequences, for example, compounds of the formula [IV]
[SEQ ID NO: 205]:
Xaa1 Xaa2 Xaa3 Xaa5 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10
Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa18
Gly Gly-Z2
Gly Gly Xaa31 Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2
Ser-Z2; [SEQ ID NO: 206]:
Preferred compounds of formula (IV) include those wherein X1 is Lys Asn, Lys-NHε—R Asn, or Lys-NHε—R Ala where R is Lys, Arg, C1-C10 straight chain or branched alkanoyl. Preferred compounds of formula (IV) include those having an amino acid sequence described in PCT application Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds” as being selected from SEQ ID NOs: 95-110 therein.
Also provided are compounds described in PCT application PCT/US98/24210, filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds”, including compounds of the formula (V)
[SEQ ID NO: 207]:
Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Ala Xaa19 Xaa20
Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 X1-Z1;
Xaa17, is Ala or Glu;
Gly Gly Xaan Ser Ser Gly Ala Xaa36 Xaa37 Xaa38-Z2;
N-alkylglycine, N-alkylpentylglycine and N-alkylalanine; and
Z2 is —OH or —NH2:
provided that no more than three of Xaa3, Xaa5, Xaa6, Xaa8, Xaa10, Xaa11, Xaa12, Xaa13, Xaa24, Xaa15, Xaa16 Xaa17, Xaa19, Xaa20, Xaa21, Xaa24, Xaa25, and Xaa26 are Ala. Also within the scope of the present invention are pharmaceutically acceptable salts of the compound of formula (V) and pharmaceutical compositions including said compounds and salts thereof.
Preferred compounds of formula (V) are those wherein Xaa24 is Leu, pentylglycine or Met.
According to one aspect, preferred are compounds of formula (V) wherein Xaa6 is Phe or naphthylalanine; and Xaa22 is Phe or naphthylalanine; and Xaa23 is Ile or Val. More preferably, Z1 is —NH2. According to one aspect, especially preferred are such compounds of formula (V) wherein Xaa31, Xaa36, Xaa27 and Xaa38 are independently selected from the group consisting of Pro, homoproline, thioproline and N-alkylalanine. More preferred, Z2 is —NH2.
Also provided are peptide compounds described in PCT Application Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds”, including compounds of the formula (VI)
[SEQ ID NO: 208]:
Preferred compounds of formula (VI) include those described in PCT Application Serial No. PCT/US96/24273, filed Nov. 13, 1998, entitled “Novel Exendin Agonist Compounds” as having an amino acid sequence selected from those identified therein as SEQ ID NOs: 95-110.
Compounds particularly useful according to the present invention are exendin agonist compounds described in U.S. patent application Ser. No. 09/003,869, filed Jan. 7, 1998, entitled “Use of Exendins And Agonists Thereof For The Reduction of Food Intake”, including compounds of the formula (VII)
[SEQ ID NO: 209]:
wherein Xaa1 is His, Arg or Tyr; Xaa2 is Ser, Gly, Ala or Thr; Xaa3 is Asp or Glu; Xaa4 is Phe, Tyr or naphthalanine; Xaa5 is Thr or Ser; Xaa6 is Ser or Thr; Xaa7 is Asp or Glu; Xaa8 is Leu, Ile, Val, pentylglycine or Met; Xaa9 is Leu, Ile, pentylglycine, Val or Met; Xaa10 is Phe, Tyr or naphthalanine; Xaa11 is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met; Xaa12 is Glu or Asp; Xaa13 is Trp, Phe, Tyr, or naphthylalanine; Xaa14, Xaa15, Xaa16 and Xaa17 are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; Xaa18 is Ser, Thr or Tyr; and Z is —OH or —NH2; with the proviso that the compound does not have the formula of either SEQ ID NOs: 1 or 2. Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine and N-alkylalanine include lower alkyl groups preferably of 1 to about 6 carbon atoms, more preferably of 1 to 4 carbon atoms. Suitable compounds include those having amino acid sequences of SEQ ID NOs: 10 to 40. Also useful in the present invention are pharmaceutically acceptable salts of the compounds of formula (VII).
According to an especially preferred aspect, especially preferred compounds include those of formula (VII) wherein: Xaa1 is His or Arg; Xaa2 is Gly; Xaa3 is Asp or Glu; Xaa4 is Phe or napthylalanine; Xaa5 is Thr or Ser; Xaa6 is Ser or Thr; Xaa7 is Asp or Glu; Xaa8 is Leu or pentylglycine; Xaa9 is Leu or pentylglycine; Xaa10 is Phe or naphthylalanine; Xaa11 is Ile, Val or t-butyltylglycine; Xaa12 is Glu or Asp; Xaa13 is Trp or Phe;
Xaa14, Xaa15, Xaa16, and Xaa17 are independently Pro, homoproline, thioproline, or N-methylalanine; Xaa18 is Ser or Tyr: and Z is —OH or —NH2; with the proviso that the compound does not have the formula of either SEQ ID NOs: 1 or 2. More preferably Z is —NH2. Especially preferred compounds include those having the amino acid sequence of SEQ ID NOs: 10, 11, 22, 23, 24, 27, 29, 36, 37 and 40.
Also provided are compounds described in PCT Application Serial No. PCT/US98/16387, filed Aug. 6, 1998, entitled “Novel Exendin Agonist Compounds”, including compounds of the formula (VIII)
[SEQ ID NO: 210]:
Pro Pro Ser-NH2
The above amidated peptide was assembled on 4-(2′-4′-dimethoxyphenyl)-Fmoc aminomethyl phenoxy acetamide norleucine MBNA resin (Novabiochem, 0.55 mmole/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), cleaved from the resin, deprotected and purified in a similar way to Exendin-3 as describe in Example 1. Used in analysis were Solvent A (0.1% TFA in water) and Solvent B (0.1% TFA in ACM). Analytical RP-HPLC (gradient 36% to 46% Solvent B in Solvent A over 30 minutes) of the lyophilized peptide gave product peptide having an observed retention time of 14.9 minutes. Electrospray Mass Spectrometry (M): calculated 4186.6. Found 4186.0 to 4186.8 (four lots).
(201) [SEQ ID NO: 211]:
(202) [SEQ ID NO: 212]:
(203) [SEQ ID NO: 213]:
(204) [SEQ ID NO. 214]:
(205) [SEQ ID NO. 215]:
(206) [SEQ ID NO. 216]:
(207) [SEQ ID NO. 217]:
(208) [SEQ ID NO. 218]:
(209) [SEQ ID NO. 219]:*
(210) [SEQ ID NO. 220]:*
(211) [SEQ ID NO. 221]:
(212) [SEQ ID NO. 222]:
(213) [SEQ ID NO. 223]:*
(214) [SEQ ID NO. 224]:
(215) [SEQ ID NO. 225]:
(216) [SEQ ID NO. 226]:
(217) [SEQ ID NO. 227]:
exendin-4 [SEQ ID NO. 2]
(218) [SEQ ID NO. 228]
(CH3)-COHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2
(219) [SEQ ID NO. 229]
(CH3)-CH2HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2
(220) [SEQ ID NO. 230]
HGEGTFTSDLSRQMEEEAVRLFIEWLK(PEG)NGGPSSGAPPPS-NH2
(221) [SEQ ID NO. 231]
HGEGTFTSDLSK(PEG)QMEEEAVRLFIEWLRNGGPSSGAPPPS-NH2
(222) [SEQ ID NO. 232]
HGEGTFTSDLSROMEEEAVRLFIEWLRNGGPSSGAPPPS-NH2
(223) [SEQ ID NO. 233]
HGEGTFTSDLSRQMEEEAVRLFIEWLIINGGPSSGAPPPK(PEG)-NH2
(224) [SEQ ID NO. 234]
HGEGTFTSDLSROMEEEAVRLFIEWLRNGK(PEG)PSSGAPPPS-NH2
(225) [SEQ ID NO. 235]
HGEGTFTSDLSROMEEEAVRLFIEWLK(PEG)NGGPSSGAPPPS-NH2
(226) [SEQ ID NO. 236]
HGEGTFTSDLSK(PEG)WEEEAVRLFIEWLRNGGPSSGAPPPS-NH2
(227) [SEQ ID NO. 237]
(PEG)COHGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSSGAPPPS-NH2
(228) [SEQ ID NO. 238]
(PEG)CH2HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSSGAPPPS-NH2
(229) [SEQ ID NO. 239]
HGEGTFTSDLSRQMEEEAVRLFIEWLRNGGPSSGAPPPK(PEG)-NH2
(230) [SEQ ID NO. 240]
The various PEG modified exendins used in Examples 5-10, below, are provided in Table I, with the corresponding results being provided in Table II (see the end of Example 9). GLP-1[(7-36]NH2 (GLP-1) was purchased from Bachem (Torrance, Calif.). All other peptides were prepared using synthesis methods such as those described herein. All chemicals were of the highest commercial grade. The cAMP SPA immunoassay was purchased from Amersham. The radioligands were purchased from New England Nuclear (Boston, Mass.). RINm5f cells (American Type Tissue Collection, Rockville, Md.) were grown in DME/F12 medium containing 10% fetal bovine serum and 2 mM L-glutamine. Cells were grown at 37° C. and 5% CO2/95% humidified air and medium was replaced every 2 to 3 days. Cells were grown to confluence then harvested and homogenized using on a Polytron homogenizer. Cell homogenates were stored frozen at −70° C. until used.
A gastric emptying study may also be carried out to examine the effects of exendin-4 and/or an exendin agonist compound on gastric emptying in rats. Such experiments typically follow a modification of the method of Scarpignato, et al., Arch. Int. Pharmacodyn. Ther. 246:286-94, 1980. Male Harlan Sprague Dawley (HSD) rats are used. All animals are housed at 22.7±0.8° C. in a 12:12 hour light:dark cycle (experiments being performed during the light cycle) and were fed and watered ad libitum (Diet LM-485, Teklad, Madison, Wis.). The determination of gastric emptying by the method described below can be performed after a fast of −20 hours to ensure that the stomach contained no chyme that would interfere with spectrophotometric absorbance measurements.
1. A compound comprising exendin-4, or agonist analog of exendin-4, linked to a polyamino acid through the C-terminal amino acid of the exendin-4 or agonist analog of exendin-4 and wherein the polyamino acid is selected from the group consisting of poly(L-lysine), poly-glutamic acid, and poly-aspartic acid.
2. The compound according to claim 1, wherein the polyamino acid is poly(L-lysine).
3. The compound according to claim 1, wherein the compound comprises the exendin-4 linked to the polyamino acid.
4. The compound according to claim 3, wherein the polyamino acid is poly(L-lysine).
5. The compound according to claim 1, wherein the compound comprises the agonist analog of exendin-4 linked to a polyamino acid through the C-terminal amino acid of the agonist analog of exendin-4 and wherein the polyamino acid is selected from the group consisting of poly(L-lysine), poly-glutamic acid, and poly-aspartic acid.
6. The compound according to claim 5, wherein the polyamino acid is poly(L-lysine).
US13/296,120 1999-04-30 2011-11-14 Modified exendins and exendin agonists Active US8445647B2 (en)
US13/296,120 US8445647B2 (en) 1999-04-30 2011-11-14 Modified exendins and exendin agonists
US13/896,937 US8951962B2 (en) 1999-04-30 2013-05-17 Modified exendins and exendin agonists
US11/174,089 Continuation US8057822B2 (en) 1999-04-30 2005-06-30 Modified exendins and exendin agonists
US13/896,937 Continuation US8951962B2 (en) 1999-04-30 2013-05-17 Modified exendins and exendin agonists
US20120135922A1 US20120135922A1 (en) 2012-05-31
US8445647B2 true US8445647B2 (en) 2013-05-21
US5176918A (en) 1990-12-20 1993-01-05 Jones Jeffry L Topical medicament
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