JP2006514649A - Composition for the treatment and prevention of nephropathy - Google Patents

Abstract

Described are compositions and methods for the prevention and treatment of nephropathy, including hypertensive and diabetic nephropathy, and nephropathy related to insulin resistance and metabolic syndrome. The composition of the present invention comprises glucagon-like peptide-1, incretin, glucagon-like peptide-1 (GLP-1), exendin, or analogs (including agonist analogs), derivatives, or variants thereof A compound that binds to a receptor for.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application is a US Provisional Application No. 60 / 434,508 filed Dec. 17, 2002, which is incorporated herein by reference in its entirety; and filed Dec. 19, 2002. Priority over US _________ entitled “Prevention and Treatment of Cardiac Arrhythmias” filed on December 17, 2003, claiming priority to US provisional application No. 60 / 434,888 Insist on the interests of rights.

The present invention relates to compositions and methods for treating nephropathy, and particularly hypertensive and diabetic nephropathy, using GLP-1 and related compounds.

BACKGROUND OF THE INVENTION End stage renal failure (ESRD) is a major health problem in the United States. The incidence has steadily increased over the past 10 years, from 155/1 million in 1988 to 296 in 1997. Illness is particularly common in racial and minority ethnic groups such as African Americans, American Indians, Alaska Natives, Hawaiian Natives and other Pacific Islanders, and Hispanic Americans.

  The four main causes of ESRD include diabetes (mainly type 2), hypertension, glomerulonephritis, and cystic kidney disease. There are significant variations in the causes of ESRD among different ethnic and racial groups. For example, while diabetic nephropathy is a major cause of ESRD in American Indian / Alaska Natives, Asian Americans, Hawaiian Natives and other Pacific Islander, Hispanic Americans, and Caucasians, hypertensive nephropathy Is the most frequently reported cause of ESRD in African Americans.

  Currently, ESRD patients must receive dialysis or receive a new kidney through transplantation. Each year, hypertension produces more than 15,000 new cases of ESRD in the United States.

  Diabetic nephropathy is a kidney disease that develops as a result of diabetes. Diabetes affects approximately 5% of the US population. Approximately 25% to 40% of diabetics eventually develop diabetic nephropathy, which progresses through five predictable stages, the final stage of which is ESRD, and then renal replacement therapy (ie blood Dialysis, peritoneal dialysis, kidney transplantation) is required.

  Hypertension and diabetes often coexist in the same patient, acting synergistically. The mechanism underlying nephropathy is not fully understood, but is hypothesized to include a period of glomerular hypertension that leads to glomerular hypertension and subsequent injury, followed by reactive vasoconstriction. Early signs of nephropathy are protein in the urine (eg proteinuria), a concentration that can be related to the extent of kidney damage. Eventually, glomerulosclerosis develops and progressive loss of functioning renal units occurs. With increasing serum creatinine and blood urea nitrogen (BUN) and excessive fluid accumulation, the kidney's ability to filter / secret waste products and maintain electrolyte and water balance is lost. At this stage, the patient is generally diagnosed with end stage renal failure (ESRD).

  Individuals with insulin resistance are also at risk, such as patients with so-called “metabolic syndrome”, regardless of whether they suffer from coexisting hypertension.

  Treatment with angiotheoncin converting enzyme inhibitors or calcium channel blockers, verapamil, can prevent the rate of nephropathy progression. However, ESRD is inevitable. ESRD that progresses to renal failure can be treated by dialysis or kidney transplantation. These are expensive therapies that are currently compensated by Medicare at an annual cost of $ 10 billion (regardless of patient age). The number of ESRD patients is increasing.

  Thus, it can be seen that there is a real and continuing need for effective treatments for kidney disorders and nephropathy, including those associated with hypertension, insulin resistance, and / or diabetes. The present invention has as its main objective to achieve this need.

Summary of the Invention The present invention describes compositions and methods for the prevention and treatment of nephropathy, including hypertensive and diabetic nephropathy, and nephropathy associated with insulin resistance and metabolic syndrome. The present invention achieves these objectives by, among other things, improving or preventing exacerbation of hypertension, endothelial function, renal function, and glomerulosclerosis. The composition of the present invention comprises glucagon-like peptide-1, incretin, glucagon-like peptide-1 (GLP-1), exendin, or any agonist, analog (preferably agonist analog), derivative, variant Or a compound that binds to a receptor for a biologically active fragment.

  In one embodiment, the present invention is for preventing or treating hypertension and diabetic nephropathy, or nephropathy including those related to insulin resistance, characterized by administering a compound of the present invention. Provide a method.

  Furthermore, the present invention provides a method for improving endothelial function in patients with reduced vasodilatory capacity, or other reductions in either glomerulosclerosis or glomerular flow. Such an improvement in endothelial function serves both to reduce hypertension and to improve the function of glomerular capillaries. In further embodiments, the molecules of the invention are useful for preventing the progression of nephropathy to ESRD, preventing proteinuria and / or glomerulosclerosis, delaying their progression, treating or ameliorating them. is there.

  In a preferred embodiment of the invention, the compound is GLP-1 or exendin-3 or exendin-4, or a biologically active analog, derivative, variant, or fragment thereof. A preferred dose is about 0.001 μg / kg / dose to about 1.0 μg / kg / dose, or a dose sufficient to achieve a therapeutic plasma level of at least 40 pg / ml.

  Means and manners for accomplishing each of the above objects will become apparent from the detailed description of the invention that follows.

Detailed Description of the Invention The present invention relates to hypertensive, diabetic, and other types of nephropathy such as analgesic nephropathy, IgA nephropathy, ischemic nephropathy, HIV-related nephropathy, membranous nephropathy, The present invention relates to a composition for the treatment of glomerulosclerosis and the use thereof. The present invention is particularly effective for use in preventing or treating hypertension and / or diabetic nephropathy and nephropathy that occurs or is likely to occur in insulin resistant patients without or with coexisting hypertension. is there.

  Without being bound by theory, the molecules of the invention may be used to improve insulin resistance, cation balance, hypertension, and / or more production of ATP for use by cells, and affected tissues. It works to some extent by promoting glucose oxidation by cells (including endothelial cells) in the kidney (and elsewhere) rather than oxidation of free fatty acids leading to reduced oxidative stress.

  The molecule of the present invention includes glucagon-like peptide-1, incretin, glucagon-like peptide-1 (GLP-1), exendin, or any agonist, analog (preferably agonist analog), derivative, mutant, Or a compound that binds to a receptor for a biologically active fragment.

  Insertion, substitution, extension, or deletion, preferably having at least 50 or 55% amino acid sequence identity with the base molecule, more preferably 70%, 80%, 90%, or 95% amino acid sequence identity with the base molecule Including any peptide of sequence derived from that of a base molecule (eg, receptor binding compound, incretin, GLP-1, or exendin), whether or not. Such analogs may contain conservative or non-conservative amino acid substitutions (including unnatural amino acids and L and D forms). “Agonist analogs” preferably have better efficacy than base molecules, or within 5 orders of magnitude (plus or minus) compared to base molecules, as assessed by measurements known in the art such as receptor binding / competition studies, More preferably, it is an analog that exhibits at least one characteristic or action of a base molecule having an efficacy of 4, 3, 2, or 1 digit.

  “Derivative” includes any base molecule or analog that is intramolecularly, attached to, attached to, or associated with a molecule. Such chemical modifications can include concomitant molecules such as internal linkers (eg, spacing or structure derivation) or molecular weight facilitating molecules (eg, polyethylene glycol (PEG), polyamino acid sites, etc.), or tissue targeting molecules. . Examples of such molecules are known in the art, for example, insulinotropic peptides including GLP-1 and exendin modified with a maleimide group are disclosed in US Pat. No. 6,593,295, incorporated herein by reference. In the issue.

  “Mutants” include any modifications to base molecules, analogs or variants not covered by the terms “analogs” and “derivatives” as known to those skilled in the art. For example, the variant may comprise a surrogate form or chimera of the selected molecule. Small molecules are included in the compounds useful in the present invention as long as they bind to the receptor for GLP-1 or exendin or have nephropathy preventive or therapeutic characteristics as described herein. Based on pharmacology that is not dependent on known GLP-1 receptors, they are useful in the present invention, but incretins, glucagon-like peptide-1 (GLP-1), exendins, or analogs, derivatives, or mutations It is understood that not all peptide molecules described as bodies bind to receptors for GLP-1. These molecules may still have the desired biological activity described herein, eg, GLP-1 (9-36), and agonists, analogs, derivatives, and variants thereof. Other exemplary compounds encompassed within the scope of the present invention are US Pat. Nos. 6,569,832; 6,528,486; 6,514,500; 6,458,924; 6,451,987, incorporated herein by reference. No .: 6,451,974; including those described in No. 6,268,343.

As the terminology is used above, examples of base molecules of the invention are amidated or not (often GLP-1 [7-36] NH ₂ ) glucagon-like peptide-1 [7- 36] GLP-1, also known as the amino acid sequence His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg- [NH _{2 as} desired] ( Product of proglucagon gene having SEQ ID NO: 1). GLP-1 is a hormone produced by L-type cells in the intestine and is released after digestion of the meal. GLP-1 improves insulin resistance and glucose utilization in patients with type 2 diabetes by increasing insulin secretion and by inhibiting glucagon secretion. Receptors for GLP-1 are expressed in islet cells, gastrointestinal tract, and lung, heart, central nervous system and kidney. GLP-1 has been reported to produce a variety of biological effects (eg, Orskov, et al., Diabetes, 42: 658-61, 1993; D'Alessio, et al., J. Clin. Invest ., 97: 133-38, 1996, Williams B, et al., J Clin Endocrinol Metab 81 (1): 327-32, 1996; Wettergren A, et al., Dig Dis Sci 38 (4): 665-73 Schjoldager BT, et al., Dig Dis Sci 34 (5): 703-8, 1989; O'Halloran DJ, 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 the carboxy terminus, also stimulates insulin secretion in humans (Orskov, et al., Diabetes, 42: 658-61, 1993).

  The composition of the present invention comprises a GLP-1 agonist analog. By “agonist analog” is meant a compound that mimics at least one effect of GLP-1. This definition of agonist analog can include compounds that bind to receptor or receptors that GLP-1 causes a particular effect. Certain GLP-1 analogs with agonist activity are described in Chen et al., US Patent No. 5,512,549, issued April 30, 1996 entitled Glucagon-Like Insulinotropic Peptide Analogs, Compositions and Methods of Use. Has been. Other GLP-1 analogs with agonist activity are described in Johnson et al., Issued November 12, 1996 entitled Biologically Active Fragments of Glucagon-Like Insulinotropic Peptide, US Pat. No. 5,574,008. . Still other GLP-1 analogs with agonist activity are described in Buckley et al., US Pat. No. 5,545,618, issued August 13, 1996 entitled GLP-1 Analogs Useful for Diabetes Treatment. . All three referenced US patents are hereby incorporated by reference. The present invention includes the use of recombinant human GLP-1 analogs and GLP-1 analogs derived from other species, whether recombinant or otherwise synthetic.

  In certain embodiments, the GLP-1 agonist analogs used in the methods of the invention are GLP-1 (7-34) as disclosed in US Pat. No. 5,118,666, incorporated herein by reference. And GLP-1 (7-35), as well as GLP-1 (7-37) as disclosed in US Pat. No. 5,120,712, incorporated herein by reference. . Also, GLP-1 analogs with a low tendency to aggregate, such as those described in WO01 / 98331; GLP-1 analogs with N-terminal truncation, US Pat. No. 5,574,008; GLP-1 analogs with acyl groups attached, US patents 5,512,549; amidated GLP-1 analogs, WO02 / 48192; and GLP-1 analogs of US Patent Application No. 10/276772, all of which are incorporated by reference.

Further exemplary analogs include GLP-1 analogs modified at position 8, eg, US Pat. No. 5,981,488, incorporated by reference. Briefly, the analog has the formula (XI), R ₁ -X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala- Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R ₂ (SEQ ID NO: 33)
[Where:
R ₁ is His, D-histidine, desaminohistidine, 2-amino-histidine, beta. Selected from the group consisting of -hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;
X is selected from the group consisting of Met, Asp, Lys, Thr, Leu, Asn, Gln, Phe, Val, and Tyr
Y and Z are independently selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; and
R ₂ is selected from the group consisting of NH _2, and Gly-OH; provided that if R ₁ is His, X is Val, if Y is Glu, and Z is Glu, R ₂ is a NH ₂ ]
Or a pharmaceutically acceptable salt thereof.

V8-GLP-1 and other position 8 analogs can be found in US Pat. No. 5,705,483, incorporated by reference. Briefly, the analog has the formula (XII), R ₁ -X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala- Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R ₂ (SEQ ID NO: 34)
[Where:
R ₁ is selected from the group consisting of L-histidine, D-histidine, desami-nohistidine, 2-amino-histidine, beta-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine Is;
X is selected from the group consisting of Ala, Gly, Val, Thr, Ile, and alpha-methyl-Ala;
Y is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly;
Z is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly;
R ₂ is selected from the group consisting of NH ₂ and Gly-OH; provided that the compound has an isoelectric point in the range of about 6.0 to about 9.0, further R ₁ is His and X is Ala R ₂ must be NH ₂ when Y is Glu and Z is Glu]
Including

In other embodiments, the GLP-1 agonist analog is, for example, Gln ⁹ -GLP-1 (7-37), D-Gln ⁹ -GLP-1 (7-37), acetyl-Lys ⁹ -GLP-1 (7 -37), Thr ¹⁶ -Lys ¹⁸ -GLP-1 (7-37), and variants or analogs of GLP-1 known in the art such as Lys ¹⁸ -GLP-1 (7-37). Derivatives of GLP-1 are also contemplated in the present invention and include, for example, acid addition salts, carboxylate salts, lower alkyl esters, and amides (see, eg, W091 / 11457). Although not required for use in the present invention, in general, various forms of GLP-1 have been found to stimulate insulin secretion (insulinotropic action) and cAMP formation (eg, Mojsov, S., Int. J. Peptide Protein Research, 40: 333-343 (1992)).

  In yet another embodiment, the present invention provides a compound of the general formula (I):

Wherein R ₁ is selected from the group consisting of 4-imidazopropionyl (des-amino-histidyl), 4-imidazoacetyl, or 4-imidazo-alpha, alphadimethyl-acetyl;
R ₂ is selected from the group consisting of C ₆ -C ₁₀ unbranched acyl or absent;
R ₃ is selected from the group consisting of Gly-OH or NH ₂ ; and
Xaa ₄₀ is Lys or Arg].

  In one embodiment, the GLP-1 agonist is a naturally occurring GLP- arising from the addition of various R groups via a peptide attached to the amino terminus of the peptide moiety of formula I (SEQ ID NO: 2). 1 (7-37). If desired, further compounds of the invention are made by acylating the epsilon amino group of the Lys34 residue and by making a limited amino acid substitution at position 26, or by modifying the carboxy terminus. be able to.

Regarding the above formula, it should be noted that the naming scheme used has evolved around the processed form of GLP-1. In this scheme, the amino terminus of the known GLP-1 (7-37) OH is assigned number 7 and the carboxy terminus is assigned number 37. Thus, the first Ala residue of formula I corresponds to residue 8 of GLP-1 (7-37) OH. Similarly, Xaa ₄₀ of formula I corresponds to residue 26 of GLP-1 (7-37) OH, and so forth.

In yet another embodiment, the present invention provides a compound of formula (II):
R ₄ -Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa _4l -Gly-Arg-R ₅ (SEQ ID NO: 3)
[Where R ₄ is:
a) H ₂ N;
b) H ₂ N-Ser;
c) H ₂ N-Val-Ser;
d) H ₂ N-Asp-Val-Ser;
e) H ₂ N-Ser-Asp-Val-Ser (SEQ ID NO: 4);
f) H ₂ N-Thr-Ser-Asp-Val-Ser (SEQ ID NO: 5);
g) H ₂ N-Phe-Thr-Ser-Asp-Val-Ser (SEQ ID NO: 6);
_{h) H 2 N-Thr-} Phe-Thr-Ser-Asp-Val-Ser ( SEQ ID NO: 7);
_{i) H 2 N-Gly-} Thr-Phe-Thr-Ser-Asp-Val-Ser ( SEQ ID NO: 8);
_{j) H 2 N-Glu-} Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser ( SEQ ID NO: 9); or _{k) H 2 N-Ala-} Glu-Gly-Thr-Phe-Thr-Ser -Asp-Val-Ser (SEQ ID NO: 10);
Selected from the group consisting of
Xaa ₄₁ is selected from the group consisting of Lys or Arg; and,
Wherein R ₅ is selected from the group consisting of NH ₂ , OH, Gly-NH ₂ , or Gly-OH]
Biologically active GLP-1 fragments of

  In still other embodiments, the invention provides GLP-1 (7-34); (7-35); (7-36) or (7-37) modified forms of human peptides or C-terminal amidation thereof Provide form. The natural peptide has the amino acid sequence (SEQ ID NO: 11):

[Wherein (G), (R), and (G) are present or absent depending on the chain length indicated]
Have Modified forms contain one or more alterations of the natural structure and have improved ability for therapeutic use. The modified form has potency over glucagon and either or both allow insulin secretion in plasma or increased stability.

The analogs of the present invention are:
(A) Neutral amino acid instead of lysine at position 26 and / or 34, arginine, or D form of lysine, and / or neutral amino acid instead of arginine at position 36, lysine, or D form of arginine Replacement;
(B) substitution of an oxidation resistant amino acid in place of tryptophan at position 31;
(C) Substitution with at least one of the following:
Y instead of V at position 16;
K instead of S at position 18;
D instead of E at position 21;
S instead of G at position 22;
R in place of Q at position 23;
R instead of A at position 24; and Q instead of K at position 26;
(D) a substitution comprising at least one of the following:
An alternative small neutral amino acid in place of A at position 8;
An alternative acidic or neutral amino acid in place of E at position 9;
An alternative neutral amino acid in place of G at position 10; and an alternative acidic amino acid in place of D at position 15; and (e) an alternative neutral amino acid in place of histidine at position 7 or a D or N-acyl of histidine It may have a previous sequence having at least one modification of SEQ ID NO: 11, selected from the group consisting of substituted or alkylated forms of substitution, or its C-terminal amide.

With respect to modifications (a), (b), (d) and (e), the substituted amino acid may be in the D form, as indicated by the superscript ^† , for example C ^† . The amino acid substituted at position 7 may also be in N-acylated or N-alkylated form.

In another embodiment, the present invention is directed to peptides that exhibit increased resistance to degradation in plasma compared to GLP-1 (7-37), where increased resistance to degradation is defined as follows: The In these analogs, any of the above truncated forms of GLP-1 (7-34) to GLP-1 (7-37) or their C-terminal amidated form is:
(A) substitution of D-neutral or D-acidic amino acid in place of H at position 7; or (b) substitution of D-amino acid in place of A at position 8; or (c) both or (d ) Modification by substitution of the N-acylated or N-alkylated form of any naturally occurring amino acid in place of H at position 7.

Thus, analogs of the invention that are resistant to degradation are (N-acyl (1-6C) AA) ⁷ GLP-1 (7-37) and (N-alkyl (1-6C) AA) ⁷ GLP- 1 (7-37), where AA is a lysyl residue, one or both nitrogens may be alkylated or acylated. AA represents any amino acid consistent with retention of insulin stimulating activity.

  With respect to substitutions of D-amino acids at positions 7 and 8 of SEQ ID NO: 11, the D residue of any acidic or neutral amino acid can be used at position 7, again consistent with insulin stimulating activity, either The D residue of this amino acid can be used at position 8, again consistent with insulin stimulating activity. One or both of positions 7 and 8 may be substituted by a D-amino acid; and the D-amino acid at position 7 may be acylated or alkylated as described above. These modified forms can be applied not only to GLP-1 (7-37) but also to the shorter truncated analogs described above.

Other modified GLP-1 as well as exendins useful in the practice of the claimed invention can be found in US Pat. No. 6,528,486, incorporated by reference. In addition, agonists of glucagon-like peptides that exhibit activity via the GLP-1 (7-36) amide receptor are described. EP 0708179 A2; Hjorth et al., J. Biol. Chem. 269; 30121 (1994); Siegel et al., Amer. Diabetes Assoc. 57th Scientific Session, Boston (1997); Hareter et al., Amer. Diabetes Assoc 57th Scientific Session, Boston (1997); Adelhorst et al., J. Biol. Chem. 269, 6275 (1994); Deacon et al., 16th International Diabetes Federation Congress Abstracts, Diabetologia Supplement (1997); Irwin et al. Natl. Acad. Sci. USA 94; 7915 (1997); Mojsov, Int. J. Peptide Protein Res. 40; 333 (1992). See Goke & Byrne, Diabetic Medicine 13; 854 (1996). Recent publications disclose black widow spider GLP-1 and Ser ² GLP-1. See Holz & Hakner, Comp. Biochem. Physiol, Part B 121; 177 (1998) and Ritzel et al., J. Endocrinol 159; 93 (1998).

  As noted above, GLP-1 analogs, as well as exendin analogs, are one or more amino acid substitutions, additions, extensions compared to GLP-1 (7-36), exendin-4 or exendin-3 Or a peptide containing a deletion. In one embodiment, the number of substitutions, deletions or additions is 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 5 amino acids or Any integer less than or between these quantities. In one embodiment of the invention, the substitution comprises one or more conservative substitutions. A “conservative” substitution refers to the replacement of an amino acid residue by another biologically active similar residue, as is well known in the art. Examples of conservative substitutions include substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine, or substitution of one polar residue instead of the other, for example, lysine It includes substitution of arginine instead of glutamic acid instead of aspartic acid or glutamine instead of asparagine.

  In addition, GLP-1 analogs have the above peptides chemically derivatized or modified, eg, unnatural amino acid residues (eg, taurine, β- and γ-amino acid residues and D-amino acid residues) C-terminal functional group modifications such as amides, esters, and cyclizations with acylated amines, C-terminal ketone modifications such as Schiff bases and N-terminal functional group modifications, or found for example with the amino acid pyroglutamic acid It is understood to include peptides having. Exendin analogs including the following may have similar modifications.

  Other compositions of the invention include exendins that refer to naturally occurring exendin peptides and related peptides found in the lizard. Preferred exendins are exendin-3 (SEQ ID NO: 12) present in the salivary secretion of Heloderma horridum, exendin-4 (SEQ ID NO: 14), a peptide present in the salivary secretion of Heloderma suspectum (Eng, J. , et al., J. Biol. Chem., 265: 20259-62, 1990; Eng., J., et al., J. Biol. Chem., 267: 7402-05, 1992), and any of them Such agonists, analogs, derivatives, or variants, as well as biologically active fragments thereof. Exendin-4, which occurs during saliva secretion in the lizard, is an amidated peptide. However, it is to be understood that the terms “exendin”, “exendin-3”, and “exendin-4” refer to both the amidated peptide of the peptide and the acid form of the peptide. Similarly, references to GLP-1 generally refer to amidated 7-36 molecules, but are also intended to include non-amidated molecules and analogs, as well as variants of these peptides are amidated. It may or may not be done.

  “Exendin agonist” refers to a compound that mimics the effects of any exendin by binding to the receptor or receptors for which the naturally occurring exendin exerts its effect. Exendin “agonist activity” in this context means having exendin biological activity, as described herein; however, the activity of an agonist is more potent than natural exendin. It is understood that it may be more powerful.

  Exendin-4 is a 39-amino acid polypeptide. The specific sequences of the molecules of the invention are compared in Table 1.

Various experiments compare the biological activities of exendin-4 and GLP-1 and show a spectrum of more favorable properties for exendin-4 against a specific measure. Exendin has been shown to lower blood glucose, reduce HbA _1c (measurement of glycosylated hemoglobin used to assess blood glucose), improve insulin sensitivity, and improve insulin response to glucose . Higher blood glucose levels are associated with greater glucose lowering effects, i.e. the observed glucose lowering effects of exendin-4 are glucose dependent and appear to be minimal if the animal is already normoglycemic Can be seen. Degradation studies with exendin-4 compared to GLP-1 show that exendin-4 is relatively resistant to degradation.

  As used herein, the term “exendin agonist” binds to or activates the receptor or receptors for which exendin exerts its effect, including one of the above. Any molecule is included, whether it is a peptide, peptidomimetic, or other chemical compound. Exendin agonists can include molecules with insulinotropic activity that bind GLP-1 receptor molecules in in vitro assays and, among other things, can induce second messenger activity against insulin-producing β-cells. It is not necessary for exendin agonists or analogs useful in the invention.

  Exendin structure-activity relationship (SAR) has been investigated for structures that may be related to exendin's activity in terms of stability to metabolism and improvement of physical characteristics, in particular it is peptide stability and submission to alternative delivery systems. Therefore, various exendin agonist peptide compounds have been invented. Exendin agonists include exendin analogs having agonist activity in which one or more naturally occurring or non-naturally occurring amino acids are added, inserted, eliminated or substituted by another amino acid (s). A preferred exendin analog is a peptide analog of exendin-4.

  An exendin analog is encoded by a polynucleotide that expresses a biologically active exendin analog having agonist activity that is functional in the present invention, as defined herein. For example, an exendin analog useful in the present invention may be a peptide containing one or more amino acid substitutions, extensions, additions or deletions compared to exendin-4 or exendin-3. In one embodiment, the number of substitutions, extensions, deletions or additions is 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 5 Amino acid or any integer less than or between these amounts. In one embodiment of the invention, the substitution comprises one or more conservative substitutions. Exendin analogs comprising chemically derivatized or modified compounds and peptides having preferred amino acid homology to SEQ ID NOs: 12 and 14 have been previously described and are within the scope of the claimed invention Is considered to be within.

  A novel exendin analog with agonist activity is disclosed in "Novel Exendin Agonist" filed on August 6, 1998, claiming the benefit of US Patent Application No. 60 / 055,404, filed August 8, 1997. PCT Application No. PCT / US98 / 16387 entitled “Compounds”, both of which are incorporated herein by reference.

  Other novel exendin analogs with agonist activity were identified in November 1998 entitled “Novel Exendin Agonist Compounds” claiming the benefit of US Provisional Application No. 60 / 065,442, filed November 14, 1997. PCT Application No. PCT / US98 / 24210 filed on the 13th, both of which are incorporated herein by reference.

  Still other novel exendin analogs with agonist activity are listed in November 1998 entitled “Novel Exendin Agonist Compounds” claiming the benefit of US Provisional Application No. 60 / 066,029, filed November 14, 1997. PCT Application No. PCT / US98 / 24273, filed on the 13th of the month, both of which are incorporated herein by reference.

  Yet another exendin analog having agonist activity was entitled “Methods for Regulating Gastrointestinal Activity”, a continuation-in-part of US patent application Ser. No. 08 / 694,954 filed Aug. 8, 1996. PCT Application No. PCT / US97 / 14199, filed August 8, 1997, both of which are incorporated herein by reference.

  Yet another exendin analog with agonist activity is claimed in US Provisional Application No. 60 / 034,905, filed January 7, 1997, "Use of Exendins and Agonists Thereof for the Reduction. PCT Application No. PCT / US98 / 00449, filed January 7, 1998, entitled “of Food Intake”, both of which are incorporated herein by reference.

  Exendin agonist activity can be assessed, for example, by confirming activity in an assay incorporated by reference in the cited application. The effects of exendins or exendin agonists are identified, evaluated, or screened using the methods described therein for determining the effects of exendins, or other methods known in the art or equivalent. Screening assays for potential exendin agonist compounds or candidate exendin agonist compounds may include in vitro GLP-1 receptor competition assays or direct binding screens, or activity screens such as increased cAMP production or insulin synthesis .

Certain preferred exendin analogs having agonist activity are:
Exendin-4 (1-30) [SEQ ID NO: 19: 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: 20: 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-NH ₂ ] ;
Exendin-4 (1-28) amide [SEQ ID NO: 21: 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-NH ₂ ];
¹⁴ Leu ²⁵ Phe exendin-4 amide [SEQ ID NO: 22: 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-NH ₂ ];
^l4 Leu, ²⁵ Phe exendin-4 (1-28) amide [SEQ ID NO: 23: 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- NH ₂ ]; and
¹⁴ Leu, ²² Ala, ²⁵ Phe Exendin-4 (1-28) amide [SEQ ID NO: 24: 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-NH ₂ ].

  Also included within the scope of the invention are pharmaceutically acceptable salts of compounds of formula (III-X) and pharmaceutical compositions comprising the compounds and salts thereof.

Formula III
Moreover, the exendin analog having agonist activity has the formula (III) [SEQ ID NO: 25]:

[In the formula
Xaa ₁ is His, Arg or Tyr;
Xaa ₂ is Ser, Gly, Ala or Thr;
Xaa ₃ is Asp or Glu;
Xaa ₅ is an Ala or Thr;
Xaa ₆ is Ala, Phe, Tyr or naphthylalanine;
Xaa ₇ is Thr or Ser;
Xaa ₈ is Ala, Ser or Thr;
Xaa ₉ is Asp or Glu;
Xaa ₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa ₁₁ is Ala or Ser;
Xaa ₁₂ is Ala or Lys;
Xaa ₁₃ is Ala or Gln;
Xaa ₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;
Xaa ₁₅ is Ala or Glu;
Xaa ₁₆ is Ala or Glu;
Xaa ₁₇ is Ala or Glu;
Xaa ₁₉ is Ala or Val;
Xaa ₂₀ is Ala or Arg;
Xaa ₂₁ is Ala or Leu;
Xaa ₂₂ is Ala, Phe, Tyr or naphthylalanine;
Xaa ₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
Xaa ₂₄ is Ala, Glu or Asp;
Xaa ₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa ₂₆ is Ala or Leu;
Xaa ₂₇ is Ala or Lys;
Xaa ₂₈ is Ala or Asn;
Z ₁ is -OH,
-NH ₂
Gly-Z ₂ ,
Gly Gly-Z ₂ ,
Gly Gly Xaa ₃₁ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂ or
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂ ;
Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and
Z ₂ is —OH or —NH ₂ ;
_{However, Xaa 3, Xaa 5, Xaa} 6, Xaa 8, Xaa 10, Xaa 11, Xaa 12, Xaa 13, Xaa 14, Xaa 15, Xaa l6, Xaa 17, Xaa l9, Xaa 20, Xaa 21, Xaa 24, 3 or more of Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ and Xaa ₂₈ are not Ala]
Including those described in US 09 / 554,533.

  Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine and N-alkylalanine are preferably lower alkyl groups having 1 to about 6 carbon atoms, more preferably 1 to 4 carbon atoms. Including.

Preferred exendin analogs include those where Xaa ₁ is His or Tyr. More preferably, Xaa ₁ is His.

A compound in which Xaa ₂ is Gly is preferred.

A compound in which Xaa ₁₄ is Leu, pentylglycine or Met is preferred.

Preferred compounds are those where Xaa ₂₅ is Trp or Phe.

Preferred compounds are those in which Xaa ₆ is Phe or naphthylalanine; Xaa ₂₂ is Phe or naphthylalanine, and Xaa ₂₃ is Ile or Val.

Preference is given to compounds in which Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently selected from Pro, homoproline, thioproline and N-alkylalanine.

Preferably Z ₁ is —NH ₂ .

Preferably Z ₂ is —NH ₂ .

According to one embodiment, formula (III) [wherein Xaa ₁ is His or Tyr; Xaa ₂ is Gly; Xaa ₆ is Phe or naphthylalanine; Xaa ₁₄ is Leu, pentylglycine or Met. Yes; Xaa ₂₂ is Phe or naphthylalanine; Xaa ₂₃ is Ile or Val; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently selected from Pro, homoproline, thioproline or N-alkylalanine Are preferred. More preferably, Z ₁ is —NH ₂ .

According to a particularly preferred embodiment, a particularly preferred compound is of formula (III) [wherein Xaa ₁ is His or Arg; Xaa ₂ is Gly or Ala; Xaa ₃ is Asp or Glu; ₅ is Ala or Thr; Xaa ₆ is Ala, Phe or neftyl align; Xaa ₇ is Thr or Ser; Xaa ₈ is Ala, Ser or Thr; Xaa ₉ is Asp or be Glu; Xaa ₁₀ is Ala, be Leu or pentylglycine glycine; Xaa ₁₁ is an Ala or Ser; Xaa ₁₂ is an Ala or Lys; Xaa ₁₃ is an Ala or Gln; Xaa ₁₄ Is Ala, Leu or pentylglycine; Xaa ₁₅ is Ala or Glu; Xaa ₁₆ is Ala or Glu; Xaa ₁₇ is Ala or Glu; Xaa ₁₉ is Ala or Val Xaa ₂₀ is Ala or Arg; Xaa ₂₁ is Ala or Leu; Xaa ₂₂ is Phe or naphthylalanine; Xaa ₂₃ is Ile, Val or Xaa ₂₄ is Ala, Glu or Asp; Xaa ₂₅ is Ala, Trp or Phe; Xaa ₂₆ is Ala or Leu; Xaa ₂₇ is Ala or Lys; in it; Xaa ₂₈ is an Ala or Asn; Z _{1 is, -OH, -NH 2, Gly-} Z 2, Gly Gly-Z 2, Gly Gly Xaa 31 -Z 2, Gly Gly Xaa 31 Ser-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂ ; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independent Pro homoproline, thioproline or N-methylalanine; and Z ₂ is —OH or —NH ₂ ; provided that Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , _{Xaa 13, Xaa 14, Xaa 15} , Xaa 16, Xaa 17, Xaa 19, Xaa 20, Xaa 21, Xaa 24, Xaa 25, Xaa 26 3 or more of the Xaa ₂₇ and Xaa ₂₈ can contain more non-Ala]. Particularly preferred compounds are those described in PCT Application No. PCT / US98 / 24210, filed November 13, 1998, entitled “Novel Exendin Agonist Compounds”, where the compound identified as compound 2-23 Including.

According to a particularly preferred embodiment, the compound wherein Xaa ₁₄ is Leu, Ile, Val or pentylglycine, more preferably Leu or pentylglycine, and Xaa ₂₅ is Phe, Tyr or naphthylalanine, more preferably Phe or naphthylalanine Is provided. These compounds will not be sensitive to oxidative degradation reactions both in vitro and in vivo, as well as during compound synthesis.

Formula IV
An exendin analog having agonist activity has the formula (IV) [SEQ ID NO: 26]:

[Where:
Xaa ₁ is His, Arg, Tyr, Ala, Norval, Val or Norleu;
Xaa ₂ is Ser, Gly, Ala or Thr;
Xaa ₃ is Ala, Asp or Glu;
Xaa ₄ is Ala, Norval, Val, Norleu or Gly;
Xaa ₅ is an Ala or Thr;
Xaa ₆ is Phe, Tyr or naphthylalanine;
Xaa ₇ is Thr or Ser;
Xaa ₈ is Ala, Ser or Thr;
Xaa ₉ is Ala, Norval, Val, Norleu, Asp or Glu;
Xaa ₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa ₁₁ is Ala or Ser;
Xaa ₁₂ is Ala or Lys;
Xaa ₁₃ is Ala or Gln;
Xaa ₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;
Xaa ₁₅ is Ala or Glu;
Xaa ₁₆ is Ala or Glu;
Xaa ₁₇ is Ala or Glu;
Xaa ₁₉ is Ala or Val;
Xaa ₂₀ is Ala or Arg;
Xaa ₂₁ is Ala or Leu;
Xaa ₂₂ is Phe, Tyr or naphthylalanine;
Xaa ₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
Xaa ₂₄ is Ala, Glu or Asp;
Xaa ₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa ₂₆ is Ala or Leu;
Xaa ₂₇ is Ala or Lys;
Xaa ₂₈ is Ala or Asn;
Z ₁ is -OH,
-NH ₂ ,
Gly-Z ₂ ,
Gly Gly-Z ₂ ,
Gly Gly Xaa ₃₁ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂ or
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ Xaa ₃₉ -Z ₂ ;
Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and
Z ₂ is —OH or —NH ₂ ;
However, Xaa ₃ , Xaa ₄ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₉ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , 3 or more of Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ and Xaa ₂₈ are not Ala; and if Xaa ₁ is His, Arg or Tyr, then Xaa ₃ , Xaa ₄ and At least one of Xaa ₉ is Ala], including those described in US Provisional Application No. 09 / 554,531.

  Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine and N-alkylalanine include lower alkyl groups having 1 to about 6 carbon atoms, more preferably 1 to 4 carbon atoms. Suitable compounds of formula (II) are described in Examples 1-89 (respectively in application number PCT / US98 / 24273 filed on November 13, 1998 entitled "Novel Exendin Agonist Compounds"). Including those identified in “Compound 1-89”) and the corresponding compounds identified in Examples 104 and 105.

Preferred such exendin analogs include those where Xaa ₁ is His, Ala or Norval. More preferably, Xaa ₁ is His or Ala. Most preferably Xaa ₁ is His.

Compounds of formula (IV) [wherein Xaa ₂ is Gly] are preferred.

Compounds of formula (IV) [wherein Xaa ₃ is Ala] are preferred.

Compounds of formula (IV) [wherein Xaa ₄ is Ala] are preferred.

Compounds of formula (IV) [wherein Xaa ₉ is Ala] are preferred.

Preference is given to compounds of the formula (IV) wherein Xaa ₁₄ is Leu, pentylglycine or Met.

Preferred compounds of formula (IV) are those wherein Xaa ₂₅ is Trp or Phe.

Preferred compounds of formula (IV) are those wherein Xaa ₆ is Ala, Phe or naphthylalanine; Xaa ₂₂ is Phe or naphthylalanine; and Xaa ₂₃ is Ile or Val.

Preference is given to compounds of the formula (IV), wherein Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently selected from Pro, homoproline, thioproline and N-alkylalanine.

Preferably Z ₁ is —NH ₂ .

Preferably Z ₂ is —NH ₂ .

According to one embodiment, formula (IV) wherein Xaa ₁ is Ala, His or Tyr, more preferably Ala or His; Xaa ₂ is Ala or Gly; Xaa ₆ is Phe or naphthylalanine Xaa ₁₄ is Ala, Leu, pentylglycine or Met; Xaa ₂₂ is Phe or naphthylalanine; Xaa ₂₃ is Ile or Val; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently , Pro, homoproline, thioproline or N-alkylalanine; and Xaa ₃₉ is Ser or Tyr, more preferably Ser]. More preferably, Z ₁ is —NH ₂ .

According to a particularly preferred embodiment, a preferred compound is of formula (IV) wherein Xaa ₁ is His or Ala; Xaa ₂ is Gly or Ala; Xaa ₃ is Ala, Asp or Glu; Xaa ₄ is Xaa ₅ is Ala or Thr; Xaa ₆ is Phe or naphthylalanine; Xaa ₇ is Thr or Ser; Xaa ₈ is Ala, Ser or Thr; Xaa ₉ is Ala; Xaa ₁₀ is Ala, Leu or pentylglycine; Xaa ₁₁ is Ala or Ser; Xaa ₁₂ is Ala or Lys; Xaa ₁₃ is Ala or Gln; Xaa ₁₄ is Ala; Xu ₁₅ is Ala or Glu; Xaa ₁₆ is Ala or Glu; Xaa ₁₇ is Ala or Glu; Xaa ₁₉ is Ala or Val; Xaa ₂₀ is Ala or It is arg; Xaa ₂₁ is an Ala or Leu; Xaa ₂₂ is Phe or naphthylalanine; Xaa ₂₃ is Ile, Val or tert- Buchiruguri Be down; Xaa ₂₄ is an Ala, Glu or Asp; Xaa ₂₅ is Ala, be Trp or Phe; Xaa ₂₆ is an Ala or Leu; Xaa ₂₇ is an Ala or Lys; Xaa ₂₈ is Ala or Asn Z ₁ is —OH, —NH ₂ , Gly-Z ₂ , Gly Gly-Z ₂ , Gly Gly Xaa ₃₁ -Z ₂ , Gly Gly Xaa ₃₁ Ser-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂ or Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ Xaa ₃₉ -Z ₂ ; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently Pro homoproline, thioproline or N-methylalanine; and Z ₂ is —OH or —NH ₂ ; provided that Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xa 3 or more of a ₂₄ , Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ and Xaa ₂₈ are not Ala; provided that if Xaa ₁ is His, Arg or Tyr, then Xaa ₃ , Xaa ₄ and Xaa _At least one of ₉ is Ala]. A particularly preferred compound of formula (IV) has application number PCT / US98 / 24273, filed on November 13, 1998 entitled "Novel Exendin Agonist Compounds" having the amino acid sequence of SEQ ID NO: 5-93. Including those described in.

According to a particularly preferred embodiment, formula (IV) wherein Xaa ₁₄ is Ala, Leu, Ile, Val or pentylglycine, more preferably Leu or pentylglycine, and Xaa ₂₅ is Ala, Phe, Tyr. Or naphthylalanine, more preferably Phe or naphthylalanine]. These compounds will be less sensitive to oxidative degradation reactions both in vitro and in vivo, and during the synthesis of the compounds.

Formula V
Also within the scope of the invention are the narrower genus concepts of compounds having peptides of various lengths, such as, for example, the genus concept of compounds that do not include peptides having a length of 28, 29 or 30 amino acid residues, respectively. Is. In addition, the present invention provides, for example, formula (V) [SEQ ID NO: 27]:
Xaa ₁ Xaa ₂ Xaa ₃ Gly Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈ Xaa ₉ Xaa ₁₀
Xaa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉
Xaa ₂₀ Xaa ₂₁ Xaa ₂₂ Xaa ₂₃ Xaa ₂₄ Xaa ₂₅ Xaa ₂₆ Xaa ₂₇ Xaa ₂₈ -Z ₁ ;
[Where:
Xaa ₁ is His or Arg;
Xaa ₂ is Gly or Ala;
Xaa ₃ is Asp or Glu;
Xaa ₅ is Ala or Thr;
Xaa ₆ is Ala, Phe or naphthylalanine;
Xaa ₇ is Thr or Ser;
Xaa ₈ is Ala, Ser or Thr;
Xaa ₉ is Asp or Glu;
Xaa ₁₀ is Ala, Leu or pentylglycine;
Xaa ₁₁ is Ala or Ser;
Xaa ₁₂ is Ala or Lys;
Xaa ₁₃ is Ala or Gln;
Xaa ₁₄ is Ala, Leu or pentylglycine;
Xaa ₁₅ is Ala or Glu;
Xaa ₁₆ is Ala or Glu;
Xaa ₁₇ is Ala or Glu;
Xaa ₁₉ is Ala or Val;
Xaa ₂₀ is Ala or Arg;
Xaa ₂₁ is Ala or Leu;
Xaa ₂₂ is Phe or naphthylalanine;
Xaa ₂₃ is Ile, Val or tert-butylglycine;
Xaa ₂₄ is Ala, Glu or Asp;
Xaa ₂₅ is Ala, Trp, or Phe;
Xaa ₂₆ is Ala or Leu;
Xaa ₂₇ is Ala or Lys;
Xaa ₂₈ is Ala or Asn;
Z ₁ is -OH,
-NH ₂ ,
Gly-Z ₂ ,
Gly Gly -Z ₂ ,
Gly Gly Xaa ₃₁ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂ or
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂ ;
Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently selected from Pro, homoproline, thioproline and N-methylylalanine; and
Z ₂ is -OH or -NH ₂ ;
However, Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ and 3 or more of Xaa ₂₈ are not Ala]; and pharmaceutically acceptable salts thereof, entitled “Novel Exendin Agonist Compounds” It includes a narrower genus concept for the compounds described in PCT Application No. PCT / US98 / 24210 filed November 13, 1998.

Formula VI
In addition, the present invention provides, for example, formula [VI] [SEQ ID NO: 28]:
Xaa ₁ Xaa ₂ Xaa ₃ Xaa ₅ Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈ Xaa ₉ Xaa ₁₀
Xaa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉
Xaa ₂₀ Xaa ₂₁ Xaa ₂₂ Xaa ₂₃ Xaa ₂₄ Xaa ₂₅ Xaa ₂₆ Xaa ₂₇ Xaa ₂₈ -Z ₁ ;
[Where:
Xaa ₁ is His or Ala;
Xaa ₂ is Gly or Ala;
Xaa ₃ is Ala, Asp or Glu;
Xaa ₄ is Ala or Gly;
Xaa ₅ is Ala or Thr;
Xaa ₆ is Phe or naphthylalanine;
Xaa ₇ is Thr or Ser;
Xaa ₈ is Ala, Ser or Thr;
Xaa ₉ is Ala, Asp or Glu;
Xaa ₁₀ is Ala, Leu or pentylglycine;
Xaa ₁₁ is Ala or Ser;
Xaa ₁₂ is Ala or Lys;
Xaa ₁₃ is Ala or Gln;
Xaa ₁₄ is Ala, Leu, Met or pentylglycine;
Xaa ₁₅ is Ala or Glu;
Xaa ₁₆ is Ala or Glu;
Xaa ₁₇ is Ala or Glu;
Xaa ₁₉ is Ala or Val;
Xaa ₂₀ is Ala or Arg;
Xaa ₂₁ is Ala or Leu;
Xaa ₂₂ is Phe or naphthylalanine;
Xaa ₂₃ is Ile, Val or tert-butylglycine;
Xaa ₂₄ is Ala, Glu or Asp;
Xaa ₂₅ is Ala, Trp or Phe;
Xaa ₂₆ is Ala or Leu;
Xaa ₂₇ is Ala or Lys;
Xaa ₂₈ is Ala or Asn;
Z ₁ is -OH,
-NH ₂ ,
Gly-Z ₂ ,
Gly Gly-Z ₂
Gly Gly Xaa ₃₁ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ Ser-Z ₂ ;
Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently Pro, homoproline, thioproline, or N-methylylalanine; and
Z ₂ is —OH or —NH ₂ ;
However, Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , Within 3 of Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ , and Xaa ₂₈ are Ala; and if Xaa ₁ is His, Arg or Tyr, then at least Xaa ₃ , Xaa ₄ and Xaa ₉ One is Ala]; and a PCT application number filed on November 13, 1998 entitled "Novel Exendin Agonist Compounds" having a specific amino acid sequence such as a pharmaceutically acceptable salt thereof; Includes a narrower genus concept of peptide compounds described in PCT / US98 / 24273.

Preferred compounds of formula (VI) include those wherein Xaa ₁ is His, Ala, Norval or 4-imidazopropionyl. Preferably Xaa ₁ is His, or 4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

  Preferred compounds of formula (VI) include those where Xaa2 is Gly.

  Preferred compounds of formula (VI) include those where Xaa4 is Ala.

  Preferred compounds of formula (VI) include those where Xaa9 is Ala.

  Preferred compounds of formula (VI) include those where Xaa14 is Leu, pentylglycine or Met.

  Preferred compounds of formula (VI) include those where Xaa25 is Trp or Phe.

  Preferred compounds of formula (VI) include those where Xaa6 is Ala, Phe or naphthylalanine; Xaa22 is Phe or naphthylalanine; and Xaa23 is Ile or Val.

  Preferred compounds of formula (VI) include those wherein Z1 is -NH2.

  Preferred compounds of formula (VI) include those wherein Xaa31, Xaa36, Xaa37 and Xaa38 are independently selected from the group consisting of Pro, homoproline, thioproline and N-alkylalanine.

  Preferred compounds of formula (VI) include those where Xaa39 is Ser or Tyr, preferably Ser.

  Preferred compounds of formula (VI) include those where Z2 is -NH2.

  Preferred compounds of formula (VI) include those wherein Z1 is -NH2.

  Preferred compounds of formula (VI) include those where Xaa21 is Lys-NH2-R, wherein R is Lys, Arg, C1-C10 linear or branched alkanoyl.

  Preferred compounds of formula (VI) are those wherein X1 is Lys Asn, Lys-NHε-R Asn, or Lys-NHε-R Ala, wherein R is Lys, Arg, C1-C10 linear or branched alkanoyl. Including some. Preferred compounds of formula (VI) are those described in PCT Application No. PCT / US98 / 24273 filed November 13, 1998 entitled "Novel Exendin Agonist Compounds" selected from SEQ ID NOs: 95-110. Including those having an amino acid sequence.

Formula VII
Also, the formula (VII) [SEQ ID NO: 29]:
Xaa ₁ Xaa ₂ Xaa ₃ Gly Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈ Xaa ₉ Xaa ₁₀
Xaa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉ Xaa ₂₀
Xaa ₂₁ Xaa ₂₂ Xaa ₂₃ Xaa ₂₄ Xaa ₂₅ Xaa ₂₆ X ₁ -Z ₁ ;
[Where:
Xaa ₁ is His, Arg or Tyr or 4-imidazopropionyl;
Xaa ₂ is Ser, Gly, Ala or Thr;
Xaa ₃ is Asp or Glu;
Xaa ₅ is Ala or Thr;
Xaa ₆ is Ala, Phe, Tyr or naphthylalanine;
Xaa ₇ is Thr or Ser;
Xaa ₈ is Ala, Ser or Thr;
Xaa ₉ is Asp or Glu;
Xaa ₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa ₁₁ is Ala or Ser;
Xaa ₁₂ is Ala or Lys;
Xaa ₁₃ is Ala or Gln;
Xaa ₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;
Xaa ₁₅ is Ala or Glu;
Xaa ₁₆ is Ala or Glu;
Xaa ₁₇ is Ala or Glu;
Xaa ₁₉ is Ala or Val;
Xaa ₂₀ is Ala or Arg;
Xaa ₂₁ is Ala, Leu or Lys-NH ^ε -R where R is Lys, Arg, C ₁ -C ₁₀ linear or branched alkanoyl or cycloalkylalkanoyl;
Xaa ₂₂ is Phe, Tyr or naphthylalanine;
Xaa ₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
Xaa ₂₄ is Ala, Glu or Asp;
Xaa ₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa ₂₆ is Ala or Leu;
X ₁ is Lys Asn, Asn Lys, Lys-NH ^ε -R Asn, Asn Lys-NH ^ε -R, Lys-NH ^ε -R Ala, Ala Lys-NH ^ε -R (where R is Lys, Arg C ₁ -C _{10 are} straight-chain or branched alkanoyl or cycloalkylalkanoyl),
Z ₁ is -OH,
-NH ₂ ,
Gly-Z ₂ ,
Gly Gly-Z ₂ ,
Gly Gly Xaa ₃₁ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂ or
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂ ;
Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkyl glycine, N-alkylpentyl glycine and N-alkyl alanine And
Z ₂ is -OH or -NH ₂ ;
However, Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , PCT Application No. PCT / US98 / 24210 filed on November 13, 1998 entitled "Novel Exendin Agonist Compounds", including Xaa ₂₅ and three or more of Xaa ₂₆ are not Ala] Are provided. Also within the scope of the invention are pharmaceutically acceptable salts and pharmaceutical compositions of compounds of formula (VII) containing said compounds and salts thereof.

Preferred exendin analogs of formula (VII) include those where Xaa ₁ is His, Tyr or 4-imidazopropionyl. More preferably, Xaa ₁ is His.

Preference is given to compounds of the formula (VII) in which Xaa ₁ is 4-imidazopropionyl.

Preference is given to compounds of the formula (VII) [wherein Xaa ₂ is Gly].

Preferred compounds of formula (VII) are those wherein Xaa ₁₄ is Leu, pentylglycine or Met.

Preferred compounds of formula (VII) are those wherein Xaa ₂₅ is Trp or Phe.

According to one embodiment, compounds of formula (VII) are preferred, wherein Xaa ₆ is Phe or naphthylalanine; and Xaa ₂₂ is Phe or naphthylalanine; and Xaa ₂₃ is Ile or Val. More preferably, Z ₁ is —NH ₂ . According to one embodiment, of formula (VII): wherein Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently selected from the group consisting of Pro, homoproline, thioproline and N-alkylalanine Such compounds are particularly preferred. More preferably, Z ₂ is —NH ₂ .

Preferred compounds of formula (VII) are those wherein X ₁ is Lys Asn, Lys-NH ^ε -R Asn, or Lys-NH ^ε -R Ala [wherein R is Lys, Arg, C ₁ -C ₁₀ linear or It is a branched alkanoyl]. Preferred compounds of formula (VII) are described in PCT Application No. PCT / US98 / 24210, filed November 13, 1998, identified as Compound No. 62-69 and entitled “Novel Exendin Agonist Compounds” Of the compound.

Preferred such exendin analogs include those where Xaa ₁ is His, Ala or NorVal. More preferably, Xaa ₁ is His or Ala. Most preferably, Xaa ₁ is His.

Preference is given to compounds of the formula (VII) [wherein Xaa ₂ is Gly].

Preference is given to compounds of the formula (VII) in which Xaa ₃ is Ala.

Preference is given to compounds of the formula (VII) in which Xaa ₄ is Ala.

Compounds of formula (VII) [wherein Xaa ₉ is Ala] are preferred.

Preference is given to compounds of the formula (VII) wherein Xaa ₁₄ is Leu, pentylglycine or Met.

Preferred compounds of formula (VII) are those wherein Xaa ₂₅ is Trp or Phe.

Preferred compounds of formula (VII) are those wherein Xaa ₆ is Ala, Phe or naphthylalanine; Xaa ₂₂ is Phe or naphthylalanine; and Xaa ₂₃ is Ile or Val.

Preference is given to compounds of the formula (VII) in which Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently selected from Pro, homoproline, thioproline and N-alkylalanine.

Preferably Z ₁ is —NH ₂ .

Preferably Z ₂ is —NH ₂ .

According to one embodiment, formula (VII): wherein Xaa ₁ is Ala, His or Tyr, more preferably Ala or His; Xaa ₂ is Ala or Gly; Xaa ₆ is Phe or naphthylalanine Xaa ₁₄ is Ala, Leu, pentylglycine or Met; Xaa ₂₂ is Phe or naphthylalanine; Xaa ₂₃ is Ile or Val; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ Are independently selected from Pro, homoproline, thioproline or N-alkylalanine; and Xaa ₃₉ is Ser or Tyr, more preferably Ser]. More preferably, Z ₁ is —NH ₂ .

According to a particularly preferred embodiment, the preferred compound is of formula (VII) wherein Xaa ₁ is His or Ala; Xaa ₂ is Gly or Ala; Xaa ₃ is Ala, Asp or Glu; Xaa ₄ is Ala or Gly; Xaa ₅ is Ala or Thr; Xaa ₆ is Phe or naphthylalanine; Xaa ₇ is Thr or Ser; Xaa ₈ is Ala, Ser or Thr Xaa ₉ is Ala, Asp or Glu; Xaa ₁₀ is Ala, Leu or pentylglycine; Xaa ₁₁ is Ala or Ser; Xaa ₁₂ is Ala or Lys; Xaa ₁₃ Is Ala or Gln; Xaa ₁₄ is Ala, Leu, Met or pentylglycine; Xaa ₁₅ is Ala or Glu; Xaa ₁₆ is Ala or Glu; Xaa ₁₇ is Ala or Glu Xaa ₁₉ is Ala or Val; Xaa ₂₀ is Ala or Arg; Xaa ₂₁ is Ala or Leu; Xaa ₂₂ is Phe or naphthylarani. Xaa ₂₃ is Ile, Val or tert-butylglycine; Xaa ₂₄ is Ala, Glu or Asp; Xaa ₂₅ is Ala, Trp or Phe; Xaa ₂₆ is Ala or Leu Xaa ₂₇ is Ala or Lys; Xaa ₂₈ is Ala or Asn; Z ₁ is —OH, —NH ₂ , Gly-Z ₂ , Gly Gly-Z ₂ , Gly Gly Xaa ₃₁ − Z ₂ , Gly Gly Xaa ₃₁ Ser-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂ , Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂ or Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ Xaa ₃₉ -Z ₂ ; Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently Pro homoproline, thioproline or N-methylalanine; and Z ₂ is- OH or —NH ₂ ; provided that Xaa ₃ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ , Xaa ₂₇ and Xaa ₂₈ are Ala; And if Xaa ₁ is His, Arg or Tyr, then at least one of Xaa ₃ , Xaa ₄ and Xaa ₉ is Ala]. A particularly preferred compound of formula (VII) is PCT Application No. PCT / US98 filed on November 13, 1998 entitled "Novel Exendin Agonist Compounds" having the amino acid sequence identified as SEQ ID NO: 5-93. Includes those described in / 24210.

  According to a particularly preferred embodiment, formula (VII) wherein Xaa14 is Ala, Leu, Ile, Val or pentylglycine, more preferably Leu or pentylglycine, and Xaa25 is Ala, Phe, Tyr or naphthylalanine, more preferably Is Phe or naphthylalanine]. These compounds will be less sensitive to oxidative degradation reactions both in vitro and in vivo and during the synthesis of the compounds.

Formula VIII
Also, the formula (VI) [SEQ ID NO: 30]:
Xaa ₁ Xaa ₂ Xaa ₃ Xaa ₄ Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈ Xaa ₉ Xaa ₁₀
Xaa ₁₁ Xaa ₁₂ Xaa ₁₃ Xaa ₁₄ Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Ala Xaa ₁₉ Xaa ₂₀
Xaa ₂₁ Xaa ₂₂ Xaa ₂₃ Xaa ₂₄ Xaa ₂₅ Xaa ₂₆ X ₁ -Z ₁ ;
[Where:
Xaa ₁ is His, Arg, Tyr, Ala, Norval, Val, Norleu or 4-imidazopropionyl;
Xaa ₂ is Ser, Gly, Ala or Thr;
Xaa ₃ is Ala, Asp or Glu;
Xaa ₄ is Ala, Norval, Val, Norleu or Gly;
Xaa ₅ is Ala or Thr;
Xaa ₆ is Phe, Tyr or naphthylalanine;
Xaa ₇ is Thr or Ser;
Xaa ₈ is Ala, Ser or Thr;
Xaa ₉ is Ala, Norval, Val, Norleu, Asp or Glu;
Xaa ₁₀ is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa ₁₁ is Ala or Ser;
Xaa ₁₂ is Ala or Lys;
Xaa ₁₃ is Ala or Gln;
Xaa ₁₄ is Ala, Leu, Ile, pentylglycine, Val or Met;
Xaa ₁₅ is Ala or Glu;
Xaa ₁₆ is Ala or Glu;
Xaa ₁₇ is Ala or Glu;
Xaa ₁₉ is Ala or Val;
Xaa ₂₀ is Ala or Arg;
Xaa ₂₁ is Ala, Leu or Lys-NH ^ε -R where R is Lys, Arg, C ^1-10 linear or branched alkanoyl or cycloarray-alkanoyl;
Xaa ₂₂ is Phe, Tyr or naphthylalanine;
Xaa ₂₃ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
Xaa ₂₄ is Ala, Glu or Asp;
Xaa ₂₅ is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa ₂₆ is Ala or Leu;
X ₁ is Lys Asn, Asn Lys, Lys-NH ^ε -R Asn, Asn Lys-NH ^ε -R, Lys-NH ^ε -R Ala, Ala Lys-NH ^ε -R (where R is Lys, Arg, C ₁ -C ₁₀ linear or branched alkanoyl or cycloalkylalkanoyl);
Z ₁ is -OH,
-NH ₂ ,
Gly-Z ₂ ,
Gly Gly-Z ₂ ,
Gly Gly Xaa ₃₁ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala-Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ -Z ₂ ,
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ -Z ₂ or
Gly Gly Xaa ₃₁ Ser Ser Gly Ala Xaa ₃₆ Xaa ₃₇ Xaa ₃₈ Xaa ₃₉ -Z ₂ ;
Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently selected from the group consisting of Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine and N-alkylalanine;
Z ₂ is -OH or -NH ₂ ;
However, Xaa ₃ , Xaa ₄ , Xaa ₅ , Xaa ₆ , Xaa ₈ , Xaa ₉ , Xaa ₁₀ , Xaa ₁₁ , Xaa ₁₂ , Xaa ₁₃ , Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , Xaa ₁₇ , Xaa ₁₉ , Xaa ₂₀ , Within 3 of Xaa ₂₁ , Xaa ₂₄ , Xaa ₂₅ , Xaa ₂₆ is Ala; and if Xaa ₁ is His, Arg, Tyr, or 4-imidazopropionyl, Xaa ₃ , Xaa ₄ and Xaa Peptide compounds described in PCT Application No. PCT / US98 / 24273 filed on November 13, 1998 entitled "Novel Exendin Agonist Compounds", comprising a compound of at least one of ₉ is Ala] Is also provided.

Preferred compounds of formula (VIII) include those where Xaa ₁ is His, Ala, Norval or 4-imidazopropionyl. Preferably Xaa ₁ is His, or 4-imidazopropionyl or Ala, more preferably His or 4-imidazopropionyl.

Preferred compounds of formula (VIII) include those where Xaa ₂ is Gly.

Preferred compounds of formula (VIII) include those where Xaa ₄ is Ala.

Preferred compounds of formula (VIII) include those where Xaa ₉ is Ala.

Preferred compounds of formula (VIII) include those where Xaa ₁₄ is Leu, pentylglycine or Met.

Preferred compounds of formula (VIII) include those where Xaa ₂₅ is Trp or Phe.

Preferred compounds of formula (VIII) include those where Xaa ₆ is Ala, Phe or naphthylalanine; Xaa ₂₂ is Phe or naphthylalanine; and Xaa ₂₃ is Ile or Val.

Preferred compounds of formula (VIII) include those wherein Z ₁ is —NH ₂ .

Preferred compounds of formula (VIII) include those wherein Xaa ₃₁ , Xaa ₃₆ , Xaa ₃₇ and Xaa ₃₈ are independently selected from the group consisting of Pro, homoproline, thioproline and N-alkylalanine.

Preferred compounds of formula (VIII) include those where Xaa ₃₉ is Ser or Tyr, preferably Ser.

Preferred compounds of formula (VIII) include those where Z ₂ is —NH ₂ .

Preferred compounds of formula (VIII) include those wherein Z ₁ is —NH ₂ .

Preferred compounds of formula (VIII) include those where Xaa ₂₁ is Lys-NH ^ε -R, wherein R is Lys, Arg, C ₁ -C ₁₀ linear or branched alkanoyl.

Preferred compounds of formula (VIII) are those wherein X ₁ is Lys Asn, Lys-NH ^ε -R Asn, or Lys-NH ^ε -R Ala, wherein R is Lys, Arg, C ₁ -C ₁₀ linear or branched That is alkanoyl].

  Preferred compounds of formula (VIII) are PCT application numbers filed November 13, 1998 entitled "Novel Exendin Agonist Compounds" having an amino acid sequence selected from those identified as SEQ ID NOs: 95-110 Includes those described in PCT / US98 / 24273.

Formula IX
According to the invention, a particularly useful compound is of formula (IX) [SEQ ID NO: 31]:
Xaa ₁ Xaa ₂ Xaa ₃ Gly Thr Xaa ₄ Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈
Ser Lys Gln Xaa ₉ Glu Glu Glu Ala Val Arg Leu
Xaa ₁₀ Xaa ₁₁ Xaa ₁₂ Xaa ₁₃ Leu Lys Asn Gly Gly Xaa ₁₄
Ser Ser Gly Ala Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Xaa ₁₈ -Z
[Where:
Xaa ₁ is His, Arg or Tyr;
Xaa ₂ is Ser, Gly, Ala or Thr;
Xaa ₃ is Asp or Glu;
Xaa ₄ is Phe, Tyr or naphthalanine;
Xaa ₅ is Thr or Ser;
Xaa ₆ is Ser or Thr;
Xaa ₇ is Asp or Glu;
Xaa ₈ is Leu, Ile, Val, pentylglycine or Met;
Xaa ₉ is Leu, Ile, pentylglycine, Val or Met;
Xaa ₁₀ is Phe, Tyr or naphthalanine;
Xaa ₁₁ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
Xaa ₁₂ is Glu or Asp; Xaa ₁₃ is Trp, Phe, Tyr, or naphthylalanine;
Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine;
Xaa ₁₈ is Ser, Thr or Tyr; and Z is —OH or —NH ₂ ;
However, the compound does not have any formula of SEQ ID NO: 12 or 14, and was filed on January 7, 1998 entitled “Use of Exendins And Agonists There of For The Reduction of Food Intake”. Exendin analogs having agonist activity described in US patent application Ser. No. 09 / 003,869. Preferred N-alkyl groups for N-alkylglycine, N-alkylpentylglycine and N-alkylalanine preferably include lower alkyl groups having 1 to about 6 carbon atoms, more preferably 1 to 4 carbon atoms. Also useful in the present invention are pharmaceutically acceptable salts of compounds of formula (IX).

Preferred exendin analogs include those where Xaa ₁ is His or Tyr. More preferably, Xaa ₁ is His.

A compound in which Xaa ₂ is Gly is preferred.

A compound in which Xaa ₉ is Leu, pentylglycine or Met is preferred.

Preferred compounds include those where Xaa ₁₃ is Trp or Phe.

Xaa ₄ is Phe or naphthalanine; Xaa ₁₁ is Il or Val, and Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ are independently selected from Pro, homoproline, thioproline or N-alkylalanine Is preferred. Preferably, the N-alkylalanine has an N-alkyl group of 1 to about 6 carbon atoms.

According to a particularly preferred embodiment, Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ are the same amino acid residue.

Preferred are compounds wherein Xaa ₁₈ is Ser or Tyr, more preferably Ser.

Preferably Z is —NH ₂ .

According to one embodiment, formula (VII) wherein Xaa ₁ is His or Tyr, more preferably His; Xaa ₂ is Gly; Xaa ₄ is Phe or naphthalanine; Xaa ₉ is Xaa ₁₀ is Phe or naphthalanine; Xaa ₁₁ is Ile or Val; Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ are independently Pro, homoproline, and Leu, pentylglycine or Met; And thioproline or N-alkylalanine; and Xaa ₁₈ is Ser or Tyr, more preferably Ser]. More preferably, Z is —NH ₂ .

According to a particularly preferred embodiment, particularly preferred compounds, formula (IX) [wherein: Xaa ₁ is an His or Arg; Xaa ₂ is an Gly; Xaa ₃ is an Asp or Glu; Xaa ₄ is Xaa ₅ is Thr or Ser; Xaa ₆ is Ser or Thr; Xaa ₇ is Asp or Glu; Xaa ₈ is Leu or pentylglycine; Xaa is Phe or naphthylalanine; ₉ is Leu or pentylglycine; Xaa ₁₀ is Phe or naphthylalanine; Xaa ₁₁ is Ile, Val or t-butyltylglycine; Xaa ₁₂ is Glu or Asp; Xaa ₁₃ is Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ , and Xaa ₁₇ are independently Pro, homoproline, thioproline, or N-methylalanine; Xaa ₁₈ is Ser or Tyr: and Z is -OH or -NH _2; provided that the compound is any of SEQ ID NO: 7 or 9 Including those of do not have even the formula]. More preferably, Z is —NH ₂ .

According to a particularly preferred embodiment, there is provided a compound wherein Xaa ₉ is Leu, Ile, Val or pentylglycine, more preferably Leu or pentylglycine and Xaa ₁₃ is Phe, Tyr or naphthylalanine, more preferably Phe or naphthylalanine Is done. These compounds exhibit an advantageous duration of action and are believed to be less susceptible to oxidative degradation reactions both in vitro and in vivo, and during compound synthesis.

Formula X
Also, the formula (X) [SEQ ID NO: 32]:
Xaa ₁ Xaa ₂ Xaa ₃ Gly Thr Xaa ₄ Xaa ₅ Xaa ₆ Xaa ₇ Xaa ₈
Ser Lys Gln Xaa ₉ Glu Glu Glu Ala Val Arg Leu
Xaa ₁₀ Xaa ₁₁ Xaa ₁₂ Xaa ₁₃ Leu X ₁ Gly Gly Xaa ₁₄
Ser Ser Gly Ala Xaa ₁₅ Xaa ₁₆ Xaa ₁₇ Xaa ₁₈ -Z
[Where:
Xaa ₁ is His, Arg, Tyr or 4-imidazopropionyl;
Xaa ₂ is Ser, Gly, Ala or Thr;
Xaa ₃ is Asp or Glu;
Xaa ₄ is, Phe, is Tyr or naphthylalanine;
Xaa ₅ is Thr or Ser;
Xaa ₆ is Ser or Thr;
Xaa ₇ is Asp or Glu;
Xaa ₈ is Leu, Ile, Val, pentylglycine or Met;
Xaa ₉ is Leu, Ile, pentylglycine, Val or Met;
Xaa ₁₀ is Phe, Tyr or naphthylalanine;
Xaa ₁₁ is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
Xaa ₁₂ is Glu or Asp;
Xaa ₁₃ is Trp, Phe, Tyr, or naphthylalanine; X ₁ is Lys Asn, Asn Lys, Lys-NH ^ε -R Asn, Asn Lys-NH ^ε -R (wherein R is Lys, Arg, which is a C ₁ -C ₁₀ linear or branched alkanoyl or cycloalkylalkanoyl);
Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ are independently Pro, homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or N-alkylalanine;
Xaa ₁₈ is Ser, Thr or Tyr; Z is —OH or —NH ₂ ;
However, the compound does not have any formula of SEQ ID NO: 7 or 9.] PCT application number PCT / US98 filed on August 6, 1998 entitled “Novel Exendin Agonist Compounds” Also provided are the compounds described in / 16387. A suitable compound of formula (X) is PCT Application No. PCT / US98 / 16387, filed August 6, 1998 entitled "Novel Exendin Agonist Compounds" having the amino acid sequence of SEQ ID NOs: 37-40. Including the compounds described in 1.

Preferred exendin analogs of formula (X) include those where Xaa ₁ is His, Tyr or 4-imidazopropionyl. More preferably, Xaa ₁ is His or 4-imidazopropionyl.

Compounds of formula (X) [wherein Xaa ₂ is Gly] are preferred.

Preference is given to compounds of the formula (X) in which Xaa ₉ is Leu, pentylglycine or Met.

Preference is given to compounds of the formula (X), wherein Xaa ₁₃ is Trp or Phe.

Formula (X) [wherein X ₁ is Lys Asn, or Lys-NH ^ε -R Asn (wherein R is Lys, Arg, C ₁ -C ₁₀ linear or branched alkanoyl)] Are preferred.

Further, the formula (X) [wherein, Xaa ₄ is Phe or naphthylalanine; Xaa ₁₀ is Phe or naphthylalanine; Xaa ₁₁ is Ile or _{Val, Xaa 14, Xaa 15,} Xaa 16 and Xaa ₁₇ Are independently selected from Pro, homoproline, thioproline or N-alkylalanine]. According to a particularly preferred embodiment, Xaa ₁₈ is Ser or Tyr. A compound in which Xaa ₁₈ is Ser is preferred. Preferably Z is —NH ₂ .

According to one preferred embodiment, the formula (X) wherein Xaa ₄ is Phe or naphthylalanine; Xaa ₁₀ is Phe or naphthylalanine; Xaa ₁₁ is Ile or Val, X ₁ is Lys Asn Or Lys-NH ^ε -R Asn (wherein R is Lys, Arg, C ₁ -C ₁₀ linear or branched alkanoyl) and Xaa ₁₄ , Xaa ₁₅ , Xaa ₁₆ and Xaa ₁₇ are independently And is selected from Pro, homoproline, thioproline or N-alkylalanine].

  Exendins and exendin agonists that are peptides, such as the exendin analogs described herein, are described, for example, in Eng, et al., J. Biol. Chem. 265, incorporated herein by reference. : 20259-62, 1990; and Eng, et al., J. Biol. Chem. 267: 7402-05, 1992. Alternatively, exendins, incretins, GLP-1, and peptides agonists, analogs, derivatives and variants are described, for example, by Raufman, et al., J. Biol. Chem. 267: 21432-37, 1992), as described above and well known in the art, one skilled in the art using standard solid phase peptide synthesis techniques and preferably an automated or semi-automated peptide synthesizer. Can be prepared by known methods.

  The peptide molecules of the present invention can also be prepared using recombinant DNA techniques using methods currently known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor (1989), adding any necessary chemical modifications to the molecule in further steps as known in the art. Alternatively, such compounds can be prepared by homogeneous phase peptide synthesis methods. Non-peptide compounds useful in the present invention can be prepared by methods known in the art. For example, phosphate-containing amino acids and peptides containing such amino acids can be prepared by methods known in the art. See, for example, Bartlett and Landen, Biorg. Chem. 14: 356-377 (1986). Also, as discussed above, exendin, its agonists, analogs, derivatives, variants, using methods for making and / or purifying GLP-1 and its agonists, analogs, derivatives, variants, and fragments , And fragments can be made and / or purified.

Also, amino acid sequence identity greater than 50% or 55%, and preferably greater than 70, 80, 90, or 95% amino acid sequence relative to SEQ ID NO: 1, 12, and 14, and its incomplete sequence Peptide sequences having identity are also included in the present invention. As used herein, sequence identity refers to a comparison made between two molecules using standard algorithms well known in the art. A preferred algorithm for calculating sequence identity for the present invention is the Smith-Waterman algorithm, eg, SEQ ID NO: 1 [ie GLP-1 (1-37)], SEQ ID NO: 12 or 14 [exendin, respectively. -3 and 4] can be used as reference sequences to define the percentage identity of homology over its length. Although some parameter values have been found to give more biologically realistic results than others, the choice of parameter values for matches, mismatches, and insertions or deletions is arbitrary. One preferred set of parameter values for the Smith-Waterman algorithm is 1 for matched residues and -1 / for mismatched residues (residues are either single nucleotides or single amino acids). It is described in the “maximum similarity segment” approach using a value of 3. Waterman, Bull. Math. Biol. 46; 473 (1984). Insertions and deletions (indels), x is weighted as x _k = 1 + 1 / 3k, where k is the number of residues in a given insert or deletion. Ibid.

によって与えられるパーセント同一性を有するだろう。 For example, except for 18 amino acid substitutions and 3 amino acid insertions, a sequence that is identical to the 37-amino acid residue sequence of SEQ ID NO: 1 is:

Will have the percent identity given by.

  This algorithm can be used with any amino acid sequence to determine sequence homology. For purposes of determining homology, truncation of the mature sequence should be ignored. Sequences with a lower degree of homology, comparable biological activity, and similar expression characteristics are considered equivalent.

  The biological activity of GLP-1 agonists and / or analogs can be determined by in vitro and in vivo animal models and human studies well known to those skilled in the art. GLP-1 biological activity is generally determined by standard methods, generally using appropriate cells expressing the GLP-1 receptor on its surface, such as RINmSF cells or insulinoma cell lines such as INS-1 cells. It can be determined by a receptor binding activity screening procedure that includes providing. See Mojsov, Int. J. Peptide Protein Res. 40; 333 (1992) and EP 0708179 A2. The GLP-1 receptor is a cell surface protein found, for example, on insulin-producing pancreatic β-cells; the GLP-1 (7-36) receptor has been characterized in the art. Moreover, it is thought that there exists an additional receptor in the position which GLP-1 and exendin act, and can mediate the effect which this invention acts. Methods of determining whether a chemical or peptide binds to or activates a particular GLP-1 receptor are known to those skilled in the art. For example, US Pat. Nos. 6,051,689, 5,846,747, and 5,670,360 describe GLP-1 receptors and methods of using them. Alternatively, cells created to express the GLP-1 receptor can be used. In addition to measuring specific binding of the tracer to the membrane using radioimmunoassay methods, cAMP activity or glucose dependent insulin production can also be measured. In one method, a polynucleotide encoding a GLP-1 receptor is used to transfect the cell so that the cell expresses the GLP-1 receptor protein. Thus, for example, these methods involve contacting such cells with a compound to be screened and determining whether such a compound generates a signal (ie, activates the receptor). It can be used for screening for agonists. Other screening techniques include the use of cells expressing GLP-1 receptor, such as transfected CHO cells, in a system that measures extracellular pH or ionic changes caused by receptor activation. For example, a potential agonist may be contacted with a cell that expresses a GLP-1 protein receptor, and a second messenger response (eg, signal transduction or ion or pH change) is measured to determine if the potential agonist is It can be determined whether it is effective or not.

  The molecules of the invention can be used in conjunction with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide and a pharmaceutically acceptable carrier or excipient. The compounds of the present invention can be administered topically to animals, including human subjects, in any effective pharmaceutically acceptable form, such as in topical, lavage, oral, suppository, parenteral, injection and / or infusion dosage forms. As a buccal, sublingual, pulmonary or spray nasal spray, it can be administered in any other manner effective to deliver the agent. The route of administration is preferably designed to optimize the delivery and / or localization of the agent, and for the peptide molecules of the invention via transdermal or other parenteral injection routes, or transmucosal delivery. Is preferred.

  In addition to administration in conjunction with conventional carriers, the active ingredient can be administered by a variety of special delivery drug techniques known to those skilled in the art, such as a portable infusion pump.

  Suitable formulations for the peptide molecules of the present invention are described in US 09 / 899,330 and related applications, all of which are hereby incorporated by reference. Additional formulations for administration are known in the art, as described in Remington's Pharmaceutical Sciences, 18th Ed., Wiley Publishing (1990), the disclosure of which is incorporated herein by reference in its entirety. And can be made according to quantity.

  The peptides of the invention are administered with a pharmaceutically acceptable carrier in an amount sufficient to prevent or treat nephropathy. The compounds of the present invention have very low toxicity and a low degree of side effects even at high doses. The dosage range of the compounds of the invention will vary depending on a number of factors such as the route and mode of administration, i.e., sustained or sustained, such as intravenous or transdermal injection, the desired dosage regimen, and the like.

  Although not limited to the following ranges and provided only as an illustration, exemplary dose ranges for the peptides of the invention may include 0.001 pmol / kg to 500 nmol / kg / day, depending on the composition selected. The lower limit of the dose range is about 0.001 pmol / kg, 0.01 pmol / kg, 0.1 pmol / kg, 1 pmol / kg, 10 pmol / kg, or 100 pmol / kg. The upper limit of the dose range can be about 10 pmol / kg, 100 pmol / kg, 1 nmol / kg, 10 nmol / kg, 100 nmol / kg, 250 nmol / kg or 500 nmol / kg. The desired dose will vary depending on the active composition selected and its relative potency, for example as compared to GLP-1 and exendin. The desired dose will also depend on other factors including bioavailability, administration and route of formulation. For example, continuous infusion as well as bolus doses and sustained release formulations are contemplated and may include administration of peptides in liquid, gel, semi-solid or solid form.

  Alternatively, depending on the mode of administration, therapeutic plasma levels (at least 5 pg / ml, preferably at least 40 pg / ml) can be achieved using from about 0.0005 μg / kg / dose to about 12000 μg / kg / dose. it can. For molecules with potency similar to exendin-4, preferably the peak plasma level does not exceed about 500 pg / ml, more preferably about 250 pg / ml, most preferably about 150 pg / ml.

  When administered parenterally, exendins and agonists in amounts of about 0.001 μg / kg / dose to about 1.0 μg / kg / dose produce a therapeutic effect.

  An exemplary dose for continuous infusion by intravenous administration (IV) is about 0.1 pmol / kg / min to 10 pmol / kg / min, and an exemplary dose for continuous infusion by transdermal administration (sc) Is about 0.1 pmol / kg / min to 75 pmol / kg / min, and is about 0.1 nmol / kg to 2.0 nmol / kg for a single injection (bolus) with IV, and a single injection with sc ( For bolus), it may be about 0.1 nmol / kg to 100 nmol / kg. The aforementioned doses may be administered as a single dose / day, or may be divided into multiple doses / day for administration. The peptide of the present invention can be administered once to several times a day.

  While the preferred method of administration of GLP-1 peptide may be via sustained application, other forms of delivery as described above are also contemplated. However, exemplary dosage rates are sustained release transdermal, intramuscular, intraperitoneal, sustained release injected drugs, pulmonary inhalation, and intravenous, buccal, patch or other sustained release delivery It may be in the range of about 1 to about 10 pmol / kg / min of GLP-1 delivered by the method. Degradation resistant GLP-1 analogs, derivatives or variants, exendins, analogs, derivatives or variants, and other molecules of the invention need not be delivered continuously but are suitable for bolus or sustained release doses And can be doses much lower than those described.

  In addition, other drugs than the inventive composition compatible with the carrier component may be incorporated into the pharmaceutical formulation. Such drugs can be readily ascertained by one skilled in the art and can include, for example, anti-inflammatory agents, diuretics, vasodilators, and the like.

  The present invention contemplates the use of the above active forms of the compositions of the present invention, as well as prodrugs (proforms) that are metabolized to the compounds and biologically active salt forms thereof, and the same pharmaceuticals Also includes optical isomers that provide effects.

  The compositions of the present invention can also be used in conjunction with agents known in the art that enhance the in vivo half-life of the peptide in order to enhance or prolong the biological activity of the peptide. For example, a molecule or chemical moiety may be covalently attached to the composition of the invention prior to its administration. Alternatively, the enhancer may be administered simultaneously with the composition. Still further, the agent can include molecules that are known to inhibit enzymatic degradation of the compositions of the invention that may be administered simultaneously with or after administration of the composition. Such molecules can be administered, for example, orally, by injection, or by any other means known in the art.

  Although there are no strict rules regarding when or how often GLP-1 must be administered in accordance with the present invention to prevent nephropathy, as a general guideline, GLP-1 Resistant, diabetic, uncontrolled hypertension history, kidney disease, increased creatinine clearance level, proteinuria, and non-white ancestry, including but not limited to the occurrence of disease 2 or It can be administered to patients with more risk factors.

  The following examples are provided as an illustration of the use of the peptide molecules of the present invention and are not intended to be limiting.

  Dahl S rats are insulin resistant and develop severe hypertension and kidney damage as soon as they eat a salty diet. Increases in mean arterial pressure (MAP) are associated with sodium retention that can be prevented by servo-controlling total body fluid volume or by using diuretics. Dahl S rats exhibit many phenotypic characteristics associated with salt-sensitive hypertension in humans. Specifically, they are salt sensitive, insulin resistant and hyperlipidemic. They also develop glomerulosclerosis following the onset of hypertension. The type of nephropathy seen in Dahl S rats fed a salty diet is the result of patients with diabetic nephropathy and African Americans with high blood pressure whose incidence of end-stage renal failure is 16 times higher than that of white hypertensive patients. Similar to what is seen.

Example 1 Method To prevent the development of hypertension, experiments were conducted on male Dahl sensitive / Jr (Dahl S) rats maintained on a low salt diet (0.1% NaCl) from birth. When rats are 9 weeks old, they are anesthetized with im injections of ketamine (40 mg / kg), xylazine (2.5 mg / kg), and acepromazine (0.6 mg / kg) and the catheters are MAP (mean arterial pressure) and For long-term measurements of iv infusion (10 ml / day), femoral arteries and blood vessels were implanted. Rats received an im injection of enrofloxacin (Baytril, 2.5 mg / kg) to prevent infection and were given 4-5 days to recover from surgery.

Evaluation of the effect of rGLP-1 on MAP and renal dysfunction
MAP was measured for 3 consecutive days, and blood and overnight urine samples were collected during the control period, during which time rats were maintained on a low salt diet (0.4 NaCl) and were combined at a rate of 10 ml / day. Vehicle is given for the modified glucagon-like peptide-1 (7-36) amide (rGLP-1) (5% mannitol solution against 0.9% saline). The rats were then replaced with a high salt diet (8% NaCl) for 14 days. One group of rats received continuous iv infusion of rGLP-1 at a dose of 1 ug / kg / min, while the other group of rats was infused with vehicle. MAP was performed on a high salt diet using a computerized data acquisition system (WINDAQ software, DataQ Instruments Inc. Akron, OH) at a sample rate of 300 Hz between 11:00 AM and 3:00 PM. Recorded directly from the catheter implanted in the femoral artery on days 7, 10, and 14, during which time the rats were conscious and moved freely in the cage. The MAP was averaged over 1 minute and converted to an average value for the recording session. In addition, blood samples are collected from arterial catheters for measurement of plasma creatinine concentration, and overnight urine samples are measured for proteinuria and microalbuminuria measurements 7 and 14 days after the start of the high salt diet. Collected for. Urine protein concentration was determined by the Bradford method (Bio-Rad Laboratories Hercules, CA) with bovine serum albumin as a standard. Urine albumin concentration was measured using albumin blue 580 method (Molecular Probes, Eugene, OR).

Kidney Histological Evaluation At the end of the experiment, rats were anesthetized with pentobarbital (60 mg / Kg, ip), kidneys were collected, weighed and fixed in 5% buffered formalin solution. Later, the tissue was embedded in paraffin, sectioned, stained with Mason's triple stain, and examined with a light microscope. The degree of glomerulosclerosis was scored on a 0-4 scale, as previously described by Raij et al, based on the percentage of glomerular capillary area filled with mesangial matrix. A score of 0 indicates no damage, a score of 2 indicates that 50% of the glomerular capillary area is filled with matrix, and a score of 4 indicates complete closure of all capillaries of a given glomerulus. In addition, kidney segments were examined for the degree of renal interstitial fibrosis and the percentage of medullary regions occupied by protein casts was determined using the Metamorph imaging program for at least 10 regions / kidney segment.

Statistical analysis Mean values represent SE. Significance of mean differences measured in the vehicle- and rGLP-1 treatment groups was determined using repeated ANOVA for repeated measures followed by Duncan's multiple range test or unpaired t-test. analyzed. The significance of within-group differences was tested using ANOVA for repeated measures. P values <0.05 were considered statistically significant.

Example 2: Effect of rGLP-1 on the development of hypertension Rats were maintained on a low salt diet (0.4% NaCl) for a 3 day control period. Rats were then transferred to a high salt diet (8% NaCl) and received either rGLP-1 (1 μg / kg / min) or vehicle. FIG. 1 shows the development of hypertension in vehicle-treated rats upon initiation of a high salt diet and the protection conferred by administration of GLP-1. Numbers in parentheses indicate the number of rats tested. * Indicates P <0.05 vs vehicle treatment, + indicates control values measured on P <0.05 vs low salt diet. Baseline MAP measured while rats were fed a low salt diet was similar in rats subsequently treated with rGLP-1 or vehicle, with an average of 122 ± 2 mmHg. MAP increased to 174 ± 6 mmHg in vehicle-treated Dahl S rats fed a high salt diet over 14 days. In contrast, in rats injected with rGLP-1, the increase in MAP was significantly attenuated, and MAP increased only to 136 ± 7 mmHg.

Example 3: Effect of rGLP-1 on renal dysfunction rGLP-1 on the onset of proteinuria, microalbuminuria and plasma creatinine concentrations (index of renal injury and nephropathy) in Dahl S rats fed a high salt diet The effect is shown in FIG. Rats were maintained on a low salt diet (0.4% NaCl) for a 3 day control period. Rats were then transferred to a high salt diet (8% NaCl) and fed rGLP-1 (1 μg / kg / min) or vehicle. LS: low salt diet, HS-7 or -14: 7 or 14 days after high salt diet. Numbers in parentheses indicate the number of rats tested. * Indicates P <0.05 vs. vehicle treatment, + indicates P <0.05 vs. control value measured on low salt diet. Protein and albumin excretion were significantly increased in vehicle-treated Dahl S rats after 14 days of high salt diet (FIGS. 2A and 2B). This was associated with a significant increase in the indicators of plasma creatinine concentration, glomerular filtration rate (GFR, FIG. 2C). Long-term administration of rGLP-1 significantly attenuated increased urinary excretion of protein and albumin by 62% and 68%, respectively (FIGS. 2A and 2B). RGLP-1 also decreased the increase in plasma creatinine concentration by 62% in Dahl S rats fed a high salt diet (FIG. 2C).

Example 4: Reduction of renal end organ damage
The effect of rGLP-1 on hypertension-induced renal end organ damage in Dahl S rats fed a 14-day high salt diet (HS-14) is shown in FIG. Rats were treated with rGLP-1 or vehicle. In FIGS. 3B and 3C, low salt (LS) shows results obtained from another group of normotensive Dahl S rats maintained on a low salt (0.4% NaCl) diet throughout the experiment. Numbers in parentheses indicate the number of rats studied. * Denotes control Dahl S rats fed with P <0.05 vs high salt diet and treated with vehicle. When Dahl S rats were treated with rGLP-1 for a long time, kidney weight (FIG. 3A) and indicators of renal hypertrophy were decreased. In vehicle-treated Dahl S rats, there is a marked enlargement of mesangial matrix in almost all glomeruli examined, the overall glomerular damage score is 3: 1 on average, and glomerular capillaries More than 75% of the area was covered with substrate. Long-term treatment of rats with rGLP-1 significantly reduced the extent of substrate expansion and glomerular damage (FIG. 3B). For comparison, the degree of glomerular injury in a group of normotensive Dahl S rats maintained on a low salt diet for 14 days was also examined. The glomerular damage score was not significantly different from that seen in Dahl S rats treated with rGLP-1 and fed a high salt diet (2.5 ± 0.04 vs 2.64 ± 0.05, FIG. 3B). These results are consistent with previous reports that Dahl S rats exhibit a high degree of glomerular damage even when maintained on a low salt diet to minimize the development of hypertension [26]. . Similarly, there was marked tubular necrosis and protein column formation in the outer medulla of vehicle-treated rats (FIG. 3C). In contrast, the number of protein casts and the extent of tubule necrosis were greatly reduced in the outer medulla of Dahl S rats injected with rGLP-1 (FIG. 3C).

Example 4: Improving Endothelial Function Thoracic aorta from vehicle and GLP-1 treated rats was collected and 119 NaCL, 4.7 KCl, 1.17 MgSO ₄ , 1.6 CaCl ₂ , 12 NaHCO ₃ , 1.18 NaH ₂ PO ₄ , 0.03 EDTA In cold saline (PSS) containing 10 glucose and 10 HEPES (pH 7.4). Connective tissue and two rings (approximately 5 mm in length) were prepared from the aorta of each rat. The rings were placed in a tungsten wire organ cage connected to a force transducer (Model FT03E, Grass Instruments, Rhode Island). Vessels were immersed in PSS, bubbled with 95% O ₂ and 5% CO ₂ and maintained at 37 ° C. The wheel was preloaded at a pressure of 2-3 g and allowed to equilibrate for 60 to 90 minutes until reproducible contraction was achieved following addition of 60 mmol / l KCl depolarizing concentration to the cage. . The blood vessels were pre-contracted with norepinephrine (NE, 10 ⁻⁷ mol / l). A cumulative dose-response curve was then constructed for acetylcholine (Ach, 10 ⁻⁹ to 10 ⁻⁴ mol / l) or NO donor, DEA NON-Oate (10 ⁻⁹ to 10 ⁻⁴ mol / l). During each dose-response experiment, the rings were immersed in fresh PSS and allowed to equilibrate again for 60 minutes. Control experiments were performed on aortic rings from a group of normotensive Sprague-Dawley (SD) rats.

FIG. 4 shows the effect of GLP-1 that helps restore endothelial function. In normotensive SD rats, Ach reduced the aortic ring pressure pre-contracted with NE by 90%. The vasodilator response to ACh was significantly less in aortic rings prepared from vehicle-treated Dahl S rats fed a high salt diet. Long-term treatment of Dahl S rats with GLP-1 partially restored endothelial function. The vasodilator response to Ach in GLP-1-treated rats was almost twice that seen in vehicle-treated rats (57 ± 4 vs 35 ± 5% relaxation at 10 ⁻⁴ M, FIG. 4a). The vasodilator response to NO donors was similar across the aortic ring from all rats (Figure 4b).

  These examples show that the exemplary molecule of the present invention, GLP-1, has antihypertensive and renal protective effects.

  From the above examples, it can be seen that the present invention achieves the stated objectives. Changes in the procedure can be made without departing from the spirit and scope of the invention. The contents of all patents cited in this specification are incorporated by reference.

FIG. 1 represents the effect of rGLP-1 on mean arterial pressure (MAP) in Dahl S rats. FIG. 2A represents the effect of rGLP-1 on proteinuria concentration in Dahl S rats. FIG. 2B represents the effect of rGLP-1 on microalbuminuria concentration in Dahl S rats. FIG. 2C represents the effect of rGLP-1 on plasma creatinine concentration in Dahl S rats. FIG. 3A represents the effect of rGLP-1 on kidney weight in Dahl S rats. FIG. 3B represents the effect of rGLP-1 on glomerular damage in Dahl S rats. FIG. 3C shows the effect of rGLP-1 on the formation of protein casts in the outer medulla of Dahl S rats. FIG. 4 represents the effect of rGLP-1 on endothelial function in the aortic ring.

Claims (14)

  In a subject in need of such treatment, glucagon-like peptide-1, or any biologically active agonist, analog, derivative, variant thereof, in the manufacture of a medicament for the prevention or treatment of nephropathy, Or use of compounds that are incretins, GLP-1, exendins that bind to receptors for fragments.   Glucagon-like peptide-1, or any of its biologically active agonists, analogs, derivatives, mutations, in the manufacture of a medicament for preventing progression of nephropathy to ESRD in a subject suffering from nephropathy Use of a compound that is an incretin, GLP-1, or exendin that binds to a receptor for a body or fragment.   Glucagon-like peptide-1, or any biologically active agonist, analog, derivative, variant, or fragment thereof in the manufacture of a medicament for improving endothelial function in a subject with reduced vasodilatory capacity Of compounds that are incretins, GLP-1, exendins that bind to the receptors for.   Receptor for glucagon-like peptide-1, or any biologically active agonist, analog, derivative, variant, or fragment thereof, in the manufacture of a medicament for reducing proteinuria in a subject in need thereof Use of compounds that are incretin, GLP-1, and exendin that bind to.   Glucagon-like peptide-1, or any biologically active agonist, analog, derivative, variant thereof, in the manufacture of a medicament for preventing or delaying the progression of glomerulosclerosis in a subject in need thereof Or use of compounds that are incretins, GLP-1, exendins that bind to receptors for fragments.   Use according to any one of claims 1 to 5, wherein the subject is also suffering from insulin resistance, diabetes or hypertension. The use according to any one of claims 1 to 6, wherein GLP-1 is GLP-1 (7-36) NH ₂ , or a biologically active analog, derivative, variant or fragment thereof.   The use according to any one of claims 1 to 6, wherein the exendin is exendin-3, exendin-4, or a biologically active analogue, derivative, variant or fragment thereof.   Use according to any one of claims 1 to 8, wherein the composition is administered at a dose of about 0.001 pmol / kg to 20 nmol / kg.   9. Use according to any one of claims 1 to 8, wherein the composition is administered at a dose of about 0.001 μg / kg / dose to about 1.0 μg / kg / dose.   11. Use according to any one of claims 1 to 10, wherein the composition is administered in a dose sufficient to achieve a therapeutic plasma level of at least 40 pg / ml.   12. Use according to any one of claims 1 to 11, wherein the compound is administered peripherally.   13. Use according to any one of claims 1 to 12, wherein the compound is administered intravenously at a dose of about 0.1 pmol / kg / min up to about 10 pmol / kg / min. 13. Use according to any one of claims 1 to 12, wherein the compound is administered subcutaneously at a dose of about 0.1 pmol / kg / min to 75 pmol / kg / min.

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