Novel polypeptide with improved protein degradation stability, and method for preparing and using the same

Abstract

To provide a method for improving protein degradation stability of polypeptide, and a chemically modified polypeptide.SOLUTION: A method includes alkylation in at least one kind selected from the group consisting of N-terminal amino group in a polypeptide, NH group of N-terminal first inner amide bond, another primary amino group, thiol group, and thioether group. A polypeptide that was chemically modified by the method provides a chemically modified polypeptide or a salt or a solvate thereof which has substantially the same biological activity and / or more tolerance to protein degradation comparing with a corresponding not chemically modified polypeptide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the following U.S. Provisional Patent Application No. 62 / 247,493, filed October 28, 2015, the entire contents of which are incorporated herein by reference: [Background technology]

[0002] Background of the Invention Polypeptides have attracted much interest as therapeutic agents. They have proven biological activity, very high affinity and excellent specificity for their biological targets, and can be prepared on a large scale using recombinant techniques or chemical synthesis. However, polypeptide-based therapeutics face major disadvantages, such as poor in vivo stability and short in vivo half-lives. Polypeptides are substrates for several in vivo peptidases, such as dipeptidyl protease 4 (DPP4) and endopeptidases, which cleave polypeptide chains into fragments that usually exhibit reduced functionality. Furthermore, polypeptides can be chemically modified in the body, marking them for excretion and / or degradation.

[0003] Attempts to minimize the proteolytic instability of polypeptides have met with limited success. Acetylation of the N-terminal amino group of a polypeptide has the general effect of slowing the rate of proteolysis, but is accompanied by a significant loss of biological activity. Furthermore, the N-acetyl group remains far from stable and is subject to hydrolysis in vivo. Stabilization against enzymes such as DPP4 can be achieved by replacing amino acids at the target cleavage site, but this modification is also almost always accompanied by a loss of biological activity.

[0004] The drawbacks of peptides as potential therapeutics, particularly their short plasma half-life, have been addressed by targeted chemical modification. For example, the insertion of unnatural amino acids into the peptide chain is a common first-pass approach to generating new peptide leads. However, modifications at specific positions in the backbone of a biologically active polypeptide can result in unacceptable loss of activity. This process of structural modification is also largely a matter of trial and error. Combined with the inherent low yields of chemical synthesis and peptide isolation, targeted chemical modification is a very expensive approach.

[0005] GLP-1 is a potent 31-residue peptide secreted by enteroendocrine cells in response to food intake. It has the remarkable properties of delayed gastric emptying and satiety induction, glucose-induced insulin secretion, increased beta-cell mass and function, and concomitant weight loss. However, its in vivo half-life is less than 2 minutes due to the action of the endogenous serine protease dipeptidyl protease 4 (DPP4), making GLP-1 unsuitable for the treatment of type 2 diabetes. Only two GLP-1 analogs have emerged as significant therapeutic approaches: exenatide, a structural GLP-1 analog, and liraglutide, a derivative of GLP-1 with a large lipid side chain added. Both of these drugs were developed in an attempt to improve the proteolytic stability of GLP-1.

[0006] Liraglutide self-assembles into a heptamer in solution, exhibits increased albumin binding, and partially protects itself from hydrolytic cleavage by DPP4. However, among FDA-approved GLP-1 mimetics, despite its outstanding ability to lower blood glucose and induce weight loss in diabetic patients, liraglutide is a relatively short-acting drug that requires daily injections. Furthermore, liraglutide is still significantly degraded and inactivated by DPP4.

[0007] The derivative N-acetyl-GLP-1 is more stable against DPP4 degradation but is 10-50 times less active than GLP-1 itself. Therefore, N-acetyl-GLP-1 cannot be used as a therapeutic agent. Furthermore, while N-acetylation confers in vitro stability to DPP4, it may be ineffective against exopeptidases ubiquitously present in serum. Furthermore, deacetylation catalyzed by specialized enzymes can occur in vivo.

[0008] Additional approaches to further extend the half-life of GLP-1 have been based on fusing the polypeptide to large carrier proteins such as albumin (albiglutide) or immunoglobulins (dulaglutide). However, these constructs are less effective than liraglutide in terms of weight loss, likely due to reduced access to the CNS. Alternatively, the penultimate residue of GLP-1 (Ala8) was replaced with a helix-inducing non-standard amino acid (α-aminoisobutyric acid, Aib). The corresponding derivative (taspoglutide) was withdrawn during clinical trials because, like exenatide, it caused antibody formation in addition to injection site reactions.

[0009] There is an urgent need to develop more effective treatments for patients with short bowel syndrome (SBS), a debilitating condition resulting from the inadequate absorption of nutrients from the intestine. This rare disease occurs in patients who have lost a large portion of their small intestine through surgery. A variety of underlying pathologies can lead to SBS, including Crohn's disease, trauma, congenital anomalies, and malignancies. SBS causes severe diarrhea, dehydration, and malnutrition, necessitating parenteral nutrition (PN) delivered via an indwelling intravenous catheter for survival. Complications of PN include sepsis, venous thrombosis, and metabolic liver disease. The cumulative cost of SBS-related PN in the United States, including hospitalizations, is nearly $5 billion annually.

[0010] After surgery, there is a prolonged period of up to 2 years during which the remaining intestine adapts and nutrient absorption increases. However, physiological compensation after adaptation is often insufficient to meet minimal nutritional requirements. Research efforts have focused on identifying pharmacological agents that enhance this adaptation process. Administration of glucagon-like peptide-2 (GLP-2), which acts via its cognate GPCR (GLP-2R), causes an increase in the intestinal epithelium, resulting in increased nutrient absorption, enhanced barrier function, and increased blood flow.

[0011] A modified GLP-2 peptide, teduglutide, or "Gatex®," was approved by the FDA for the treatment of SBS in 2012. Gatex® contains a single amino acid change at position 2 (His-Gly instead of His-Ala), which increases the polypeptide's resistance to dipeptidyl peptidase-4 (DPP4) and extends its in vivo half-life from 7 minutes to approximately 3 hours. Gatex® represents an important but limited therapeutic advance. After 52 weeks of treatment, 68% of SBS patients were able to reduce their PN regimen by at least 1 day per week. Despite this progress, only 4 of 52 treated patients became completely PN-independent, and 32% were unable to reduce their PN regimen even by 1 day per week. However, the cost of Gatex® per patient is approximately $300,000 per year. Furthermore, the introduction of a Gly residue at position 2 leads to the formation of an unexpected synthetic impurity (aspartimide contamination), which must be avoided by utilizing manufacturing workarounds that make the synthesis longer and more costly. Therefore, more effective / competitive drugs, including improved GLP-2 analogs, are critically needed for SBS to improve patients' quality of life and reduce treatment costs.

[0012] There is a need in the art to identify new methods for improving the proteolytic stability of polypeptides without adversely affecting the biological activity of the polypeptide. The present invention satisfies this need. Summary of the Invention

[0013] The present invention relates to chemically modified polypeptides, salts or solvates thereof, characterized by increased proteolytic stability without adversely affecting their biological activity. In some embodiments, the chemically modified polypeptide or salt or solvate thereof, wherein the polypeptide has one of the following chemical modifications: (i) C1 to C6, in which at least one selected from the group consisting of the N-terminal amino group, the NH of the N-terminal first internal amide bond, other free primary amino groups, thiol groups, and thioether groups of the polypeptide is independently optionally substituted; 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C3-C 16 Aryl, optionally substituted C2-C 16 Alkenyl or optionally substituted C2-C 16 derivatized with alkynyl; and (ii) at least one NH group selected from the group consisting of the N-terminal amino group and the N-terminal first internal amide bond is derivatized with X, where each X is an optionally substituted phenyl, an optionally substituted benzyl, an optionally substituted -(CR2) 1~6 -phenyl, →O, -OH, -OR, alkoxy, NH, optionally substituted NH(C 16 alkyl), and optionally substituted N(C1-C 16 Alkyl) (C1-C 16 alkyl), wherein R, at each occurrence, is independently selected from the group consisting of hydrogen or optionally substituted C1-C 16 alkyl) wherein the chemically modified polypeptide has essentially the same biological activity and / or is more resistant to proteolysis compared to the corresponding unmodified polypeptide, or a salt or solvate thereof. In some embodiments, the proteolysis can be catalyzed by at least one selected from the group consisting of acylpeptide hydrolase, DPP4, DPP2, DPP8, DPP9, fibroblast activation protein (FAP), S9B family oligopropylpeptidase, and a protease having 50% or greater homology to DPP4 and / or DPP2. In some embodiments, the unmodified polypeptide comprises an incretin.

[0014] The chemically modified polypeptide may have higher serum stability than the corresponding unmodified polypeptide. The chemically modified polypeptide may have a longer in vivo half-life than the corresponding unmodified polypeptide. The chemically modified polypeptide of claim 1 may have higher blood-brain barrier permeability or higher oral bioavailability than the corresponding unmodified polypeptide. In some embodiments, the half-life of the modified polypeptide may be increased by at least 10 times compared to the corresponding unmodified polypeptide. In some embodiments, the modified peptide may retain at least about 5% of the biological activity of the corresponding unmodified polypeptide.

[0015] Various derivatives of chemically modified polypeptides are also included in the present invention. In some embodiments, the polypeptide comprises at least one of unsubstituted C1-C6 alkyl, unsubstituted C3-C 16 Cycloalkyl, unsubstituted C2-C 16 Alkenyl, unsubstituted aryl, or unsubstituted C-C 16 In some embodiments, one or more of the N-terminal amino group, other free amino groups, and / or thiol groups of the polypeptide can be independently derivatized with an optionally substituted C1-C alkynyl. 16Alkyl, C1-C 16 Alkylaryl (e.g., benzyl, etc.), optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl and optionally substituted C2-C 16 In some embodiments, the N-terminal amino group is derivatized with a first and second substituent independently selected from the group consisting of an optionally substituted C1-C alkynyl. 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl and optionally substituted C2-C 16 and alkynyl, wherein the first and second substituents are independently the same or different. The alkyl, and / or alkylaryl, and / or cycloalkyl, and / or aryl, and / or alkenyl, and / or alkylaryl, and / or alkynyl groups are independently C1-C 16 Alkyl, C3-C 16 Cycloalkyl, C2-C 16 Alkenyl, C2-C 16 Alkynyl, heteroaryl, heterocyclyl, C1-C6 alkoxy, azido, diaziryl TIFF2025120243000001.tif10128, -CHO, 1,3-dioxol-2-yl, halo, haloalkyl, haloalkoxy, cyano, nitro, triflyl, mesyl, tosyl, heterocyclyl, aryl, heteroaryl, -SR, -S(=O)(C1-C6 alkyl), -S(=O)2(C1-C6 alkyl), -S(=O)2NRR, -C(=O)R, -OC(=O)R, -C(=O)OR, -OC(=O)O(C1-C6 alkyl), -NRR, -C(=O)NRR, -N(R)C(=O)R, -C(=NR)NRR, and -P(=O)(OR)2, wherein R, at each occurrence, is independently H or C1-C6 alkyl.16 In some embodiments, the alkyl, and / or alkylaryl, and / or cycloalkyl, and / or aryl, and / or alkenyl, and / or alkylaryl, and / or alkynyl groups may be fluorinated or perfluorinated. In some embodiments, the alkyl group may be a halogenated C1-C 16 In some embodiments, the alkyl group can be 2,2,2-trifluoroethyl. In some embodiments, at least one (e.g., two, three, four, five, etc.) alkyl, cycloalkyl, alkenyl, aryl, or alkynyl group is TIFF2025120243000002.tif224160TIFF2025120243000003.tif204147TIFF2025120243000004.tif212152TIFF2025120243000005.tif65170 (wherein n is an integer ranging from 1 to 6, and R is hydrogen or an optionally substituted alkyl or aryl). In some embodiments, the alkyl, and / or alkylaryl, and / or cycloalkyl, and / or aryl, and / or alkenyl, and / or alkylaryl, and / or alkynyl groups are selected from the group consisting of 2,2,2-trifluoro-1-ethyl, 2,2,3,3,3-pentafluoro-1-propyl, 2,2,3,3,4,4,4-heptafluoro-1-butyl, ethyl, isopropyl, benzyl, substituted benzyl, adamant-1-yl-methyl, quinolin-4- 17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl-ethynyl-methyl, 2-amino-1-propyl, 4-phenyl-benzyl, 1H-imidazol-4-yl-methyl, 4-hydroxy-benzyl, 4-[3-(trifluoromethyl)-3H-diazirine]-benzyl, and (8R,9R,13S,14R)-3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl-ethynyl-methyl.

[0016] The group attached to the N-terminal amino group can occupy a binding pocket on the receptor close to the membrane-water interface where the polypeptide binds to the receptor. Chemical modifications of the polypeptide can have a specific volume or shape. In some embodiments, the polypeptide has a volume of about 240 Å. 3 The chemically modified polypeptide may be chemically modified by groups occupying a volume of less than about 190 Å (e.g., less than about 190 Å). The chemically modified polypeptide may contain from about 3 to about 100 amino acids, or from about 3 to about 49 amino acids, or from about 80 to about 100 amino acids.

[0017] In some embodiments, at least the N-terminal amino group is an optionally substituted C1-C 16 Alkyl, optionally substituted C1-C 16 Aryl alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl or optionally substituted C2-C 16 In some embodiments, the polypeptide is chemically modified. In some embodiments, the amino acid or amino acid residue is It may contain at least one amino acid residue selected from the group consisting of TIFF2025120243000006.tif96145.

[0018] In some embodiments, the chemically modified polypeptide has the structure: TIFF2025120243000007.tif25155, or at least one derivatized amino acid, salt, or solvate thereof.

[0019] In some embodiments, the polypeptide comprises: GLP-1 TIFF2025120243000008.tif4128; Exenatide TIFF2025120243000009.tif4131; Liraglutide TIFF2025120243000010.tif5170; Semaglutide TIFF2025120243000011.tif4128 (In the sequence, X attached to the ε-amino group of lysine is TIFF2025120243000012.tif24128); Taspoglutide TIFF2025120243000013.tif4128; Lixisenatide TIFF2025120243000014.tif4161; Triagonists TIFF2025120243000015.tif5170; Exendin TIFF2025120243000016.tif4128; VIP TIFF2025120243000017.tif4128; PACAP TIFF2025120243000018.tif4128; GIP TIFF2025120243000019.tif4131; Met-enkephalin TIFF2025120243000020.tif4128; BNP TIFF2025120243000021.tif4128; Substance P TIFF2025120243000022.tif4128; Tyr-MIF-1 TIFF2025120243000023.tif4128; Tyr-W-MIF-1 TIFF2025120243000024.tif4128; Glucagon TIFF2025120243000025.tif4128; growth hormone-releasing hormone (ghrh); pituitary adenylate cyclase-activating polypeptide (PACAP) ADCYAP1; Glucagon (GCG); gastric inhibitory polypeptide (GIP); Secretin (SCT); vasoactive intestinal peptide (Peptid) (VIP); OXM (oxyntomodulin); PTH (parathyroid hormone); Peptide YY3-36 and peptide YY1-36 (PYY); NPY (neuropeptide Y); VIP peptide (vasoactive intestinal peptide); at least one dual agonist selected from the group consisting of GLP-1+GIP; GLP-1+amylin; GLP-1+gastrin; GLP-1+estrogen; GLP-1+PYY; and GLP-1+cholecystin kinase (CCK); Dual agonist of GLP-1R + glucagon receptor; mixed agonists; Albiglutide; Dulaglutide; other GLP-1R agonists; Amylin; other substrates of DPP4, DPP2, and / or proteases having 50% or more homology to DPP4 and / or DPP2; GLP-1 analogs stabilized by other modifications; or A sequence that has at least 75% identity to any of these sequences and wherein at least one of the residues marked by * is R or a hydrocarbon R (e.g., C 1~16 The aryl group may be alkylated (-R) or acylated (-C(O)R) with an alkyl group such as an alkyl.

[0020] In some embodiments, the chemically modified polypeptide may be selected from the group consisting of parathyroid hormone (PTH), PYY3-36, cholecystokinin, PYY1-36, corticotropin-releasing hormone receptor 1 or 2, growth hormone-releasing factor, glucagon, exendin, GLP-1, gastric inhibitory peptide, liraglutide, prealbumin, peptide HI-27, PACAP, secretin, and vasoactive intestinal peptide (VIP), wherein the N-terminal amino group is (i) an optionally substituted C1-C6 16 Alkyl, optionally substituted arylalkyl, optionally substituted C3-C 16 Aryl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl and optionally substituted C2-C 16 or (ii) optionally substituted phenyl, optionally substituted benzyl, → O, —OH, alkoxy, NH, NH(C 16 alkyl), and N(C1-C 16 Alkyl) (C1-C 16 The polypeptide may be derivatized with one selected from the group consisting of acylamino acid releasing enzymes, DPP4, DPP2, DPP8, DPP9, all S9B family oligopropylpeptidases, and proteases having 50% or greater homology to DPP4 and / or DPP2, without reducing the biological activity of the polypeptide relative to the corresponding unmodified protein.

[0021] Pharmaceutical compositions comprising the chemically modified polypeptides are also provided. In some embodiments, a pharmaceutically acceptable composition can comprise any of the chemically modified polypeptides described herein.

[0022] Methods for treating or preventing at least one disease or disorder are also provided. In some embodiments, the method may comprise administering at least one chemically modified polypeptide to a patient in need thereof. In some embodiments, the method may comprise administering a pharmaceutical composition comprising any of the chemically modified polypeptides described herein. In some embodiments, the disease or disorder may be selected from the group consisting of short bowel syndrome, non-alcoholic steatohepatitis, smoking cessation, neurodegeneration, Alzheimer's disease, Parkinson's disease, congenital hyperinsulinism, and / or hypoglycemia. In some embodiments, the disease or disorder may be selected from the group consisting of diabetes, weight gain, obesity, and / or metabolic syndrome.

[0023] Also provided are methods for increasing the in vivo half-life of a polypeptide, and / or improving the blood-brain barrier permeability of a polypeptide, and / or improving the oral bioavailability of a polypeptide. These changes (e.g., increasing half-life, improving permeability, improving bioavailability) are compared to the corresponding unchemically modified polypeptide. In some embodiments, these methods can include any of the chemical modifications described herein. In some embodiments, methods for increasing the in vivo half-life of a polypeptide, and / or improving the blood-brain barrier permeability of a polypeptide, and / or improving the oral bioavailability of a polypeptide, compared to the corresponding unchemically modified polypeptide, include: (i) chemically modifying at least one substituent selected from the group consisting of the N-terminal amino group, the NH group of the N-terminal first internal amide bond, other free primary amino groups, a thiol group, and a thioether group of a polypeptide, wherein each chemical modification is performed by modifying the substituent with an optionally substituted C1-C 16 Alkyl, optionally substituted C4-C 16 Aryl alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C3-C 16 Aryl, optionally substituted C2-C 16 Alkenyl or optionally substituted C2-C 16chemically modifying, independently including derivatizing with alkynyl; and / or (ii) chemically modifying at least one substituent selected from the N-terminal amino group and NH, independently comprising derivatizing the substituent with a group X, where X, in each occurrence, is independently selected from optionally substituted phenyl, optionally substituted benzyl, optionally substituted -(CR2) 1~6 -Phenyl, →O, -OH, alkoxy, NH2, NH(C1-C 16 alkyl), and N(C1-C 16 Alkyl) (C1-C 16 alkyl), wherein R, at each occurrence, is independently hydrogen or an optionally substituted C1-C 16 alkyl) In some embodiments, the alkyl group is 2,2,2-trifluoroethyl. In some embodiments, at least one alkyl, cycloalkyl, aryl, arylalkyl, alkenyl, or alkynyl group is TIFF2025120243000026.tif224153TIFF2025120243000027.tif204140TIFF2025120243000028.tif212152TIFF2025120243000029.tif65170 (wherein "n" is an integer ranging from 1 to 6, and R can be an optionally substituted alkyl, an optionally substituted aryl, or an optionally substituted arylacryl). In some embodiments, at least one alkyl, arylalkyl, arylcycloalkyl, alkenyl, or alkynyl group is 2,2,2-trifluoro-1-ethyl, 2,2,3,3,3-pentafluoro-1-propyl, 2,2,3,3,4,4,4-heptafluoro-1-butyl, ethyl, isopropyl, benzyl, substituted benzyl, adamant-1-yl-methyl, quinolin-4-yl-methyl, 2-amino-1 ... In some embodiments, the polypeptide is selected from the group consisting of 8R,9R,13S,14R)-3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl-ethynyl-methyl. 3 In some embodiments, the polypeptide may contain about 5 to about 100 amino acids, or about 3 to about 49 amino acids, or about 80 to about 100 amino acids. In some embodiments, the N-terminal amino group is optionally substituted with a C1 to C6 group. 16 Alkyl, C1-C 16 Aryl alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl or optionally substituted C2-C 16 In some embodiments, the polypeptide may be derivatized with an alkynyl group. TIFF2025120243000030.tif56145.

[0024] In some embodiments, the polypeptide has the structure: TIFF2025120243000031.tif25170, its salts, or solvates.

[0025] Also provided are methods for derivatizing a polypeptide, hi some embodiments, a method for derivatizing a polypeptide comprising a free amino group comprises contacting a derivatized amino acid with the polypeptide under conditions in which the free carboxylic acid of the derivatized amino acid forms an amide bond with the free amino acid of the polypeptide.

[0026] A method for imaging cells or tissues in a subject is also possible. In some embodiments, the method for imaging cells or tissues in a subject comprises administering to a subject in need thereof an effective amount of a chemically modified polypeptide, wherein the polypeptide is labeled with a detectable isotope and / or conjugated to a detectable label. In some embodiments, the detectable label may comprise a chromophore, a fluorescent group, a bioluminescent group, a chemiluminescent group, or a radioactive group.

[0027] The present invention also includes methods for characterizing a gastrinoma in a subject. In some embodiments, the methods for characterizing a gastrinoma in a subject can include administering a chemically modified polypeptide (such as a chemically modified secretin polypeptide) to the subject and detecting an increase in gastrin in the subject, thereby revealing the presence or absence of a gastrinoma in the subject.

[0028] In some embodiments, modification of the polypeptide increases binding to a plasma component. In some embodiments, the plasma component can be vitamin D3 binding protein, albumin, or transthyretin. In some embodiments, the chemically modified polypeptide is not GLP2. [The present invention 1001] A chemically modified polypeptide or a salt or solvate thereof, wherein the polypeptide has one of the following chemical modifications: (i) at least one C1 to C6 optionally substituted independently selected from the group consisting of the N-terminal amino group, the NH of the N-terminal first internal amide bond, other free primary amino groups, a thiol group, and a thioether group of the polypeptide; 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C3-C 16 Aryl, optionally substituted C2-C 16 Alkenyl or optionally substituted C2-C 16 derivatized with alkynyl; and (ii) at least one NH group selected from the group consisting of the N-terminal amino group and the N-terminal first internal amide bond is derivatized with X, wherein each X is an optionally substituted phenyl, an optionally substituted benzyl, an optionally substituted -(CR2) 1~6 -phenyl, →O, -OH, -OR, alkoxy, NH, optionally substituted NH(C 16 alkyl), and optionally substituted N(C1-C 16 Alkyl) (C1-C 16 alkyl), wherein R, at each occurrence, is independently selected from the group consisting of hydrogen or optionally substituted C1-C 16 alkyl) wherein the chemically modified polypeptide has essentially the same biological activity and / or is more resistant to proteolysis compared to a corresponding non-chemically modified polypeptide, or a salt or solvate thereof. [The present invention 1002] The chemically modified polypeptide of the present invention 1001, wherein the proteolysis is catalyzed by at least one selected from the group consisting of acylpeptide hydrolase, DPP4, DPP2, DPP8, DPP9, fibroblast activation protein (FAP), S9B family oligopropylpeptidase, and proteases having 50% or more homology to DPP4 and / or DPP2. [The present invention 1003] 1001. The chemically modified polypeptide of claim 10, wherein said unmodified polypeptide comprises an incretin. [The present invention 1004] 1001. A chemically modified polypeptide of the present invention, having higher serum stability compared to the corresponding unmodified polypeptide. [The present invention 1005] 1001. A chemically modified polypeptide of the present invention, having a longer in vivo half-life compared to said corresponding non-chemically modified polypeptide. [The present invention 1006] 1001. A chemically modified polypeptide of the present invention, having higher blood-brain barrier permeability or higher oral bioavailability compared to the corresponding non-chemically modified polypeptide. [The present invention 1007] In (i), the polypeptide comprises at least one unsubstituted C1-C6 alkyl, unsubstituted C3-C 16 Cycloalkyl, unsubstituted C2-C 16 Alkenyl, unsubstituted aryl, or unsubstituted C-C 16 1001. A chemically modified polypeptide of the present invention, wherein the polypeptide is alkynyl-derivatized. [The present invention 1008] In (i), one or more of the N-terminal amino group, other free amino groups, and / or thiol groups of the polypeptide are independently optionally substituted C1 to C6 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16Alkenyl and optionally substituted C2-C 16 1001. The chemically modified polypeptide of the present invention, wherein the polypeptide is derivatized with a first substituent and a second substituent independently selected from the group consisting of alkynyl, wherein said first substituent and said second substituent are independently the same or different. [The present invention 1009] In (i), the N-terminal amino group is an optionally substituted C1-C 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl and optionally substituted C2-C 16 1001. The chemically modified polypeptide of the present invention, wherein the polypeptide is derivatized with a first substituent and a second substituent independently selected from the group consisting of alkynyl, wherein said first substituent and said second substituent are independently the same or different. [The present invention 1010] In (i), the alkyl, cycloalkyl, alkenyl, or alkynyl groups are independently C1 to C 16 Alkyl, C3-C 16 Cycloalkyl, C2-C 16 Alkenyl, C2-C 16 Alkynyl, heteroaryl, heterocyclyl, C1-C6 alkoxy, azido, diaziryl TIFF2025120243000032.tif10128, -CHO, 1,3-dioxol-2-yl, halo, haloalkyl, haloalkoxy, cyano, nitro, triflyl, mesyl, tosyl, heterocyclyl, aryl, heteroaryl, -SR, -S(=O)(C1-C6 alkyl), -S(=O)2(C1-C6 alkyl), -S(=O)2NRR, -C(=O)R, -OC(=O)R, -C(=O)OR, -OC(=O)O(C1-C6 alkyl), -NRR, -C(=O)NRR, -N(R)C(=O)R, -C(=NR)NRR, and -P(=O)(OR)2, wherein R, at each occurrence, is independently H or C1-C6 alkyl. 161001. The chemically modified polypeptide of the present invention, wherein the polypeptide is alkyl. [The present invention 1011] The alkyl group is a halogenated C1-C 16 The chemically modified polypeptide of the present invention 1010, wherein the polypeptide is alkyl. [The present invention 1012] 1011. The chemically modified polypeptide of the present invention, wherein the alkyl group is 2,2,2-trifluoroethyl. [The present invention 1013] At least one alkyl, cycloalkyl, alkenyl, aryl, or alkynyl group is TIFF2025120243000033.tif131150TIFF2025120243000034.tif216147TIFF2025120243000035.tif219150TIFF2025120243000036.tif127170 (wherein n is an integer ranging from 1 to 6, and R is hydrogen or an optionally substituted alkyl or aryl). [The present invention 1014] At least one alkyl, cycloalkyl, alkenyl, or alkynyl group is selected from the group consisting of 2,2,2-trifluoro-1-ethyl, 2,2,3,3,3-pentafluoro-1-propyl, 2,2,3,3,4,4,4-heptafluoro-1-butyl, ethyl, isopropyl, benzyl, substituted benzyl, adamant-1-yl-methyl, quinolin-4-yl-methyl, 2-amino-1-propyl, 4-phenyl-benzyl, 1H-imidazoline ... 1001. The chemically modified polypeptide of the present invention, wherein the modified polypeptide is selected from the group consisting of 4-[3-(trifluoromethyl)-3H-diazirine]-benzyl, 4-[3-(trifluoromethyl)-3H-diazirine]-benzyl, and (8R,9R,13S,14R)-3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl-ethynyl-methyl. [The present invention 1015] Approximately 240Å 31001. A chemically modified polypeptide of the present invention, wherein the polypeptide is chemically modified by a group occupying a volume of: [The present invention 1016] At the N-terminal amino group, about 190 Å 3 1015. A chemically modified polypeptide of the present invention, wherein the polypeptide is chemically modified by a group occupying a volume of: [The present invention 1017] 1001. A chemically modified polypeptide of the present invention, comprising from about 3 to about 100 amino acids. [The present invention 1018] 1001. A chemically modified polypeptide of the present invention, comprising from about 3 to about 49 amino acids. [The present invention 1019] 1001. A chemically modified polypeptide of the present invention, comprising about 80 to about 100 amino acids. [The present invention 1020] At least the N-terminal amino group is an optionally substituted C1-C 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl or optionally substituted C2-C 16 1001. A chemically modified polypeptide of the present invention, wherein the polypeptide is alkynyl-derivatized. [The present invention 1021] A chemically modified polypeptide of the present invention 1001, comprising at least one amino acid residue selected from the group consisting of TIFF2025120243000037.tif56145TIFF2025120243000038.tif37144. [The present invention 1022] the polypeptide GLP-1 TIFF2025120243000039.tif4128; Exenatide TIFF2025120243000040.tif4131; Liraglutide TIFF2025120243000041.tif5170; Semaglutide TIFF2025120243000042.tif4128 (In the sequence, X attached to the ε-amino group of lysine is TIFF2025120243000043.tif23128); Taspoglutide TIFF2025120243000044.tif4128; Lixisenatide TIFF2025120243000045.tif4161; Triagonists TIFF2025120243000046.tif4170; Exendin TIFF2025120243000047.tif4128; VIP TIFF2025120243000048.tif4128; PACAP TIFF2025120243000049.tif4128; GIP TIFF2025120243000050.tif4131; Met-enkephalin TIFF2025120243000051.tif4128; BNP TIFF2025120243000052.tif4128; Substance P TIFF2025120243000053.tif4128; Tyr-MIF-1 TIFF2025120243000054.tif4128; Tyr-W-MIF-1 TIFF2025120243000055.tif4128; Glucagon TIFF2025120243000056.tif4128; growth hormone-releasing hormone (GHRH); pituitary adenylate cyclase-activating polypeptide (PACAP) ADCYAP1; Glucagon (GCG); gastric inhibitory polypeptide (GIP); Secretin (SCT); vasoactive intestinal peptide (VIP); OXM (oxyntomodulin); PTH (parathyroid hormone); Peptide YY3-36 and peptide YY1-36 (PYY); NPY (neuropeptide Y); VIP peptide (vasoactive intestinal peptide); at least one dual agonist selected from the group consisting of GLP-1+GIP; GLP-1+amylin; GLP-1+gastrin; GLP-1+estrogen; GLP-1+PYY; and GLP-1+cholecystin kinase (CCK); Dual agonist of GLP-1R+glucagon receptor; mixed agonists; Albiglutide; Dulaglutide; other GLP-1R agonists; Amylin; other substrates of DPP4, DPP2, and / or proteases having 50% or more homology to DPP4 and / or DPP2; GLP-1 analogs stabilized by other modifications; or A sequence that has at least 75% identity to any of the above sequences 1001. The chemically modified polypeptide of claim 1001, wherein at least one of said residues marked by * is alkylated or acylated by R. [The present invention 1023] 1001. The chemically modified polypeptide of the present invention, wherein the half-life of said polypeptide is increased by at least 10-fold relative to said corresponding non-chemically modified polypeptide. [The present invention 1024] 1001. A chemically modified polypeptide of the present invention, which retains at least about 5% of the biological activity of the corresponding unmodified polypeptide. [The present invention 1025] a chemically modified polypeptide selected from the group consisting of parathyroid hormone (PTH), PYY3-36, cholecystokinin, PYY1-36, corticotropin-releasing hormone receptor 1 or 2, growth hormone-releasing factor, glucagon, exendin, GLP-1, gastric inhibitory peptide, liraglutide, prealbumin, peptide HI-27, PACAP, secretin, and vasoactive intestinal peptide (VIP); The N-terminal amino group is (i) an optionally substituted C1-C 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl and optionally substituted C2-C 16 or (ii) optionally substituted phenyl, optionally substituted benzyl, → O, —OH, alkoxy, NH, NH(C 16 alkyl), and N(C1-C 16 Alkyl) (C1-C 16 alkyl), the derivatization stabilizes the polypeptide against degradation by at least one selected from the group consisting of acylamino acid releasing enzymes, DPP4, DPP2, DPP8, DPP9, all S9B family oligopropylpeptidases, and proteases having 50% or more homology to DPP4 and / or DPP2, without reducing the biological activity of the polypeptide relative to the corresponding unmodified protein; Chemically modified polypeptides. [The present invention 1026] A pharmaceutically acceptable composition comprising at least one polypeptide of any one of 1001 to 1025 of the present invention. [The present invention 1027] A method for treating or preventing at least one disease or disorder, comprising administering at least one polypeptide according to any one of claims 1001 to 1025 of the present invention, wherein the disease or disorder is selected from the group consisting of short bowel syndrome, non-alcoholic steatohepatitis, smoking cessation, neurodegeneration, Alzheimer's disease, Parkinson's disease, congenital hyperinsulinism, hypoglycemia, diabetes, weight gain, obesity, and metabolic syndrome. [The present invention 1028] 1. A method for increasing the in vivo half-life of a polypeptide compared to a corresponding non-chemically modified polypeptide, comprising: (i) chemically modifying at least one substituent selected from the group consisting of the N-terminal amino group, the NH group of the N-terminal first internal amide bond, other free primary amino groups, a thiol group, and a thioether group of the polypeptide, wherein each chemical modification converts the substituent into an optionally substituted C1-C 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C3-C 16 Aryl, optionally substituted C2-C 16 Alkenyl or optionally substituted C2-C 16 chemically modifying, independently including derivatizing with alkynyl; and (ii) chemically modifying at least one substituent selected from the N-terminal amino group and the NH, independently comprising derivatizing the substituent with a group X, wherein X, in each occurrence, is independently optionally substituted phenyl, optionally substituted benzyl, optionally substituted -(CR2). 1~6 -Phenyl, →O, -OH, alkoxy, NH2, NH(C1-C 16 alkyl), and N(C1-C 16 Alkyl) (C1-C 16 alkyl), wherein R, at each occurrence, is independently hydrogen or an optionally substituted C1-C 16 alkyl, The method includes at least one of: [The present invention 1029] 1. A method for improving the blood-brain barrier permeability or oral bioavailability of a polypeptide compared to a corresponding unmodified polypeptide, comprising: (i) chemically modifying at least one substituent selected from the group consisting of the N-terminal amino group, the NH group of the N-terminal first internal amide bond, other free primary amino groups, a thiol group, and a thioether group of the polypeptide, wherein each chemical modification converts the substituent into an optionally substituted C1-C 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl or optionally substituted C2-C 16 chemically modifying, independently including derivatizing with alkynyl; and (ii) chemically modifying at least one substituent selected from the N-terminal amino group and the NH, independently comprising derivatizing the substituent with a group X, wherein X, in each occurrence, is independently optionally substituted phenyl, optionally substituted benzyl, optionally substituted -(CR2). 1~6 -Phenyl, →O, -OH, alkoxy, NH2, NH(C1-C 16 alkyl), and N(C1-C 16 Alkyl) (C1-C 16 alkyl), wherein R, at each occurrence, is independently hydrogen or an optionally substituted C1-C 16 alkyl, The method includes at least one of: [The present invention 1030] In (i), the polypeptide comprises at least one unsubstituted C1-C6 alkyl, unsubstituted C3-C 16 Cycloalkyl, unsubstituted C2-C 16 Alkenyl or unsubstituted C2-C 16 The method of any one of claims 1027 to 1029, wherein the compound is derivatized with an alkynyl. [The present invention 1031] In (i), the alkyl, cycloalkyl, alkenyl, or alkynyl groups are independently C1 to C 16 Alkyl, C3-C 16 Cycloalkyl, C2-C 16 Alkenyl, C2-C 16 Alkynyl, heteroaryl, heterocyclyl, C1-C6 alkoxy, azido, diaziryl TIFF2025120243000057.tif10128, -CHO, 1,3-dioxol-2-yl, halo, haloalkyl, haloalkoxy, cyano, nitro, triflyl, mesyl, tosyl, heterocyclyl, aryl, optionally substituted phenyl heteroaryl, optionally substituted benzyl heteroaryl, optionally substituted -(CR2) 1~6 heteroaryl of -phenyl, -SR, -S(=O)(C1-C6 alkyl), -S(=O)2(C1-C6 alkyl), -S(=O)2NRR, -C(=O)R, -OC(=O)R, -C(=O)OR, -OC(=O)O(C1-C6 alkyl), -NRR, -C(=O)NRR, -N(R)C(=O)R, -C(=NR)NRR, and -P(=O)(OR)2, where R, in each occurrence, is independently H or C1-C 16 The method of any one of claims 1027 to 1029, wherein the substituted group is at least one independently selected from the group consisting of alkyl. [The present invention 1032] The method according to any one of claims 1033 to 1036, wherein the alkyl group is a halogenated C1 to C6 alkyl. [The present invention 1033] The method according to any one of claims 1033 to 1036, wherein the alkyl group is 2,2,2-trifluoroethyl. [The present invention 1034] the at least one alkyl, cycloalkyl, alkenyl, or alkynyl group being TIFF2025120243000058.tif82150TIFF2025120243000059.tif221147TIFF2025120243000060.tif253150TIFF2025120243000061.tif158170 (wherein n is an integer ranging from 1 to 6, and R is an optionally substituted alkyl or an optionally substituted aryl). [This invention 1035] At least one alkyl, cycloalkyl, alkenyl, or alkynyl group is 2,2,2-trifluoro-1-ethyl, 2,2,3,3,3-pentafluoro-1-propyl, 2,2,3,3,4,4,4-heptafluoro-1-butyl, ethyl, isopropyl, benzyl, substituted benzyl, adamant-1-yl-methyl, quinolin-4-yl-methyl, 2-amino-1-propyl, 4-phenyl-benzyl, 1H-imidazole ... 1027. The method of any of claims 1027 to 1029, wherein the hydroxybenzoate is selected from the group consisting of 4-hydroxybenzoate, 4-[3-(trifluoromethyl)-3H-diazirine]-benzyl, 4-hydroxybenzoate, 4-[3-(trifluoromethyl)-3H-diazirine]-benzyl, and (8R,9R,13S,14R)-3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl-ethynyl-methyl. [The present invention 1036] The polypeptide is about 240 Å 3 1029. The method of any one of claims 1027 to 1029, wherein the compound is chemically modified with a group that occupies a volume of: [This invention 1037] the polypeptide has a length of about 240 Å at the N-terminal amino group 3 1029. The method of any one of claims 1027 to 1029, wherein the compound is chemically modified with a group that occupies a volume of: [The present invention 1038] 1029. The method of any of claims 1027 to 1029, wherein said polypeptide comprises about 5 to about 100 amino acids. [This invention 1039] 1029. The method of any of claims 1027 to 1029, wherein said polypeptide comprises from about 3 to about 49 amino acids. [The present invention 1040] 1029. The method of any of claims 1027 to 1029, wherein said polypeptide comprises about 80 to about 100 amino acids. [The present invention 1041] At least the N-terminal amino group is an optionally substituted C1-C 16 Alkyl, optionally substituted C3-C 16 Cycloalkyl, optionally substituted C2-C 16 Alkenyl or optionally substituted C2-C 16 The method of any one of claims 1027 to 1029, wherein the compound is derivatized with an alkynyl. [The present invention 1042] the polypeptide Any of the methods of claims 1027 to 1029, comprising at least one amino acid residue selected from the group consisting of TIFF2025120243000062.tif56145. [This invention 1043] 10. The polypeptide of claim 1, wherein the polypeptide has the structure: TIFF2025120243000063.tif25155, or a salt or solvate thereof. [This invention 1044] A method for derivatizing a polypeptide containing a free amino group, comprising contacting a derivatized amino acid of the present invention with said polypeptide under conditions in which the free carboxylic acid of said derivatized amino acid forms an amide bond with the free amino acid of said polypeptide. [This invention 1045] A method for imaging cells or tissues in a subject, comprising administering to said subject in need thereof an effective amount of any of the polypeptides of the present inventions 1001 to 1026, wherein said polypeptide is labeled with a detectable isotope and / or conjugated to a detectable label. [The present invention 1046] 1045. The method of claim 1045, wherein said detectable label comprises a chromophore, a fluorescent group, a bioluminescent group, a chemiluminescent group, or a radioactive group. [This invention 1047] A method for characterizing a gastrinoma in a subject, comprising administering to the subject a chemically modified secretin polypeptide of the present invention and detecting an increase in gastrin in the subject, thereby determining the presence or absence of a gastrinoma in the subject. [This invention 1048] 1001. The chemically modified polypeptide of the present invention, wherein said modification increases binding to a plasma component. [This invention 1049] The chemically modified polypeptide of the present invention 1048, wherein the plasma component is vitamin D3 binding protein, albumin, or transthyretin. [The present invention 1050] A chemically modified polypeptide of the present invention 1048 that is not GLP2. [This invention 1051] structure: A chemically modified polypeptide of the present invention 1001 comprising a derivatized amino acid, a salt thereof, or a solvate thereof having TIFF2025120243000064.tif25155. [Brief explanation of the drawings]

[0029] The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, specific embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0030] [Figure 1]Figure 1 shows HPLC traces of native and modified GLP-1 variants obtained simultaneously from the same batch of resin during SPPS. Two products were observed following direct on-resin alkylation of GLP-1 with reagent 1a (see Figures 2 and 4), corresponding to N-terminal decoration of GLP-1 with either one (G-4) or two ("bis-G-4") CHCF3 groups. In the latter case, the second alkylation occurs at the π-nitrogen of His. The peptides were purified to >95% purity before being assayed against the GLP-1 receptor (Figure 3). The nomenclature, according to Figure 6, is as follows: G-1 (unmodified GLP-1); G-2 (Ac-GLP-1); and G-4 (mono-C2-GLP-1). "Bis-C2-GLP-1" is GLP-1 with two CH2CF3 moieties attached to the N-terminal histidine. [Figure 2] Figure 2 provides a schematic diagram of an exemplary fluoroalkylation scheme. A trivalent iodonium salt coordinates with a free amine, and the addition of a base triggers the alkylation reaction. The resulting trifluoroethyl functionality is unreactive, and in its presence, different chemical modifications of the side chain can be performed both on and off the resin. At the end of the synthesis, the peptide is cleaved from the resin and worked up as usual. [Figure 3]Figure 3 provides exemplary stability assay data for native and fluoroalkyl-modified GLP-1 (referred to as "G-4" in Figure 6). Briefly, HEK293 cells were seeded into white, clear-bottom 96-well plates at a density of 2–3 × 10 cells per well and grown for 2 days until approximately 80% confluent. Cells were then transiently transfected using Lipofectamine reagent (Invitrogen) with cDNA encoding (i) a GPCR (or an empty expression vector); (ii) a cAMP response element-luciferase reporter gene (CRE6X-luc); and (iv) β-galactosidase as a control. Cells were incubated with or without selected peptide agonists in serum-free medium for 6 hours. Luciferase activity was quantified using Steadylite reagent (Perkin-Elmer). β-galactosidase assays were then performed after adding the enzyme substrate 2-nitrophenyl β-d-galactopyranoside. After 30–60 min of incubation at 37°C, substrate cleavage was quantified by measuring the OD at 420 nM using a SpectraMax microplate reader (Molecular Devices). Top panel: Native GLP-1 is degraded after incubation with DPP4, resulting in a significant reduction in potency compared to GLP-1 without DPP4 incubation. Bottom panel: In contrast, GLP-1 is resistant to DPP4-induced degradation. [Figure 4] FIG. 4 provides a schematic showing the synthesis of reagents useful in the methods of the present invention. [Figure 5] Figure 5 provides a set of images showing that GLP-1 is rapidly inactivated by the enzyme DPP4, which cleaves the dipeptide HA from its N-terminus. Left: Schematic showing the inactive peptide missing the amino-terminal HA dipeptide fragment. Right: ESI LC-MS analysis of GLP-1 and G-4 (an N-trifluoroethyl analogue that remains unchanged by enzymatic action) incubated overnight at 37°C with DPP4. [Figure 6A-1]Figure 6A provides a schematic representation of the chemical structures of certain analogs of the invention. X corresponds to the moiety at the N-terminus, represented by a number. All unmodified peptides have X=1 (corresponding to hydrogen; e.g., unmodified GLP-1=G-1). The abbreviated nomenclature used in the text and other figures is as follows: GX (GLP-1 analog); IX (GIP analog); GCG-X (glucagon analog); DA-X (dual agonist analog); TA-X (triagonist analog); Lira-X (liraglutide analog); EXE-X (exendin analog); oxy-X (oxyntomodulin analog); GH29-X or GH44-X (growth hormone-releasing hormone analog). [Figure 6A-2] See legend to Figure 6A-1. [Figure 6A-3] See legend to Figure 6A-1. [Figure 6B-1] Figure 6B provides a schematic representation of the chemical structures of the GLP-2 analogs of the present invention. X corresponds to the moiety at the N-terminus, represented by a number. Peptides without N-terminal modification have X = 1 (corresponding to hydrogen; e.g., unmodified GLP-2 = GLP2-1). Some analogs are further modified by lysine substitution and attachment of a lipid-linker moiety at this site. Examples of sites in GLP-2 where such modifications are made include L17 and N24, and examples of attachments include l1, l2, or l3. Gatex is a pre-existing GLP-2-based drug containing an A2G substitution. [Figure 6B-2] See legend to Figure 6B-1. [Figure 7] Figure 7 provides graphs showing concentration-response curves for liraglutide and Lira-4 (see Figure 6, corresponding to N-trifluoroethyl-modified liraglutide) with or without overnight DPP4 incubation. Under these conditions, more than 98% of liraglutide was degraded, while Lira-4 remained unaffected. The corresponding EC50 values are shown in pM. [Figure 8] FIG. 8 provides a table summarizing exemplary results of stability assays of certain GLP-1 analogs (incubated with or without DPP4). [Figure 9] Figure 9A provides a table summarizing exemplary results of stability assays of specific GLP-1 or GIP (incubated with or without DPP4, DPP2, or FAP). Native GLP-1 (G-1) and GIP (I-4) exhibit a significant decrease in potency (reflected by an increased EC50) upon enzyme exposure, while the corresponding alkylated analogs (G4 and I4, respectively) are stable under the same conditions. [Figure 9A] Figure 9B provides a table showing that native GLP-2 (GLP2-1) is degraded by DPP4, while the trifluoroethyl-decorated analog (GLP2-4) is completely resistant to degradation. Peptides were incubated with vehicle or recombinant DPP4. Serial dilutions of GLP2-1 and GLP2-4 were then applied to HEK293 cells transfected with cDNA encoding the human GLP-2R and a cAMP-responsive reporter gene. DPP4 induced a significant increase in the EC50 of GLP-2 (indicating reduced potency), which was not observed for the GLP2-4 derivative. [Figure 9B] Figure 9C provides a table showing that selected N-terminal decorations are tolerated in GLP-1. Compound potency was assessed in a bioassay in HEK293 cells expressing the GLP-1 receptor and a cAMP-responsive luciferase reporter gene. [Figure 10A] Figures 10A-10D show a series of graphs reflecting stability assays of acetyl GLP-1 (="G-2"; Figure 10A), ethyl GLP-1 (="G-5"; Figure 10B), isobutyl GLP-1 (="G-6"; Figure 10C), and C-phenyl GLP-1 (="G-11"; Figure 10D, nomenclature from Figure 6) (incubated overnight with or without DPP4). "ON": overnight incubation. Corresponding EC50 values are shown in pM. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 11]Figure 11 provides a schematic showing exemplary GLP-1 analogs. The nomenclature in brackets refers to Figure 6. [Figure 12] Figure 12 provides a series of graphs showing the results of a stability assay of unmodified GIP versus CHCF-GIP (compound I-4) (incubated with or without DPP4). While the unmodified peptide is degraded by DPP4, I-4 is resistant to this enzyme. EC values are shown in pM. [Figure 13] Figure 13 provides a series of graphs showing the results of a stability assay of unmodified glucagon versus CHCF-glucagon (compound GCG-4) (incubated with or without DPP4). While the unmodified peptide is degraded by DPP4, GCG-4 is resistant to this enzyme. EC values are shown in pM. [Figure 14] Figure 14 provides a graph showing the results of a stability assay of unmodified exendin (Exe) versus CHCF-exendin (compound EXE-4) (incubated with or without DPP4). While unmodified exendin shows detectable susceptibility to DPP4 degradation, compound Exe-4 is completely resistant to this enzyme. EC values are shown in pM. [Figure 15] Figure 15 provides a graph showing that a triagonist decorated at the N-terminus with CHCF ("F-TA" or TA-4 in Figure 6) co-activates GLP-1, GIP, and glucagon receptors with similar potency. EC values are shown in pM. [Figure 16] FIG. 16 provides a schematic showing modifications made to amino acids found at the N-terminus of secretin family peptides. [Figure 17] FIG. 17 provides a schematic diagram showing the molecular structures of various charged modifications. [Figure 18]Figure 18 provides a series of graphs comparing the susceptibility of GLP-1 (left panel) to enzymatic degradation of CHCF3-GLP-1 (right panel; G-4 in Figure 6). The activity of these peptides was measured by luciferase assay in cells expressing the GLP-1 receptor after overnight incubation without enzyme (control) or with either DPP4, DPP9, or FAP. In contrast to unmodified GLP-1, the G-4 derivative is resistant to enzyme-induced loss of potency (no shift to the right in the concentration-response curve). [Figure 19] Figure 19 provides a series of graphs comparing the susceptibility of natural GHRH (top) to CHCF3-GHRH ("C2-GHRH"; bottom, compound GH29-4 in Figure 6) to enzymatic degradation. The activity of these peptides, tested as fresh stocks or after overnight incubation with or without DPP4, was measured by luciferase assay in cells expressing the GHRH receptor (GHRHR). In contrast to natural GHRH, GH29-4 derivatives are resistant to enzyme-induced loss of potency (no shift to the right of the concentration-response curve). [Figure 20] Figure 20 provides a helical wheel diagram of the interaction of the extracellular domains (ECDs) of GLP-2 and GLP-2R. The predicted extracellular domains are shown as gray ellipses. Black arrows point to modified sites, and white arrows point to proposed future modifications (arginine substitution; linker and lipid attachment; see Figure 6A). L17K and N24K modifications / acylation have already been tested in combination with N-terminal modifications. Other potential modification sites for attaching lipid-linker moieties include positions I13, R20, I27, and I31. [Figure 21]Figure 21 provides a series of graphs showing that native GLP-2 is degraded by DPP4, while a trifluoroethyl-decorated analog (GLP2-4) is completely resistant to degradation. Peptides were incubated with recombinant DPP4. Serial dilutions of GLP-2 and GLP2-4 were then added to HEK293 cells transfected with cDNA encoding the human GLP-2R and a cAMP-responsive reporter gene. DPP4 induced a greater than 40-fold decrease in GLP-2 potency, reflecting greater than 97% peptide degradation. In contrast, no potency loss was observed with GLP2-4. N=4, mean + SEM. [Figure 22] Figure 22A provides a graph comparing the time-dependent decrease in drug activity in plasma after a bolus injection of either liraglutide or its CHCF3-decorated derivative, Lira-4. Rats received a bolus injection of either drug via a central catheter, followed by serial blood sampling and measurement of drug activity in plasma by agonism bioassay at the indicated intervals. The efficacy of each peptide immediately after injection was defined as 100%. Liraglutide's plasma persistence is prolonged by the CHCF3 modification of Lira-4. [Figure 22A] Figure 22B shows the sustained hypoglycemic activity of Lira-4 after an oral glucose tolerance test. Mice received subcutaneous injections of either vehicle, G-4, liraglutide, or Lira-4 (shown in bars from left to right in each group). Thirty minutes and 5.5 hours later, two consecutive oral glucose loads were administered by oral gavage. Blood glucose levels were measured immediately before drug injection (-30 minutes) and at the indicated time intervals after the glucose load. A single injection of compound G-4 attenuated the glycemic excursion after the first glucose load and remained active several hours later, attenuating the second glucose challenge. [Figure 23]Figure 23A provides a graph depicting the extended in vivo bioactivity of GLP2-L17K(l1)-4, a fluorinated / acylated GLP-2 analog shown in Figure 6A. The clearance of this compound was compared to that of the existing GLP-2-based drug, Gatex. The compound (1.25 μg) was injected subcutaneously (sc) into mice. Plasma was collected 24 hours later and analyzed for GLP-2R agonist activity by luciferase reporter gene assay. [Figure 23A] Figures 23B-D show that GLP2-L17K(l1)-4 (alternatively referred to as oTTx-88 in these figures) induces intestinal mucosal growth. C57BL / 6J mice were injected subcutaneously once daily for 5 days with either vehicle, GLP2-L17K(l1)-4, or Gatex at the indicated doses. Animals were sacrificed on day 6, followed by analysis of intestinal tissue. Figure 23B shows that GLP2-L17K(l1)-4(oTTx-88) increases intestinal weight. N=6 animals / group, mean±SEM. *p<0.05 vs. vehicle. [Figure 23B] FIG. 23C provides histology images performed by hematoxylin and eosin staining of mucosal sections. [Figure 23C] Figure 23D provides a graph showing quantification of villus height in mice treated with vehicle or GLP2-L17K(l1)-4(oTTx-88) (daily dosing was 25 μg / mouse). N=6 animals / group, mean±SEM. **p<0.01 vs. vehicle. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description of the Invention The present invention generally provides modified peptides that are resistant to proteolysis while retaining the biological activity of the unmodified peptides, methods for synthesizing such modified peptides, and therapeutic methods featuring the modified peptides.

[0032] The present invention is based, at least in part, on the discovery that certain chemical modifications of a polypeptide improve its resistance to proteolysis in vitro or in vivo without significantly altering its biological activity.

[0033] In certain embodiments, chemical modifications include derivatization of the N-terminal amino group of a polypeptide, the NH of the amide bond formed between the N-terminal amino acid and the next amino acid in the polypeptide (i.e., the N-terminal first internal amide bond), other free primary amino groups of the polypeptide, thiol groups, and thioether groups, where derivatization includes alkylation, cycloalkylation, alkenylation, or alkynylation. In other embodiments, each of at least one alkyl, alkenyl, or alkynyl group modifying the polypeptide is independently unsubstituted or substituted. In yet other embodiments, at least one of the alkyl, alkenyl, or alkynyl groups modifying the polypeptide is inert to in vivo metabolism.

[0034] In certain embodiments, the chemical modification of the N-terminal amino group and / or the NH of the N-terminal first internal amide bond comprises replacement of the NH with N-X, where X is, independently at each occurrence, optionally substituted phenyl, optionally substituted benzyl, optionally substituted -(CR2) 1~6 -phenyl, →O, -OH, -OR, alkoxy, NH, optionally substituted NH(C 16 alkyl), and optionally substituted N(C1-C 16 Alkyl) (C1-C 16 alkyl), wherein R, at each occurrence, is independently selected from hydrogen or optionally substituted C1-C6 alkyl.

[0035] In certain embodiments, the chemically modified polypeptides of the present invention have improved properties compared to the corresponding non-chemically modified polypeptides, wherein the properties are at least one selected from the group consisting of log P, CNS permeability, bioabsorption, renal clearance, efficacy (receptor stimulation), delivery / formulation, storage stability (non-protease mediated), solubility, reduced tendency to aggregate / oligomerize, resistance to cytochrome P450 and other enzymes, ability to be used in combination with other drugs (such as C4 agonists, melanocortin MC4 receptors, and / or serotonin), reduced immunogenicity, route of administration, and pharmacokinetic parameters.

[0036] It should be understood that the present disclosure contemplates the sequences disclosed herein and sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequences disclosed herein.

[0037] As described herein, the present invention provides a novel method for increasing proteolytic resistance in polypeptides using minimal chemical modification. In certain embodiments, a 2,2,2-trifluoroethyl group is chemically attached to the N-terminal amino group and / or other amino groups, and / or thiol and / or thioether groups in the polypeptide. The chemically modified N-terminal amino group is uncharged at physiological pH, similar to N-terminal acetylated analogs, which are known to have good proteolytic stability. The 2,2,2-trifluoroethyl group is chemically inert. Unlike acetyl groups, the 2,2,2-trifluoroethyl group is not removed by enzymatic processes in vivo. Exemplary results for N-2,2,2-trifluoroethyl GLP-1 demonstrate no loss of binding or activity in cell-based assays, in stark contrast to N-acetyl-GLP-1, which is approximately 10-50 times less active than GLP-1.

[0038] In certain embodiments, the GLP-1 analogs of the present invention have a low risk of hypoglycemia, have cardioprotective and neuroprotective effects, and stimulate GLP-1R in the brain, reducing appetite in administered subjects. In other embodiments, the GLP-1 analogs of the present invention promote weight loss or maintenance or prevent weight gain in administered subjects.

[0039] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2002); nd Ed. 1994); The Cambridge Dictionary of Science and Technology (Walker, Ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger, et al. (Eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Generally, the nomenclature used herein and the laboratory procedures in pharmaceutical, organic, and polymer chemistry are those well known and commonly employed in the art.

[0040] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0041] As used herein, the term "about" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, when referring to a measurable value such as an amount, duration, etc., the term "about" is meant to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, as such variations are appropriate for performing the disclosed methods.

[0042] As used herein, the term "administering" means providing a compound and / or composition of the invention to a subject by any suitable method.

[0043] As used herein, the term "Aib" refers to 2-aminoisobutyric acid.

[0044] As used herein, the term "alkenyl," used alone or in combination with other terms, means, unless otherwise specified, a stable mono- or di-unsaturated, straight- or branched-chain hydrocarbon radical having the stated number of carbon atoms. Examples include vinyl, propenyl (or allyl), crotyl, isopentenyl, butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, and the higher homologs and isomers. A functional group representing an alkene is exemplified by -CH-CH=CH.

[0045] The term "alkoxy," as used herein, alone or in combination with other terms, unless otherwise specified, refers to an alkyl group having the specified number of carbon atoms as defined above, attached to the remainder of the molecule by an oxygen atom, e.g., methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy), and higher homologs and isomers. Preferred are (C1-C3)alkoxy, including, but not limited to, ethoxy and methoxy.

[0046] As used herein, the term "alkyl," by itself or as part of another substituent, unless otherwise specified, refers to an alkyl group having the specified number of carbon atoms (i.e., C1-C 10means a straight or branched chain hydrocarbon of 1 to 10 carbon atoms, including straight, branched, or cyclic substituents. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. Most preferred are (C1-C6)alkyl, including, but not limited to, ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl, and cyclopropylmethyl.

[0047] The term "alkynyl," as used herein, alone or in combination with other terms, means, unless otherwise specified, a stable straight or branched chain hydrocarbon group having a carbon-carbon triple bond and having the stated number of carbon atoms. Non-limiting examples include ethynyl and propynyl, and higher homologs and isomers. The term "propargyl" refers to a group exemplified by -CH-C≡CH. The term "homopropargyl" refers to a group exemplified by -CHCH-C≡CH. The term "substituted propargyl" refers to a group exemplified by -CR-C≡CR, where R, in each instance, is independently H, alkyl, substituted alkyl, alkenyl, or substituted alkenyl, provided that at least one R group is not hydrogen. The term "substituted homopropargyl" refers to a group exemplified by -CR2CR2-C≡CR, where R, at each occurrence, is independently H, alkyl, substituted alkyl, alkenyl, or substituted alkenyl, provided that at least one R group is not hydrogen.

[0048] By "ameliorate" is meant to reduce, inhibit, attenuate, lessen, arrest, or stabilize the occurrence or progression of a disease or disorder.

[0049] In this specification, "amino acids" are represented by their full names, three-letter abbreviations, and corresponding one-letter abbreviations, as shown in the table below. The structures of amino acids and their abbreviations are described in Stryer, 1988, "Biochemistry", 3 rdEd., W.H. Freeman and Co., New York.

[0050] As used herein, the term "aromatic" refers to a carbocyclic or heterocyclic ring having one or more polyunsaturated rings and having aromaticity, i.e., (4n+2) delocalized π (pi) electrons, where n is an integer.

[0051] As used herein, the terms "aryl" or "arene," used alone or in combination with other terms, mean, unless otherwise specified, a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), which rings may be linked together in a pendant manner, such as biphenyl, or fused, such as naphthalene. Examples include phenyl, anthracyl, and naphthyl (including 1- and 2-naphthyl). Preferred are phenyl and naphthyl, and most preferred is phenyl.

[0052] As used herein, the term "aryl-(C1-C3)alkyl" refers to a functional group in which a 1-3 carbon alkylene chain is attached to an aryl group, e.g., -CH2CH2-phenyl or -CH2-phenyl(benzyl). Preferred are aryl-CH2- and aryl-CH(CH3)-. The term "substituted aryl-(C1-C3)alkyl" refers to an aryl-(C1-C3)alkyl functional group in which the aryl group is substituted. Preferred is substituted aryl(CH2)-. Similarly, the term "heteroaryl-(C1-C3)alkyl" refers to a functional group in which a 1-3 carbon alkylene chain is attached to a heteroaryl group, e.g., -CH2CH2-pyridyl. Preferred is heteroaryl-(CH2)-. The term "substituted heteroaryl-(C1-C3)alkyl" refers to a heteroaryl-(C1-C3)alkyl functional group in which the heteroaryl group is substituted. Preferred is substituted heteroaryl-(CH2)-.

[0053] In one aspect, the terms "co-administered" and "co-administration" in relation to a subject refer to administering to a subject a compound and / or composition of the present invention, or a salt thereof, together with a compound and / or composition that can also treat any of the diseases contemplated in the present invention. In one embodiment, the co-administered compounds and / or compositions are administered separately or as part of a single therapeutic approach in any kind of combination. The co-administered compounds and / or compositions can be formulated in any kind of combination, as a mixture of solid and liquid under various solid, gel, and liquid formulations, and as a liquid.

[0054] As used herein, the terms "comprise," "contain," "have," and the like may have the meaning ascribed to them in U.S. patent law and may mean "include," "comprising," etc. Similarly, "consisting essentially of" may have the meaning ascribed to it in U.S. patent law, and the term is open-ended, allowing for the presence of more than what is recited, but excluding prior art aspects, so long as the basic or novel characteristics of what is recited are not changed by the presence of more than what is recited.

[0055] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound useful in the present invention with a pharmaceutically acceptable carrier. A pharmaceutical composition facilitates administration of the compound to a subject.

[0056] As used herein, the term "cycloalkyl," by itself or as part of another substituent, means, unless otherwise specified, a monocyclic or polycyclic chain hydrocarbon having the specified number of carbon atoms (i.e., C3-C6 means a ring group containing a ring group of 3 to 6 carbon atoms), including straight-chain, branched-chain, or cyclic substituents. As used herein, the term "cycloalkyl" further includes cycloalkenyl and cycloalkynyl compounds. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. An example is (C3-C6)cycloalkyl, such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl further includes decalin, bicyclodecane, bicyclooctane, bicycloheptane, bicyclohexane, adamantyl, and other polycyclic alkyl groups.

[0057] As used herein, the term "δ" refers to delta (ppm).

[0058] By "reduce / reduce" is meant a negative change of at least about 10%, 25%, 50%, 75%, 100%, or more.

[0059] "Detecting" refers to determining the presence, absence, or amount of the analyte to be detected.

[0060] By "disease" or "disorder" is meant any condition that damages or interferes with the normal function of a cell, tissue, or organ.

[0061] As used herein, the term "acyl amino acid releasing enzyme," also known as "acylpeptide hydrolase," refers to the enzyme encoded by the APEH gene in humans. The enzyme is an acylpeptide hydrolase, which preferentially catalyzes the hydrolysis of terminal acetylated amino acids from small acetylated peptides.

[0062] As used herein, the term "DMSO" refers to dimethyl sulfoxide.

[0063] As used herein, the term "DPP2" refers to dipeptidyl protease 2.

[0064] As used herein, the term "DPP4" refers to dipeptidyl protease 4.

[0065] As used herein, the term "DPP8" refers to dipeptidyl protease 8.

[0066] As used herein, the term "DDP9" refers to dipeptidyl protease 9.

[0067] As used herein, the term "FAP" refers to fibroblast activation protein, also known as seprase.

[0068] As used herein, the term "S9B family" refers to all S9B oligopropyl peptidases.

[0069] As used herein, the term "vitamin D binding protein" refers to gc-globulin. Along with human serum albumin and α-fetoprotein, it belongs to the albumin gene family. Vitamin D binding protein is a multifunctional protein present in plasma, ascites, cerebrospinal fluid, and on the surface of many cell types.

[0070] As used herein, "transferrin" refers to an iron-binding plasma glycoprotein that regulates the level of free iron in body fluids. Human transferrin is encoded by the TF gene.

[0071] As used herein, the term "GHRH" refers to growth hormone-releasing hormone. This gene encodes several proteins of the glucagon family. The encoded preproprotein is produced in the hypothalamus and cleaved to generate a mature factor known as somatoliberin, which acts to stimulate growth hormone release from the pituitary gland. Variant receptors for somatoliberin have been found in several tumors, and antagonists of these receptors can inhibit tumor growth. An exemplary GHRH amino acid sequence is available under NCBI Reference Number AAB37758.1 and is provided below. TIFF2025120243000065.tif30138

[0072] As used herein, the term "GHRHR" refers to the receptor for growth hormone-releasing hormone.

[0073] As used herein, the term "GLP-1" refers to glucagon-like peptide-1, a neuropeptide and incretin derived from the transcription product of the proglucagon gene. Biologically active forms of GLP-1 are GLP-1-(7-37) and GLP-1-(7-36)NH2. These peptides result from selective cleavage of the proglucagon molecule. GLP-1 is a potent hypoglycemic hormone that induces pancreatic beta cells to release the hormone insulin in response to elevated glucose levels while suppressing glucagon secretion. An exemplary GLP-1 amino acid sequence is provided below. TIFF2025120243000066.tif18128

[0074] As used herein, the term "GLP-2" refers to glucagon-like peptide-2. GLP-2 is produced by specific post-translational proteolytic cleavage of proglucagon in a process that liberates glucagon-like peptide-1 (GLP-1). Intestinal GLP-2 is secreted simultaneously with GLP-1 upon nutrient ingestion. The activities of GLP-2 may include intestinal growth, increased intestinal function, reduced bone destruction, and neuroprotection. GLP-2 and related analogs may be used as treatments for short bowel syndrome, Crohn's disease, osteoporosis, and as adjunctive therapy in cancer chemotherapy. An exemplary GLP-2 amino acid sequence is provided below. TIFF2025120243000067.tif4128

[0075] As used herein, the term "PACAP" refers to pituitary adenylate cyclase-activating polypeptide.

[0076] As used herein, the term PYY (peptide YY3-36 and also YY1-36) refers to the peptide tyrosine, or pancreatic peptide YY3-36, which in humans is encoded by the PYY gene.

[0077] As used herein, the term "GCG" refers to glucagon.

[0078] As used herein, the term "GIP" refers to gastric inhibitory polypeptide.

[0079] As used herein, the term "SCT" refers to secretin.

[0080] As used herein, the term "VIP" refers to vasoactive intestinal peptide.

[0081] By "effective amount" is meant the amount of compound required to ameliorate the symptoms of a disease in an untreated patient. The effective amount of active compound used to practice the present invention for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as an "effective" amount.

[0082] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, which portion preferably comprises at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may comprise about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotides or amino acids.

[0083] As used herein, the term "GLP-1" refers to glucagon-like peptide-1 or a peptide having 75% identity, preferably at least 90% identity to its sequence.

[0084] As used herein, the term "GLP-2" refers to glucagon-like peptide-2 or a peptide having 75% identity, preferably at least 90% identity to its sequence.

[0085] As used herein, the terms "halo" or "halogen," employed alone or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably fluorine, chlorine, or bromine, more preferably fluorine or chlorine.

[0086] As used herein, the term "heteroalkyl," by itself or in combination with another term, unless otherwise specified, refers to a stable, linear or branched alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, wherein the nitrogen and sulfur atoms thereof may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom may be positioned at any position on the heteroalkyl group, including between the remainder of the heteroalkyl group and the fragment to which it is attached, or may be attached to the most distal carbon atom in the heteroalkyl group. Examples include -O-CH-CH-CH, -CH-CH-CH-OH, -CH-CH-NH-CH, -CH-S-CH-CH, and -CHCH-S(=O)-CH. Up to two heteroatoms may be consecutive, for example, -CH-NH-OCH or -CH-CH-SS-CH.

[0087] As used herein, the terms "heterocycle" or "heterocyclyl" or "heterocyclic," by themselves or as part of another substituent, mean, unless otherwise specified, a stable, unsubstituted or substituted, mono- or polycyclic heterocyclic ring system consisting of carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, wherein the nitrogen and sulfur heteroatoms are optionally oxidized and the nitrogen atom is optionally quaternized. A heterocyclic ring system may be attached at any heteroatom or carbon atom that results in a stable structure, unless otherwise specified. A heterocycle can be aromatic or non-aromatic in nature. In one embodiment, a heterocycle is heteroaryl.

[0088] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. Polycyclic heteroaryls can contain one or more rings that are partially saturated. Examples include tetrahydroquinoline and 2,3-dihydrobenzofuryl.

[0089] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepine, and hexamethylene oxide.

[0090] Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (including, but not limited to, 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

[0091] Examples of polycyclic heterocycles include indolyl (including, but not limited to, 3-, 4-, 5-, 6-, and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (including, but not limited to, 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (including, but not limited to, 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl, and the like. (including but not limited to 3-, 4-, 5-, 6-, and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (including but not limited to 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (including but not limited to 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolidinyl, and quinolizidinyl.

[0092] The above lists of heterocyclyl and heteroaryl moieties are intended to be representative and not limiting.

[0093] "Identity" refers to the amino acid or nucleic acid sequence identity between a subject sequence and a reference sequence. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program is used to -3 ~e -100 The probability scores can be used to indicate closely related sequences.

[0094] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0095] By "increase" is meant a positive change of at least about 10%, 25%, 50%, 75%, 100%, or more.

[0096] As used herein, the term "instructional material" includes publications, recordings, diagrams, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. In some examples, the instructional material can be part of a kit useful for specifically alkylating N-terminal amino groups, other amino groups, thiol groups, and / or thioether groups of polypeptides. The kit's instructional material can, for example, be attached to a container that holds the composition of the present invention or shipped with the container that holds the composition. Alternatively, the instructional material can be shipped separately from the container, with the intention that the recipient use the instructional material and the composition in conjunction with each other. For example, the instructional material is for use of the kit; instructions for use of the composition; or instructions for use of a formulation of the composition.

[0097] The terms "isolated," "purified," or "biologically pure" refer to material that is free, to varying degrees, from components that normally accompany it when found in its native state. "Isolated" refers to some degree of separation from the original source or environment. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials so that the impurities do not significantly affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA techniques, or chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can refer to a nucleic acid or protein that yields essentially one band in an electrophoretic gel. For proteins that can undergo modifications, such as phosphorylation or glycosylation, different modifications will yield different isolated proteins that can be separated separately.

[0098] As used herein, the phrase "N-terminal first internal amide bond" refers to an amide bond formed between the N-terminal amino acid of a polypeptide and the next amino acid in the polypeptide (i.e., between the first and second residues from the N-terminus of the polypeptide).

[0099] As used herein, "naturally occurring amino acids" includes the L-isomers of the 20 amino acids (plus cystine) that occur naturally in proteins as shown in Table 1. Unless otherwise specified, all amino acids referred to in this application are in the L form.

[0100] Table 1. L-isomers of 20 naturally occurring amino acids in proteins TIFF2025120243000068.tif70136

[0101] As used herein, the terms "peptide," "polypeptide," or "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and to the many types of longer chains, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. Polypeptides can be naturally occurring, recombinant, synthetic, or a combination thereof. Non-cyclic peptides have an N-terminus and a C-terminus. The N-terminus has an amino group, which may be free (i.e., as an NH group) or suitably protected (e.g., by a BOC or Fmoc group). The C-terminus has a carboxyl group, which may be free (i.e., as a COOH group) or suitably protected (e.g., as a benzyl or methyl ester). Cyclic peptides do not necessarily have free N- or C-termini, as they are covalently bonded to form a cyclic structure by amide bonds.

[0102] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not interfere with the biological activity or properties of the compounds useful in the invention and that is relatively non-toxic, i.e., the material can be administered to a subject without causing undesired biological effects or interfering in a deleterious manner with any of the components of the composition in which it is contained.

[0103] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of the administered compound, its solvate, hydrate, or clathrate, prepared from a pharmaceutically acceptable non-toxic acid or base, including an inorganic acid or base, an organic acid or base. The peptides described herein may form salts with acids or bases, and such salts are included in the present invention. In one embodiment, the salt is a pharmaceutically acceptable salt. The term "salt" encompasses free acid or base addition salts that are useful in the methods of the present invention. The term "pharmaceutically acceptable salt" refers to a salt that has a toxicity profile within a range that confers usefulness in pharmaceutical applications. Nevertheless, pharmaceutically unacceptable salts may have properties such as high crystallinity, which are useful in the practice of the present invention, such as usefulness in the process of synthesizing, purifying, or formulating peptides useful in the methods of the present invention.

[0104] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids, examples of which include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric (including hydrogen phosphate and dihydrogen phosphate). Suitable organic acids may be selected from the aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, malonic acid, saccharinic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid.

[0105] Suitable pharmaceutically acceptable base addition salts of the peptides of the present invention include metal salts, including, for example, alkali metal, alkaline earth metal, and transition metal salts, such as, for example, calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts prepared from basic amines, such as N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. All of these salts can be prepared from the corresponding peptide, for example, by reacting the peptide with an appropriate acid or base.

[0106] The terms "prevent" or "prevention," as used herein, mean avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who does not experience such symptoms at the time administration of an agent or compound begins. Disease, condition, and disorder are used interchangeably herein.

[0107] As used herein, the term "reaction conditions" refers to physical treatments, chemical reagents, or a combination thereof, required or optionally required to promote a reaction. Non-limiting examples of reaction conditions are electromagnetic radiation, heat, catalysts, chemical reagents (such as, but not limited to, acids, bases, electrophiles, or nucleophiles), and buffers.

[0108] "Reference" means a standard or control condition.

[0109] A "reference sequence" is a defined sequence used as a basis for sequence comparison.

[0110] As used herein, the terms "subject," "patient," or "individual" can be a human, a non-human mammal, or a bird. Non-human mammals include, for example, livestock and pets, such as sheep, cows, horses, pigs, dogs, cats, and murine mammals. Preferably, the subject is a human.

[0111] As used herein, the term "substituted" means that an atom or group of atoms replaces a hydrogen atom as a substituent bonded to another group. Unless otherwise specified, any group recited in the present invention can be substituted.

[0112] In a substituted alkyl, alkenyl, alkynyl, or cycloalkyl group, the substituents are C1-C 16 Alkyl, C3-C 16 Cycloalkyl, C2-C 16 Alkenyl, C2-C 16 Alkynyl (including -C≡CH), heteroaryl, heterocyclyl, C1-C6 alkoxy, azido, diaziryl TIFF2025120243000069.tif10128, -CHO, 1,3-dioxol-2-yl, halo, haloalkyl (such as trifluoromethyl, difluoromethyl, and fluoromethyl), cyano, nitro, triflyl, mesyl, tosyl, haloalkoxy (such as trifluoromethoxy, difluoromethoxy, and fluoromethoxy), heterocyclyl, aryl, heteroaryl, -SR, -S(=O)(C1-C6 alkyl) and -P(=O)(OR)2, wherein R is, in each occurrence, independently H or C1-C6 alkyl.

[0113] For substituted aryl, aryl-(C1-C3)alkyl, and heterocyclyl groups, the term "substituted" as applied to the rings of these groups refers to any level of substitution, i.e., mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution can be at any chemically accessible site. In one embodiment, the number of substituents varies from 1 to 4. In another embodiment, the number of substituents varies from 1 to 3. In yet another embodiment, the number of substituents varies from 1 to 2. In yet another embodiment, the substituents are C1-C3 alkyl, C1-C3 aryl ... 16 Alkyl, C3-C 16 Cycloalkyl, C2-C 16 Alkenyl, C2-C 16 Alkynyl (including -C≡CH), heteroaryl, heterocyclyl, C1-C6 alkoxy, azido, diaziryl TIFF2025120243000070.tif10128, -CHO, 1,3-dioxol-2-yl, halo, haloalkyl (such as trifluoromethyl, difluoromethyl, and fluoromethyl), cyano, nitro, triflyl, mesyl, tosyl, haloalkoxy (such as trifluoromethoxy, difluoromethoxy, and fluoromethoxy), heterocyclyl, aryl, heteroaryl, -SR, -S(=O)(C and -P(=O)(OR) (wherein R is, independently at each occurrence, H or C1-C6 alkyl). As used herein, when a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight, or cyclic, with straight chain being preferred.

[0114] As used herein, the terms "treat" and "treatment" mean to reduce the frequency or severity with which symptoms of a disease or condition are experienced by a subject by administering an agent or compound to the subject.

[0115] Throughout this disclosure, various aspects of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values within that range and, where appropriate, partial integers of numerical values within the range. The ranges provided herein are understood to be shorthand expressions for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0116] Any compound, composition, or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0117] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

[0118] Polypeptides In one aspect, the present invention provides chemically modified polypeptides, wherein the chemical modification is: (i) at least one selected from the group consisting of the N-terminal amino group, the NH of the N-terminal first internal amide bond, other free primary amino groups, thiol groups, and thioether groups of the polypeptide is independently alkylated, cycloalkylated, alkenylated, or alkynylated (wherein each individual alkyl, cycloalkyl, alkenyl, or alkynyl group is independently optionally substituted); and (ii) at least one NH selected from the group consisting of the N-terminal amino group and the N-terminal first internal amide bond is derivatized with X (wherein each X is an optionally substituted phenyl, →O (thus generating an N-oxide), —OH, alkoxy, NH, NH(C1-C2). 16 alkyl), and N(C1-C 16 Alkyl) (C1-C 16 alkyl).

[0119] In certain embodiments, the N-terminal amino group, the NH of the N-terminal first internal amide bond, other free primary amino groups, thiol groups, and / or thioether groups of the polypeptide are each independently alkylated, cycloalkylated, alkenylated, or alkynylated with a corresponding optionally substituted group.

[0120] In certain embodiments, one or more of the N-terminal amino group, other free amino groups, and / or thiol groups of the polypeptide are independently alkylated with a first and second substituent contemplated herein, wherein the first and second substituents are independently the same or different. In other embodiments, one or more of the N-terminal amino group, other free amino groups, and / or thiol groups of the polypeptide are independently alkylated with a first optionally substituted C1-C 16 Alkyl and a second optionally substituted C1-C 16In still other embodiments, one or more of the N-terminal amino group, other free amino groups, and / or thiol groups of the polypeptide are independently alkylated with a first optionally substituted methyl and a second optionally substituted methyl, wherein the first and second methyls are independently the same or different. In still other embodiments, one or more of the N-terminal amino group, other free amino groups, and / or thiol groups of the polypeptide are independently alkylated with two optionally substituted C1-C 16 In yet another embodiment, one or more of the N-terminal amino group, other free amino groups, and / or thiol groups of the polypeptide are independently alkylated with two methyls. In yet another embodiment, at least the N-terminal amino group of the polypeptide is derivatized with a group contemplated herein, wherein the group is substituted or unsubstituted.

[0121] Non-limiting examples of alkyl, cycloalkyl, alkenyl, or alkynyl groups contemplated in the present invention are: TIFF2025120243000071.tif211153TIFF2025120243000072.tif213146TIFF2025120243000073.tif214137TIFF2025120243000074.tif199145 (wherein n is an integer ranging from 1 to 6, and R is an optionally substituted alkyl or aryl).

[0122] Without wishing to be limited by any theory, the group attached to the N-terminal amino group occupies a binding pocket on the receptor that is close to the membrane-water interface where the polypeptide binds to the receptor. The dimensions of the binding pocket are expected to vary between receptors. In certain embodiments, in the GLP-1R, the binding pocket is approximately 190 Å, as confirmed by the fact that an N-1-adamantyl-methyl derivative of GLP-1 was found to have activity comparable to underivatized GLP-1. 3 It has the following volume:

[0123] In certain embodiments, the polypeptides of the invention are more resistant to proteolytic activity by DPP4, DPP2, and / or proteases having 50% or more homology to DPP4 and / or DPP2 than the corresponding unmodified polypeptides. In certain embodiments, the polypeptides of the invention are more resistant to endoprotease activity than the corresponding unmodified polypeptides. In yet other embodiments, the chemically modified polypeptides have improved overall proteolytic stability in human serum than the corresponding unmodified polypeptides.

[0124] In certain embodiments, the chemically modified polypeptide has an improved in vivo half-life compared to the corresponding non-chemically modified polypeptide. In other embodiments, the chemically modified polypeptide has improved blood-brain barrier permeability compared to the corresponding non-chemically modified polypeptide. In still other embodiments, the chemically modified polypeptide has improved pharmacokinetics compared to the corresponding non-chemically modified polypeptide.

[0125] In certain embodiments, the chemical modifications contemplated in the present invention are C1-C 16 Alkyl, C3-C 16 Cycloalkyl, C2-C 16 Alkenyl, or C2-C 16 alkynyl, which contains a substituent selected from the group consisting of C1-C 16 Alkyl, C3-C 16Cycloalkyl, C2-C 16 Alkenyl, C2-C 16 Alkynyl (including -C≡CH), heteroaryl, heterocyclyl, C1-C6 alkoxy, azido, diaziryl TIFF2025120243000075.tif10128, -CHO, 1,3-dioxol-2-yl, halo, haloalkyl (such as trifluoromethyl, difluoromethyl, and fluoromethyl), cyano, nitro, triflyl, mesyl, tosyl, haloalkoxy (such as trifluoromethoxy, difluoromethoxy, and fluoromethoxy), heterocyclyl, aryl, heteroaryl, -SR, -S(=O)(C1-C6 alkyl) and -P(=O)(OR)2, wherein R, in each occurrence, is independently H or C1-C6 alkyl. 16 In other embodiments, the alkyl group contemplated is —CH 2 CF 3 in at least one instance.

[0126] Polypeptides of the present invention can be prepared using any method known to those skilled in the art. In certain embodiments, polypeptides can be prepared using standard peptide synthesis, where at least one of the residues to be chemically modified is introduced into the reaction mixture already in its chemically modified form. In other embodiments, polypeptides can be prepared using standard peptide synthesis, where all of the modified residues are introduced into the reaction mixture already in their chemically modified form. In still other embodiments, polypeptides of the present invention or fragments thereof are chemically modified in situ, whereby the N-terminal amino group, other free amino groups, thiol groups (such as from cysteine residues), and / or thioether groups (such as from methionine residues) are reacted with reagents that promote alkylation of the groups. The resulting chemically modified polypeptide or fragment thereof can be used itself for therapeutic treatment and / or further chemical reactions. Alternatively, the resulting chemically modified polypeptide or fragment thereof can be purified and then used as a therapeutic agent and / or subjected to further chemical reactions.

[0127] In certain embodiments, peptides are assembled by standard automated peptide synthesis using Fmoc chemistry. In other embodiments, the lysine side chain amino group is protected with an allyloxycarbonyl (alloc) group, which can be selectively removed using, for example, PhSiH3 and Pd(PPh3)4. In yet other embodiments, the lysine side chain amino group can then be coupled to a lipid using existing chemistries.

[0128] In certain embodiments, a free primary amino group in a polypeptide (such as the N-terminal amino group) is reacted with an aldehyde or ketone under reducing conditions (e.g., in the presence of a borohydride) to yield the corresponding derivatized amine. Alternatively, a derivatized amino acid is prepared separately and, as needed, coupled to a growing polypeptide chain.

[0129] In certain embodiments, the polypeptide is cleaved from the resin using, for example, CF3CO2H:TIPS:H2O (95:2.5:2.5), purified using, for example, reverse-phase HPLC, and analyzed using, for example, ESI and MALDI-MS.

[0130] In certain embodiments, at least one free amino group, thiol group, and / or thioether group of a polypeptide is modified with a 2,2,2-trifluoroethyl group. Such modification can be performed on a solid phase, whereby the immobilized polypeptide or a fragment thereof is reacted with a 2,2,2-trifluoroethyl iodonium salt, such as (2,2,2-trifluoroethyl)phenyl iodonium salt, (2,2,2-trifluoroethyl)(1,3,5-tri-R-phenyl) iodonium salt (wherein R is H or methyl). The salts shown below transfer -CHCFCF or -CHCFCFH. TIFF2025120243000076.tif20128

[0131] In other embodiments, modification of a polypeptide with a trifluoromethyl group can be achieved by, for example, the following non-limiting reaction: It can be performed in either solid or liquid phase using TIFF2025120243000077.tif22134.

[0132] The present invention further contemplates intramolecular and / or intermolecular cross-linking of the polypeptides of the present invention. In certain embodiments, at least two functional groups selected from the group consisting of free amino groups, thiol groups, and / or thioether groups of the polypeptide are: Cross-linking is performed using bis(iodonium) salts such as TIFF2025120243000078.tif21128.

[0133] In certain embodiments, the polypeptides of the present invention contain the following chemically modified amino acids: TIFF2025120243000079.tif89148 (Figure 29).

[0134] In certain embodiments, the chemically modified polypeptide of the present invention comprises one of the following sequences, in which in each polypeptide at least one of the residues marked with an asterisk (*) is chemically modified as contemplated in the present invention (Aib stands for 2-aminoisobutyric acid), and the polypeptide may be lipidated: GLP-1: TIFF2025120243000080.tif4128 Exenatide: TIFF2025120243000081.tif4129 Liraglutide: TIFF2025120243000082.tif5170Semaglutide: TIFF2025120243000083.tif4128 (wherein X bonded to the ε-amino group of lysine is TIFF2025120243000084.tif27128) Taspoglutide: TIFF2025120243000085.tif4128 Lixisenatide: TIFF2025120243000086.tif4158 Triagonist: TIFF2025120243000087.tif4170GLP-2: TIFF2025120243000088.tif4128Exendin: TIFF2025120243000089.tif4128VIP: TIFF2025120243000090.tif4128PACAP: TIFF2025120243000091.tif4128GIP: TIFF2025120243000092.tif4128Met-enkephalin: TIFF2025120243000093.tif3128BNP: TIFF2025120243000094.tif4128 Substance P: TIFF2025120243000095.tif4128Tyr-MIF-1: TIFF2025120243000096.tif3128Tyr-W-MIF-1: TIFF2025120243000097.tif3128Glucagon TIFF2025120243000098.tif4128Growth hormone-releasing hormone (GHRH); pituitary adenylate cyclase-activating polypeptide (PACAP) ADCYAP1; Glucagon (GCG); gastric inhibitory polypeptide (GIP); Secretin (SCT); vasoactive intestinal peptide (VIP); OXM (oxyntomodulin) (a dual agonist of GLP-1R and GIPR) PTH (parathyroid hormone)--hPTH(1-34); PYY (also peptides YY3-36, and YY1-36); NPY (neuropeptide Y); VIP peptide (vasoactive intestinal peptide); GLP-1 + GIP; GLP-1 + amylin; GLP-1 + gastrin; GLP-1 + estrogen; GLP-1 + GLP-2 dual agonists (e.g., as detailed in Finan, et al., 2015, Mol. Cell Endocrinol., Jul. 4, p.i.:S0303-7207(15)30012-5. doi:10.1016 / j.mce.2015.07.003) GLP-1R + glucagon dual agonists, including but not limited to LY2944876 / TT-401 [Eli Lilly]; ZP2929 [Zealand]; HM12525A [Hamni Pharmaceuticals]; MEDI0382 [MedImmune]; SAR425899 [Sanofi]; G530L / NN9030 + liraglutide [Novo Nordisk]; VPD-107 [Spitfire Pharma]; MOD-6030 / 1 [Prolor / OPKO Biologics]; Mixed agonists, including but not limited to MAR709 / RO6811135 [MB-2]; Cpd86 {Eli Lilly]; ZP-DI-70 [Zealand]; IUB447 [MB-2]; ZP-GG-23 [Zealand]; ZP3022 [Zealand]; albiglutide (GLP-1 covalently bound to albumin [GlaxoSmithKline]); dulaglutide (GLP-1 conjugated to an Fc antibody fragment / Eli Lilly); Other GLP-1R agonists, including but not limited to: ITCA 650[Intarcia];CJC-1134-PC[ConjuChem];Langlenatide / HM11260C[Hamni Pharmaceuticals];PB1023(Glymera)[PhaseBio];VRS 859[Diartis Pharmaceuticals];TTP054[Trans Tech Pharma];ZYOG1[Zydus] Cadila];NN9924 / OG217SC[Novo Nordisk];NN9926 / OG987GT[Novo Nordisk];NN9927 / OG987SC[Novo Nordisk];ARI-1732TS[Arisaph Pharmaceuticals] etc; Other DPP4 substrates, including but not limited to: GHRH; MCP-3; MCP-4; LEC; endomorphin 1; endomorphin 2; I-TAC; SDF-1α; SDF-1β; GRP; PACAP38; GH(1-43); CART(55-102); IP-10; PHM; ghrelin; Gro-β; LIX; pancreatic polypeptide; eotaxin; TIFF2025120243000099.tif44128 (from Penchala, et al., 2015, Nat. Chem. Biol. 11:793-800) GLP-1 analogs stabilized by other modifications such as, but not limited to, biotinylation, vitamin B12 conjugation, and P' modifications (e.g., as detailed in Clardy-James, et al., 2013, ChemMedChem, Apr;8(4):582-6. doi:10.1002 / cmdc.201200461. Epub 2012 Nov 30; and Heard, et al., 2013, J Med Chem., Nov 14;56(21):8339-51. doi:10.1021 / jm400423p. Epub 2013 Oct 16).

[0135] Polypeptides of the invention may possess one or more stereocenters, and each stereocenter may independently exist in either the (R) or (S) configuration. In one embodiment, the polypeptides described herein exist in optically active or racemic form. The polypeptides described herein include racemic, optically active, regioisomeric, and stereoisomeric forms, or combinations thereof, that possess the therapeutically useful properties described herein. Preparation of optically active forms is accomplished by any suitable method, including, but not limited to, resolution of racemic forms by recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In one embodiment, a mixture of one or more isomers is utilized as a therapeutic polypeptide described herein. In another embodiment, the compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis, and / or separation of a mixture of enantiomers and / or diastereomers. Resolution of polypeptides and their isomers can be accomplished by any means, including, but not limited to, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.

[0136] The methods and formulations described herein include the use of N-oxides (where appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and / or pharmaceutically acceptable salts of polypeptides having the structure of any of the polypeptides of the invention, as well as metabolites and active metabolites of these polypeptides having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran or methyl tert-butyl ether), or alcohol (e.g., ethanol) solvates, acetate, and the like. In certain embodiments, the polypeptides described herein exist in solvated form with pharmaceutically acceptable solvents such as water and ethanol. In other embodiments, the compounds described herein exist in unsolvated form.

[0137] In one aspect, the polypeptides of the present invention may exist as tautomers, and all tautomers are included within the scope of the compounds recited herein.

[0138] In one embodiment, the compounds described herein are prepared as prodrugs. A "prodrug" is a drug that is converted into the parent drug in vivo. In one embodiment, upon administration in vivo, the prodrug is chemically converted into a biologically, pharmaceutically, or therapeutically active form of the polypeptide. In another embodiment, the prodrug is enzymatically metabolized by one or more steps or processes to produce a biologically, pharmaceutically, or therapeutically active form of the polypeptide.

[0139] In one embodiment, for example, sites on the aromatic ring portion of the polypeptides of the present invention are susceptible to various metabolic reactions. Incorporation of appropriate substituents into the aromatic ring structure can reduce, minimize, or eliminate this metabolic pathway. In one embodiment, suitable substituents for reducing or eliminating the susceptibility of the aromatic ring to metabolic reactions are, by way of example only, deuterium, halogen, or alkyl groups.

[0140] The compounds described herein also include isotopically labeled compounds in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for inclusion in the polypeptides described herein include: 2 H, 3 H, 11 C. 13 C. 14 C. 36 Cl, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O. 17 O. 18 O. 32 P, 35 S, 111 In, 99m Tec, 68 Ga, 18 F, 64 Cu, 125 I, and / or 131 I. In certain embodiments, isotopically labeled compounds are useful for drug and / or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium confers greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements). In yet another embodiment, 11 C. 18 F, 15 O, and 13 Substitution with positron emitting isotopes, such as N, is useful in Positron Emission Topography (PET) studies to examine substrate receptor occupancy. Isotopically labeled polypeptides are prepared by any suitable method or process in which an appropriate isotopically labeled reagent is substituted for a non-labeled reagent that would otherwise be used.

[0141] In one aspect, the polypeptides described herein are labeled by other means, including but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0142] The present invention further includes pharmaceutical compositions comprising at least one polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0143] In certain embodiments, the pharmaceutical composition further comprises at least one additional agent useful for treating a disease or disorder contemplated herein, hi certain embodiments, the polypeptide of the invention and the additional agent are formulated together in the composition.

[0144] salt The polypeptides described herein may form salts with acids or bases, and such salts are included in the present invention. In certain embodiments, the salts are pharmaceutically acceptable salts. The term "salt" encompasses free acid or base addition salts that are useful in the methods of the present invention. The term "pharmaceutically acceptable salt" refers to a salt that has a toxicity profile within a range that confers usefulness in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which are useful in the practice of the present invention, for example, in the process of synthesizing, purifying, or formulating polypeptides useful in the methods of the present invention.

[0145] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids, examples of which include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acid (hydrogen phosphate and dihydrogen phosphate). Suitable organic acids can be selected from the aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, malonic acid, saccharinic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid.

[0146] Suitable pharmaceutically acceptable base addition salts of the polypeptides of the present invention include, for example, ammonium salts and alkali metal, alkaline earth metal, and transition metal salts, e.g., calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts prepared from basic amines, such as N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. All of these salts can be prepared from the corresponding polypeptide, for example, by reacting the appropriate acid or base with the compound.

[0147] method In one aspect, the present invention includes a method for increasing the proteolytic resistance of a polypeptide to a protease relative to a corresponding unmodified polypeptide. In a specific embodiment, the protease is DPP4, DPP2, and / or another protease having 50% or more homology to DPP4 and / or DPP2.

[0148] In another aspect, the invention includes a method for increasing the serum stability of a polypeptide relative to a corresponding unmodified polypeptide.

[0149] In yet another aspect, the invention includes a method for improving the in vivo half-life of a polypeptide relative to the corresponding non-chemically modified polypeptide.

[0150] In yet another aspect, the invention includes a method for improving the blood-brain barrier permeability of a polypeptide relative to a corresponding unmodified polypeptide.

[0151] In yet another aspect, the invention includes a method for improving the pharmacokinetic properties of a polypeptide relative to the corresponding unmodified polypeptide.

[0152] In certain embodiments, the method comprises (i) chemically modifying at least one substituent selected from the group consisting of the N-terminal amino group, the NH group of the N-terminal first internal amide bond, other free primary amino groups, a thiol group, and a thioether group of the polypeptide, wherein each chemical modification independently comprises derivatizing the substituent with an optionally substituted alkyl, cycloalkyl, alkenyl, or alkynyl group, and (ii) chemically modifying at least one substituent selected from the N-terminal amino group and NH, wherein the chemical modification independently comprises derivatizing the substituent with a group X, wherein X is, in each occurrence, independently →O, —OH, alkoxy, NH, NH(C1-C 16 alkyl), and N(C1-C 16 Alkyl) (C1-C 16alkyl).

[0153] In certain embodiments, the present invention provides C1-C 16 Alkyl, C3-C 16 Cycloalkyl, C2-C 16 Alkenyl, or C2-C 16 The independent existence of alkynyl is contemplated, which is C1-C 16 Alkyl, C3-C 16 Cycloalkyl, C2-C 16 Alkenyl, C2-C 16 Alkynyl (including -C≡CH), heteroaryl, heterocyclyl, C1-C6 alkoxy, azido, diaziryl TIFF2025120243000100.tif10128 (including methyldiaziryl or trifluoromethyldiaziryl), -CHO, 1,3-dioxol-2-yl, halo (such as trifluoromethyl, difluoromethyl, and fluoromethyl), cyano, nitro, triflyl, mesyl, tosyl, haloalkoxy (such as trifluoromethoxy, difluoromethoxy, and fluoromethoxy), heterocyclyl, aryl, heteroaryl, -SR, -S(=O)( and -P(=O)(OR) (wherein R is, independently at each occurrence, H or C1-C6 alkyl). In other embodiments, a contemplated alkyl group is -CHCF in at least one instance.

[0154] In yet another aspect, the present invention includes a method for treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one polypeptide of the present invention, optionally formulated as a pharmaceutically effective composition. In certain embodiments, the polypeptide or composition is administered to the subject by at least one route selected from oral, rectal, mucosal (e.g., by oral or intranasal inhalation), transmucosal, topical (transdermal), and intravenous, intradermal, intramuscular, subcutaneous, intradermal, intrauterine, epidural, and intracerebroventricular injection. In other embodiments, the subject is further administered at least one additional agent useful for treating or preventing the disorder or disease. In still other embodiments, the subject is a mammal. In still other embodiments, the mammal is a human. In still other embodiments, the disease is congenital hyperinsulinism, nonalcoholic steatohepatitis (NASH), short bowel syndrome, Alzheimer's disease, Parkinson's disease, and smoking cessation.

[0155] In another aspect, the present invention provides therapeutic compositions comprising modified forms of GLP-2, alone or in combination with valproic acid and / or CHIR99021, and methods of using such compositions to increase intestinal cell proliferation and / or augment intestinal growth for the treatment of short bowel syndrome, Crohn's disease, intestinal radiation damage, chemotherapy-induced enteritis, or necrotizing enterocolitis.

[0156] CHIR99021 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile: TIFF2025120243000101.tif37128

[0157] In yet another aspect, the present invention includes a method for imaging cells or tissues in a subject. In certain embodiments, the method comprises administering an effective amount of a polypeptide of the present invention to a subject in need thereof, wherein the polypeptide is labeled with a detectable isotope and / or conjugated to a detectable label. In other embodiments, the detectable label comprises a chromophore, a fluorescent group, a bioluminescent group, a chemiluminescent group, or a radioactive group. In yet other embodiments, the polypeptides of the present invention can be used to image insulinoma and / or pancreatic neuroendocrine tumors.

[0158] Formulation / Administration The composition of the present invention may comprise a pharmaceutically acceptable carrier, excipient, and / or diluent, and may be administered to a subject by a suitable method.The composition of the present invention may be formulated into various forms, including oral dosage forms or sterile injectable solutions, according to any conventional method known in the art.In another embodiment, the composition may also be used as an inhalation-type drug delivery system.In yet another embodiment, the composition of the present invention may be formulated for injectable solutions.

[0159] The compositions can be formulated as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, topical preparations, suppositories, and sterile injectable solutions. Suitable formulations known in the art are disclosed, for example, in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA). Carriers, excipients, and diluents that can be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil.

[0160] Tablets may also contain standard ingredients such as binders and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g., swellable cross-linked polymers such as cross-linked carboxymethylcellulose), lubricants (e.g., stearates), preservatives (e.g., parabens), antioxidants (e.g., BHT), buffers (e.g., phosphate or citrate buffers), and effervescent agents such as citrate / bicarbonate mixtures. Such excipients are well known and need not be discussed in detail herein. Capsule formulations may be of the hard or soft gelatin variety and may contain the active ingredient in solid, semi-solid, or liquid form. Gelatin capsules can be formed from animal gelatin or its equivalents of synthetic or vegetable origin. Solid dosage forms (e.g., tablets, capsules, etc.) may be coated or uncoated, but typically have a coating, such as a protective film coating (e.g., wax or varnish) or a release-controlling coating. Coatings (e.g., Eudragit™-type polymers) can be designed to release the active ingredient at a desired location in the gastrointestinal tract. Thus, coatings can be selected to degrade under specific pH conditions in the gastrointestinal tract, thereby selectively releasing the compound in the stomach or the ileum or duodenum. Alternatively, or additionally, coatings can be used as taste-masking agents to mask unpleasant tastes, such as bitter-tasting drugs. Coatings can contain sugars or other agents that help mask unpleasant tastes. Instead of or in addition to a coating, the antibiotic can be provided in a solid matrix containing a release-controlling agent, e.g., a release-retarding agent that can be adapted to selectively release the compound under conditions of varying acidity or alkalinity in the gastrointestinal tract. Alternatively, the matrix material or release-retarding coating can take the form of an erodible polymer (e.g., maleic anhydride polymer) that erodes substantially continuously as the dosage form passes through the gastrointestinal tract. As a further option, the active compound can be formulated in a delivery system that provides osmotic control of the release of the compound. Osmotic and other delayed- or sustained-release formulations can be prepared according to methods well known to those skilled in the art. Pharmaceutical formulations may be provided to patients in "patient packs" in which the entire therapeutic regimen is contained in a single package, usually a blister pack.Patient packs have the advantage over traditional prescription drugs, in which a pharmacist separates a patient's medication supply from a bulk supply, in that patients always have access to the package insert contained in the patient pack, which is not typically present on the patient's prescription. The inclusion of a package insert has been shown to improve patient compliance with physician instructions. Each tablet, capsule, caplet, pill, etc., may be a single dose, e.g., as discussed herein, or the dose may be two or more tablets, capsules, caplets, pills, etc. For example, if a tablet, capsule, etc. is 125 mg and the dose is 250 mg, the patient may take two tablets, capsules, etc., at each interval present for administration.

[0161] The composition of the present invention can be formulated with commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, or surfactants.Solid preparations for oral administration include tablets, pills, powders, granules, or capsules, and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin, in addition to the composition.In addition to simple excipients, lubricants such as magnesium stearate or talc can also be used.Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to the commonly used simple diluents, water and liquid paraffin, various excipients, such as wetting agents, flavorings, fragrances, and preservatives, can be included.

[0162] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents or suspending agents may be used, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate. Suppository bases may be used, such as witepsol, macrogol, Tween 61, cocoa butter, laurin butter, or glycerogelatin.

[0163] The dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the type of drug, and the route and duration of administration, and can be appropriately selected by those skilled in the art. For specific effects, the pharmaceutical composition of the present invention can be administered at a dosage of 0.01 to 100 mg / kg / day. Administration can generally be 1 to 4 times daily, for example, once, twice, three times, or four times daily. The maximum amount administered in a 24-hour period can be up to 1500 mg. Administration can be over a course of 2 to 30 days, for example, 3 to 21 days, such as 7, 10, or 14 days. Those skilled in the art can adjust the dosage depending on the subject's weight and overall health condition and the purpose of administering the antibiotic. Depending on the response obtained, repeated courses of treatment can be performed.

[0164] The compositions of the present invention can be administered to a subject by a variety of routes. All modes of administration are contemplated, e.g., oral, rectal, mucosal (e.g., by oral or intranasal inhalation), transmucosal, topical (transdermal), or by intravenous, intradermal, intramuscular, subcutaneous, intradermal, intrauterine, epidural, or intracerebroventricular injection.

[0165] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0166] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0167] It will be understood that whatever values and ranges are provided herein, all values and ranges encompassed by those values and ranges are intended to be within the scope of the present invention. Furthermore, all values within those ranges and upper or lower limits of value ranges are also contemplated by this application.

[0168] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the present invention and covered by the claims appended hereto. For example, it should be understood that variations of reaction conditions, including but not limited to reaction time, reaction size / volume, and solvents, experimental reagents such as catalysts, pressure, atmospheric conditions, e.g., nitrogen atmosphere, and reducing / oxidizing agents, using art-recognized choices and no more than routine experimentation, are within the scope of this application.

[0169] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the present invention and are not intended to limit the scope of what the inventors regard to be their invention. [Example]

[0170] The present invention will now be described with reference to the following examples, which are given for illustrative purposes only, and the present invention is not limited to these examples, but rather encompasses all variations that become apparent as a result of the teachings provided herein.

[0171] Unless otherwise noted, starting materials for the syntheses described herein were obtained from commercial sources or known synthetic procedures and used without further purification.

[0172] animal: We will investigate the acute effects of GLP-1 analogs on improving glucose metabolism using 20-24 week-old C57blk6J wild-type male mice. Mice will be fed a chow diet and housed individually for the duration of the study (up to 8 weeks). Mice will be treated with GLP-1 analogs and subjected to a glucose tolerance test to evaluate treatment outcomes.

[0173] Ten mice are used per experimental condition and endpoint to detect statistically significant differences in the parameters to be measured. Male mice are housed individually to avoid handling and fighting during the study. Two cohorts of mice are used: a first cohort of 40 mice (four experimental groups of 10 mice each) to determine the dosage and time interval for the action of the reference product liraglutide, and a second cohort of 50 mice (five experimental groups of 10 mice each) to determine the properties of new GLP-1 analogs.

[0174] A total of 130 mice will be used for the GLP2 study. Five-week-old male C57BL / 6J mice will be acclimated for one week prior to the experiment. Animals will be administered a GLP-2 analog (experimental group) or Gatex® (control group). Four doses of each compound will be tested. Mice will be euthanized at two time points (6 or 10 days) after the start of injections, and intestinal segments will be collected for histomorphometric analysis. Additional groups of animals (one group per compound) will undergo PET / CT imaging at various time points after the start of injections. Animals will be housed and maintained in a ventilated facility with a constant ambient temperature of 19-22°C and humidity of 40-60% on a 12-h light-dark cycle (lights on at 7:00).

[0175] Activity and Stability Assays: Cellular assays of receptor binding and intracellular signaling were performed with the compounds of the present invention. Briefly, HEK293 cells were cultured in a 200-well plate containing (i) GLP-1R (or an empty expression vector), (ii) CRE 6xThe cells were transiently transfected with cDNA encoding the -LUC reporter gene (to detect cAMP production induced by receptor stimulation) and (iii) β-galactosidase (as a control for transfection efficiency). After 24 h of transfection, the cells were incubated with various concentrations of peptide (or control / blank) in serum-free medium for 6 h. Cells were then lysed, SteadyLite reagent (PerkinElmer) was added, and luciferase activity was measured using a TopCount NXT HTS plate luminometer. After further incubation with a chromogenic substrate (2-nitrophenyl β-D-galactopyranoside), the optical density at 420 nm was measured spectrophotometrically as a control to correct for well-to-well transfection variability. Potency was determined by calculating a sigmoidal concentration-response curve of GLP-1R ligand activity.

[0176] Plotting concentration versus luciferase activity yielded a quantitative parameter that combined binding (potency) and signaling (efficacy) in a single step. Cells were stimulated with DPP4-treated and control peptides for 4 hours. DPP4-treated peptides: 10 μl of 0.26 mM peptide was incubated with DPP4 in 2 μL of buffer ± DMSO for 18 hours. Control peptide: 10 μl of 0.26 mM peptide was added to buffer ± DMSO. The final DMSO concentration was the same for all peptides, and all peptides were diluted to a 0.26 mM concentration for the DPP4 assay.

[0177] Detection of peptide inactivation by DPP4: Compounds are incubated overnight at 37°C in the presence of recombinant DPP4 or vehicle (Tris buffer). Samples are then evaluated in a GLP-1R agonist assay. Because DPP4-induced N-terminal cleavage of the peptide results in virtually complete loss of function, a decrease in intact peptide is reflected in a corresponding decrease in potency (DPP4 vs. vehicle treatment; e.g., a 10-fold decrease in potency indicates 90% degradation). An example of this analysis is shown in Figure 8. A complementary evaluation is performed by ESI LC-MS (as in Figure 6) to detect potential minor degrees of DPP4-mediated hydrolysis that would be difficult to detect by comparing potencies in functional assays.

[0178] Example 1: Modified Glucagon-Like Peptide 1 (GLP-1) As demonstrated herein, the present invention provides methods for ameliorating, preventing, or minimizing the problem of proteolytic instability ubiquitous in peptides. Such methods involve the preparation of novel chemically modified polypeptides.

[0179] In certain embodiments, the modification comprises covalently attaching at least one 2,2,2-trifluoroethyl group to the N-terminal amino group, other free amino groups, thiol groups, and / or thioether groups in the polypeptide. Such modifications can be achieved using a variety of synthetic groups. In a non-limiting example, this chemical derivatization can be carried out using a rapid and efficient fluoroalkylation reaction, as demonstrated with two analogs of glucagon-like peptide 1 (GLP-1).

[0180] GLP-1 synthesized by solid-phase peptide synthesis was derivatized while resin-bound to yield the desired trifluoroethyl analog with predictable ease and speed comparable to the well-established acetylation of peptides. In addition to GLP-1 itself, an N-acetyl analog of GLP-1 was prepared as a control. A bis-trifluoroethyl analog of GLP-1 was also obtained. HPLC traces of the native and modified GLP-1 variants are shown in Figure 1. The chemistry involved in this modification is depicted in Figure 2.

[0181] Two exemplary chemically modified peptides were synthesized along with the corresponding control polypeptide using a single automated synthesis. The sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (GLP-1 7-36 amide) was prepared on an automated synthesizer using solid-phase Fmoc chemistry. The solid support was MBHA-Rink amide resin, loaded at 0.35 mmol / gram. 0.1 meq. of resin (280 mg) yielded 629 mg of resin containing crude GLP-1.

[0182] Three 100 mg portions of derivatized resin, each with a theoretical 0.016 mmol of GLP-1, were treated as follows.

[0183] Portion 1: Deprotection with 20% piperidine in dimethylformamide (DMF); washing with DMF; washing with dichloromethane (DCM); cleavage with trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water (95 / 2.5 / 2.5); evaporation to a small volume; and crystallization in ether gave 26 mg of crude GLP-1 after lyophilization.

[0184] Part 2: Deprotection with 20% piperidine in DMF; washing with DMF; treatment with acetic anhydride (1.5 mL) and diisopropylethylamine (DIEA, 0.15 mL) in DMF (10 mL); washing with DMF; washing with DCM; cleavage with TFA / TIPS / water 95 / 2.5 / 2.5; evaporation to a small volume; and crystallization in ether gave 21 mg of crude N-acetyl-GLP-1 after lyophilization.

[0185] Portion 3: Deprotection with 20% piperidine in DMF; washing with DMF; washing with DCM; addition of phenyl(trifluoroethyl)iodonium triflimide 1a (9 mg, 0.016 mmol, in 2 mL of DCM) for 3 min; then addition of collidine (10 mg, 0.08 mmol, in 4 mL of DCM) for 5 min; then washing with DMF; washing with DCM; cleavage with TFA / TIPS / water 95 / 2.5 / 2.5; evaporation to a small volume; and crystallization in ether gave 30 mg of crude N-trifluoroethyl-GLP-1 after lyophilization. This crude material contained 2,2,2-trifluoroethyl-GLP-1 (Figure 4) as the major component and (bis-2,2,2-trifluoroethyl)-GLP-1 as a minor component (by ESI-MS).

[0186] Each crude material was purified by HPLC. Analytical HPLC showed greater than 96% purity. Retention times (Vydac 218TP104 C-18 column, 35-55% solvent B in solvent A over 20 min) were 9.6 min (GLP-1), 10.9 min (N-acetyl-GLP-1), 11.6 min (N-(2,2,2-trifluoroethyl)-GLP-1), and 13.4 min ((Nα,Nπ-bis-2,2,2-trifluoroethyl)-GLP-1). ESI-MS data were consistent with pure compounds. Solvent A = 99% HO, 1% CH3CN, 0.1% TFA; Solvent B = 90% CH3CN, 10% HO, 0.07% TFA.

[0187] Example 2: Sensitivity to DPP4 Compounds were incubated overnight at 37°C in the presence of recombinant DPP4 or vehicle (Tris buffer, pH 8.0). Samples were then evaluated in the GLP1R agonist assay, as described elsewhere herein. Because DPP4-induced N-terminal cleavage of peptides results in virtually complete loss of function, a decrease in intact peptide is reflected in a corresponding decrease in potency (DPP4 vs. vehicle treatment; e.g., a 10-fold decrease in potency indicates 90% degradation). A non-limiting example is shown in Figure 3. Complementary evaluation can be performed by ESI LC-MS to detect potential minor degrees of DPP4-mediated hydrolysis, which may be difficult to detect by comparing potencies in functional assays.

[0188] Table 2. Agonist potency and susceptibility to DPP4-mediated hydrolysis TIFF2025120243000102.tif152154TIFF2025120243000103.tif212154GLP-1 corresponds to GLP-1(7-36); GIP corresponds to GIP(1-42). nd = not determined.

[0189] Non-limiting constructs contemplated herein are shown in Scheme 1 (FIG. 6), where X represents a chemical modification contemplated herein: TIFF2025120243000104.tif42155GLP-1(7-36)amide: X=H; GIP(1-42) acid: X = H; Dual agonist: X=H, Lys40 is derivatized with acyl C16, and Ala2 and / or Ala20 are independently and optionally Aib; Liraglutide: GLP(7-37) acid, where residue 34 = Arg, residue 37 = Glycine, and Lys26 is acylated with γ-Glu-C16 acid.

[0190] Assessing the role of size and other molecular properties of residue modifications: Constructs are tested with respect to various attributes such as size, hydrophobicity, charge, polarizability, stereochemistry, and electronegativity to establish molecular parameters that best complement the receptor binding site (which can be plastic depending on the ligand). Predicted pKa of accepted appendages in GLP-1 a Although the values indicate that a positive charge is not essential, the natural ligand is partially charged at physiological pH. The electronic factors are evaluated by varying the electron-withdrawing and electron-donating properties of the substituent at the 4-position of the aryl ring of the N-benzyl derivative, assuming that the attached group is accommodated in the binding pocket.

[0191] Photoaffinity probes to identify sites of residue interaction with the receptor: The N-terminal regions of GLP-1 and related peptides, which directly interact with their cognate receptors, have been excluded from structural characterization using either crystallization or cross-linking approaches. A limitation of the latter is the lack of suitable photoaffinity probes with His7 modifications compatible with GLP-1 binding to its receptor. To generate such tools, we exploited modest structural perturbations of previously characterized ligands that exhibit potency and efficacy similar to the native ligand. To this end, we prepared a para-trifluoromethyldiazirine benzyl derivative. This compound retains full agonist potency and is 123-fold less potent than GLP-1, likely due to steric bulk at the 4-position of the aryl group. Further preparation and testing of para-azidobenzyl and methyldiazirine ethyl derivatives was undertaken. The photoactive groups diazirine and phenyl azide were chosen for their spectroscopic properties and their ability to rapidly insert reactive carbenes or nitrenes into closely spaced C–H bonds upon generation. In certain embodiments, the polypeptide construct further comprises another affinity tag (e.g., biotin) attached to Lys26, a site well-tolerated for conjugation (e.g., lipidation in the case of liraglutide). After incubation with isolated cell membranes containing GLP1R and photocrosslinking, the receptor-ligand complex is affinity purified and subsequently digested with a protease. The site of labeling is then identified by mass spectrometry, which can be performed either on a gel by MALDI MS or LC ESI-MS.

[0192] The data presented herein demonstrate that a variety of modifications can be tolerated at the N-terminus of ligands without impairing receptor activation. Together, these changes confer complete protection against DPP4-mediated hydrolysis. Overall, these findings open new opportunities to elucidate the structural determinants of receptor activation that can be applied to other class B GPCRs.

[0193] Example 3: DPP4-resistant analogs Resistant analogs are shown in Figures 6-10, 12, 13, 14, 18, 19, and 21. A palmitoylated form of GLP-1, a DPP4-insensitive analog of liraglutide, is generated by derivatization of the His7 residue. Palmitoylation promotes peptide multimerization and increases reversible serum albumin binding as a mechanism for partially protecting liraglutide. The potential synergistic effect of N-alkylation and lipidation of His7 in liraglutide is investigated. Another lipid-mediated His7 modification in a GLP-1 analog (compound "A6"), which may also increase albumin binding, is also explored.

[0194] In certain embodiments, currently contemplated modifications of the terminal His7 in GLP-1 and analogs selectively abolish cleavage by DPP4 without impairing agonist activity. In other embodiments, currently contemplated modifications of the terminal His7 in GLP-1 and analogs essentially maintain the native non-helical N-terminal conformation of GLP-1 and its interaction with the receptor, which is important for affinity and agonist function. In yet other embodiments, currently contemplated modifications of the terminal His7 in GLP-1 and analogs allow for exploration of the details of the GLP-1R ligand-binding pocket versus recognition by DPP4, and also allow for fine-tuning of polypeptides to facilitate access to different biological compartments (e.g., mucosal uptake after oral or intranasal application; a future direction potentially optimizing drug-induced weight loss by crossing the blood-brain barrier).

[0195] As shown herein, liraglutide is indeed significantly inactivated by DPP4. The assessment of active drug levels in serum by an improved, sensitive bioassay represents a significant technological advance over conventional peptide-specific RIA / ELISA (typically used in industry, e.g., during drug discovery for liraglutide and exenatide). While conventional sandwich immunoassays rely on specific antibodies simultaneously detecting the intact N- and C-termini of a peptide, this approach is universally applicable to novel potent agonists, regardless of structural modifications.

[0196] In vitro testing As part of a structure-function analysis with native GLP-1, seven analogs with different chemical attributes are created. A His7 modification is introduced into either liraglutide or an analog with an alternative lipid moiety. Cell-based assays are used to assess the potency and efficacy of these peptides at the GLP-1R. Parallel studies are used to quantify inactivation after prolonged exposure to DPP4 in vitro. Direct comparison with liraglutide is made. Potent / stable analogs can be selected for in vivo testing.

[0197] In vitro experiments confirmed that unmodified GLP-1 is highly susceptible to DPP4 degradation. After overnight incubation with the enzyme, apparent potency was reduced by more than 500-fold, indicating that more than 99.8% of the peptide was degraded / inactivated. Liraglutide, a lipidated GLP-1 analog used as an antidiabetic, has been reported to be more DPP4-resistant, but is still degraded in vivo by this enzyme. Indeed, liraglutide was shown to undergo a more than 50-fold decrease in potency after incubation with DPP4, indicating that more than 98% of the peptide was degraded.

[0198] The N-terminal His7 of GLP-1 plays a key role in GLP-1R activation while simultaneously enabling peptide recognition and degradation by DPP4. Modification at His7 has been considered impractical due to the stabilization of GLP-1. Previous attempts to modify His7 by substitution or, for example, acetylation, confer DPP4 resistance but also result in a significant reduction in agonist potency (Figures 8, 9, and 9a). The functional assays demonstrated that this analog possesses stability against DPP4, combined with an approximately 50-fold reduction in potency compared to GLP-1b.

[0199] However, as demonstrated herein, the present modifications selectively increase the stability of GLP-1 without perturbing its agonist function. The trifluoroethyl, isobutyl, or benzyl analogs conferred virtually complete DPP4 resistance while producing insignificant, barely detectable changes in potency.

[0200] Furthermore, trifluoroethylation did not affect liraglutide efficacy. Furthermore, this analog exhibited complete resistance to DPP4 degradation. The remaining analogs contemplated in the present invention can be evaluated for agonist activity and enzyme stability using similar assays. Such analogs include the liraglutide analog "A6," which contains an alternative lipid moiety at the R2 position (lipid 12), which confers significantly higher albumin binding. A6 is cleared as rapidly as liraglutide, likely due to degradation of the remaining A6 by DPP4.

[0201] The peptide is assembled by standard automated peptide synthesis using Fmoc chemistry. Lys26 is side-chain protected with an allyloxycarbonyl (alloc) group that can be selectively removed using PhSiH3 and Pd(PPh3)4, followed by R-protection using conventional chemistry. 2 The R 1 N-terminal modifications at position 1 are introduced by reductive amination on solid phase using the corresponding aldehyde and NaBH4. For trifluoroethyl-containing compounds (GLP-1c, MG1, and MG4; Figure 7), the histidine derivatives are synthesized independently. The constructs are cleaved from the resin using CF3CO2H:TIPS:HO (95:2.5:2.5), purified using reverse-phase HPLC, and analyzed using ESI and MALDI-MS.

[0202] In vivo testing In one aspect, the attenuation of glycemic excursions by GLP-1 analogs was analyzed (Figure 22A). Figure 21 provides a series of graphs showing that native GLP-2 is degraded by DPP4, while a trifluoroethyl-decorated analog (GLP2-4) is completely resistant to degradation. Peptides were incubated with recombinant DPP4. Serial dilutions of GLP-2 and GLP2-4 were then applied to HEK293 cells transfected with cDNA encoding the human GLP-2R and a cAMP-responsive reporter gene. DPP4 induced a greater than 40-fold decrease in GLP-2 potency, reflecting that more than 97% of the peptide was degraded. In contrast, no potency loss was observed with GLP2-4. N=4, mean + SEM.

[0203] Figure 22A provides a graph comparing the time-dependent decrease in drug activity in plasma after a bolus injection of either liraglutide or its CHCF3-decorated derivative, Lira-4. Rats received a bolus injection of either drug via a central catheter, followed by serial blood sampling at indicated intervals and measurement of drug activity in plasma by agonism bioassay. The potency of each peptide immediately after injection was defined as 100%. Liraglutide's plasma persistence is prolonged by the CHCF3 modification of Lira-4.

[0204] Figure 22B shows the sustained hypoglycemic activity of Lira-4 after an oral glucose tolerance test. Mice received subcutaneous injections of either vehicle, G-4, liraglutide, or Lira-4 (bars are displayed from left to right within each group). 30 minutes and 5.5 hours later, two consecutive oral glucose loads were administered by oral gavage. Blood glucose levels were measured immediately before drug injection (-30 minutes) and at the indicated time intervals after the glucose load. A single injection of compound G-4 attenuated blood glucose excursions after the first glucose load but remained active several hours later, attenuating the second glucose challenge.

[0205] Example 4: Modified forms of GLP-2 Increased DPP4 resistance can be achieved using simple chemical modifications of the amino terminus of GLP-2. Furthermore, certain sites in GLP-2 tolerate amino acid substitutions (Leu17 to Lys) and palmitoylation without impairing activity. These modifications resulted in the compound GLP-2 L17K[L1-4] shown in Figure 6B, which contains an Asp3→Glu substitution compared to GLP-2 (Figure 6A). Lipid addition generally increases binding to serum albumin. In certain embodiments, the GLP-2 analogs of the present invention have improved protease resistance, reduced renal clearance, and improved pharmacokinetics compared to GLP-2 (Figure 18). In other embodiments, the GLP-2 analogs of the present invention can be used to treat diseases such as mucositis, radiation-induced intestinal damage, and Crohn's disease.

[0206] As presented herein, novel lipidated stable GLP-2 analogs are prepared by sequentially introducing different lipid anchors into stable GLP-2, each replacing palmitic acid in the prototype compound, GLP-2 L17K[L1-4]. The corresponding ligands are compared with F-GLP2-palmGLP-2 L17K[L1-4] and Gatex® using existing in vitro cell-based assays. Potency, efficacy, protease resistance, albumin binding, and oligomerization are evaluated and used to select compounds for in vivo testing. The intestinal growth-promoting effects of GLP-2 analogs are evaluated by subcutaneously administering lipidated stable GLP-2 analogs and Gatex® to mice. The efficacy of the peptides is assessed using histomorphometric analysis, including quantification of intestinal weight, villus height, and crypt depth. Additionally, proliferation index (Ki67 staining) and glucose uptake (PET scanning) are measured.

[0207] F-GLP2-Palm is a novel, high-potency (EC 50= 44 ± 13 pM (mean ± SEM, n = 4), a protease (DPP4)-resistant lipidated GLP-2 peptide analog. A Leu to Lys substitution was made at position 17 (designated "K*") to facilitate conjugation of a lipid tail (palmitic acid, "C16") with a β-alanine spacer. DPP4 resistance was conferred by the introduction of a trifluoromethyl-modified N-terminal histidine.

[0208] Example 5: Modification of the N-terminus of GLP-1 The following experiments address a continuing challenge in designing therapeutics based on agonism at the GLP-1R: combining resistance to DPP4 with retention of potency and efficacy during receptor activation (see Table 3). The data demonstrate that the GLP-1 / GLP-1R system tolerates a wide range of derivatives that exhibit both desirable properties. First, we explore the structural space available for creating GLP-1 analogs. A two-pronged approach is utilized to narrow the potentially large landscape of potential candidates (see below). Chemical decoration of the α-amine of His7, located at the N-terminus of GLP-1, spans a variety of functional groups and sizes. From the known SAR in the literature and the data presented herein, several requirements are known: (i) the presence of a methylimidazole side chain at His7 is important; (ii) N-acyl (including N-acetyl) derivatives reduce potency and efficacy; and (iii) large polar adducts on the nitrogen are not tolerated (e.g., N-mannitol or N-glucitol). Nevertheless, the data indicate that a variety of modifications, including charged groups, are tolerated at physiological pH. Here, a practical model envisions a binding pocket on the receptor close to the membrane-water interface that accommodates chemical changes made to histidines. The exact dimensions and precise functional makeup of this pocket remain to be defined, but it is estimated to be approximately 240 Å. 3A GLP-1R binding box with a volume of 100 or slightly larger can be hypothesized (based on the activity of the N-methyladamantyl derivative G-10). To test this hypothesis and explore the nature of putative receptor-ligand interactions within this binding box, a library of 50 GLP-1 derivatives will be prepared, exploring the criteria of size, hydrophobicity, charge, polarity, stereochemistry (G17-21), and polarizability. Exemplary GLP-1 derivatives are shown in Figure 6A.

[0209] Example 6: Chemical synthesis of the conjugate Peptides are assembled by standard automated peptide synthesis using Fmoc chemistry. Lys26 in GLP-1 is side-chain protected with an allyloxycarbonyl (alloc) group, which can be selectively removed using PhSiH3 and Pd(PPh3)4, and then optionally conjugated to lipids using established chemistry. N-terminal modifications ("X", Figures 6A and 6B) are introduced by reductive amination on solid phase using the corresponding aldehyde and NaBH4 (89). For compounds containing fluoroalkyl (X = 4, 8, 9) and azide (X = 29, 32) functional groups, histidine derivatives are synthesized independently using previously reported procedures (90-95). Constructs are cleaved from the resin using CF3CO2H:TIPS:HO (95:2.5:2.5), purified using reverse-phase HPLC, and analyzed using ESI and MALDI-MS.

[0210] Example 7: Photoaffinity probe to identify the site of His7 interaction in GLP-1 with the receptor The N-terminal regions of GLP-1 and related peptides, which directly interact with their cognate receptors, have been excluded from structural characterization using crystallization or cross-linking approaches. A significant limitation of the latter is the lack of suitable photoaffinity probes with His7 modifications compatible with GLP-1 binding to its receptor. To generate such tools, structural perturbations are performed on ligands that exhibit potency and efficacy similar to the native ligand. Compound G-22 exhibited significantly reduced potency compared to unmodified GLP-1, likely due to steric bulk at the 4-position of the aryl group (Figure 6A), while maintaining full potency. Agonist potency was preserved in photoprobe G-30, suggesting that the latter recapitulates the interaction of unmodified GLP-1 with its receptor. To facilitate detection in cross-linking studies, a biotin moiety can be further attached to residue K26 in GLP-1. Data indicate that this addition does not affect GLP-1 affinity, consistent with the fact that the same residue is also used for acylation of liraglutide (Figure 6A).

[0211] Example 8: Estimating the role of size and other molecular properties of His7 To establish molecular parameters that best complement the receptor binding site (which is likely to be plastic in response to ligand), 50 constructs are tested with respect to various attributes, including size (G-23, G-24), hydrophobicity, charge (G-17, G-18, G-23, polarizability (G-4, G-5, G-12, G-11, G-27)), stereochemistry (G-17, G-18, G-20, G-21), and electronegativity (G-4, G-8, G-9). The predicted pK of adducts tolerated in GLP-1 are shown. a The natural ligand is partially charged at physiological pH, although the values indicate that a positive charge is not essential. Assuming that the "X" group is accommodated in the binding pocket, the electron-withdrawing and electron-donating nature of the substituents at the 4-position of the aryl ring of the N-benzyl derivatives is varied (a total of eight compounds ranging from the most electron-withdrawing -NO2 (Hammett σ = 0.81) to the most electron-donating -N(CH3)2 (σ = -0.83)). log(EC 50,X / EC 50,H ) vs. σ XThe plot is used to determine the slope ρ (a measure of the sensitivity of the equilibrium to substituents relative to the ionization of benzoic acid) as the thermodynamic parameter to be evaluated. a is estimated to be 6.91 and can be varied in either direction depending on the identity of the X adduct.

[0212] (Table 3 and Figure 8) Agonist potency and susceptibility to DPP4-mediated hydrolysis TIFF2025120243000105.tif98128

[0213] Example 9: Effect of alkylation on glucagon stability and function The N-terminus of glucagon (His-Ser) differs from both GLP-1 (His-Ala) and GIP (Tyr-Ala). Although the serine residue at the penultimate position of glucagon reduces the DPP4 sensitivity of this peptide relative to either incretin, glucagon is still degraded by DPP4 and would benefit from N-terminal protection, extending its half-life. Experimental data show that the attachment of a trifluoroethyl group confers complete DPP4 resistance to glucagon without compromising agonist potency (Figure 13 and Table 3). The glucagon receptor was recently crystallized (at limited resolution, including only a portion of the molecule), but it remains unclear how the N-terminus of glucagon fits into its cognate GPCR. The experimental design outlined for GIP will determine which of the 30 selected N-terminal decorations are tolerated by the glucagon receptor, allowing for an initial profile comparison of the agonist pockets of all three glucagon family peptides to be tested.

[0214] Example 10: A novel strategy allows for the generation of GLP-2 and related peptides that are refractory to DPP4 degradation without compromising biological activity. The data showed that the traditional modification, Ala2Gly substitution (as applied to GLP-2), confers only partial DPP4 resistance (Table 4). Identifying an N-terminal modification that confers complete DPP4 resistance while maintaining receptor potency has been a significant challenge. To address this limitation, a broadly applicable strategy was developed in which the amino-terminal residue of the corresponding peptide can be decorated with members of a large library of candidate moieties to abolish DPP4 susceptibility. Providing proof of concept, the data showed that approximately 97% of native GLP-2 is degraded by DPP4, whereas decoration of His1 with fluoroethyl confers complete enzyme resistance (Figure 21). In addition to conferring DPP4 resistance, fluoroethyl and alternative decorations at the N-terminus of GLP-2 analogs offer novel means to optimize stability and in vivo efficacy.

[0215] Figure 21 shows data demonstrating that native GLP-2 is degraded by DPP4, while trifluoroethyl-decorated analogs are resistant to degradation. Peptides were incubated with recombinant DPP4. Serial dilutions of GLP-2 and GLP-2-4 were then applied to HEK293 cells transfected with cDNA encoding the human GLP-2R and a cAMP-responsive reporter gene. DPP4 induced a greater than 40-fold decrease in GLP-2 potency, reflecting that more than 97% of the peptide was degraded. In contrast, no potency loss was observed with GLP-2-4 (Figure 6A). N=4, mean + SEM.

[0216] Example 11: Attachment of lipid side chains to GLP-2 in combination with N-terminal stabilization prolongs biological activity. In addition to the N-terminal trifluoroethyl decoration, GLP2-L17K[L1]-4 (Figure 6A) also incorporates a lysine-linked lipid moiety as a strategy to increase albumin binding, reduce renal clearance, and further prolong peptide retention in plasma. Data demonstrated that GLP2-L17K[L1]-4 is refractory to DPP4 degradation in vitro by bioassay. The structure of GLP2-L17K[L1]-4 is shown in Figure 6A. To form GLP2-L17K[L1]-4, GLP-2 was modified with N-terminal trifluoroethyl decoration, Leu17Lys substitution, and attachment of a C16 palmitate ester via a β-alanine linker.

[0217] Furthermore, the data demonstrated the prolonged bioactivity of GLP2-L17K[L1]-4 (against Gatex). GLP2-L17K[L1]-4 remained at high levels in the circulation 24 hours after subcutaneous (sc) injection in mice. The prolonged bioactivity of GLP2-L17K[L1]-4 against Gatex in vivo is shown in Figure 23. The compound (1.25 μg) was subcutaneously injected into mice. Plasma was collected 24 hours later and analyzed for GLP-2R agonist activity by luciferase reporter gene assay.

[0218] Example 12: Identification of two sites at which lipids conjugated to Lys can be introduced into GLP-2. In GLP2-L17K[L1]-4, amino acid position 17 was identified as a site where lysine could be introduced (i.e., Leu17Lys) without disrupting potency or efficacy, providing a lipid conjugation site for enhanced pharmacokinetic properties as summarized above. An alternative site was identified at position 24, where lysine substitution (Asn24Lys) and lipidation were well tolerated (Figure 20). Because the biophysical and pharmacological properties of GLP-2 analogs are modified by both N-terminal decoration and lipidation, identifying position 24 provides a means to generate additional GLP2-L17K[L1]-4 derivatives that may exhibit further optimized in vivo efficacy. The compounds shown in Figure 6A were characterized in vitro by luciferase reporter gene assays and DPP4-induced potency shifts, as shown in Figure 20 (n = 3).

[0219] Example 13: In vivo studies showing that GLP2-L17K[L1]-4 increases intestinal and mucosal growth. After five daily subcutaneous (sc) injections in C57BL / 6J mice, the prototype compound GLP2-L17K[L1]-4 dose-dependently increased intestinal weight (Figures 23A-23C). GLP2-L17K[L1]-4 increases intestinal weight. C57BL / 6J mice were subcutaneously injected once daily for 5 days with either vehicle, GLP2-L17K[L1]-4, or Gatex at the indicated doses. Animals were sacrificed on day 6, and intestinal weights were subsequently measured. N = 6 animals / group, mean ± SEM. *p < 0.05 vs. vehicle. Furthermore, GLP2-L17K[L1]-4 has a trophic effect on the intestinal mucosa, which is a predictor of efficacy in treating SBS (Figures 23B and 23C). C57BL / 6J mice were subcutaneously injected once daily for 5 days with either 25 μg of GLP2-L17K[L1]-4 or vehicle. Animals were sacrificed on day 6. Figure 23B shows hematoxylin and eosin staining of mucosal sections. Figure 23C shows quantification of villus height. N=6 animals / group, mean±SEM. **p<0.01 vs. vehicle.

[0220] Example 14: Comparison of GLP-2 analogue pharmacological properties. The pharmacological properties of synthetic GLP-2 analogs are characterized using in vitro assays with transfected HEK293 cells expressing GLP-2R. The methodology parallels that used to characterize GLP2-L17K[L1]-4 (assayed in parallel with Gαs as the benchmark). The following are examined: (i) Gas-mediated signaling to determine potency and efficacy, and (ii) DPP4 resistance.

[0221] HEK293 cells are transiently transfected with cDNA encoding GLP-2R, a CRE luciferase reporter gene, and a β-galactosidase construct (allowing for normalization of transfection efficiency). The efficacy and potency of each peptide are determined as shown in Figure 36A. DPP4 resistance is tested after overnight incubation (16 hours) in the presence or absence of DPP4, and receptor-mediated function is assessed as shown in Figure 21A. Parallel experiments are performed using GLP-2 as a DPP4-sensitive control.

[0222] Example 15: Comparison of GLP-2 analogue plasma stability in mice. In addition to DPP4 resistance, many other parameters affect the pharmacokinetics and bioactivity of compounds in vivo, including digestion by other proteases, albumin binding / oligomerization, and renal filtration. The gradual decline in plasma agonist activity after subcutaneous (sc) injection in mice was tracked, mimicking the clinical route of Gatex administration and anticipating that the same administration mode would be used for second-generation drugs. As shown in Figure 23, 12.5 μg of Gatex, GLP2-L17K[L1]-4 (control), or each of the seven proposed new derivatives was subcutaneously injected into 6-week-old male C57 / BL6 mice (3 animals / compound / time point). Mice were sacrificed after 24 or 72 hours, and plasma samples were collected. Bioactivity was measured using routine methods known in the art and described herein.

[0223] Example 16: GLP-2 Analogue Analysis Figures 23A-C show that GLP2-L17K(l1)-4 (alternatively referred to as oTTx-88 in these figures) induces intestinal mucosal growth. C57BL / 6J mice were injected subcutaneously once daily for 5 days with either vehicle, GLP2-L17K(l1)-4, or Gatex at the indicated doses. Animals were sacrificed on day 6, and subsequent analysis of intestinal tissue was performed.

[0224] Figure 23A shows that GLP2-L17K(l1)-4(oTTx-88) increases intestinal weight. N=6 animals / group, mean±SEM. *p<0.05 vs. vehicle.

[0225] FIG. 23B provides images of histology performed by hematoxylin and eosin staining of mucosal sections.

[0226] Example 17: GHRH Analogues (Figure 6A) Figure 19 provides a series of graphs comparing the susceptibility of natural GHRH to CHCF3-GHRH ("C2-GHRH", bottom row, compound GH29-4 in Figure 6) to enzymatic degradation. The activity of these peptides was measured by luciferase assay in cells expressing the GHRH receptor (GHRHR), tested as fresh stocks or after overnight (O / N) incubation with or without DPP4. In contrast to natural GHRH, GH29-4 derivatives are resistant to enzyme-induced loss of potency (no shift to the right in the concentration-response curve). GHRH analogues are useful in treating growth failure due to growth hormone deficiency (GHD), growth failure in girls due to gonadal dysgenesis (Turner syndrome), growth retardation in prepubertal children due to chronic kidney disease, and growth failure in small-for-gestational age (SGA) children whose birth weight and / or height was less than -2 SD and who have not shown any rebound growth after age 4 (HV SDS < 0 within the last year) (current height SDS < -2.5 and parental adjusted height SDS < -1).

[0227] Example 18: Helical wheel diagram of GLP2 shows the spatial identity and orientation of amino acid residues suitable for lipidation. To determine potential sites for lipid addition, alanine mutagenesis data was analyzed along with predicted GLP-2 and GLP-2R interaction data. From the NMR data, GLP-2 appears to have an α-helical secondary structure between residues Phe6 and Ile27 in aqueous systems and between residues Phe6 and Arg24 in micellar systems. At the C-terminus, there is a loose helical segment, while the N-terminus to Ser6 is not fully organized. 35 Because the GLP-2 structure is largely α-helical, a helical wheel diagram was constructed to visualize the positions and nature of the residues (Figure 20). The amino acid sequence is plotted around the helical axis, with each amino acid at a 100° angle from the previous one, due to the secondary structure having 3.6 amino acids per helical turn. The nature of each residue is indicated by color, allowing confirmation of the pattern.

[0228] By utilizing the known GLP-1R extracellular domain surface interactions with GLP-1 and comparing the helical wheel diagrams of GLP-1 and GLP-2, we can estimate the extracellular domain surface of the GLP-2R. This correlates with NMR observations that the GLP-2 binding interface occurs between Leu17 and Lys30 on the hydrophobic face of the helix. 35 Hydrogen bonds may form between Asp21 and Lys30 of GLP-2 and the ECD of GLP-2R residues Thr72, Asp101, and Asp151. 35 Furthermore, exposure of Leu17, Ala18, Ala19, Phe22, Ile23, Trp25, Il27, and Thr29 may enable hydrophobic interactions between GLP-2 and the GLP-2R. 35 Mutagenesis of Leu17 to Ala showed no reduction in GLP-2R binding but reduced GLP-2R activation. 45 However, acylation at this site after mutation to lysine was found to maintain potency. 45,167 Notably, GLP-2 with a Leu17Lys modification, a β-alanine linker, and palmitic acid attached to the ε-amine had potency similar to that of native GLP-2 as measured by luciferase assay. 167 Therefore, the first site for acylation of GLP-2 was performed at residue 17 after the L17K modification. Because Arg24 resides on the helical surface away from the ECD of the GLP-2R and does not significantly affect GLP-2 binding or potency, Arg24 was also a target for acylation after modification to lysine.

[0229] Various analogs of GLP-2 can also be produced by acylation at the 17th position with different linkers and lipids. The hydrophobicity of GLP-2 limits its solubility in water, which is not improved by the β-alanine linker in the original construct. To improve solubility in the future, two oligoethylene glycol (OEG) linkers and γ-glutamic acid could be employed. Studies with semaglutide suggest that this modification would also improve albumin binding. The goal is to optimize the equilibrium between serum albumin and GLP-2R to enable better circulation and durability, and the exploration of other lipids is warranted.

[0230] Example 19: Analog Stability The analogs of the present invention are resistant to various proteases. Figure 18 provides a series of graphs comparing the susceptibility of GLP-1 (left panel) to CHCF3-GLP-1 (right panel; G-4 in Figure 6) to enzymatic degradation. The activity of these peptides after overnight incubation without enzyme (control) or with either DPP4, DPP9, or FAP was measured by luciferase assay in cells expressing the GLP-1 receptor. In contrast to unmodified GLP-1, the G-4 derivatives are resistant to enzyme-induced loss of potency (no shift to the right in the concentration-response curve).

[0231] The analogs of the present invention are not only resistant to proteases in vitro, but also exhibit resistance compared to liraglutide in vivo. Figure 22 provides a graph comparing the time-dependent decrease in drug activity in plasma after bolus injection of either liraglutide or the CHCF3-decorated derivative, Lira-4.

[0232] Rats received a bolus injection of either drug via a central catheter, followed by serial blood sampling at indicated intervals and measurement of drug activity in plasma by receptor agonism bioassay. The efficacy of each peptide immediately after injection was defined as 100%. The plasma persistence of liraglutide is prolonged by the CHCF3 modification of Lira-4.

[0233] Figure 22A shows the sustained hypoglycemic activity of Lira-4 after an oral glucose tolerance test. Mice received subcutaneous injections of either vehicle, G-4, liraglutide, or Lira-4 (represented from left to right in each group of bars). 30 minutes and 5.5 hours later, two consecutive oral glucose loads were administered by oral gavage. Blood glucose levels were measured immediately before drug injection (-30 minutes) and at the indicated time intervals after the glucose load. A single injection of compound G-4 attenuated blood glucose excursions after the first glucose load and remained active several hours later, attenuating the second glucose challenge.

[0234] Figure 23A provides a graph depicting the extended in vivo bioactivity of GLP2-L17K(l1)-4, a fluorinated / acylated GLP-2 analog shown in Figure 6A. The clearance of this compound was compared to that of Gatex, an existing GLP-2-based drug. The compound (1.25 μg) was injected subcutaneously (sc) into mice. Plasma was collected 24 hours later and analyzed for GLP-2R agonist activity by luciferase reporter gene assay.

[0235] The results described herein above were obtained utilizing the following methods and materials.

[0236] In vitro testing Different lipid anchors are successively introduced into the prototype compound, F-GLP2-palm, replacing palmitic acid, respectively. The pharmacological properties of the corresponding compounds are characterized and compared with F-GLP2-palm and GATEX®. In certain embodiments, lipid addition can affect potency, efficacy, albumin binding, cell interaction, oligomerization, and protease resistance. Exemplary lipids contemplated in the present invention include myristic acid, stearic acid, palmitoleic acid, oleic acid, and linoleic acid. Due to the cis-unsaturation in the chain, these lipids have a higher two-dimensional diffusion rate in membranes and a faster koff for their binding to albumin and cell membranes.

[0237] The overall methodology for generating these lipidated peptides and modifying the histidines is well established below.

[0238] An in vitro assay using transfected HEK293 cells expressing GLP-2R will be used to characterize the pharmacological properties of the synthetic lipidated constructs. As a benchmark, the assay will include a prototypical lipidated stable GLP-2 analog (F-GLP2-palm).

[0239] Evaluating potency and efficacy of Gas signaling: HEK293 cells are transiently transfected with cDNAs encoding GLP-2R, a CRE luciferase reporter gene, and an α-galactosidase construct (allowing for normalization of transfection efficiency). The potency and efficacy of each lipidated peptide are determined.

[0240] Wash-resistant activity / albumin binding: Lipidated peptides are in equilibrium between binding to cell membranes and circulating proteins (e.g., albumin). The success of liraglutide as a drug is partially attributed to its ability to bind to albumin, thereby favorably altering multiple pharmacological parameters. Studies using a GPCR peptide ligand, protease-resistant chemerin ("stable chemerin"), were performed to establish a sensitive assay that reflects the equilibrium between cell membranes and albumin (Figure 11). The study allowed for evaluation of how lipidation of the ligand and / or the presence of albumin in the medium alters the binding equilibrium to cells (using receptor-mediated signaling as an indicator of ligand availability; Figure 12). For stable chemerin (non-lipidated), washing after ligand addition significantly reduced activity (Figure 12, panel A). For lipidated stable chemerin (without albumin in the medium), activity persisted despite washing, reflecting membrane anchoring (Figure 12, panel B). For lipidated stable chemerin with albumin in the medium, washing tended to reduce activity (Figure 12, panel C), reflecting lipidated ligand binding to albumin and resulting in loss of ligand (and subsequent reduced signaling) upon washing. In this method, CMKLR1-expressing cells were incubated with the ligand for 4 hours. A luciferase reporter gene assay was used to monitor Gαi-coupled second messenger signaling. "Wash": 15 minutes after ligand addition, cells were washed three times. After an additional 4 hours of incubation, luciferase activity was measured. For albumin interaction studies, a physiological concentration of albumin (4.5 g / dL) was dissolved in the cell medium. This assay provides a sensitive indication of how the lipidated peptide reaches equilibrium between the cell membrane and albumin.

[0241] DPP4 resistance (Figure 13): The lipidated peptide is compared to the prototype, F-GLP2-palm. After overnight incubation (16 h) in the presence or absence of DPP4, receptor-mediated function is assessed as described elsewhere herein. Parallel experiments are performed using GLP-2 as a DPP4-sensitive control. Similar to the DPP4 assay, F-GLP2-palm and native GLP-2 (81 μM) were preincubated overnight at 37°C with or without DPP4 (27.5 μg / ml). After overnight preincubation, serial dilutions of the ligand were prepared and added to GLP-2R-expressing cells for 4 h. A luciferase reporter gene assay was used to monitor Gas-coupled second messenger signaling.

[0242] Multimerization: Stable lipidated GLP-2 can be administered subcutaneously. Lipidated GLP-1 (liraglutide) forms a heptamer, allowing for slow absorption. To investigate this feature of lipidated GLP-2 analogs, analytical sedimentation equilibrium studies were performed to determine the apparent molecular weight in solution. Compounds were investigated in the 5-100 μM range at three different rotor speeds in a Beckman XL-I analytical ultracentrifuge. Equilibrium was determined to be complete when successive absorbance scans were superimposable. The presence of tryptophan allowed monitoring at 275 nm. Data were fit to a single ideal species model. Other equilibria (e.g., monomer vs. n-mer) could be included as needed.

[0243] Species Differences: Recognizing that species differences may potentially alter receptor-mediated functions, lipidated stable GLP-2 analogs are tested at both mouse and human receptors.

[0244] In vivo testing To evaluate the intestinal growth-promoting effects of GLP-2 compounds, lipidated stable GLP-2 analogs are subcutaneously administered to mice, and histomorphometric analysis, proliferation index, and intestinal function assessment are evaluated in comparison with GATEX®.

[0245] Administration of GLP-2 results in intestinal proliferation, expansion of the mucosal epithelial surface, and increased nutrient absorption. Numerous rodent models have been used to evaluate the effects of GLP-2, Gatex®, and other GLP-2 analogs on the gastrointestinal tract. One highly informative yet straightforward model involves twice-daily administration of GLP-2 or a stable analog to wild-type mice. Corresponding experiments with GLP-2 were performed over a 10-day period with twice-daily administration. A significant increase in small intestinal weight was observed after 6 days. Daily administration of Gatex® to mice also resulted in a significant increase (normalized to body weight) in both colon and small intestinal weight. Additional parameters increased by GLP-2 analogs included small intestinal epithelial height (crypt plus villus height) and proliferation index, particularly in the lower villous regions. The use of mouse models allows for a direct comparison of the physiological effects of lipidated stable GLP-2 analogs and Gatex®.

[0246] Histomorphometry / Proliferation Index. Groups of seven C57 / B6 mice are tested for each condition (drug / time point). Compounds are administered as 100 μl subcutaneous injections twice daily at 12-hour intervals for 6 and 10 days. The initial dose is adjusted to the equivalent of 25 μg of Gatex® (molar equivalent), which is highly effective in inducing intestinal growth at 6 days. The 10-day time point defines the maximum effect. Mice are then euthanized and weighed. The small and large intestines are removed, flushed with PBS, and the length / wet weight of the intestinal segments is recorded. Many established indices are used to evaluate the in vivo efficacy of the GLP-2 analogs under investigation. Histomorphometric analysis (Figure 14) includes quantification of intestinal weight / thickness, villus height, and crypt depth. Proliferation indices include Ki67 staining and mitotic index (Figure 15).

[0247] To assess the duration of drug activity, each drug (two lipidated GLP-2 analogs and Gatex® as a control) is administered subcutaneously to three groups of seven mice over a period of six days. Group 1 receives daily dosing (6 doses), group 2 receives drug every two days (3 doses), and group 3 receives drug every three days (2 doses). Animals are sacrificed and analyzed.

[0248] Changes in intestinal metabolic function in response to stabilized lipidated GLP-2 analogs were assessed using 2-deoxy-2[ 18 The dose schedule of the GLP-2 analogue is selected based on histomorphometric analysis. Overnight fasted mice are administered 2 mCi / kg of [F]-D-glucose, [18F]FDG, as a tracer, using positron emission tomography (PET). 18 [F]FDG is administered via tail vein injection. 50 minutes after injection, mice are anesthetized with isoflurane and imaged using a microPET Focus 220 scanner (Siemens Medical Solutions USA, Inc., Malvern, PA), followed by a whole-body CT scan (portable CereTom CT scanner; NeuroLogica Inc., Danvers, MA). PET and CT images are overlaid to accurately calculate FDG uptake in each intestinal section. Direct comparison with Gatex® is performed.

[0249] Depending on the potency of the drug, PET may not provide sufficient resolution. When this situation occurs, biodistribution imaging is used instead of PET imaging. In biodistribution studies, after administration of FDG (using methods similar to those described for PET imaging), mice are euthanized, and intestinal segments are collected, weighed, and quickly transferred to a gamma counter to measure radioactivity. 18 [F]FDG tissue uptake levels are expressed as a percentage of the injected dose per gram of tissue.

[0250] There is a distinction between promoting healthy epithelial regeneration and accelerating the growth of early adenomas or cancers. In animal studies, histological sections are examined for abnormal growths.

[0251] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0252] While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention, and the appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0253] Sequence information SEQUENCE LISTING <110> TUFTS UNIVERSITY TUFTS MEDICAL CENTER <120> NOVEL POLYPEPTIDES WITH IMPROVED PROTEOLYTIC STABILITY, AND METHODS OF PREPARING AND USING SAME <150> US 62 / 247,493 <151> 2015-10-28 <160> 33 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Polyp <220> <221> MOD_RES <222> (30)..(30) <223> unmodified or amidation <400> 1 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 <210> 2 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (39)..(39) <223> unmodified or amidation <400> 2 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 3 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 3 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 30 <210> 4 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (2)..(2) <223> 2-Aminoisobutyric acid <400> 4 His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 30 <210> 5 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (2)..(2) <223> 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (29)..(29) <223> 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (30)..(30) <223> unmodified or amidation <400> 5 His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg 20 25 30 <210> 6 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (44)..(44) <223> unmodified or amidation <400> 6 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Ser Lys Lys Lys Lys Lys Lys 35 40 <210> 7 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (2)..(2) <223> 2-Aminoisobutyric acid <220> <221> MOD_RES <222> (39)..(39) <223> unmodified or amidation <400> 7 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 8 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn 20 25 <210> 9 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys 20 25 30 Gln Arg Val Lys Asn Lys 35 <210> 10 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys 1 5 10 15 Ile His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys 20 25 30 Lys Asn Asp Trp Lys His Asn Ile Thr Gln 35 40 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 11 Tyr Gly Gly Phe Met 1 5 <210> 12 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 20 25 30 Arg Gln Arg Tyr 35 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10 <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Tyr Pro Trp Gly 1 <210> 15 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 16 <211> 235 <212> PRT <213> Homo sapiens <400> 16 Asp Glu Ser Ala Cys Leu Gln Ala Ala Glu Glu Met Pro Asn Thr Thr 1 5 10 15 Leu Gly Cys Pro Ala Thr Trp Asp Gly Leu Leu Cys Trp Pro Thr Ala 20 25 30 Gly Ser Gly Glu Trp Val Thr Leu Pro Cys Pro Asp Phe Phe Ser His 35 40 45 Phe Ser Ser Glu Ser Gly Ala Val Lys Arg Asp Cys Thr Ile Thr Gly 50 55 60 Trp Ser Glu Pro Phe Pro Pro Tyr Pro Val Ala Cys Pro Val Pro Leu 65 70 75 80 Glu Leu Leu Ala Glu Glu Glu Ser Tyr Phe Ser Thr Val Lys Ile Ile 85 90 95 Tyr Thr Val Gly His Ser Ile Ser Ile Val Ala Leu Phe Val Ala Ile 100 105 110 Thr Ile Leu Val Ala Leu Arg Arg Leu His Cys Pro Arg Asn Tyr Val 115 120 125 His Thr Gln Leu Phe Thr Thr Phe Ile Leu Lys Ala Gly Ala Val Phe 130 135 140 Leu Lys Asp Ala Ala Leu Phe His Ser Asp Asp Thr Asp His Cys Ser 145 150 155 160 Phe Ser Thr Val Leu Cys Lys Val Ser Val Ala Ala Ser His Phe Ala 165 170 175 Thr Met Thr Asn Phe Ser Trp Leu Leu Ala Glu Ala Val Tyr Leu Asn 180 185 190 Cys Leu Leu Ala Ser Thr Ser Pro Ser Ser Arg Arg Ala Phe Trp Trp 195 200 205 Leu Val Leu Ala Gly Trp Gly Leu Pro Val Leu Phe Thr Gly Thr Trp 210 215 220 Val Ser Cys Lys Leu Ala Phe Glu Asp Ile Ala 225 230 235 <210> 17 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MOD_RES <222> (29)..(29) <223> unmodified or amidation <400> 17 Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln 1 5 10 15 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg 20 25 <210> 18 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MOD_RES <222> (44)..(44) <223> unmodified or amidation <400> 18 Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln 1 5 10 15 Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly 20 25 30 Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu 35 40 <210> 19 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 19 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 <210> 20 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 20 His Ala Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 21 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 21 Glu Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu 1 5 10 <210> 22 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 22 Glu His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 <210> 23 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 23 Tyr Ala Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Asp Lys 1 5 10 15 Gln Ala Ala Ala Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Lys 35 40 <210> 24 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 24 His Ala Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 25 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 25 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn 20 25 30 Arg Asn Asn Ile Ala 35 <210> 26 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 26 His Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 27 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 27 His Ala Glu Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Lys Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 28 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 28 His Ala Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Lys Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 29 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15 Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 <210> 30 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 30 Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile 1 5 10 15 His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys Lys 20 25 30 Asn Asp Trp Lys His Asn Ile Thr Gln 35 40 <210> 31 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 31 Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser Arg 1 5 10 15 Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25 <210> 32 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 32 Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu 1 5 10 15 Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser 20 25 30 Gly Ala Pro Pro Pro Ser 35 <210> 33 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 33 Ala Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu Arg 1 5 10 15 Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser Ser 20 25 30 Gly Ala Pro Pro Pro Ser 35

Claims

1. A chemically modified polypeptide or a salt or solvate thereof, wherein the polypeptide has one of the following chemical modifications: (i) at least one selected from the group consisting of the N-terminal amino group, the NH of the N-terminal first internal amide bond, other free primary amino groups, thiol groups, and thioether groups of the polypeptide, independently optionally substituted; １ ~C １６ Alkyl, optionally substituted C ３ ~C １６ Cycloalkyl, optionally substituted C ３ ~C １６ aryl, optionally substituted C ２ ~C １６ Alkenyl, or optionally substituted C ２ ~C １６ derivatized with alkynyl; and (ii) at least one NH group selected from the group consisting of the N-terminal amino group and the N-terminal first internal amide bond is derivatized with X, wherein each X is an optionally substituted phenyl, an optionally substituted benzyl, an optionally substituted -(CR ２ ) １～６ -phenyl, →O, -OH, -OR, alkoxy, NH ２ , optionally substituted NH(C １ ~C １６ alkyl), and optionally substituted N(C １ ~C １６ alkyl) (C １ ~C １６ wherein R is independently selected at each occurrence from the group consisting of hydrogen or optionally substituted C １ ~C １６ alkyl) wherein the chemically modified polypeptide has essentially the same biological activity and / or is more resistant to proteolysis compared to a corresponding non-chemically modified polypeptide, or a salt or solvate thereof.

2. The chemically modified polypeptide of claim 1, wherein the proteolysis is catalyzed by at least one selected from the group consisting of acylpeptide hydrolase, DPP4, DPP2, DPP8, DPP9, fibroblast activation protein (FAP), S9B family oligopropylpeptidase, and proteases having 50% or more homology to DPP4 and / or DPP2.

3. The chemically modified polypeptide of claim 1 , wherein the unmodified polypeptide comprises an incretin.

4. The chemically modified polypeptide of claim 1, which has higher serum stability compared to the corresponding unmodified polypeptide.

5. 2. The chemically modified polypeptide of claim 1, which has a longer in vivo half-life compared to the corresponding unmodified polypeptide.

6. The chemically modified polypeptide of claim 1, which has higher blood-brain barrier permeability or higher oral bioavailability compared to the corresponding unmodified polypeptide.

7. In (i), the polypeptide comprises at least one unsubstituted C １ ~C ６ Alkyl, unsubstituted C ３ ~C １６ Cycloalkyl, unsubstituted C ２ ~C １６ Alkenyl, unsubstituted aryl, or unsubstituted C ２ ~C １６ The chemically modified polypeptide of claim 1 , which is derivatized with an alkynyl.

8. In (i), one or more of the N-terminal amino group, other free amino groups, and / or thiol groups of the polypeptide are independently optionally substituted C １ ~C １６ Alkyl, optionally substituted C ３ ~C １６ Cycloalkyl, optionally substituted C ２ ~C １６ Alkenyl, and optionally substituted C ２ ~C １６ 2. The chemically modified polypeptide of claim 1, wherein the polypeptide is derivatized with a first and second substituent independently selected from the group consisting of alkynyl, wherein the first and second substituents are independently the same or different.

9. In (i), the N-terminal amino group is an optionally substituted C １ ~C １６ Alkyl, optionally substituted C ３ ~C １６ Cycloalkyl, optionally substituted C ２ ~C １６ Alkenyl, and optionally substituted C ２ ~C １６ 2. The chemically modified polypeptide of claim 1, wherein the polypeptide is derivatized with a first and second substituent independently selected from the group consisting of alkynyl, wherein the first and second substituents are independently the same or different.

10. In (i), the alkyl, cycloalkyl, alkenyl, or alkynyl groups are independently selected from the group consisting of: １ ~C １６ Alkyl, C ３ ~C １６ Cycloalkyl, C ２ ~C １６ Alkenyl, C ２ ~C １６ Alkynyl, heteroaryl, heterocyclyl, C １ ~C ６ Alkoxy, azido, diaziryl , —CHO, 1,3-dioxol-2-yl, halo, haloalkyl, haloalkoxy, cyano, nitro, triflyl, mesyl, tosyl, heterocyclyl, aryl, heteroaryl, —SR, —S(═O)(C １ ~C ６ alkyl), -S(=O) ２ (C １ ~C ６ alkyl), -S(=O) ２ NRR, -C(=O)R, -OC(=O)R, -C(=O)OR, -OC(=O)O(C １ ~C ６ alkyl), —NRR, —C(═O)NRR, —N(R)C(═O)R, —C(═NR)NRR, and —P(═O)(OR) ２ wherein R, at each occurrence, is independently H or C. １ ~C １６ The chemically modified polypeptide of claim 1, wherein the hydroxyl group is alkyl.

11. The alkyl group is a halogenated C １ ~C １６ The chemically modified polypeptide of claim 10, wherein the hydroxyl group is alkyl.

12. The chemically modified polypeptide of claim 11, wherein the alkyl group is 2,2,2-trifluoroethyl.

13. At least one alkyl, cycloalkyl, alkenyl, aryl, or alkynyl group is 2. The chemically modified polypeptide of claim 1, wherein n is an integer ranging from 1 to 6, and R is hydrogen or an optionally substituted alkyl or aryl.

14. At least one alkyl, cycloalkyl, alkenyl, or alkynyl group is selected from the group consisting of 2,2,2-trifluoro-1-ethyl, 2,2,3,3,3-pentafluoro-1-propyl, 2,2,3,3,4,4,4-heptafluoro-1-butyl, ethyl, isopropyl, benzyl, substituted benzyl, adamant-1-yl-methyl, quinolin-4-yl-methyl, 2-amino-1-propyl, 4-phenyl-benzyl, 1H-imidazoline ...

2. The chemically modified polypeptide of claim 1, wherein the modified polypeptide is selected from the group consisting of 4-azol-4-yl-methyl, 4-hydroxy-benzyl, 4-[3-(trifluoromethyl)-3H-diazirine]-benzyl, and (8R,9R,13S,14R)-3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl-ethynyl-methyl.

15. Approximately 240 Å ３ 2. The chemically modified polypeptide of claim 1, which is chemically modified by a group that occupies the following volume:

16. At the N-terminal amino group, about 190 Å ３ 16. The chemically modified polypeptide of claim 15, which is chemically modified by a group that occupies the following volume:

17. 2. The chemically modified polypeptide of claim 1, comprising from about 3 to about 100 amino acids.

18. 2. The chemically modified polypeptide of claim 1, comprising from about 3 to about 49 amino acids.

19. 2. The chemically modified polypeptide of claim 1, comprising from about 80 to about 100 amino acids.

20. At least the N-terminal amino group is an optionally substituted C １ ~C １６ Alkyl, optionally substituted C ３ ~C １６ Cycloalkyl, optionally substituted C ２ ~C １６ Alkenyl, or optionally substituted C ２ ~C １６ The chemically modified polypeptide of claim 1 , which is derivatized with an alkynyl.

21. A chemically modified polypeptide as described in claim 1, comprising at least one amino acid residue selected from the group consisting of:

22. the polypeptide GLP-1 ； Exenatide ； Liraglutide ； Semaglutide (In the sequence, X attached to the ε-amino group of lysine is ); Taspoglutide ； Lixisenatide ； Triagonists ； Exendin ； VIP ； PACAP ； GIP ； Met-enkephalin ； BNP ； Substance P ； Tyr-MIF-1 ； Tyr-W-MIF-1 ； Glucagon ； Growth hormone-releasing hormone (GHRH); pituitary adenylate cyclase-activating polypeptide (PACAP) ADCYAP1; Glucagon (GCG); Gastric inhibitory polypeptide (GIP); Secretin (SCT); vasoactive intestinal peptide (VIP); OXM (oxyntomodulin); PTH (parathyroid hormone); Peptide YY3-36 and peptide YY1-36 (PYY); NPY (neuropeptide Y); VIP peptide (vasoactive intestinal peptide); GLP-1+GIP; GLP-1+amylin; GLP-1+gastrin; GLP-1+estrogen; GLP-1+PYY; GLP-1+cholecystin kinase (CCK) and at least one dual agonist selected from the group consisting of: Dual agonist of GLP-1R + glucagon receptor; mixed agonists; Albiglutide; Dulaglutide; Other GLP-1R agonists; Amylin; DPP4, DPP2, and / or other substrates of proteases having 50% or more homology to DPP4 and / or DPP2; GLP-1 analogs stabilized by other modifications; or A sequence having at least 75% identity to any of the above sequences 2. The chemically modified polypeptide of claim 1, wherein at least one of the residues marked by * is selected from the group consisting of:

23. 2. The chemically modified polypeptide of claim 1, wherein the half-life of the polypeptide is increased by at least 10-fold relative to the corresponding unmodified polypeptide.

24. 2. The chemically modified polypeptide of claim 1, which retains at least about 5% of the biological activity of the corresponding unmodified polypeptide.

25. A chemically modified polypeptide selected from the group consisting of parathyroid hormone (PTH), PYY3-36, cholecystokinin, PYY1-36, corticotropin-releasing hormone receptor 1 or 2, growth hormone-releasing factor, glucagon, exendin, GLP-1, gastric inhibitory peptide, liraglutide, prealbumin, peptide HI-27, PACAP, secretin, and vasoactive intestinal peptide (VIP), The N-terminal amino group is (i) optionally substituted C １ ~C １６ Alkyl, optionally substituted C ３ ~C １６ Cycloalkyl, optionally substituted C ２ ~C １６ Alkenyl, and optionally substituted C ２ ~C １６ or (ii) optionally substituted phenyl, optionally substituted benzyl, →O, —OH, alkoxy, NH ２ , NH(C １ ~C １６ alkyl), and N(C １ ~C １６ alkyl) (C １ ~C １６ alkyl), the derivatization stabilizes the polypeptide against degradation by at least one selected from the group consisting of acylamino acid releasing enzymes, DPP4, DPP2, DPP8, DPP9, all S9B family oligopropylpeptidases, and proteases having 50% or more homology to DPP4 and / or DPP2, without reducing the biological activity of the polypeptide relative to the corresponding unmodified protein; Chemically modified polypeptides.

26. A pharmaceutically acceptable composition comprising at least one polypeptide according to any one of claims 1 to 25.

27. 26. A method for treating or preventing at least one disease or disorder comprising administering at least one polypeptide according to any one of claims 1 to 25, wherein said disease or disorder is selected from the group consisting of short bowel syndrome, non-alcoholic steatohepatitis, smoking cessation, neurodegeneration, Alzheimer's disease, Parkinson's disease, congenital hyperinsulinism, hypoglycemia, diabetes, weight gain, obesity, and metabolic syndrome.

28. 1. A method for increasing the in vivo half-life of a polypeptide compared to a corresponding non-chemically modified polypeptide, comprising: (i) chemically modifying at least one substituent selected from the group consisting of the N-terminal amino group, the NH group of the N-terminal first internal amide bond, other free primary amino groups, a thiol group, and a thioether group of the polypeptide, wherein each chemical modification converts the substituent into an optionally substituted C １ ~C １６ Alkyl, optionally substituted C ３ ~C １６ Cycloalkyl, optionally substituted C ３ ~C １６ aryl, optionally substituted C ２ ~C １６ Alkenyl, or optionally substituted C ２ ~C １６ chemically modifying, independently including derivatizing with alkynyl; and (ii) chemically modifying at least one substituent selected from the N-terminal amino group and the NH, independently comprising derivatizing the substituent with a group X, wherein X, in each occurrence, is independently selected from optionally substituted phenyl, optionally substituted benzyl, optionally substituted -(CR ２ ) １～６ -phenyl, →O, -OH, alkoxy, NH ２ , NH(C １ ~C １６ alkyl), and N(C １ ~C １６ alkyl) (C １ ~C １６ alkyl), wherein R, at each occurrence, is independently hydrogen or optionally substituted C １ ~C １６ alkyl, The method includes at least one of:

29. 1. A method for improving the blood-brain barrier permeability or oral bioavailability of a polypeptide compared to a corresponding unmodified polypeptide, comprising: (i) chemically modifying at least one substituent selected from the group consisting of the N-terminal amino group, the NH group of the N-terminal first internal amide bond, other free primary amino groups, a thiol group, and a thioether group of the polypeptide, wherein each chemical modification converts the substituent into an optionally substituted C １ ~C １６ Alkyl, optionally substituted C ３ ~C １６ Cycloalkyl, optionally substituted C ２ ~C １６ Alkenyl, or optionally substituted C ２ ~C １６ chemically modifying, independently including derivatizing with alkynyl; and (ii) chemically modifying at least one substituent selected from the N-terminal amino group and the NH, independently comprising derivatizing the substituent with a group X, wherein X, in each occurrence, is independently selected from optionally substituted phenyl, optionally substituted benzyl, optionally substituted -(CR ２ ) １～６ -phenyl, →O, -OH, alkoxy, NH ２ , NH(C １ ~C １６ alkyl), and N(C １ ~C １６ alkyl) (C １ ~C １６ alkyl), wherein R, at each occurrence, is independently hydrogen or optionally substituted C １ ~C １６ alkyl, The method includes at least one of:

30. In (i), the polypeptide comprises at least one unsubstituted C １ ~C ６ Alkyl, unsubstituted C ３ ~C １６ Cycloalkyl, unsubstituted C ２ ~C １６ Alkenyl, or unsubstituted C ２ ~C １６ 30. The method of any one of claims 27 to 29, wherein the alkynyl is derivatized.

31. In (i), the alkyl, cycloalkyl, alkenyl, or alkynyl groups are independently selected from the group consisting of: １ ~C １６ Alkyl, C ３ ~C １６ Cycloalkyl, C ２ ~C １６ Alkenyl, C ２ ~C １６ Alkynyl, heteroaryl, heterocyclyl, C １ ~C ６ Alkoxy, azido, diaziryl , —CHO, 1,3-dioxol-2-yl, halo, haloalkyl, haloalkoxy, cyano, nitro, triflyl, mesyl, tosyl, heterocyclyl, aryl, optionally substituted phenyl heteroaryl, optionally substituted benzyl heteroaryl, optionally substituted —(CR ２ ) １～６ -phenyl heteroaryl, -SR, -S(=O)(C １ ~C ６ alkyl), -S(=O) ２ (C １ ~C ６ alkyl), -S(=O) ２ NRR, -C(=O)R, -OC(=O)R, -C(=O)OR, -OC(=O)O(C １ ~C ６ alkyl), —NRR, —C(═O)NRR, —N(R)C(═O)R, —C(═NR)NRR, and —P(═O)(OR) ２ wherein R, in each occurrence, is independently H or C １ ~C １６ 30. The method of any one of claims 27 to 29, wherein the substituted aryl group is substituted with at least one independently selected from the group consisting of alkyl, aryl, aryl and alkyl.

32. The alkyl group is a halogenated C １ ~C ６ The method of any one of claims 33 to 36, wherein the alkyl is alkyl.

33. 37. The method of any one of claims 33 to 36, wherein the alkyl group is 2,2,2-trifluoroethyl.

34. the at least one alkyl, cycloalkyl, alkenyl, or alkynyl group being 37. The method of any one of claims 33 to 36, wherein n is an integer ranging from 1 to 6, and R is an optionally substituted alkyl or an optionally substituted aryl.

35. At least one alkyl, cycloalkyl, alkenyl, or alkynyl group is selected from the group consisting of 2,2,2-trifluoro-1-ethyl, 2,2,3,3,3-pentafluoro-1-propyl, 2,2,3,3,4,4,4-heptafluoro-1-butyl, ethyl, isopropyl, benzyl, substituted benzyl, adamant-1-yl-methyl, quinolin-4-yl-methyl, 2-amino-1-propyl, 4-phenyl-benzyl, 1H-imidazoline ...

30. The method of any one of claims 27 to 29, wherein the hydroxybenzoate is selected from the group consisting of 4-azol-yl-methyl, 4-hydroxy-benzyl, 4-[3-(trifluoromethyl)-3H-diazirine]-benzyl, and (8R,9R,13S,14R)-3,17-dihydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl-ethynyl-methyl.

36. The polypeptide has a length of about 240 Å ３ 30. The method of any one of claims 27 to 29, wherein the compound is chemically modified by a group that occupies a volume of:

37. The polypeptide has a length of about 240 Å at the N-terminal amino group. ３ 30. The method of any one of claims 27 to 29, wherein the compound is chemically modified by a group that occupies a volume of:

38. 30. The method of any one of claims 27 to 29, wherein the polypeptide comprises from about 5 to about 100 amino acids.

39. 30. The method of any one of claims 27 to 29, wherein the polypeptide comprises from about 3 to about 49 amino acids.

40. 30. The method of any one of claims 27 to 29, wherein the polypeptide comprises from about 80 to about 100 amino acids.

41. At least the N-terminal amino group is an optionally substituted C １ ~C １６ Alkyl, optionally substituted C ３ ~C １６ Cycloalkyl, optionally substituted C ２ ~C １６ Alkenyl, or optionally substituted C ２ ~C １６ 30. The method of any one of claims 27 to 29, wherein the alkynyl is derivatized.

42. the polypeptide The method of any one of claims 27 to 29, comprising at least one amino acid residue selected from the group consisting of:

43. 10. The polypeptide of claim 1, wherein the polypeptide has the structure:

30. The method of any one of claims 27 to 29, comprising a derivatized amino acid having the formula:

44. 44. A method for derivatizing a polypeptide containing a free amino group, comprising contacting a derivatized amino acid of claim 43 with the polypeptide under conditions in which the free carboxylic acid of the derivatized amino acid forms an amide bond with the free amino acid of the polypeptide.

45. 27. A method for imaging cells or tissues in a subject, comprising administering to said subject in need thereof an effective amount of a polypeptide according to any one of claims 1 to 26, wherein said polypeptide is labelled with a detectable isotope and / or conjugated to a detectable label.

46. 46. The method of claim 45, wherein the detectable label comprises a chromophore, a fluorescent group, a bioluminescent group, a chemiluminescent group, or a radioactive group.

47. 25. A method for characterizing a gastrinoma in a subject, comprising administering to the subject a chemically modified secretin polypeptide of claim 24 and detecting an increase in gastrin in the subject, thereby determining the presence or absence of a gastrinoma in the subject.

48. The chemically modified polypeptide of claim 1 , wherein the modification increases binding to a plasma component.

49. 49. The chemically modified polypeptide of claim 48, wherein the plasma component is vitamin D3 binding protein, albumin, or transthyretin.

50. 49. The chemically modified polypeptide of claim 48, which is not GLP2.

51. structure:

2. The chemically modified polypeptide of claim 1, comprising a derivatized amino acid having the formula:

Images