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BR-112018008681-B1 - New polypeptides with improved proteolytic stability, and methods for preparing and using them.

BR112018008681B1BR 112018008681 B1BR112018008681 B1BR 112018008681B1BR-112018008681-B1

Abstract

NEW POLYPEPTIDES WITH IMPROVED PROTEOLYTIC STABILITY, AND METHODS FOR PREPARING AND USING THEM. The present invention includes methods for improving the proteolytic stability of a polypeptide, comprising the alkylation of at least one selected group consisting of an N-terminal amino group, the NH group of the first N-terminal internal amide bond, another primary amino group, a thiol group, and a thioether group in the polypeptide. The present invention further includes polypeptides incorporating such chemical modifications.

Inventors

  • Krishna Kumar
  • Vittorio Montanari
  • Martin Beinborn
  • VENKATA RAMAN

Assignees

  • TUFTS MEDICAL CENTER
  • TUFTS UNIVERSITY

Dates

Publication Date
20260310
Application Date
20161028
Priority Date
20151028

Claims (11)

  1. 1. A GLP-1 analog polypeptide, characterized in that it comprises a modified N-terminal amino acid residue, wherein the nitrogen of the N-terminal histidine residue is replaced by a group selected from: (a) fluorinated C2-C4 alkyls selected from N-trifluoroethyl or perfluoroalkyls; (b) N-alkyl-adamantyl, wherein the alkyl radical has from 1 to 4 carbon atoms, optionally N-methyl-adamantyl; (c) a non-planar aromatic ring selected from N-benzyl or hydroxybenzyl; (d) an N-methylimidazole; or (e) an alanine aldehyde derivative, consisting of a short-chain aminoalkyl containing up to 4 carbon atoms, wherein the chemically modified polypeptide has essentially the same biological activity and is more resistant to proteolytic degradation compared with the corresponding non-chemically modified polypeptide; and wherein the polypeptide has the amino acid sequence of: GLP-1 (SEQ ID NO: 1), Taspoglutide (SEQ ID NO: 5); Liraglutide (SEQ ID NO: 3), or Semaglutide (SEQ ID NO: 4).
  2. 2. GLP-1 analog polypeptide, according to claim 1, characterized in that proteolysis is catalyzed by at least one selected from the group consisting of Acyl-Hydrolase Peptide, DPP4, DPP2, DPP8, DPP9, Fibroblast Activating Protein (FAP), an oligopropyl peptidase of the S9B family and a protease with homology > 50% to DPP4 and/or DPP2.
  3. 3. GLP-1 analog polypeptide, according to claim 1, characterized in that the chemically unmodified polypeptide comprises an incretin.
  4. 4. GLP-1 analogous polypeptide according to claim 1, characterized in that the alkyl group attached to the N-terminal amino group is a fluorinated C2-C4 alkyl group.
  5. 5. GLP-1 analog polypeptide according to claim 4, characterized in that the alkyl group is 2,2,2-trifluoroethyl.
  6. 6. GLP-1 analog polypeptide according to claim 1, characterized in that the N-terminal amino group of the polypeptide is replaced by a substituent selected from the group consisting of 2,2,2-trifluoro-1-ethyl, benzyl, adamant-1-yl-methyl, 1H-imidazol-4-yl-methyl, 4-hydroxy-benzyl.
  7. 7. Pharmaceutical composition characterized in that it comprises at least one polypeptide as defined in any of claims 1 to 6 and one or more pharmaceutically acceptable carriers.
  8. 8. Use of a chemically modified polypeptide as defined in any one of claims 1 to 6, or of a pharmaceutical composition as defined in claim 7, characterized in that it is for the preparation of a medicament to treat or prevent at least one disease or disorder selected from the group consisting of short bowel syndrome, non-alcoholic steatohepatitis, congenital hyperinsulinism, hypoglycemia, diabetes, weight gain, obesity and metabolic syndrome.
  9. 9. Use of a chemically modified polypeptide as defined in any one of claims 1 to 6, or of a pharmaceutical composition as defined in claim 7, characterized in that it is for preparing a composition to identify a gastrinoma in an individual.
  10. 10. Method for increasing the stability of a polypeptide compared to the corresponding unmodified polypeptide, characterized in that it comprises at least one chemical modification as defined in any one of claims 1 to 6.
  11. 11. Method for increasing the in vivo half-life of a polypeptide compared to the corresponding unmodified polypeptide, characterized in that it comprises at least one chemical modification as defined in any one of claims 1 to 6.

Description

CROSS-REFERENCE TO THE RELATIVE REQUEST [001] This application claims the benefit of the following Interim Application U.S. No. 62/247,493, filed October 28, 2015, the full content of which is incorporated herein by reference. FUNDAMENTALS OF THE INVENTION [002] Polypeptides have attracted much interest as therapeutic agents. They have proven biological activity, 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 key responsibilities, such as poor in vivo stability and short in vivo half-life. Polypeptides are substrates for several peptidases in vivo, such as dipeptidyl protease 4 (DPP4) and endopeptidases, which cleave the polypeptide chain into fragments that usually exhibit diminished function. In addition, polypeptides can be chemically modified within the body, marking them for excretion and/or degradation. [003] Attempts to minimize the instability of polypeptides with respect to proteolysis have been met with limited success. Acetylation of the N-terminal amino group of the polypeptide has the overall effect of reducing proteolysis rates, but with a marked loss of biological activity. Furthermore, the N-acetyl group is still far from stable, undergoing hydrolysis in vivo. Stabilization against enzymes, such as DPP4, can be achieved by amino acid substitution at the target cleavage site, but this modification also leads to a concomitant loss of biological activity in most cases. [004] The drawbacks of peptides as developable therapeutic agents, especially short half-lives in plasma, have been addressed by targeted chemical modifications. For example, the insertion of non-natural amino acids into the peptide chain is a common first-pass approach to new-generation peptides. However, modifying a biologically active polypeptide at certain positions in the backbone can result in an unacceptable loss of activity. This structural modification process is also largely a matter of trial and error. Combined with the inherently low yields of chemical synthesis and peptide isolation, targeted chemical modification is a very expensive approach. [005] GLP-1 is a potent 31-residue peptide secreted by enteroendocrine cells in response to food intake, and possesses the salient properties of delayed gastric emptying and induced satiety, insulin secretion after glucose ingestion, increased β-cell mass and function, and concomitant weight loss. However, its in vivo half-life is less than two minutes due to the action of the serine protease dipeptidyl protease 4 (DPP4), making GLP-1 unsuitable for the treatment of type II diabetes. Only two GLP-1 analogs have emerged as important therapeutic agents: Exenatide, a structural analog of GLP-1; and Liraglutide, a GLP-1 derivative with an added large lipid side chain. Both drugs were developed in an effort to improve the proteolytic stability of GLP-1. [006] Liraglutide self-assembles into heptamers in solution and exhibits enhanced albumin binding, partially protecting it from hydrolytic cleavage by DPP4. However, despite its unsurpassed ability among FDA-approved GLP-1 mimetics to reduce blood glucose in diabetics and induce weight loss, liraglutide is a relatively short-acting drug requiring daily injections. Furthermore, liraglutide is still markedly degraded by DPP4, leading to its inactivation. [007] The N-acetyl-GLP-1 derivative is more stable to DPP4 degradation, but is 10-50 times less active than GLP-1 itself. Thus, N-acetyl-GLP-1 cannot be used as a therapeutic agent. Furthermore, N-acetylation confers in vitro stability against DPP4, but may not be effective against exopeptidases that are ubiquitous in serum. In addition, deacetylation catalyzed by dedicated enzymes can occur in vivo. [008] Additional approaches to further prolong the half-life of GLP-1 have relied on fusing the polypeptide to large carrier proteins, such as albumin (albiglutide) or immunoglobulin (dulaglutide). However, probably due to reduced access to the CNS, these constructs are less effective than liraglutide in reducing weight. Alternatively, the penultimate residue (Ala8) in GLP-1 has been replaced with a non-canonical helix-inducing amino acid (α-aminoisobutyric acid, Aib). A corresponding derivative (taspoglutide) triggered the formation of exenatide-like antibodies, in addition to injection site reactions, and was therefore withdrawn during clinical trials. [009] There is an urgent need to develop more effective treatments for patients with short bowel syndrome (SBS), a debilitating medical condition resulting from the inability to adequately absorb nutrients from the intestine. This orphan disease occurs in patients who suffer a large loss of small intestine with surgery. A variety of underlying pathologies can lead to SBS, including Crohn's disease, trauma, congenital abnormalities, and malignancy. Short bowel syndrome results in severe