# US 2019/0375786 A1 · Stable peptides and methods of use thereof

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- Number: US 2019/0375786 A1
- Document ID: US20190375786A1
- Status: Published application
- Kind: A1
- Application number: 16/330,069
- Filed: 2017-09-09
- Published: 2019-12-12
- Earliest claimed: 2016-09-09
- Assignees: Fred Hutchinson Cancer Research Center; Blaze Bioscience, Inc.
- Applicants: Fred Hutchinson Cancer Research Center; Blaze Bioscience, Inc.
- Inventors: Emily June GIRARD; Colin CORRENTI; James OLSON; Natalie Winblade NAIRN; Mesfin Mulugeta GEWE; Christopher MEHLIN; Zachary CROOK; Roland STRONG; Scott Ronald PRESNELL
- Web page: https://patentgrep.com/patent/US20190375786A1

## CPC

- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- C07K 5/1002: {with the first amino acid being neutral}
- A61K 38/17: from animals; from humans {(enzyme inhibitors A61K38/005)}
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/10: Laxatives
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 13/12: of the kidneys
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 31/10: Antimycotics
- A61P 35/00: Antineoplastic agents
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/001: {by chemical synthesis}
- C07K 14/435: from animals; from humans
- C12Q 1/37: involving peptidase or proteinase
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Peptides that are stable to denaturation and degradation by reducing agents, proteases, temperature, and low pH environments are disclosed. Pharmaceutical compositions and uses for peptides, peptide-active agent conjugates, and peptide-detectable agent conjugates comprising such peptides are also disclosed. Peptide compositions, peptide conjugate compositions, and pharmaceutical compositions can be formulated for various routes of delivery, such as oral delivery, and for deliver to various compartments of the body. Peptides of this disclosure are stable and display enhanced pharmacokinetics after such delivery.

## Claims

### Claim at position 1

1-372. (canceled)

### Claim 373

A method comprising administering a compound comprising a peptide to a subject, wherein at least 70% of the peptide remains intact after exposure to pepsin at a temperature of at least 20° C. for at least 5 minutes and the peptide comprises at least 6 cysteine residues.

### Claim 374 (depends on 373)

The method of claim 373, wherein the peptide remains intact when exposed to pepsin at a concentration of 47 U/ml and a temperature of 23° C. for 5 minutes as measured by high performance liquid chromatography.

### Claim 375 (depends on 373)

The method of claim 373, wherein the peptide has at least one of the following characteristics:
(a) at least 70% of the peptide remains intact after exposure to pepsin at a temperature of at least 23° C. for at least 5 minutes;
(b) at least 70% of the peptide remains intact after exposure to dithiothreitol (DTT) at a concentration of 5 mM and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(c) at least 70% of the peptide remains intact after exposure to reduced glutathione (GSH) at a concentration of 5 mM and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(d) at least 70% of the peptide remains intact after exposure to trypsin at a concentration of 0.5 U/ml and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(e) at least 70% of the peptide remains intact after exposure to simulated gastric fluid (SGF; pH 1.05; 2% (w/v) sodium chloride in 0.7% (v/v) hydrochloric acid) and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(f) at least 70% of the peptide remains intact after exposure to a pH of 5 and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(g) at least 70% of the peptide remains intact after passage through the mouth, stomach, small intestine, or the large intestine, as measured by tandem high performance liquid chromatography and liquid scintillation counting;
(h) at least 70% of the peptide remains intact after exposure to the combination of simulated gastric fluid (SGF; pH 1.05; 2% (w/v) sodium chloride in 0.7% (v/v) hydrochloric acid) with 0.5 U/ml pepsin, 100 mM Tris, and 10 mM DTT and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(i) at least 70% of the peptide remains intact after exposure to at least 70° C. for at least 5 minutes, as measured by high performance liquid chromatography; or
(j) at least 70% of the peptide remains intact after exposure to at least 100° C. for at least 5 minutes, as measured by high performance liquid chromatography.

### Claim 376 (depends on 373)

The method of claim 373, wherein the peptide comprises three or more disulfide bridges formed between cysteine residues, wherein one of the disulfide bridges passes through a loop formed by two other disulfide bridges.

### Claim 377 (depends on 373)

The method of claim 373, wherein the peptide comprises a structural proline amino acid residue.

### Claim 378 (depends on 373)

The method of claim 373, wherein the peptide comprises at least 3 positively charged amino acid residues.

### Claim 379 (depends on 373)

The method of claim 373, wherein the peptide comprises a sequence motif of leucine-X1-X2-leucine-phenylalanine (LX1X2LF).

### Claim 380 (depends on 373)

The method of claim 373, wherein the peptide comprises:
a) at least 90% sequence identity to SEQ ID NO: 114; or
b) at least 90% sequence identity to any one of SEQ ID NO: 110, SEQ ID NO: 140, SEQ ID NO: 89, SEQ ID NO: 85; SEQ ID: 107, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 94, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 100, SEQ ID NO: 92, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 85, SEQ ID NO: 101, SEQ ID NO: 115, SEQ ID NO: 108, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 112, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 106, or SEQ ID NO: 88.

### Claim 381 (depends on 373)

The method of claim 373, wherein the peptide achieves an average t½ of 0.1 hours-168 hours in a subject after administering the peptide to the subject.

### Claim 382 (depends on 373)

The method of claim 373, wherein the compound is administered via oral administration, inhalation, intranasal administration, topical administration, intravenous administration, subcutaneous administration, intra-articular administration, intramuscular administration, intraperitoneal administration, intra-synovial administration, vaginal administration, rectal administration, pulmonary administration, ocular administration, buccal administration, sublingual administration, intrathecal administration, or any combination thereof, to a subject.

### Claim 383 (depends on 373)

The method of claim 373, wherein the peptide is linked to an active agent.

### Claim 384 (depends on 383)

The method of claim 383, wherein the active agent is: a peptide, an oligopeptide, a polypeptide, a polynucleotide, a polyribonucleotide, a DNA, a cDNA, a ssDNA, a RNA, a dsRNA, a micro RNA, an oligonucleotide, an antibody, an antibody fragment, an aptamer, a cytokine, an enzyme, a growth factor, a chemokine, a neurotransmitter, a chemical agent, a fluorophore, a metal, a metal chelate, an X-ray contrast agent, a PET agent, a radioisotope, a photosensitizer, a radiosensitizer, a radionuclide chelator, a therapeutic small molecule, a steroid, a corticosteroid, an anti-inflammatory agent, an immune modulator, a protease inhibitor, an amino sugar, a chemotherapeutic agent, a cytotoxic chemical, a toxin, a tyrosine kinase inhibitor, an anti-infective agent, an antibiotic, an anti-viral agent, an anti-fungal agent, an aminoglycoside, a nonsteroidal anti-inflammatory drug (NSAID), a statin, a nanoparticle, a liposome, a polymer, a biopolymer, a polysaccharide, a proteoglycan, a glycosaminoglycan, a glucocorticoid, an anti-cytokine agent, a pain-reducing agent, a dendrimer, a fatty acid, an Fc region, siderocalin, or any combination thereof.

### Claim 385 (depends on 384)

The method of claim 384, wherein the steroid is triamcinolone, triamcinolone hexacetonide, budesonide, or dexamethasone.

### Claim 386 (depends on 373)

The method of claim 373, wherein administering the compound treats the subject.

### Claim 387 (depends on 373)

The method of claim 373, wherein the subject has a gastrointestinal infection or chronic gastrointestinal disease.

### Claim 388 (depends on 387)

The method of claim 387, wherein the gastrointestinal infection is a bacterial infection, prokaryotic infection, yeast infection, or fungal infection.

### Claim 389 (depends on 387)

The method of claim 387, wherein the chronic gastrointestinal disease is irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, gastroesophageal reflux disease, ulcerative colitis, or constipation.

### Claim 390 (depends on 373)

The method of claim 373, wherein the subject has a cancer, and wherein the cancer is colorectal cancer, stomach cancer, or esophageal cancer.

### Claim 391 (depends on 373)

The method of claim 373, wherein the subject has an inflammation, a cancer, a degradation, a growth disturbance, genetic, a tear, an infection, an injury, a rheumatic condition, an immune system disorder, a kidney disease, lung disease, a condition of aging, a degenerative brain condition, a degenerative body condition, a childhood condition, a hepatic disease, a pulmonary disease, a pancreatic condition, or a gastrointestinal condition.

### Claim 392 (depends on 391)

The method of claim 391, wherein the kidney disease is acute kidney injury or chronic kidney disease.

### Claim 393 (depends on 373)

The method of claim 373, wherein the peptide homes to cartilage, kidneys, proximal tubules of the kidneys, or tumors.

### Claim 394 (depends on 373)

The method of claim 373, wherein the peptide enters a cell, and wherein the peptide is active intracellularly.

### Claim 395 (depends on 373)

The method of claim 373, wherein the peptide is formulated in a pharmaceutical composition.

### Claim 396 (depends on 395)

The method of claim 395, wherein the pharmaceutical composition further includes a permeation enhancer and the permeation enhancer increases oral absorption.

### Claim 397 (depends on 396)

The method of claim 396, wherein the permeation enhancer is SNAC, 5-CNAC, sodium caprylate, an aromatic alcohol, EDTA, a sodium alkyl sulfate, or a citrate.

### Claim 398 (depends on 383)

The method of claim 383, wherein the peptide linked to the active agent brings or enters a cell and wherein the active agent is delivered to the cell or is active intracellularly.

### Claim 399 (depends on 383)

The method of claim 383, wherein the peptide linked to the active agent is formulated in a pharmaceutical formulation.

### Claim 400 (depends on 399)

The method of claim 399, wherein the pharmaceutical composition further includes a permeation enhancer and the permeation enhancer increases oral absorption.

### Claim 401 (depends on 400)

The method of claim 400, wherein the permeation enhancer is SNAC, 5-CNAC, sodium caprylate, an aromatic alcohol, EDTA, a sodium alkyl sulfate, or a citrate.

### Claim 402

A compound comprising a peptide for use in a treatment of a gastrointestinal disorder, wherein at least 70% of the peptide remains intact after exposure to pepsin at a temperature of at least 20° C. for at least 5 minutes and the peptide comprises at least 6 cysteine residues.

### Claim 403 (depends on 402)

The peptide of claim 402 having at least one of the following characteristics:
(a) at least 70% of the peptide remains intact after exposure to pepsin at a temperature of at least 23° C. for at least 5 minutes;
(b) at least 70% of the peptide remains intact after exposure to dithiothreitol (DTT) at a concentration of 5 mM and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(c) at least 70% of the peptide remains intact after exposure to reduced glutathione (GSH) at a concentration of 5 mM and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(d) at least 70% of the peptide remains intact after exposure to trypsin at a concentration of 0.5 U/ml and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(e) at least 70% of the peptide remains intact after exposure to simulated gastric fluid (SGF; pH 1.05; 2% (w/v) sodium chloride in 0.7% (v/v) hydrochloric acid) and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(f) at least 70% of the peptide remains intact after exposure to a pH of 5 and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(g) at least 70% of the peptide remains intact after passage through the mouth, stomach, small intestine, or the large intestine, as measured by tandem high performance liquid chromatography and liquid scintillation counting;
(h) at least 70% of the peptide remains intact after exposure to the combination of simulated gastric fluid (SGF; pH 1.05; 2% (w/v) sodium chloride in 0.7% (v/v) hydrochloric acid) with 0.5 U/ml pepsin, 100 mM Tris, and 10 mM DTT and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(i) at least 70% of the peptide remains intact after exposure to at least 70° C. for at least 5 minutes, as measured by high performance liquid chromatography; or
(j) at least 70% of the peptide remains intact after exposure to at least 100° C. for at least 5 minutes, as measured by high performance liquid chromatography.

### Claim 404

A compound comprising a peptide and a permeation enhancer, wherein at least 70% of the peptide remains intact after exposure to pepsin at a temperature of at least 20° C. for at least 5 minutes and the peptide comprises at least 6 cysteine residues.

### Claim 405 (depends on 404)

The peptide of claim 404 having at least one of the following characteristics:
(a) at least 70% of the peptide remains intact after exposure to pepsin at a temperature of at least 23° C. for at least 5 minutes;
(b) at least 70% of the peptide remains intact after exposure to dithiothreitol (DTT) at a concentration of 5 mM and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(c) at least 70% of the peptide remains intact after exposure to reduced glutathione (GSH) at a concentration of 5 mM and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(d) at least 70% of the peptide remains intact after exposure to trypsin at a concentration of 0.5 U/ml and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(e) at least 70% of the peptide remains intact after exposure to simulated gastric fluid (SGF; pH 1.05; 2% (w/v) sodium chloride in 0.7% (v/v) hydrochloric acid) and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(f) at least 70% of the peptide remains intact after exposure to a pH of 5 and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(g) at least 70% of the peptide remains intact after passage through the mouth, stomach, small intestine, or the large intestine, as measured by tandem high performance liquid chromatography and liquid scintillation counting;
(h) at least 70% of the peptide remains intact after exposure to the combination of simulated gastric fluid (SGF; pH 1.05; 2% (w/v) sodium chloride in 0.7% (v/v) hydrochloric acid) with 0.5 U/ml pepsin, 100 mM Tris, and 10 mM DTT and a temperature of at least 20° C. for at least 5 minutes, as measured by high performance liquid chromatography;
(i) at least 70% of the peptide remains intact after exposure to at least 70° C. for at least 5 minutes, as measured by high performance liquid chromatography; or
(j) at least 70% of the peptide remains intact after exposure to at least 100° C. for at least 5 minutes, as measured by high performance liquid chromatography.

### Claim 406 (depends on 404)

The peptide of claim 404, wherein the permeation enhancer is SNAC, 5-CNAC, sodium caprylate, an aromatic alcohol, EDTA, a sodium alkyl sulfate, or a citrate.