# US 10,500,287 B2 · Amphipathic peptide

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- Number: US 10,500,287 B2
- Document ID: US10500287B2
- Status: Granted
- Kind: B2
- Application number: 16/215,183
- Filed: 2018-12-10
- Published: 2019-03-28
- Granted: 2019-12-10
- Earliest claimed: 2012-12-07
- Assignees: The Queen's University of Belfast
- Applicants: The Queen's University of Belfast
- Inventors: Helen McCarthy; Aleksey Zholobenko; Ashley Davison; Tracy Robson
- Web page: https://patentgrep.com/patent/US10500287B2

## CPC

- 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)
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 33/242: Gold; Compounds thereof
- A61K 33/42: Phosphorus; Compounds thereof
- A61K 38/44: Oxidoreductases (1)
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/6455: {Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids}
- A61K 47/6929: {the form being a nanoparticle, e.g. an immuno-nanoparticle}
- A61K 48/0025: {wherein the non-active part clearly interacts with the delivered nucleic acid}
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/001: {by chemical synthesis}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C07K 2319/00: Fusion polypeptide
- C07K 2319/09: containing a nuclear localisation signal
- C07K 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/3513: Protein; Peptide
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12Y 114/13039: Nitric-oxide synthase (NADPH dependent) (1.14.13.39)
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The present invention is directed to an amphipathic peptide and methods of using the amphipathic peptide for delivering small molecule agents to a cell. Ideally, the amphipathic cell penetrating peptide comprises less than approximately 50 amino acid residues with at least 6 arginine residues, at least 12 Alanine Residues, at least 6 leucine residues, optionally at least one cysteine residue, and at least two but no greater than three glutamic acids wherein the arginine residues are evenly distributed along the length of the peptide; and the peptide has a defined ratio of arginine to negatively charged amino acid residues and a defined ratio of hydrophilic amino acid residues to hydrophobic amino acid residues. The present invention is also directed to a nanoparticle and cell delivery system comprising the amphipathic cell penetrating peptide of the invention. The peptide, nanoparticle or cell delivery system of the invention may be used in therapy. For example, the peptide may be used as a therapeutic agent delivery system, in which the therapeutic agent may include nucleic acids or other small molecules.

## Claims

### Claim 1

A nanoparticle comprising an amphipathic cell penetrating peptide of less than 50 amino acid residues comprising at least 6 arginine residues (R), at least 12 alanine residues (A), at least 6 leucine residues (L), at least one cysteine residue (C), and at least two but no greater than three glutamic acids (E) wherein
the arginine (R) residues are evenly distributed along the length of the peptide;
the ratio of arginine (R) to negatively charged glutamic acid (E) residues is from at least 6:2 to 8:2; and
the ratio of hydrophilic amino acid residues to hydrophobic amino acid residues at pH 7 is at least 30:70 to 40:60;
wherein said peptide is complexed with a nucleic acid or other agent.

### Claim 2 (depends on 1)

The nanoparticle according to claim 1 wherein the arginine (R) residues are evenly distributed at every third or fourth amino acid position along the entire length of the peptide.

### Claim 3 (depends on 1)

The nanoparticle according to claim 1 wherein the peptide comprises a consensus sequence selected from EARLARALARALAR (SEQ ID No. 15) and LARALARALRA (SEQ ID No. 16).

### Claim 4 (depends on 1)

The nanoparticle according to claim 1 wherein the peptide comprises or consists of the amino acid sequence X-EARLARALARALAR-Y-LARALARALRA-Z-EA (SEQ ID No. 17), wherein
X is W or R;
Y is optional or selected from H or E; and
Z is C or R;
or a sequence with at least 80% sequence identity or homology.

### Claim 5 (depends on 1)

The nanoparticle according to claim 1 wherein the peptide comprises or consists of one of the following amino acid sequences:
(SEQ ID No. 1)
WEARLARALARALARHLARALARALRACEA;
(SEQ ID No. 2)
WEARLARALARALARLARALARALRACEA;
(SEQ ID No. 5)
REARLARALARALARLARALARALRACEA;
or a fragment thereof.

### Claim 6 (depends on 1)

The nanoparticle according to claim 1 wherein the peptide comprises or consists of:
WEARLARALARALARHLARALARALRACEA (SEQ ID No. 1);
or a fragment thereof.

### Claim 7 (depends on 1)

The nanoparticle according to claim 1 wherein the nucleic acid is one or more of DNA, RNA, shRNA, and siRNA.

### Claim 8 (depends on 7)

The nanoparticle according to claim 7 wherein the siRNA or shRNA inhibits the expression of a disease causing gene.

### Claim 9 (depends on 7)

The nanoparticle according to claim 7 wherein the DNA is inducible nitric oxide synthase (iNOS) plasmid DNA under the control of a tumour specific promoter.

### Claim 10 (depends on 9)

The nanoparticle according to claim 9 wherein the tumour specific promoter is selected from the human osteocalcin (hOC) promoter, osteopontin promoter, WAF1, CARG and a prostate membrane specific antigen promoter.

### Claim 11 (depends on 1)

The nanoparticle according to claim 1 wherein the other agent is a small molecule agent.

### Claim 12 (depends on 11)

The nanoparticle according to claim 11 wherein the small molecule agent is a phosphate based drug selected from alendronate, etidronate, zolendrate or any other nitrogen or non-nitrogen based bisphosphonate drug.

### Claim 13 (depends on 1)

The nanoparticle according to claim 1 wherein the other agent is gold.

### Claim 14 (depends on 1)

The nanoparticle of claim 1 complexed with a nucleic acid to form discrete spherical nanoparticles, each nanoparticle with a diameter less than 150 nm.

### Claim 15 (depends on 1)

The nanoparticle of claim 1 complexed with a nucleic acid selected from DNA or siRNA to form discrete spherical nanoparticles, each nanoparticle with a diameter less than 150 nm.

### Claim 16 (depends on 1)

The nanoparticle of claim 1 complexed with a nucleic acid to form discrete spherical nanoparticles, each nanoparticle with a diameter less than or equal to 100 nm.

### Claim 17 (depends on 1)

The nanoparticle of claim 1 complexed with a nucleic acid selected from DNA or siRNA to form discrete spherical nanoparticles, each nanoparticle with a diameter less than or equal to 100 nm.

### Claim 18 (depends on 1)

The nanoparticle of claim 1 wherein the other agent is a negatively charged or hydrophilic compound.

### Claim 19 (depends on 1)

A pharmaceutical composition comprising the nanoparticle of claim 1 and a suitable pharmaceutical excipient.

### Claim 20 (depends on 1)

A method of (i) treating an individual in need of therapy, (ii) improving the bioavailability of a phosphate based drug or (iii) improving the cellular uptake of gold comprising the administration of the nanoparticle of claim 1, when the peptide is complexed with (i) a nucleic acid or other agent, (ii) a phosphate based drug or (iii) gold, to an individual in need thereof.

### Claim 21 (depends on 20)

The method of claim 20 wherein said therapy is gene therapy.

### Claim 22 (depends on 1)

A method for the delivery of a nucleic acid or other agent, wherein the other agent is a negatively charged or hydrophilic compound, to a cell comprising providing and administering the nanoparticle according to claim 1 to said cell.