# US 7,745,390 B2 · Antimicrobial peptides

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- Number: US 7,745,390 B2
- Document ID: US7745390B2
- Status: Granted
- Kind: B2
- Application number: 11/438,563
- Filed: 2006-05-22
- Published: 2006-12-21
- Granted: 2010-06-29
- Earliest claimed: 2005-05-23
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Carol Clayberger; Alan M. Krensky
- Web page: https://patentgrep.com/patent/US7745390B2

## CPC

- C07K 14/47: from mammals
- C07K 7/06: having 5 to 11 amino acids
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 14/4723: {Cationic antimicrobial peptides, e.g. defensins}
- 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)
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Artificial antimicrobial peptides are obtained by alterations in alpha helical portions of a known antimicrobial protein, granulysin. The peptides obtained have significantly improved antimicrobial activity and lack the ability to lyse mammalian cells, which may be toxic to a host. The peptides may be designed according to certain guidelines, and may further be modified by the addition of altered residues and alterations in normal peptide secondary and tertiary structure, including modifications in quaternary (multimeric) structure.

## Claims

### Claim 1

An artificial antimicrobial peptide having:
at least 90% sequence identity to peptide G11.46 (SEQ ID NO: 93), G12.21 (SEQ ID NO: 64) or G14.15 (SEQ ID NO: 83), but further having at least one non-identical amino acid when compared to SEQ ID NO: 2, an MIC against at least one of a pathogenic bacterium selected from the group consisting of E. coli, S. typhimurium and B. cereus of less than 10 μM; and
having minimal lytic activity against mammalian cells.

### Claim 2 (depends on 1)

The artificial antimicrobial peptide of claim 1 further comprised in a pharmaceutically acceptable composition.

### Claim 3 (depends on 2)

The pharmaceutical preparation of claim 2 further comprising a sterile lyophilized powder.

### Claim 4 (depends on 1)

The artificial antimicrobial peptide of claim 1 having a net positive charge of at least 8.

### Claim 5 (depends on 1)

The artificial peptide of claim 1 having more D-amino acids than L-amino acids.

### Claim 6 (depends on 1)

The artificial peptide of claim 1 having at least 3 hydrophobic residues.

### Claim 7

An artificial antimicrobial peptide selected from the group consisting of:
G8.17, (SEQ ID NO: 9), G8.3, (SEQ ID NO: 12), G8.15, (SEQ ID NO: 13), G8.11, (SEQ ID NO: 18), G11.6, (SEQ ID NO: 16), G11.3, (SEQ ID NO: 26), G11.24, (SEQ ID NO: 26), G11.30, (SEQ ID NO: 39), G11.32, (SEQ ID NO: 41), G11.34, (SEQ ID NO: 26), G11.35, (SEQ ID NO: 48), G11.55, (SEQ ID NO: 35), G11.46, (SEQ ID NO: 93), G12.5, (SEQ ID NO: 51), G12.10, (SEQ ID NO: 61), G12.19, (SEQ ID NO: 65), G12.20, (SEQ ID NO: 66), G12.21, (SEQ ID NO: 64), G14.2, (SEQ ID NO: 81), G14.10, (SEQ ID NO: 81), G14.11, (SEQ ID NO: 83), and G14.15 (SEQ ID NO: 83), wherein said peptide has an MIC (μM) in at least two microbial organisms selected from the group consisting of: E. coli, S. typhimurium, V. cholerae and B. cereus of less than about 2.0, and further has minimal lysis of mammalian cells.

### Claim 8 (depends on 7)

The peptide of claim 7 wherein said peptide is selected from: G8.3, (SEQ ID NO: 12), G8.15, (SEQ ID NO: 13), G 8.11, (SEQ ID NO: 18), G11.46, (SEQ ID NO: 93), G12.21 (SEQ ID NO: 64), and G14.15 (SEQ ID NO: 83).

### Claim 9 (depends on 7)

The peptide of claim 7 selected from the group consisting of G11.46, (SEQ ID NO: 93), G12.21 (SEQ ID NO: 64), and G14.15 (SEQ ID NO: 83).

### Claim 10 (depends on 7)

The peptide of claim 7 comprising at least 50% D-amino acids.

### Claim 11 (depends on 7)

The peptide of claim 7 comprising the sequence arg-arg-val-ser-arg-arg (SEQ ID NO: 92).

### Claim 12 (depends on 7)

The peptide of claim 7 comprising at least 50% D-amino acids.

### Claim 13 (depends on 7)

The peptide of claim 7 comprising the sequence arg-arg-val-ser-arg-arg (SEQ ID NO: 92).

### Claim 14 (depends on 7)

A method of inhibiting microbial growth in an infected subject, comprising administering to said subject a composition comprising a peptide having a formula according to claim 7.

### Claim 15 (depends on 4)

A method of inhibiting microbial growth in an infected subject, comprising administering to said subject a composition comprising a peptide having a formula according to claim 4.

### Claim 16 (depends on 1)

A method for treating bacterial or fungal infection, comprising administering to a living body in need of such treatment a therapeutically effective amount of a peptide according to claim 1.

### Claim 17

A method for preparing an antimicrobial peptide, comprising:
(a) selecting a sequence of between 10 and 20 residues comprised in SEQ ID NO: 2;
(b) modifying the sequence by increasing a selected number of charged residues to be at least 8 residues;
(c) modifying the sequence to include at least one D-amino acid;
(d) preparing the peptide;
(e) testing the peptide for antimicrobial activity against at least 3 microorganisms and selecting only a peptide with an MIC less than 1.0 μM against at least one of the microorganisms, and selecting as the antimicrobial peptide one having at least 90% sequence identity to peptide G11.46, (SEQ ID NO: 93), G12.21 (SEQ ID NO: 64), or 90% sequence identity to G14.15 (SEQ ID NO: 83), having at least one non-identical amino acid when compared to SEQ ID NO: 2 and having minimum lytic activity against mammalian cells.

### Claim 18 (depends on 17)

The method of claim 17 further comprising the step of preparing a dendrimer containing the peptide.

### Claim 19 (depends on 17)

The method of claim 17 further comprising the step of including at least one chemically modified amino acid.

### Claim 20 (depends on 1)

The peptide of claim 1 further comprised in a dendrimer.

### Claim 21 (depends on 1)

The peptide of claim 1 comprising at least 7 arg residues.

### Claim 22 (depends on 1)

The peptide of claim 1 further comprising at least one chemically modified amino acid.

### Claim 23 (depends on 1)

The peptide of claim 1 wherein the MIC against at least one of a pathogenic bacterium is less than 1 μM.