# US 2010/0286367 A1 · Conjugates of soluble peptidic compounds with membrane-binding agents

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- Number: US 2010/0286367 A1
- Document ID: US20100286367A1
- Status: Published application
- Kind: A1
- Application number: 12/192,560
- Filed: 2008-08-15
- Published: 2010-11-11
- Earliest claimed: 1996-07-15
- Assignees: AdProTech Limited
- Inventors: Richard Anthony Godwin SMITH; Ian Dodd; Danuta Ewa Irena Mossakowkska
- Web page: https://patentgrep.com/patent/US20100286367A1

## CPC

- C12N 9/6462: {u-Plasminogen activator (3.4.21.73), i.e. urokinase}
- A61K 47/54: the modifying agent being an organic compound
- 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 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- C07C 323/41: Y being a hydrogen or an acyclic carbon atom
- C07C 323/59: with acylated amino groups bound to the carbon skeleton
- C07K 14/3153: {Streptokinase}
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C12Y 304/21073: u-Plasminogen activator (3.4.21.73), i.e. urokinase
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- 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)
- C12Y: ENZYMES

## Abstract

The present invention provides, among other things, soluble derivatives of soluble polypeptides that incorporate membrane binding elements. Methods of making these soluble derivatives, and methods of using these soluble derivatives also are provided.

## Claims

### Claim at position 1

1-49. (canceled)

### Claim 50

A membrane localization reagent for directing a molecule to an outer membrane of a cell, wherein the membrane localization reagent is soluble and comprises:
(a) at least one lipophilic binding element comprising aliphatic acyl groups;
(b) a hydrophilic peptide binding element comprising basic amino acids, wherein the hydrophilic binding element is bound to the lipophilic element; and
(c) a linker that covalently binds (i) the molecule to be directed to the outer membrane of a cell to (ii) the hydrophilic peptide binding element of the membrane localization reagent.

### Claim 51 (depends on 50)

The membrane localization reagent of claim 50, wherein the hydrophilic peptide binding element comprises lysine residues.

### Claim 52 (depends on 51)

The membrane localization reagent of claim 51, wherein the hydrophilic peptide binding element comprises three to ten lysine residues.

### Claim 53 (depends on 51)

The membrane localization reagent of claim 51, wherein the hydrophilic peptide binding element comprises four to seven lysine residues.

### Claim 54 (depends on 50)

The membrane localization reagent of claim 50, wherein the hydrophilic peptide binding element comprises arginine residues.

### Claim 55 (depends on 54)

The membrane localization reagent of claim 54, wherein the hydrophilic peptide binding element comprises three to ten arginine residues.

### Claim 56 (depends on 55)

The membrane localization reagent of claim 55, wherein the hydrophilic peptide binding element comprises four to seven arginine residues.

### Claim 57

A membrane localization reagent for directing a molecule to an outer membrane of a cell, wherein the membrane localization reagent is soluble and comprises a hydrophilic peptide binding element, wherein the peptide comprises a sequence selected from the group consisting of:
(a) DGPKKKKKKSPSKSSG;
(SEQ ID NO. 8)
(b) GSSKSPSKKKKKKPGD;
(SEQ ID NO. 9)
(c) SPSNETPKKKKKRFSFKKSG;
(SEQ ID NO. 10)
(d) DGPKKKKKKSPSKSSK;
(SEQ ID NO. 11)
and
(e) SKDGKKKKKKSKTK.
(SEQ ID NO. 12)

### Claim 58 (depends on 57)

The membrane localization reagent of claim 57, wherein the hydrophilic peptide binding element comprises GSSKSPSKKKKKKPGD (SEQ ID NO. 9).

### Claim 59 (depends on 50)

The membrane localization reagent of claim 50, wherein the lipophilic binding element comprises 8 to 18 methylene units.

### Claim 60 (depends on 59)

The membrane localization reagent of claim 59, wherein the lipophilic binding element comprises 10 to 14 methylene units.

### Claim 61 (depends on 58)

The membrane localization reagent of claim 58, wherein the membrane localization reagent comprises a lipophilic binding element, and wherein the lipophilic binding element comprises myristoyl.

### Claim 62 (depends on 60)

The membrane localization reagent of claim 60, wherein the lipophilic binding element comprises myristoyl.

### Claim 63 (depends on 50)

The membrane localization reagent according to claim 50, wherein the linker is selected from the group consisting of a cysteine residue; an N-haloacetyl group (where halo signifies chlorine, bromine or iodine); a haloacetyl group (where halo signifies chlorine, bromine or iodine) at an 6-amino group of a lysine residue; a bond; an amide group at the C-terminus; an N-terminal blocking group; and a fatty acid N-acyl group at the N-terminus or at an E-amino group of a lysine residue.

### Claim 64 (depends on 50)

The membrane localization reagent according to claim 50, wherein the molecule is a therapeutic agent.

### Claim 65

The soluble compound according to 50, wherein the linker is selected from the group consisting of a cysteine residue; an N-haloacetyl group (where halo signifies chlorine, bromine or iodine); a haloacetyl group (where halo signifies chlorine, bromine or iodine) at an ε-amino group of a lysine residue; a bond; an amide group at the C-terminus; an N-terminal blocking group; and a fatty acid N-acyl group at the N-terminus or at an ε-amino group of a lysine residue.

### Claim 66

A soluble derivative of a soluble polypeptide, wherein said derivative consisting of a conjugate or fusion of the polypeptide with a unit further consisting of two or more covalently linked heterologous membrane binding elements which elements are not all identical, which are capable of interacting independently with components of cellular or artificial membranes exposed to extracellular fluids and which are selected from the group consisting of:
a) a membrane-insertive hydrophobic group selected from aliphatic acyl groups with between 8 and 18 methylene units and aliphatic amines with between 8 and 18 methylene units;
b) a membrane-associative charged peptide consisting of between 3 and 25 amino acids of which no less than 2 and no more than 12 are basic residues; and
c) a membrane component ligand further consisting of a peptide containing between 3 and 13 amino acids derived from known ligands for membrane components such as transmembrane proteins and identified by screening of chemical, bacteriophage, or other displayed libraries against specific membrane-derived molecular targets.

### Claim 67 (depends on 66)

A soluble derivative of a soluble polypeptide according to claim 66, wherein the basic residues comprise lysine.

### Claim 68 (depends on 66)

A soluble derivative of a soluble polypeptide according to claim 66, wherein the membrane-associative charged peptide comprises basic amino acids; and other amino acids selected from the group consisting of Serine, Threonine, Glycine, Proline, Alanine, Cysteine, Aspartic Acid, Glutamic Acid, Asparagine, and Glutamine.

### Claim 69 (depends on 66)

A soluble derivative of a soluble polypeptide according to claim 66, wherein the membrane-associative charged peptide comprises lysine.