Intracellular delivery of biological effectors

• US 20030032594A1
• Filed: 06/07/2002
• Published: 02/13/2003
• Est. Priority Date: 10/13/2000
• Status: Active Grant

• Current Assignee: Xigen SA
• Original Assignee: Xigen SA
• Inventors: Bonny, Christophe
• Granted Patent: US 7,033,597 B2
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: 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
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 5/1013 the side chain containing O or S as heteroatoms, e.g. Cys, Ser
• CPC Class Codes: C07K 5/1016 and aromatic or cycloaliphatic
• CPC Class Codes: C07K 5/1019 with the first amino acid being basic
• CPC Class Codes: C07K 5/1021 with the first amino acid being acidic
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 : having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: Y10S 530/81 : Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier

Abstract

The invention relates to sequences of amino acids with the capacity to facilitate transport of an effector across a biological membrane. More specifically, the present invention relates to novel peptide transporters that specifically target certain cell types for the intracellular delivery of drugs and therapeutic agents.

Claims

1. A multimeric transporter peptide according to Formula I,

[P]_v−

[Spacer]_x−

Core−

[Linker]_y−

[Reporter]_z 

 

(I)

wherein P is a transporter peptide, comprising at least one amino acid sequence selected from the group consisting of;

a) (X_mRX_oRX_n);

b) (X_mRRRX_n);

c) (X_mRRXRX_n); and

d) (X_mRXRRX_n), wherein

X is a non-basic amino acid;

m is an integer from zero to fourteen;

n is an integer, independent of m, between zero and fourteen;

o is an integer, independent of m and n, between zero and five;

v is an integer from two to eight;

x, y and z, are, independently, zero or one; and

wherein each P can be the same or different, and said transporter peptide is capable of translocating across a biological membrane.

2. The transporter peptide of claim 1, wherein at least one P has the amino acid sequence R-X-X-R.

3. The transporter peptide of claim 1, wherein each P is selected from the group consisting of SEQ ID NOs:

1-34.

4. The transporter peptide of claim 1, wherein v is selected from the group consisting of two, four, six, and eight.

5. The transporter peptide of claim 4, wherein v is four.

6. The transporter peptide of claim 4, wherein v is eight.

7. The transporter peptide of claim 1, wherein the Spacer is succinimidyl-polyethylene glycol.

8. The transporter peptide of claim 1, wherein the Core is selected from the group consisting of C₄-K₂K-K(succ-peg-S)-aimide;

C-GGG-[K(C)]₃-K(succ-peg-S)-amide;

(NH₂OCH₂CO)₄-K₂K-K(succ-peg)-amide; and

(NH₂OCH₂CO)₈-K₄K₂K-K(GGG)-amide.

9. A transporter unit comprising the transporter peptide of claim 1 conjugated to an effector.

36. A method of producing a translocatable conjugate between the transporter peptide of claim 1 and an effector, said method comprising conjugating said effector to said transporter peptide to form a transporter peptide-effector conjugate.

37. A method of translocating an effector into the cytoplasm and nucleus of a eukaryotic cell, said method comprising:

conjugating said effector to the transporter peptide of claim 1 to form a transporter peptide-effector conjugate; and

introducing said transporter peptide-effector conjugate to the cell.

38. The method of claim 37, wherein the introducing step is achieved by incubating a cell culture in the presence of said transporter peptide-effector conjugate or by injecting said transporter peptide-effector conjugate into the cell.

39. The method of claim 37, wherein the eukaryotic cell is a human cell.

40. The method of claim 37, wherein the eukaryotic cell is a β

-cell.

41. A method of increasing the intracellular concentration of an effector within a eukaryotic cell, said method comprising;

conjugating said effector to the transporter peptide of claim 1 to form a transporter peptide-effector conjugate;

incubating said cell in the presence of said transporter peptide-effector conjugate, under conditions promoting active metabolism of said eukaryotic cell.

42. The method of claim 41, wherein the eukaryotic cell is a human cell.

10. A transporter unit of Formula II:

([P]_v−

[Spacer]_x−

Core−

[Linker]_y−

[Reporter]_z)·

E 

 

(II)

wherein;

E is an effector;

P is a transporter peptide, comprising at least one amino acid sequence selected from the group consisting of;

e) (X_mRX_oRX_n);

f) (X_mRRRX_n);

g) (X_mRRXRX_n); and

h) (X_mRXRRX_n), wherein

X is a non-basic amino acid;

m is an integer from zero to fourteen;

n is an integer, independent of m, between zero and fourteen;

o is an integer, independent of m and n, between zero and five;

v is an integer from two to eight;

x, y and z, are, independently, zero or one; and

wherein each P can be the same or different, and wherein said transporter peptide is capable of translocating across a biological membrane.

11. The transporter unit of claim 10, wherein at least one P has the amino acid sequence R-X-X-R.

12. The transporter unit of claim 10, wherein each P is selected from the group consisting of SEQ ID NOs:

1-34.

13. The transporter unit of claim 10, wherein v is selected from the group consisting of two, four, six, and eight.

14. The transporter unit of claim 13, wherein v is four.

15. The transporter unit of claim 13, wherein v is eight.

16. The transporter unit of claim 10, wherein the core is selected from the group consisting of C₄-K₂K-K(succ-peg-S)-amide;

C-GGG-[K(C)]₃-K(succ-peg-S)-amide;

(NH₂OCH₂CO)₄-K₂K-K(succ-peg)-amide; and

(NH₂OCH₂CO)₈-K₄K₂K-K(GGG)-amide.

17. The transporter unit of claim 10, wherein the effector is covalently fused to the linker.

18. The transporter unit of claim 10, wherein the effector is selected from the group consisting of:

a nucleic acid;

a peptide; and

a pharmaceutically active agent.

19. The transporter unit of claim 18, wherein said effector is a nucleic acid.

20. The transporter unit of claim 19 wherein said nucleic acid is DNA.

21. The transporter unit of claim 19, wherein said nucleic acid is RNA.

22. The transporter unit of claim 18, wherein said effector is a peptide.

23. The transporter unit of claim 18, wherein said effector is a pharmaceutically active agent.

24. The transporter unit of claim 23, wherein said pharmaceutically active agent is selected from the group consisting of:

a toxin, an antibiotic, an antipathogenic agent, an antigen, an antibody fragment, an immunomodulator, an enzyme, and a therapeutic agent.

25. The transporter unit of claim 10 wherein each peptide is less than 50 amino acids long.

26. The transporter unit of claim 10 wherein each peptide is less than 25 amino acids long.

27. The transporter unit of claim 10 wherein each peptide is less than 15 amino acids long.

28. The transporter unit of claim 10 wherein translocation occurs within a tissue selected from the group consisting of:

pancreatic B-cells;

hepatocytes;

colon cells;

muscle cells; and

lung cells.

29. A method of translocating the transporter unit of claim 12 across the membrane of pancreatic β

-cells, wherein at least one transporter peptide is selected from the group consisting of SEQ ID NOS;

1-6.

30. The method of claim 29, wherein at least one transporter peptide is SEQ ID NO:

1.

31. A method of translocating the transporter unit of claim 12 across the membrane of hepatocytes, wherein at least one peptide is selected from the group consisting of SEQ ID NOS:

7-10.

32. A method of translocating the transporter unit of claim 12 across the membrane of colon cells, wherein at least one peptide is SEQ ID NO:

11.

33. A method of translocating the transporter unit of claim 12 across the membrane of muscle cells, wherein at least one peptide is selected from the group consisting of SEQ ID NOS:

12-20.

34. A method of translocating the transporter unit of claim 12 across the membrane of lung cells, wherein at least one peptide is selected from the group consisting of SEQ ID NOS:

21-34.

35. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the transporter unit according to claim 10, and a pharmaceutically acceptable carrier.

43. A kit comprising in one or more containers, a therapeutically or prophylactically effective amount of the pharmaceutical composition of claim 35.

44. A method of treating or preventing a disease, said method comprising administering to a subject in which such treatment or prevention is desired the pharmaceutical composition of claim 35, in an amount sufficient to treat or prevent said disease in said subject.

45. The method of claim 44, wherein said disease is selected from a group consisting of:

diabetes, colon cancer, respiratory ailments, neurodegenerative disorders, cardiplegia, and viral infections.

46. A multimeric transporter peptide, wherein each transporter peptide amino acid sequence is selected from the group consisting of SEQ ID NOS:

1-34.