Cyclotides as immunosuppressive agents

• US 9,453,052 B2
• Filed: 12/21/2012
• Issued: 09/27/2016
• Est. Priority Date: 12/22/2011
• Status: Active Grant

• Current Assignee: Medizinische Uni Wien, Universitätsklinikum Freiburg
• Original Assignee: Medizinische Uni Wien, Universitätsklinikum Freiburg
• Inventors: Gruber, Christian Werner, Gruendemann, Carsten
• Primary Examiner(s): LIEB, JEANETTE M
• Application Number:
  US14/366,427
• Publication Number:
  US 20140369930A1
• Time in Patent Office: 1,376 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 38/12 Cyclic peptides , e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C A61K38/043 - A61K38/046 take precedence
• CPC Class Codes: A61K 38/16 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/168 from plants
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 51/08 Peptides, e.g. proteins , carriers being peptides, polyamino acids, proteins
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: 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

Abstract

The present invention relates to a pharmaceutical composition comprising a cyclotide for use in immune suppression as well as to a method for immune suppression comprising the step of administering an effective amount of a pharmaceutical composition comprising such a cyclotide to a subject in need thereof. The present invention also relates to a pharmaceutical composition comprising a cyclotide for use in treating or preventing a disorder selected from the group consisting of (i) an autoimmune disorder; (ii) a hypersensitivity disorder; and (iii) a lymphocyte-mediated inflammation. Likewise, the present invention also relates to a method for treating or preventing a disorder selected from the group consisting of (i) an autoimmune disorder; (ii) a hypersensitivity disorder; and (iii) a lymphocyte-mediated inflammation. The present invention further relates to a method of screening for and/or selecting an immunosuppressive cyclotide or a mutation which results in a mutated cyclotide having an induced or enhanced immunosuppressive activity. The present invention further relates to a method of producing an immunosuppressive cyclotide or an immunosuppressive pharmaceutical composition. The present invention further relates to a mutated cyclotide having immunosuppressive activity and a pharmaceutical composition comprising the same.

Claims

1. A mutated cyclotide having immunosuppressive activity, said cyclotide comprising an amino acid sequence of formula IL wherein formula II comprises:

Xxx₁-Leu-Pro-Val-Cys-Gly-Glu-Xxx₂-Cys-Xxx₃-Gly-Gly-Thr-Cys-Asn-Thr-Pro-Xxx₁-Cys-Xxx₁-Cys-Xxx₁-Trp-Pro-Xxx₁-Cys-Thr-Arg-Xxx₁ (SEQ ID NO;

     17);

wherein each Xxx₁ position independently comprises any amino acid, non-natural amino acid or peptidomimetic;

wherein Xxx₂ comprises an amino acid non-natural amino acid or peptidomimetic but not Lys;

wherein Xxx₃ comprises any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys;

wherein the mutated cyclotide comprises one or more mutations from the naturally occurring kalata B1 cyclotide (SEQ ID NO;

1),

wherein said cyclotide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO;

7, and

wherein the amino acid sequence of said cyclotide carries a mutation at least one amino acid position corresponding to an amino acid position which has been mutated in any one of the mutated cyclotides as depicted in SEQ ID NO;

5, 6 or 7.

2. The mutated cyclotide of claim 1, wherein said mutation is G→

K at position 1, T→

K at position 20, and/or N→

K at position 29.

6. The mutated cyclotide of claim 1, wherein the amino acid sequence of said cyclotide is radio labelled, fluorescence-labelled or biotin-labelled.

7. A pharmaceutical composition comprising the mutated cyclotide of claim 1 and a pharmaceutically acceptable carrier, excipient or diluent.

8. The composition of claim 7, wherein said pharmaceutical composition further comprises one or more additional immunosuppressants.

9. The composition of claim 8, wherein said additional immunosuppressant comprises Cyclosporine A, Muromonab-CD3 or Basiliximab.

24. A method of producing an immunosuppressive pharmaceutical composition comprising mixing a mutated cyclotide of claim 1 with a pharmaceutically acceptable carrier.

32. A method for immunosuppression, the method comprising administering an effective amount of a mutated cyclotide of claim 1 to a subject in need thereof.

33. A method for immunosuppression, the method comprising administering an effective amount of a mutated cyclotide of claim 2 to a subject in need thereof.

3. A cyclotide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:

5, 6, and 7.

34. A method for immunosuppression, the method comprising administering an effective amount of a cyclotide of claim 3 to a subject in need thereof.

4. A mutated cyclotide having immunosuppressive activity, said cyclotide comprising an amino acid sequence of formula IL wherein formula II comprises:

Xxx₁-Leu-Pro-Val-Cys-Gly-Glu-Xxx₂-Cys-Xxx₃-Gly-Gly-Thr-Cys-Asn-Thr-Pro-Xxx₁-Cys-Xxx₁-Cys-Xxx₁-Trp-Pro-Xxx₁-Cys-Thr-Arg-Xxx₁ (SEQ ID NO;

17);

wherein each Xxx₁ position independently comprises any amino acid, non-natural amino acid or peptidomimetic;

wherein Xxx₂ comprises an amino acid non-natural amino acid or peptidomimetic but not Lys;

wherein Xxx₃ comprises any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys;

wherein the cyclotide comprises one or more mutations from the naturally occurring kalata B1 cyclotide (SEQ ID NO;

1),

wherein the cyclotide comprises an amino acid sequence having one or more of amino residues at amino acid positions 1, 18, 20, 22, 25, and/or 29 of SEQ ID NO;

17 representing a different amino acid from the same position of SEQ ID NO;

1, and

wherein said at least one amino acid position corresponds to;

(i) amino acid position 29 of the mutated cyclotide SEQ ID NO;

5;

(ii) amino acid position 1 of the mutated cyclotide SEQ ID NO. 6;

(iii) amino acid position 20 of the mutated cyclotide SEQ ID NO. 6;

or

(iv) amino acid position 20 of the mutated cyclotide as depicted in SEQ ID NO;

7.

35. A method for immunosuppression, the method comprising administering an effective amount of a mutated cyclotide of claim 4 to a subject in need thereof.

5. A mutated cyclotide having immunosuppressive activity, said cyclotide comprising an amino acid sequence of formula IL wherein formula II comprises:

Xxx₁-Leu-Pro-Val-Cys-Gly-Glu-Xxx₂-Cys-Xxx₃-Gly-Gly-Thr-Cys-Asn-Thr-Pro-Xxx₁-Cys-Xxx₁-Cys-Xxx₁-Trp-Pro-Xxx₁-Cys-Thr-Arg-Xxx₁ (SEQ ID NO;

     17);

wherein each Xxx₁ position independently comprises any amino acid, non-natural amino acid or peptidomimetic;

wherein Xxx₂ comprises an amino acid non-natural amino acid or peptidomimetic but not Lys;

wherein Xxx₃ comprises any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys; and

wherein the mutated cyclotide comprises one or more mutations from the naturally occurring kalata B1 cyclotide (SEQ ID NO;

1), and

wherein said mutation is G→

K, T→

K, S→

K, N→

K, or D→

K.

36. A method for immunosuppression, the method comprising administering an effective amount of a mutated cyclotide of claim 5 to a subject in need thereof.

10. A method for immunosuppression, the method comprising administering an effective amount of a non-grafted mutated cyclotide to a subject in need thereof, said cyclotide comprising an amino acid sequence of formula IL wherein formula II comprises:

Xxx₁-Leu-Pro-Val-Cys-Gly-Glu-Xxx₂-Cys-Xxx₃-Gly-Gly-Thr-Cys-Asn-Thr-Pro-Xxx₁-Cys-Pro-Xxx₁-Cys-Thr-Arg-Xxx₁ (SEQ ID NO;

     17);

wherein each Xxx₁ position independently comprises any amino acid, non-natural amino acid or peptidomimetic;

wherein Xxx₂ comprises any amino acid, non-natural amino acid or peptidomimetic but not Lys;

wherein Xxx₃ comprises any amino acid, non-natural amino acid or peptidomimetic but not Ala or Lys; and

wherein the mutated cyclotide comprises one or more mutations from the naturally occurring kalata B1 cyclotide (SEQ ID NO;

1).

11. The method of claim 10, wherein said mutation is X→

K, wherein X is any amino acid.

12. The method of claim 10, wherein said cyclotide has an anti-proliferative effect on (an) immune cell(s) and/or suppresses/reduces the effector function(s) of (an) immune cell(s).

13. The method of claim 10, wherein said cyclotide is administered so that cytostatic but no cytotoxic activity occurs.

14. The method of claim 10, wherein said cyclotide is administered in an amount to reach a serum concentration in the range of 1 to 50 μ

M.

15. The method of claim 10, wherein said pharmaceutical composition further comprises one or more additional immunosuppressants.

16. The method of claim 15, wherein said additional immunosuppressant comprises Cyclosporine A, Muromonab-CD3 or Basiliximab.

17. The method of claim 10, for the treatment of a subject suffering from a disorder selected from the group consisting of an autoimmune disorder, a hypersensitivity disorder, and a lymphocyte-mediated inflammation.

18. The method of claim 17, wherein said autoimmune disorder is selected from the group consisting of Multiple Sclerosis, Psoriasis, Systemic Lupus Erythematosus, Sjö

gren'"'"'s syndrome, Rheumatoid Arthritis, Idiopathic Thrombocytopenic Purpura, Diabetes, Vasculitis, and Crohn'"'"'s disease.

19. The method of claim 17, wherein said lymphocyte-mediated inflammation comprises a T cell-mediated inflammation.

20. The method of claim 17, wherein said lymphocyte-mediated inflammation comprises Keratoconjunctivitis sicca or Dry Eye Syndrome (DES).

21. The method of claim 10, wherein:

the proliferation of (an) immune cell(s);

the effector function(s) of (an) immune cell(s);

the degranulation/cytotoxicity of (an) immune cell(s);

the expression of a cytokine surface receptor on (an) immune cell(s);

the proliferation of (primary) activated lymphocytes;

the proliferation of peripheral blood mononuclear cells (PBMC);

secretion/production of IL-2, IFN-gamma and/or TNF-alpha;

degranulation/cytotoxicity of CD107a+ CD8+ PBMCs; and

/or

expression of IL-2 surface receptor CD25 is/are suppressed.

22. The method of claim 10, wherein said cyclotide

suppresses/reduces secretion/production of IL-2, IFN-gamma and/or TNF-alpha;

suppresses/reduces degranulation/cytotoxicity of CD107a+ CD8+ PBMCs; and

/or

suppresses/reduces expression of IL-2 surface receptor CD25.

23. The method of claim 10, wherein the anti-proliferative effect or suppression/reduction is mediated in an IL-2-, IFN-gamma- and/or TNF-alpha-depending manner and/or can be antagonized by IL-2.

25. The method of claim 10, said cyclotide comprising an amino acid sequence having one or more of amino residues at amino acid positions 1, 18, 20, 22, 25, and/or 29 of SEQ ID NO:

17 representing a different amino acid from the same position of SEQ ID NO;

1.

26. The method of claim 10, said cyclotide comprising an amino acid sequence that is:

at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs;

5, 6, or 7;

or

at least 90% identical to an amino acid sequence encoded by a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs;

5, 6, or 7;

wherein the amino acid sequence of said cyclotide carries a mutation at least one amino acid position corresponding to an amino acid position which has been mutated in any one of the mutated cyclotides as depicted in SEQ ID NO;

5, 6 or 7.

27. The method of claim 10, said cyclotide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO:

7.

28. The method of claim 10, said cyclotide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence SEQ ID NO:

7.

29. The method of claim 14, wherein said cyclotide is administered in an amount to reach a serum concentration in the range of 3 to 10 μ

M.

30. The method of claim 14, wherein said cyclotide is administered in an amount to reach a serum concentration in the range of 4 to 9 μ

M.

31. The method of claim 14, wherein said cyclotide is administered in an amount to reach a serum concentration in the range of 5 to 9 μ

M.