Receptor(SSTR4)-selective somatostatin analogs

• US 20050245438A1
• Filed: 01/24/2005
• Published: 11/03/2005
• Est. Priority Date: 07/24/2002
• Status: Active Grant

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Riek, Roland, Reubi, Jean Claude, Rivier, Jean E. F., Erchegyi, Judit
• Granted Patent: US 7,238,775 B2
• Time in Patent Office: Days
• US Class Current: 530/350
• 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: C07K 14/655 Somatostatins

Abstract

Analogs of SRIF which are selective for SSTR4 in contrast to the other cloned SRIF receptors are useful in determining tissue and cellular expression of the receptor SSTR4 and its biological role in regulating tumor growth. SRIF analog peptides, such as des-AA^{1,2,4,5,12,13}[Ala⁷]-SRIF; des-AA^{1,2,4,5,12,13}[Aph⁷]-SRIF, des-AA^{1,2,4,5,12,13}[Aph⁷]Cbm-SRIF; des-AA^{1,2,4,5,12,13}[Tyr²,Ala⁷]-Cbm-SRIF, and des-AA^{1,2,4,5,12,13}[Tyr⁷,C^βMe-L-2Nal⁸]-SRIF, and counterparts incorporating D-Cys³ and/or D-Trp⁸ and/or Ala¹¹, bind with high affinity to the cloned human receptor SSTR4 and activate the receptor, but they do not bind with significant affinity to human SSTR1, SSTR2, SSTR3 or SSTR5. By incorporating an iodinated tyrosine in position-2 in these SSTR4-selective SRIF analogs, a labeled compound useful in drug-screening methods is provided. Alternatively, for use in therapy, cytotoxins or highly radioactive elements can be N-terminally coupled or complexed thereto.

Claims

1. A cyclic somatostatin (SRIF) analog peptide which selectively binds the SRIF receptor SSTR4, which peptide comprises the amino acid sequence (cyclo)Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Phe-Xaa₇-Xaa₈-Lys-Thr-Xaa₁₁-Xaa₁₂-Xaa₁₃-Cys (SEQ ID NO:

2) wherein Xaa₁ is des-Xaa, D-Ala, Ala, L-Hor, Cbm, an acyl group having up to 20 carbon atoms or alkyl(C₁ to C₆);

Xaa₂ is Tyr, D-Tyr, Gly or des-Xaa;

Xaa₃ is Cys or D-Cys;

Xaa₄ is Lys or des-Xaa;

Xaa₅ is Asn or des-Xaa;

Xaa₇ is an amino acid selected from the group consisting of Ala, Aph, Amp, Iamp, Val and Tyr;

Xaa₈ is an amino acid selected from the group consisting of (A)Trp, (A)D-Trp, 2Nal and threo-L-C^β

Me-2Nal, wherein A is H, Cl, F, Br, Me, NO₃, OMe or formyl;

Xaa₁₁ is Ala, Gly, Val, Phe or Tyr;

Xaa₁₂ is Thr or des-Xaa; and

Xaa₁₃ is Ser, D-Ser or des-Xaa;

provided that when Xaa₇ is Tyr, Xaa₈ is threo-L-C^β

Me-2Nal.

2. The peptide according to claim 1 wherein Xaa₁, Xaa₂, Xaa₄, Xaa₅, and Xaa₁₂ are des-Xaa.

3. The peptide according to claim 1 wherein Xaa₇ is Ala, Aph or Amp.

4. The peptide according to claim 3 wherein Xaa₇ is Ala and Xaa₁₁ is Ala.

5. The peptide according to claim 3 wherein Xaa₁ is Cbm;

Xaa₂, Xaa₄, and Xaa₅ are des-Xaa;

Xaa₇ is Aph;

Xaa₈ is Trp;

Xaa₁₁ is Phe and Xaa₁₂ is des-Xaa.

6. The peptide according to claim 1 wherein Xaa₇ is Tyr and Xaa₈ is threo-L-C^β

Me-2Nal.

7. The peptide according to claim 1 wherein Xaa₂ is Tyr or D-Tyr and is radioiodinated.

8. The peptide according to claim 1 wherein there is also present at the N-terminus a complexing agent or a conjugating agent.

9. The peptide according to claim 8 wherein a complexing agent is present which is capable of joining to a radioactive nuclide.

10. The peptide according to claim 8 wherein a conjugating agent is present which is capable of linking to a cytotoxin.

11. A method for destroying SSTR4-containing cells, which method comprises administering an amount of the peptide according to claim 8 which includes a radioactive nuclide or a cytotoxin, which amount is effective to destroy such cells.

12. A method for treating diseases of organs expressing SSTR4, which method comprises administering an amount of a nonradioactive peptide according to claim 1, which amount is effective to mitigate such disease.

13. A method of detecting, in the body of a human being, tumors having SSTR4 and their metastases in tissues, which in healthy condition and in non-neoplastic conditions of chronic inflammation do not contain substantial quantities of SSTR4, which method comprises (i) administering to said human, in a quantity sufficient for external imaging, a composition comprising a peptide according to claim 1, said peptide being labeled with (a) a radioactive metal isotope or (b) a paramagnetic metal atom or (c) a radioactive halogen isotope, and thereupon (ii) subjecting said human to external imaging, by radioactive scanning or by magnetic resonance imaging, to determine the targeted sites in the body thereof in relation to the background activity, in order to allow detection and localization of said tumors in the body.

14. A pharmaceutical composition comprising a mixture of the peptide according to claim 1 and at least one pharmaceutically acceptable carrier.

15. A method of stimulating the biological action of somatostatin upon SSTR4 receptors, which method comprises administering an amount of the composition according to claim 14, which amount is effective to reach desired tissue having SSTR4 receptors and bind to said receptors on said tissue.

16. A method for screening for ligands that bind with high affinity to SSTR4, which method comprises carrying out a competitive binding assay with (1) SSTR4, (2) the peptide according to claim 1 which is labeled and (3) a candidate ligand, and determining the ability of the said candidate ligand to displace said labeled peptide.

17. The peptide according to claim 1 comprising the amino acid sequence:

(cyclo)Xaa₁-Tyr-Xaa₃-Xaa₄-Xaa₅-Phe-Xaa₇-Xaa₈-Lys-Thr-Xaa₁₁-Xaa₁₂-Xaa₁₃-Cys (SEQ ID NO;

2), where Xaa₁ is Ac, Cbm or des-Xaa;

Xaa₃ is Cys or D-Cys;

Xaa₇ is Ala, Aph, Amp or lamp;

Xaa₈ is Trp, D-Trp or 2Nal, Xaa₁₁ is Ala, Phe or Tyr, and Xaa₄, Xaa₅, Xaa₁₂ and Xaa₁₃ are des-Xaa.

18. A cyclic somatostatin (SRIF) analog peptide having specific affinity for the SRIF receptor SSTR4, which peptide has an amino acid sequence at least 8 residues in length, contains a Cys-Cys disulfide bond with a sequence of at least 6 residues located between said Cys residues as a ring, and contains Phe-Xaa₇-Xaa₈-Lys-Thr-Xaa₁₁ (SEQ ID NO:

10) or its equivalent adjacent the Cys residue near the N-terminus, which analog peptide is characterized by the presence of Xaa₇ in the form of Ala, Aph, Amp or lamp;

Xaa₈ being (A)Trp, (A)D-Trp or 2Nal, wherein A is H, Cl or F and B is H, Cl, F or NO; and

Xaa₁₁ being Ala, Phe or Tyr.

19. A method of identifying a ligand that is selective for the SRIF receptor SSTR4 which is patterned after the structure of the cyclic ligand SRIF, which method comprises:

providing a pharmacophore model embodying precise 3-dimensional characteristics wherein the distances (a) between the Cγ

of the side chain of residue 8 or its equivalent and the Cγ

of that of an aromatic residue is 5.5-9.5 Å

;

(b) between the Cγ

of said side chain of residue 8 or its equivalent and the Cγ

of the side chain of Lys⁹ or its equivalent is 4.5-6.5 Å

; and

(c) between the Cγ

of said side chain of said aromatic residue or its equivalent and the Cγ

of said side chain of Lys⁹ or its equivalent is 4.5-6.5 Å

, and

using said pharmacophore model and 3-D computer analysis to predict the construction of a ligand which binds selectively and with high affinity to SSTR4.

20. A ligand which binds with selectivity and high affinity to SSTR4 which is constructed according to identification obtained by the method of claim 19.