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Details for Patent: 5,989,519

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Details for Patent: 5,989,519

Title: Technetium-99m labeled peptides for imaging inflammation
Abstract:This invention relates to radiolabeled peptides and methods for producing such peptides. Specifically, the invention relates to technetium-99m (Tc-99m) labeled leukocyte-binding peptides, methods and kits for making such peptides, and methods for using such peptides to image sites of infection and inflammation in a mammalian body.
Inventor(s): Dean; Richard T. (Bedford, NH), Lees; Robert S. (Brookline, MA), Buttram; Scott (Derry, NH), Lister-James; John (Bedford, NH)
Assignee: Diatide, Inc. (Londonderry, NH)
Filing Date:Oct 11, 1994
Application Number:08/290,853
Claims:1. A reagent for preparing a scintigraphic imaging agent comprising a peptide that specifically binds to leukocytes, said peptide being covalently linked to a technetium-99m complexing moiety having a formula selected from the group consisting of:

wherein Cp is a protected cysteine and (aa) is any primary .alpha.- or .beta.-amino acid not having a thiol-group containing sidechain;

a technetium-99m complexing moiety comprising a single thiol having a formula:

wherein

A is H, HOOC, H.sub.2 NOC, (peptide)-NHOC, (peptide)-OOC or R.sup.4 ;

B is H, SH, --NHR.sup.3, --N(R.sup.3)-(peptide), or R.sup.4 ;

X is H, SH, --NHR.sup.3, --N(R.sup.3)-(peptide) or R.sup.4 ;

Z is H or R.sup.4 ;

R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently H or lower straight or branched chain or cyclic alkyl;

n is 0, 1 or 2; and

where B is --NHR.sup.3 or --N(R.sup.3)-(peptide), X is SH, and n is 1 or 2;

where X is --NHR.sup.3 or --N(R.sup.3)-(peptide), B is SH, and n is 1 or 2;

where B is H or R.sup.4, A is HOOC, H.sub.2 NOC, (peptide)-NHOC or (peptide)-OOC, X is SH, and n is 0 or 1;

where A is H or R.sup.4, then where B is SH, X is --NHR.sup.3 or --N(R.sup.3)-(peptide) and

where X is SH, B is --NHR.sup.3 or --N(R.sup.3)-(peptide);

where X is H or R.sup.4, A is HOOC, H.sub.2 NOC, (peptide)-NHOC or (peptide)-OOC and B is SH;

where Z is methyl, X is methyl, A is HOOC, H.sub.2 NOC, (peptide)-NHOC or (peptide)-OOC, B is SH and n is 0;

and wherein the thiol moiety is in the reduced form; ##STR5## wherein X=H or a protecting group;

(amino acid)=any amino acid; ##STR6## wherein X=H or a protecting group;

(amino acid)=any amino acid; ##STR7## wherein each R.sup.5 is independently H, CH.sub.3 or C.sub.2 H.sub.5 ;

each (pgp).sup.s is independently a thiol protecting group or H;

m, n and p are independently 2 or 3;

A=linear lower alkyl, cyclic lower alkyl, aryl, heterocyclyl, or a combination thereof; and ##STR8## wherein each R.sup.5 is independently H, lower alkyl having 1 to 6 carbon atoms, phenyl, or phenyl substituted with lower alkyl or lower alkoxy;

m, n and p are independently 1 or 2;

A=linear lower alkyl, cyclic lower alkyl, aryl, heterocyclyl, or a combination thereof;

V=H or --CO-peptide;

R.sup.6 =H or peptide;

and wherein when V=H, R.sup.6 =peptide and when R.sup.6 =H, V=--CO-peptide.

2. The reagent of claim 1 wherein the peptide and the technetium-99m complexing moiety are covalently linked through one or more amino acids.

3. The reagent of claim 2 wherein the technetium-99m complexing moiety is Cp(aa)Cp and wherein Cp has a protecting group of the formula

wherein R is lower alkyl having 1 to 6 carbon atoms, a 2-pyridyl, a 3-puridyl, a 4-pyridyl, a phenyl, or a phenyl substituted with a lower alkyl, a hydroxy, a lower alkoxy, a carboxy, or a lower alkoxycarbonyl.

4. The reagent of claim 1 wherein the technetium-99m complexing moiety is: ##STR9##

5. A scintigraphic imaging agent comprising the reagent of claim 1 radiolabeled with technetium-99m.

6. The reagent of claim 1 wherein the peptide is platelet factor 4 .

7. The reagent of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of formylMLF and formyl.Nle.LF.Nle.

8. The reagent of claim 1, wherein the peptide is tuftsin.

9. The reagent of claim 1, wherein the peptide is selected from the group consisting of fibrinopeptide B and fibrinogen B.beta.-chain.

10. The reagent of claim 1, having an amino acid sequence selected from the group consisting of: C.sub.Mob GC.sub.Acm PLYKKIIKKLLES (SEQ ID No.: 36);

Pic.GC.sub.Acm PLYKKIIKKLLES (SEQ ID No.: 9);

C.sub.Acm GC.sub.Acm GGPLYKKIIKKLLES (SEQ ID No.: 10);

acetyl.CGGGPLYKKIIKKLLES (SEQ ID No.: 16);

[BAT].GGPLYKKIIKKLLES (SEQ ID No.: 20);

[DTPA].C.sub.Acm GC.sub.Acm PLYKKIIKKLLES (SEQ ID No.: 29);

[BAT].KKLLKKLYKKIIKKLLES (SEQ ID No.: 30);

AC.C.sub.Acm GC.sub.Acm QAPLYKKIIKKLLES (SEQ ID No.: 37); ##STR10##

11. The reagent of claim 7, having an amino acid sequence selected from the group consisting of: formyl.MLFC.sub.Acm GC.sub.Acm (SEQ ID No.: 1);

formyl.MIFLC.sub.Acm GC.sub.Ac (SEQ ID No.: 3);

formyl.MLFC.sub.Acm GPica (SEQ ID No.: 5);

formyl.Nle.LF.Nle.YKC.sub.Acm GC.sub.Acm (SEQ ID No.: 6);

formyl.MLFK.[BAT].amide (SEQ ID No.: 21);

formyl.Thp.LF.[BAM];

formyl.MLFK.[BAT] (SEQ ID No.: 22);

formyl.MLFK.[BAT].KKKKK.amide;

formyl.MLFK.[BAT].GSGS.amide;

formyl.MLFK.[BAT].E (SEQ ID No.: 25);

formyl.M.Dpg.F.[BAM] (SEQ ID No.: 26);

formyl.MLFK.[BAT].EGE; ##STR11##

12. The reagent of claim 2, having an amino acid sequence selected from the group consisting of: C.sub.Acm GC.sub.Acm (VGVAPG).sub.3 amide (SEQ ID No. 2);

Pic.GC.sub.Acm (VGVAPG).sub.3 amide (SEQ ID No. 7);

Pic.GC.sub.Acm (VPGVG).sub.4 amide (SEQ ID No. 8);

Pic.GC(VGVAPG).sub.3 amide (SEQ ID No. 17);

[BAT].(VGVAPG).sub.3 amide (SEQ ID No. 28); and

[BAT].(VPGVG).sub.4 amide (SEQ ID No. 23).

13. The reagent of claim 9, having an amino acid sequence selected from the group consisting of:

pGlu.GVNDNEEGFFSARGGC.amide (SEQ ID No. 18);

Pic.GC.sub.Acm GHRPLDKKREEAPSLRPAPPPISGGGYR (SEQ ID No. 35); and [BAT].GHRPLDKKREEAPSLRPAPPPISGGGYR.amide (SEQ ID No. 33).

14. The reagent of claim 8, having a formula:

C.sub.Acm GC.sub.Acm TKPR (SEQ ID No.: 4).

15. A reagent comprising

a) a multiplicity of synthetically prepared, leukocyte-binding peptides, each peptide comprising from three to 100 amino acids and being covalently linked to a polyvalent linker; and

b) a technetium complexing moiety; wherein the technetium complexing moiety is covalently linked to a specific amino acid of each of a plurality of the peptides, the linker, or both.

16. The reagent of claim 15, wherein the linker is selected from the group consisting of bis-succinimidyl-methylether, 4-(2,2-dimethylacetyl)benzoic acid, tris(succinimidylethyl)amine, a derivative of bis-succinimidyl-methylether, and a derivative of 4-(2,2-dimethylacetyl)benzoic acid, tris(succinimidylethyl)amine.

17. A complex formed by reacting the reagent of claim 1 with technetium-99m in the presence of a reducing agent.

18. The complex of claim 17, wherein the reducing agent is selected from the group consisting of a dithionite ion, a stannous ion, and a ferrous ion.

19. A complex formed by labeling the reagent of claim 1 with technetium-99m by ligand exchange of a prereduced technetium-99m complex.

20. A kit for preparing a radiopharmaceutical preparation, said kit comprising a sealed vial containing a predetermined quantity of the reagent of claim 1 and a sufficient amount of a reducing agent to label the reagent with technetium-99m.

21. A method for imaging a site of inflammation within a mammalian body comprising the steps of

a) administering an effective diagnostic amount of the scintigraphic imaging agent of claim 5; and

b) detecting the Tc-99m localized at the site of inflammation.

22. A process for preparing the reagent according to claim 1, wherein the reagent is chemically synthesized in vitro.

23. The process of claim 22, wherein the peptide is synthesized by solid phase peptide synthesis.

24. The reagent of claim 1, wherein the technetium-99m complexing moiety is covalently linked to the peptide during solid phase peptide synthesis.

25. The reagent of claim 1, wherein the technetium-99m complexing moiety is covalently linked to the peptide during in vitro chemical synthesis.

26. A method for labeling a reagent according to claim 1, comprising the step of reacting the reagent with Tc-99m in the presence of a reducing agent.

27. The method of claim 26, wherein the reducing agent is selected from the group consisting of a dithionite ion, a stannous ion, and a ferrous ion.

28. A reagent for preparing a scintigraphic imaging agent comprising a peptide that specifically binds to leukocytes, covalently linked to a-technetium-99m complexing moiety comprising a single thiol containing moiety of formula:

wherein

A is H, HOOC, H.sub.2 NOC, (amino acid or peptide)-NHOC,(amino acid or peptide)-OOC or R.sup.4

B is H, SH, --NHR.sup.3,--N(R.sup.3)-(amino acid or peptide), or R.sup.4 ;

X is H, SH,--NHR.sup.3,--N(R.sup.3)-(amino acid or peptide or R.sup.4 ;

Z is H or R.sup.4

R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently H or lower straight or branched chain or cyclic alkyl;

n is 0, 1 or 2; and

where B is --NHR.sup.3 or --N(R.sup.3)-(amino acid or peptide), X is SH, and n is 1 or 2;

where X is --NHR.sup.3 or --NHR.sup.3 or --N(R.sup.3)-(amino acid or peptide), B is SH, and n is 1 or 2;

where B is H or R.sup.4, A is HOOC, H.sub.2 NOC, (amino acid or peptide)-NHOC or (amino acid or peptide)-OOC, X is SH, and n is 0 or 1;

where A is H or R.sup.4, then where B is SH, X is --NHR.sup.3 or --N(R.sup.3)-(amino acid peptide) and where X is SH, B is --NHR.sup.3 or --N(R.sup.3)-(amino acid or peptide);

where X is H or R.sup.4, A is HOOC, H.sub.2 NOC, (amino acid or peptide), .sub.-- NHOC or (amino acid or peptide)-OOC and B is SH;

where Z is methyl, X is methyl, A is HOOC, H.sub.2 NOC,(amino acid or peptide)-NHOC or (amino acid or peptide)-OOC, B is SH and n is 0;

and wherein the thiol moiety is in the reduced form and (amino acid) is any primary .alpha.- or .beta.-amino acid not containing a thiol group.

29. The reagent of claim 28, wherein the technetium-99m complexing moiety is selected from the group consisting of:

IIa. -(amino acid).sup.1 -(amino acid).sup.2 -{A--CZ(B)--(C(R.sup.1 R.sup.2)).sub.n X},

IIb. -{A--CZ(B)--[C(R.sup.1 R.sup.2)].sub.n --X}--(amino acid).sup.1 -(amino acid).sup.2,

IIc. -(a primary a, .omega.- or .beta., .omega.-diamino acid)-(amino acid).sup.1 -{A--CZ (B)--{C(R.sup.1 R.sup.2)).sub.n --X}, and

IId. -{A--CZ(B)--(C(R.sup.1 R.sup.2).sub.n --X}-(amino acid).sup.1 -(a primary .alpha.,.omega.- or .beta., .omega.-diamino acid),

wherein (amino acid).sup.1 and (amino acid).sup.2 are each independently any naturally-occurring, modified, substituted or altered .alpha.- or .beta.-amino acid not containing a thiol group.

30. A pharmaceutical composition for imaging a site of inflammation within a mammalian body comprising the scintigraphic imaging agent of claim 5 in a pharmaceutically acceptable carrier.

31. The method of claim 21, wherein the imaging agent is elastin.
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