Probes for In Vivo Targeting of Active Cysteine Proteases

• US 20090252677A1
• Filed: 04/03/2009
• Published: 10/08/2009
• Est. Priority Date: 04/03/2008
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Gambhir, Sanjiv S., Blum, Galia, Berger, Alicia, Cheng, Zhen, Bogyo, Matthew S.
• Granted Patent: US 8,343,458 B2
• Time in Patent Office: Days
• US Class Current: 424/1.650
• CPC Class Codes: A61B 6/508 for non-human patients
• CPC Class Codes: A61K 49/0032 Methine dyes, e.g. cyanine dyes
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino acids
• CPC Class Codes: A61K 51/0495 Pretargeting
• CPC Class Codes: A61K 51/08 Peptides, e.g. proteins , carriers being peptides, polyamino acids, proteins
• CPC Class Codes: A61K 51/088 conjugates with carriers being peptides, polyamino acids or proteins antibodies A61K51/10
• CPC Class Codes: A61P 35/00 Antineoplastic agents

Abstract

Activity-based probes, which are specific for certain active cysteine proteases (caspase, cathepsin and legumain) and carry radioactive labels, are disclosed. The present probes comprise an acyloxymethyketone (AOMK) “warhead” that binds only to active enzyme. The probes further comprise peptide-like structure that targets the probe to a specific cysteine protease or protease family, and a radiolabel on the probe, which is bound to the targeted enzyme. It has been found that the present probes are stable in vivo and give specific target images distinguishable over background. The preferred probes are labeled with a positron-emitting agent such as ⁶⁴Cu, ¹²⁵I (SPECT) and ^99mTc (PET). The probes show in vivo half-life and stability well suited for imaging.

Claims

1. A composition useful for in vivo imaging of tissue containing an active cysteine protease, said composition comprising a compound according to the formula:

where

n is an integer between 0 and 2, P₃ being absent when n=0, and there being a P₄ when n=2;

R₁ and R₃ are independently selected from the groups consisting of H, NH₂, aminocarbonyl, aryl, substituted aryl (including 2-nitro, 3-hydroxy, methyl and dimethyl substituted benzyl and benzyloxycarbonyl), amino, lower alkyl, or cycloalkyl;

P₁, P₂, P₃ and P₄ are independently amino acid side chains selected from naturally occurring amino acid side chains and nonnatural amino acid side chains 3, 6 8, 23, 29, 31, 34 and 38; and

one of R₁, R₃ or a P₁, P₂, P₃ or P₄ further is bonded to a chelator for a radiolabel or directly to a radiolabel,

which compound specifically binds a predetermined active cysteine protease.

2. The composition of claim 1 where R₁ and R₃ of Formula I are each an optionally substituted benzyl or phenyl.

3. The composition of claim 2 where one of R₁ or P₁ further comprises said chelator.

4. The composition of claim 3 where the chelator of Formula I is on P₁, and where P₁ contains a linker of 2-5 carbon atoms.

5. The composition of claim 1 where a chelator is linked by an amide linkage to a lower alkyl group in P₁, P₂, P₃ or P₄ of Formula I.

6. The composition of claim 4 where the chelator is selected from the group consisting of:

diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,N′

,N″

,N′

″

-tetraacetate (DOTA), tetraazacyclotetradecane-N,N′

,N″

,N′

″

-tetraacetic acid (TETA), hydroxyethylidene diphosphonate (HEDP), dimercaptosuccinic acid (DMSA), diethylenetriaminetetramethylenephosphonic acid (DTTP) and 1-(p-aminobenzyl)-DTPA, 1,6-diamino hexane-N,N,N′

,N′

-tetraacetic acid, DPDP, and ethylenebis (oxyethylenenitrilo)-tetraacetic acid.

7. The composition of claim 1 further comprising a radiolabel which is a positron emitter.

8. The composition of claim 6 where the radiolabel is selected from the group consisting of ¹³¹I, ^99mTC, ¹¹¹In, ¹⁸F, ⁶⁴Cu, ⁷⁶Br, ⁸⁶Y, ⁵⁵Co, ¹²⁴I and ¹²⁵.

9. The composition of claim 1 where P₁ of Formula I is D or R.

10. The composition of claim 1 where P₂ of Formula I is P or aryl.

11. The composition of claim 1 where P₃ of Formula I is aryl.

12. The composition of claim 1 where P₂, P₃ and P₄ of Formula I are, respectively, Arg, Phe and none.

13. The composition of claim 12 where R₁ and R₃ of Formula I are aryl.

14. The composition of claim 1 where the compound is selective for human cathepsin and does not substantially bind to any other cysteine proteases.

15. The composition of claim 1 where in Formula I R₁ is benzyl, n is 0, P₂ is tyrosine, P₁ is lower alkyl aminocarbonyl DOTA, and R₃ is dimethyl phenyl.

16. A kit for the preparation of the composition of claim 1, comprising a compound according to Formula I, and a radioisotope.

17. The kit of claim 16 wherein the radioisotope is a positron emitter.

20. A method of imaging an active cysteine protease in a subject, comprising the steps of:

(a) obtaining a compound according to Formula I of claim 1, wherein said Formula is defined to include a radiolabel which is a radioactive compound;

(b) intravenously administering to the subject a composition comprising a compound of step (a); and

(c) obtaining an image of the administered compound in step (b) after a time sufficient to allow the compound to bind to and be cleaved by an active cysteine protease selected from the group consisting of cathepsin B, cathepsin L, caspase 3, caspase 7, caspase 8, and caspase 9, where said obtaining is done using a camera which detects radiation by location,

whereby portions of the subject which have higher levels of said active cysteine protease can be distinguished from other portions.

21. The method of claim 20 using a compound of the formula

22. The method of claim 20 where n is between 1 and 3 and X is a chelator.

23. The method of claim 20 where P₂ is aryl.

24. The method of claim 20 where P₂, R₁ and R₃ are aryl.

25. The method of claim 20 where the radiolabel is on P₁ or R₁.

26. The method of claim 20 further comprising the step of reacting a macrocyclic chelator with a sulfonosuccimimide in order to couple it to the compound.

27. The method of claim 20 further comprising the step of heating the compound in the presence of a radioactive metal to bind the radioactive metal to the macrocyclic chelator, which is DOTA.

18. A method of in vivo imaging of an active protease, comprising the step of administering a composition comprising a compound having the formula

DOTA-P3-P2-P1 (-label)-AOMK, where DOTA is a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and comprises a linker, P3, P2, P1 are amino acid residues, -label is a cyanine dye attached to a linker, and AOMK is an acyloxymethyl ketone warhead for binding to the active protease.

19. A method of diagnosis of tumor activity in a mammalian body, comprising administration of a compound according to the formula:

where

n is an integer between 0 and 2, P₃ being absent when n=0, and there being a P₄ when n=2;

R₁ and R₃ are independently selected from the groups consisting of H, NH₂, aminocarbonyl, aryl, substituted aryl (including 2-nitro, 3-hydroxy, methyl and dimethyl substituted benzyl and benzyloxycarbonyl), amino, lower alkyl, or cycloalkyl;

P₁, P₂, P₃ and P₄ are independently amino acid side chains selected from naturally occurring amino acid side chains and nonnatural amino acid side chains 3, 6 8, 23, 29, 31, 34 and 38; and

one of R₁, R₃ or a P₁, P₂, P₃ or P₄ further is bonded to a chelator for a radiolabel or directly to a radiolabel,

which compound specifically binds a predetermined active cysteine protease, wherein said mammal is previously administered a compound which binds selectively to cathepsins B and L and an area having a more active tumor shows higher amount of active enzyme.