Caspase-triggered nano-aggregation probes and methods of use

Patent metadata

Field	Value
Publication number	US-9642922-B2
Application number	US-201414464111-A
Country	US
Kind code	B2
Filing date	Aug 20, 2014
Priority date	Aug 23, 2013
Publication date	May 9, 2017
Grant date	May 9, 2017

Abstract

Provided is an activatable probe that undergoes intramolecular cyclization and subsequent aggregation in apoptotic tumor cells upon peptidase-initiated, and most advantageously caspase-3, activation. These caspase-sensitive nano-aggregation probes (C-SNAFs) are generally biocompatible, possess NIR spectral properties or may serve as PET or MRI imaging agents, and have a mechanism of target-mediated nanostructure self-assembly amenable to in vivo use. The probes encompass biocompatible condensation chemistry products that comprise D-cysteine and 2-cyano-6-hydroxyquinoline (CHQ) moieties linked to an amino-luciferin scaffold, and which can be activated by a two-step reaction requiring caspase-3/7-mediated cleavage of an aspartate-glutamate-valine-aspartate (L-DEVD) capping peptide and the free intracellular thiol-mediated reduction of the disulfide bond.

First claim

What is claimed:

1. An activatable probe comprising a self-cyclizing molecule that comprises a cysteine moiety, a 2-cyano-6-hydroxyquinoline moiety, and an amino luciferin scaffold, and wherein the activatable probe further comprises a detachable capping moiety, wherein the detachable capping moiety is a peptide having an amino acid sequence consisting of L-aspartate-glutamate-valine-aspartate and conjugated to the cysteine moiety of the self-cyclizing molecule by a peptidase-cleavable bond, and wherein the activatable probe further comprises a detectable imaging moiety attached thereto.

2. The activatable probe of claim 1 , wherein the peptidase-cleavable bond conjugating the detachable capping moiety to the cysteine moiety is selected from a caspase-3-cleavable bond or a caspase-7-cleavable bond.

3. An activatable probe comprising a detachable capping moiety conjugated to a self-cyclizing molecule comprising a cysteine moiety, a 2-cyano-6-hydroxyquinoline moiety, and an amino luciferin scaffold, and a detectable imaging moiety, wherein the detachable capping moiety is a peptide having an amino acid sequence consisting of L-aspartate-glutamate-valine-aspartate and conjugated to the cysteine moiety by a peptidase-cleavable bond.

4. The activatable probe of claim 1 , wherein the self-cyclizing molecule has the formula I:

5. The activatable probe of claim 1 , wherein the detectable imaging moiety is an optically detectable label, a positron electron transmission detectable label, or a magnetic resonance imaging (MRI) detectable label.

6. The activatable probe of claim 5 , wherein the detectable imaging moiety is a fluorophore.

7. The activatable probe of claim 5 , where the detectable imaging moiety detectable by positron electron transmission is selected from the group consisting of: 11 C, 13 N, 15 O, 17 F, 18 F, 75 Br, 76 Br, and 124 I.

8. The activatable probe of claim 5 , wherein the activatable probe further comprises a chelating agent and the detectable imaging moiety is a magnetic resonance imaging (MRI) detectable label, and wherein said detectable label is a metal ion chelated to the chelating agent.

9. The activatable probe of claim 8 , wherein the metal ion is gadolinium (Gd), thulium (Tm), europium (Eu), or a combination thereof.

10. The activatable probe of claim 6 , further comprising a quenching moiety attached to the detachable capping moiety.

11. The activatable probe of claim 1 having the formula II or III or IV:

wherein, in Formula IV, M is a detectable metal ion.

12. The activatable probe of claim 11 , wherein M is gadolinium (Gd), thulium (Tm), europium (Eu), or a combination thereof.

13. A composition comprising the activatable probe of claim 1 and a pharmaceutically acceptable carrier.

14. A nano-aggregation probe, wherein said nano-aggregation probe is an aggregate of an activated probe, wherein said activated probe comprises a self-cyclized molecule having the formula V:

and a detectable imaging moiety attached thereto.

15. A method of forming a nano-aggregation probe comprising delivering to an apoptotic cell a pharmaceutically acceptable composition comprising an activatable probe and a pharmaceutically acceptable carrier, wherein said activatable probe comprises a detachable capping moiety conjugated to a self-cyclizing molecule, said self-cyclizing molecule comprising a cysteine moiety, a 2-cyano-6-hydroxyquinoline moiety, an amino luciferin scaffold, and a detectable imaging moiety, wherein the detachable capping moiety has an amino acid sequence consisting of L-aspartate-glutamate-valine-aspartate and is selectively cleavable from the self-cyclizing molecule by caspase 3/7, whereupon the activatable probe enters the apoptotic cell, an apotopically-induced caspase 3/7 cleaves the detachable capping moiety from the activatable probe, and said probe aggregates to form the nano-aggregation probe.

16. The method of claim 15 , wherein the activatable probe has the formula II, III, or IV:

wherein, in Formula IV, M is a detectable metal ion.

17. The method of claim 16 , wherein in the activatable probe having the formula IV, M is gadolinium (Gd), thulium (Tm), or europium (Eu).

18. The method of claim 15 , wherein the detectable imaging moiety is a fluorophore and the activatable probe further comprises a quenching moiety attached to the detachable capping moiety.

19. A method of detecting an apoptotic cell, the method comprising the steps of:
(i) delivering to the cytoplasm of an animal cell a pharmaceutically acceptable composition comprising an activatable probe, wherein said activatable probe comprises a detachable capping moiety conjugated to a self-cyclizing molecule, said self-cyclizing molecule comprising a cysteine moiety, a 2-cyano-6-hydroxyquinoline moiety, an amino luciferin scaffold, and a detectable imaging moiety, wherein the detachable capping moiety has an amino acid sequence consisting of L-aspartate-glutamate-valine-aspartate and is selectively cleavable from the self-cyclizing molecule by caspase 3/7, whereupon the activatable probe enters the apoptotic cell, an apotopically-induced caspase 3/7 cleaves the detachable capping moiety from the activatable probe, and said probe aggregates to form a nano-aggregation probe; and
(ii) detecting the nano-aggregation probe, thereby determining that the animal cell recipient of the activatable probe is an apoptotic animal cell having an apoptosis-induced caspase 3/7 that cleaved the detachable capping moiety from the activatable probe to form the nano-aggregation probe,

wherein, when the detectable imaging moiety is a fluorophore step (ii) further comprises irradiating the recipient cell with an incident excitation energy, optically detecting an emitted fluorescence, measuring the intensity of said emission, and optionally generating an image of the fluorescence, when the detectable imaging moiety is detectable by positron electron transmission step (ii) further comprises detecting the nano-aggregation probe by PET imaging, and when the detectable imaging moiety is detectable by magnetic resonance imaging step (ii) further comprises detecting the nano-aggregation probe by MRI imaging.

Assignees

• Univ Leland Stanford Junior

Inventors

• Rao Jianghong
• Ye Deju
• Shuhendler Adam
• Chin Frederick Te-Ning
• Jeon Jongho
• Shen Bin

Classifications

• A61K51/0455

  having six-membered rings with one nitrogen as the only ring hetero atom · CPC title

• A61K49/0032

  Methine dyes, e.g. cyanine dyes · CPC title

• G01N33/5005

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title

• A61K49/0002 Primary

  General or multifunctional contrast agents, e.g. chelated agents · CPC title

• A61K49/101

  the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals · CPC title