Protease assay

Patent metadata

Field	Value
Publication number	US-9731034-B2
Application number	US-201514605083-A
Country	US
Kind code	B2
Filing date	Jan 26, 2015
Priority date	Mar 3, 2008
Publication date	Aug 15, 2017
Grant date	Aug 15, 2017

Abstract

The present invention provides a diagnostic reagent or assay for assessing the activity of a protease in vivo or in vitro and methods of detecting the presence of a cancerous or precancerous cell. The assays are comprised of two particles linked via an oligopeptide linkage that comprises a consensus sequence specific for the target protease. Cleavage of the sequence by the target protease can be detected visually or using various sensors, and the diagnostic results can be correlated with cancer prognosis.

First claim

We claim:

1. An in vivo method for detecting the activity of a protease associated with a cancerous or precancerous cell in a mammal comprising:
(a) administering to the mammal in vivo a composition comprising a nanoplatform assembly and a pharmaceutically-acceptable carrier, said nanoplatform assembly comprising:
a first particle, wherein said first particle is a core/shell nanoparticle, said core being selected from the group consisting of Au, Ag, Cu, Co, Fe, and Pt, and said shell being selected from the group consisting of Au, Ag, Cu, Co, Fe, Pt, the metal oxides thereof, and combinations thereof;
a second particle, wherein said second particle is selected from the group consisting of nanoparticles, chromophores/luminophores, quantum dots, viologens, and combinations thereof; and
a linkage between said first and second particles, wherein said linkage comprises a protease consensus sequence;

(b) exposing a region of the mammal suspected of having a cancerous or precancerous cell to an energy source; and
(c) detecting changes in an absorption or emission spectrum of the assembly, wherein said changes correspond to protease activity, and wherein said linkage separates said first and second particles by a distance such that said first particle quenches an excited state of said second particle prior to said exposing (b).

2. The method of claim 1 , wherein said administering (a) comprises injecting said composition directly into said region of the mammal suspected of having a cancerous or precancerous cell.

3. The method of claim 1 , wherein said administering (a) comprises injecting said composition into the bloodstream of said mammal.

4. The method of claim 3 , wherein said composition reaches said region suspected of having a cancerous or precancerous cell within one hour after injection.

5. The method of claim 1 , further comprising activating said assembly before said exposing (b), wherein said activating comprises heating said composition to a temperature sufficient to release said assembly from said pharmaceutically-acceptable carrier.

6. The method of claim 1 , wherein said energy source is selected from the group consisting of a tungsten lamp, laser diode, laser, bioluminescence, and combinations thereof.

7. The method of claim 1 , wherein said exposing occurs at a wavelength of from about 400 nm to about 500 nm or from about 800 nm to about 900 nm.

8. The method of claim 1 , wherein a blue-shift in absorption or emission spectrum maximum between about 5 nm and about 200 nm indicates the presence of a cancerous or precancerous cell in the mammal.

9. The method of claim 1 , wherein said changes comprise the appearance of a new luminescence band relative to the absorption or emission spectrum of said assembly prior to said exposing (b), said luminescence band indicating the presence of a cancerous or precancerous cell in the mammal.

10. The method of claim 1 , further comprising correlating said protease activity with a prognosis for cancer progression, wherein protease activity detected within 10 minutes after exposing said assembly is correlated with a high probability that the cancerous or precancerous cell is aggressive.

11. The method of claim 1 , further comprising correlating said protease activity with a prognosis for cancer progression, wherein the absence of protease activity detection within the first 30 minutes after exposing said assembly is correlated with a low probability that the cancerous or precancerous cell is aggressive.

12. The method of claim 1 , further comprising correlating said protease activity with a prognosis for cancer progression, wherein the detection of activity of both uPA and MMP-7, and the absence of activity of both MMP-1 and MMP-2, is correlated with a prognosis for angiogenesis.

13. The method of claim 1 , further comprising correlating said protease activity with a prognosis for cancer progression, wherein the detection of activity of all of uPA, MMP-1, MMP-2, MMP-7 is correlated with a prognosis for cell invasion.

14. The method of claim 1 , wherein a change in absorption or emission spectrum indicates the activity of a protease selected from the group consisting of uPA, MMP-1, MMP-2, MMP-7, and combinations thereof.

15. The method of claim 1 , wherein said cancerous or precancerous cell is associated with a cancer selected from the group consisting of an AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, extrahepatic bile duct cancer, childhood brain stem glioma, adult brain tumor, childhood malignant glioma, childhood ependymoma, childhood medulloblastoma, childhood supratentorial primitive neuroectodermal tumors, childhood visual pathway and hypothalamic glioma, breast cancer, pregnancy-related breast cancer, childhood breast cancer, male breast cancer, childhood carcinoid tumor, gastrointestinal carcinoid tumor, primary central nervous system lymphoma, cervical cancer, colon cancer, childhood colorectal cancer, esophageal cancer, childhood esophageal cancer, intraocular melanoma, retinoblastoma, adult glioma, adult (primary) hepatocellular cancer, childhood (primary) hepatocellular cancer, adult Hodgkin lymphoma, childhood Hodgkin lymphoma, islet cell tumors, Kaposi Sarcoma, kidney (renal cell) cancer, childhood kidney cancer, adult (primary) liver cancer, childhood (primary) liver cancer, Non-small cell liver cancer, small cell liver cancer, AIDS-related lymphoma, Burkitt lymphoma, adult Non-Hodgkin lymphoma, childhood Non-Hodgkin lymphoma, primary central nervous system lymphoma, melanoma, adult malignant mesothelioma, childhood mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, childhood multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, adult acute myeloid leukemia, childhood acute myeloid leukemia, multiple myeloma, neuroblastoma, non-small cell lung cancer, childhood ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, childhood pancreatic cancer, islet cell pancreatic cancer, parathyroid cancer, penile cancer, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, childhood renal cell cancer, renal pelvis and ureter, transitional cell cancer, adult soft tissue sarcoma, childhood soft tissue sarcoma, uterine sarcoma, skin cancer (nonmelanoma), childhood skin cancer, melanoma, Merkel cell skin carcinoma, small cell lung cancer, small intestine cancer, squamous cell carcinoma, stomach cancer, childhood stomach cancer, cutaneous T-Cell lymphoma, testicular cancer, thyroid cancer, childhood thyroid cancer, and vaginal cancer.

16. The method of 1 , wherein said core/shell nanoparticle is a stabilized nanoparticle comprising an organic monolayer surrounding the nanoparticle.

17. The method of claim 1 , wherein said second particle is a chromophore or luminophore selected from the group consisting of organic dyes, inorganic dyes, fluorophores, phosphophores, light absorbing nanoparticles, combinations thereof, and the metalated complexes thereof.

18. The method of claim 1 , said nanoplatform assembly further comprising multiple of said second particle, each of said second particles being linked to said first particle by respective linkages, wherein each of said linkages comprises a protease consensus sequence.

19. The method of claim

Assignees

• Univ Kansas State

Inventors

• Bossmann Stefan H
• Troyer Deryl L
• Basel Matthew T

Classifications

• A61K49/0067 Primary

  quantum dots, fluorescent nanocrystals · CPC title

• Y10S977/774

  Exhibiting three-dimensional carrier confinement, e.g. quantum dots · CPC title

• A61K49/0093

  Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle · CPC title

• A61K49/0056

  Peptides, proteins, polyamino acids · CPC title

• Y10S977/92

  Detection of biochemical · CPC title