Methods and products for in vivo enzyme profiling

• US 8,673,267 B2
• Filed: 03/02/2010
• Issued: 03/18/2014
• Est. Priority Date: 03/02/2009
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Bhatia, Sangeeta N., von Maltzahn, Geoffrey A., Kwong, Gabriel
• Primary Examiner(s): DESAI, ANAND U
• Application Number:
  US12/715,965
• Publication Number:
  US 20100240050A1
• Time in Patent Office: 1,477 Days
• Field of Search: None
• US Class Current: 424/9.1
• 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 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/56 involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

Abstract

The present invention relates to methods and products associated with in vivo enzyme profiling. In particular, the invention relates to methods of in vivo processing of exogenous molecules followed by detection of signature molecules as representative of the presence of active enzymes associated with diseases or conditions. The invention also relates to products, kits, and databases for use in the methods of the invention.

Claims

1. A method comprising

administering to a subject a pro-diagnostic reagent, wherein the pro-diagnostic reagent comprises a carrier domain linked to a signature producing domain, wherein the signature producing domain comprises

(i) an enzyme susceptible domain which is linked to a signature molecule;

or

(ii) an enzyme that acts on an endogenous substrate to produce a signature molecule;

identifying a biological sample for detection of the signature molecule, wherein the biological sample is at a site remote from the production of the signature molecule; and

subjecting the biological sample to a multiplex analysis method in order to detect the presence of the signature molecule, wherein the presence of the signature molecule in the biological sample is indicative of a biological predictor molecule within the subject.

2. The method of claim 1, wherein the signature producing domain comprises an enzyme susceptible domain linked to a signature molecule, wherein the biological predictor molecule is an enzyme, wherein the enzyme susceptible domain is susceptible to modification by the enzyme in the subject, and wherein the presence of the signature molecule in the biological sample is indicative of an active enzyme within the subject.

3. The method of claim 1, wherein the enzyme is a protease or a glycosidase.

4. The method of claim 1, wherein the pro-diagnostic reagent further comprises an implantable microdelivery device that houses the carrier domain linked to the signature producing domain.

5. The method of claim 4, wherein the implantable microdelivery device is an implantable capsule with a semi-permeable membrane that encapsulates the carrier domain linked to the signature producing domain.

6. The method of claim 4, wherein the implantable microdelivery device is a chip having the carrier domain linked to the signature producing domain attached thereto.

7. The method of claim 4, wherein the implantable microdelivery device is a sustained-release formulation.

8. The method of claim 2, wherein the enzyme susceptible domain is susceptible to modification by an enzyme associated with a disease or condition.

9. The method of claim 2, wherein the enzyme susceptible domain is susceptible to cleavage by a protease associated with a disease or condition.

10. The method of claim 1, wherein the carrier domain is a microparticle, a nanoparticle, a RGD peptide, an antibody, a targeting molecule, or an aptamer.

11. The method of claim 2, wherein the enzyme susceptible domain is a peptide, wherein the peptide includes a MMP sensitive site, a kallikrein sensitive site, a cathepsin sensitive site, a plasminogen activator sensitive site, or an ADAM sensitive site.

12. The method of claim 1, wherein the signature molecule is a peptide, nucleic acid, small molecule, fluorophore/quencher, carbohydrate, particle, radiolabel, MRI-active compound, inorganic material, or organic material, with encoded characteristics to facilitate optimal detection.

13. The method of claim 1, wherein the analysis method involves mass spectrometry.

14. The method of claim 1, wherein the analysis method is liquid chromatography-mass spectrometry.

15. The method of claim 1, wherein the presence of the signature molecule is detected via non-fluorescent detection.