Methods and products for in vivo enzyme profiling

• US 9,970,941 B2
• Filed: 01/28/2014
• Issued: 05/15/2018
• 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 A.
• Primary Examiner(s): DESAI, ANAND U
• Application Number:
  US14/166,481
• Publication Number:
  US 20140234431A1
• Time in Patent Office: 1,568 Days
• Field of Search: None
• 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

collecting a urine sample from a subject suspected of having a disorder or condition associated with dysregulated enzymatic activity, wherein the subject has been administered a pro-diagnostic reagent, the pro-diagnostic reagent comprising a nanoparticle carrier domain linked to a signature producing domain, wherein the signature producing domain comprises an enzyme susceptible domain which is linked to a signature molecule, wherein the signature molecule comprises a peptide, small molecule, fluorophore, quencher, or carbohydrate;

and,

subjecting the urine sample to a multiplex analysis method in order to detect the presence of the signature molecule, and wherein the cleavage of the enzyme susceptible domain results in the presence of the signature molecule in the biological sample and is indicative of the disorder or condition within the subject.

2. The method of claim 1, further comprising

administering a therapeutic agent to the subject to treat the disorder or condition.

3. The method of claim 1, wherein the carrier domain is a particle and is greater than 5 nm in size and,

wherein the signature molecule is a peptide.

4. The method of claim 1, wherein the administration is via an implantable microdelivery device housing a modular structure having the carrier domain linked to the signature producing domain.

5. The method of claim 1, wherein the signature producing domain comprises an active signature producing agent, wherein the active signature producing agent is capable of modifying a biological predictor molecule to produce a signature molecule.

6. The method of claim 5, wherein the active signature producing agent is an enzyme.

7. The method of claim 6, wherein the enzyme is a protease or a glycosidase.

8. The method of claim 4, wherein the implantable microdelivery device is an implantable capsule with a semi-permeable membrane that encapsulates the modular structure.

9. The method of claim 4, wherein the implantable microdelivery device is a chip having the modular structure attached thereto.

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

11. The method of claim 3, wherein the enzyme susceptible domain is a peptide, a nucleotide, or a carbohydrate.

12. The method of claim 3, wherein the enzyme susceptible domain is a peptide and the peptide comprises GGPQGIWGQC (SEQ ID NO:

1), GGPLGVRGKC (SEQ ID NO;

     2), GGPLANvaDpaARGC (SEQ ID NO;

     3), GGPVGLIGL (SEQ ID NO;

     4), GGPVPLSLVMC (SEQ ID NO;

     5), GGSGGPLGLRSWC (SEQ ID NO;

     6), GGGPWGIWGQGC (SEQ ID NO;

     7), GGdFPipRSGGGC (SEQ ID NO;

     8), or GGLVPRGSGC (SEQ ID NO;

     9).

13. The method of claim 3, wherein the signature molecule comprises a peptide, nucleic acid, small molecule, fluorophore, quencher, carbohydrate, and/or particle.

14. The method of claim 13, wherein signature molecule comprises a peptide.

15. The method of claim 14, wherein the peptide is GGPQG (SEQ ID NO:

10), GGPLG (SEQ ID NO;

     11), GGPLA (SEQ ID NO;

     12), GGPVG (SEQ ID NO;

     13), GGPVPLS (SEQ ID NO;

     14), GGSGGPLG (SEQ ID NO;

     15), GGGPWG (SEQ ID NO;

     16), GGdFPipR (SEQ ID NO;

     17), or GGLVP (SEQ ID NO;

     18).

16. The method of claim 1, wherein the signature molecule is optimized for LC/MS detection.

17. The method of claim 16, wherein the signature molecule comprises a peptide comprising two basic amino acid residues.

18. The method of claim 17, wherein the two basic amino acid residues are dR residues.

19. The method of claim 17, wherein the two basic amino acid residues are separated by at least one non-basic amino acid residue.

20. The method of claim 17, wherein the signature molecule comprises at least three but not more than five amino acid residues.

21. The method of claim 17, wherein the signature molecule is hydrophilic.

22. The method of claim 17, wherein the signature molecule consists of the signature molecule given in SEQ ID NOs 20, 21, 35, or 36.

23. The method of claim 3, wherein the carrier domain is a polymer based microparticle, an iron oxide microparticle, or nanoparticle, an inorganic carrier, an organic carrier, a small molecule, a peptide, a nucleic acid, or a carbohydrate.

24. The method of claim 3, wherein the carrier domain comprises a RGD peptide.

25. The method of claim 3, wherein the carrier domain comprises an antibody.

26. The method of claim 3, wherein the carrier domain includes a targeting domain.

27. The method of claim 3, wherein the carrier domain is a therapeutic agent.

28. The method of claim 3, wherein the carrier domain further comprises a therapeutic agent incorporated into a particle carrier.

29. A method comprising

collecting a urine sample from a subject suspected of having a disorder or condition associated with dysregulated enzymatic activity, wherein the subject has been administered a pro-diagnostic reagent, the pro-diagnostic reagent comprising a nanoparticle carrier domain linked to a signature producing domain, wherein the signature producing domain comprises an enzyme susceptible domain which is linked to a signature molecule, wherein the signature molecule comprises a nucleic acid;

and,

subjecting the urine sample to a multiplex analysis method in order to detect the presence of the signature molecule, and wherein the cleavage of the enzyme susceptible domain results in the presence of the signature molecule in the biological sample and is indicative of the disorder or condition within the subject.

30. The method of claim 29, further comprising

administering a therapeutic agent to the subject to treat the disorder or condition.