Method of determining metabolism of a test agent

• US 8,173,361 B2
• Filed: 02/17/2010
• Issued: 05/08/2012
• Est. Priority Date: 01/16/2003
• Status: Active Grant

• Current Assignee: Charles Stark Draper Laboratory Incorporated, The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: Charles Stark Draper Laboratory Incorporated, The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Vacanti, Joseph P., Rubin, Robert, Cheung, Wing, Borenstein, Jeffrey T.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US12/707,469
• Publication Number:
  US 20110008765A1
• Time in Patent Office: 811 Days
• Field of Search: 435/4
• US Class Current: 435/4
• CPC Class Codes: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: G01N 33/5038 involving detection of metabolites per se
• CPC Class Codes: G01N 33/5088 of vertebrates

Abstract

A method for detecting one or more metabolites of a test agent in a tissue including: A) incubating a test agent and an enzyme within a three-dimensional structure comprising a first mold or polymer scaffold, a semi-permeable membrane, and a second mold or polymer scaffold, wherein the semi-permeable membrane is disposed between the first and second molds or polymer scaffolds, and wherein the first mold or polymer scaffold has microchannels that form a fluidic branching network that mimics the forces and transport of natural vasculatures and wherein the second mold or polymer scaffold comprises cells; B) forming an enzyme-substrate complex between the enzyme and the test agent; and C) detecting one or more metabolites of the test agent.

Claims

1. A method for detecting one or more metabolites of a test agent in a tissue comprising:

A) incubating a test agent and an enzyme within a three-dimensional structure comprising a first mold or polymer scaffold, a semi-permeable membrane, and a second mold or polymer scaffold, wherein the semi-permeable membrane is disposed between the first and second molds or polymer scaffolds, and wherein the first mold or polymer scaffold has microchannels that form a fluidic branching network that mimics the forces and transport of natural vasculatures and wherein the second mold or polymer scaffold comprises cells;

B) forming an enzyme-substrate complex between the enzyme and the test agent; and

C) detecting one or more metabolites of the test agent.

2. The method of claim 1, wherein the structure comprises liver tissue, kidney tissue, cardiac tissue, cartilage tissue, or bone marrow tissue, and combinations thereof.

3. The method of claim 1, wherein the test agent comprises a compound selected from the group consisting of opioid analgesics, anti-inflammatory drugs, diuretics, cardiovascular drugs, cardiac drugs, agents that affect hyperlipoproteinemias, anti-neoplastic agents, immunomodulators, anti-diabetic agents, and anti-microbial agents.

4. The method of claim 1, wherein the enzyme is selected from the group consisting of cytochrome P450, alkaline phosphatase, α

-galactosidase, β

-galactosidase, α

-glucosidase, β

-glucosidase, α

-glucuronidase, β

-glucuronidase, α

-amylase, NADPH-cytochrome P450 reductase, cytochrome b₅, N-demethylase, O-demethylase, acetylcholinesterase, pseudocholinesterase, epoxide hydrolase, amidases, uridine diphosphate (UDP)-glucuronosyltransferases, phenol sulfotransferase, alcohol sulfotransferase, sterid sulfotransferase, and arylamine sulfotransferase, UDP-glycosyltransferases, purine phosphoribosyltransferase, N-acetyltransferases, glutathione S-transferase, phenylethanolamine N-methyltransferase, non-specific N-methyltransferase, imidazole N-methyltransferase, catechol-O-methyltransferase, hydroxyindole-O-methyltransferase, S-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase, xanthine oxidase, monoamine oxidases, diamine oxidases, flavoprotein N-oxidases, hydroxylases, aromatases, cysteine conjugate β

-lyase, and alkylhydrazine oxidase.

5. The method of claim 1, wherein the enzyme is endogenously expressed in the tissue.

6. The method of claim 1, wherein the enzyme has normal enzymatic activity compared to an enzyme containing a polymorphism or mutation and having altered enzymatic activity.

7. The method of claim 1, wherein the enzyme contains a polymorphism or mutation.

8. The method of claim 1, wherein the enzyme is a recombinant enzyme.

9. The method of claim 1, wherein the enzyme is cytochrome P450.

10. The method of claim 1, wherein the one or more metabolites are detected by liquid chromatography, mass spectrometry, nuclear magnetic resonance, or spectrophotometry.

11. The method of claim 1, wherein the membrane is endothelialized.

12. The method of claim 1, further comprising circulating a physiological fluid through the microchannels, wherein the physiological fluid is selected from the group consisting of media, blood, plasma and combinations thereof.

13. The method of claim 3, wherein the anti-inflammatory drugs are selected from the group consisting of antihistamines and non-steroidal anti-inflammatory drugs (nsaids).

14. The method of claim 3, wherein the diuretics are selected from the group consisting of carbonic anhydrase inhibitors, loop diuretics, high-ceiling diuretics, thiazides and potassium-sparing diuretics.

15. The method of claim 3, wherein the cardiovascular drug is an angiotensin converting enzyme inhibitor.

16. The method of claim 3, wherein the cardiac drugs are selected from the group consisting of organic nitrates, calcium channel blockers, sympatholytic agents, vasodilators, β

-adrenergic receptor agonists, β

-adrenergic receptor antagonists, cardiac glycosides and anti-arrhythmic drugs.

17. The method of claim 3, wherein the agent that affects hyperlipoproteinemias is a 3-hydroxymethylglutaryl-coenzyme a (hmg-coa) inhibitor.

18. The method of claim 3, wherein the anti-neoplastic agents are selected from the group consisting alkylating agents and antimetabolites.

19. The method of claim 3, wherein the anti-microbial agents are selected from the group consisting antibiotics, antibacterial agents, antiviral agents, antifungal agents, antiprotozoal agents and antihelminthic agents.