Use of three-dimensional microfabricated tissue engineered systems for pharmacologic applications

• US 20060019326A1
• Filed: 07/15/2005
• Published: 01/26/2006
• 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
• Inventors: Rubin, Robert, Vacanti, Joseph P., Cheung, Wing
• Granted Patent: US 7,670,797 B2
• Time in Patent Office: Days
• US Class Current: 435/18
• 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

The present invention generally relates to a combination of the fields of tissue engineering, drug discovery and drug development. It more specifically provides new methods and materials for testing the efficacy and safety of experimental drugs, defining the metabolic pathways of experimental drugs and characterizing the properties (e.g., side effects, new uses) of existing drugs. Preferably, evaluation is carried out in three-dimensional tissue-engineered systems, wherein drug toxicity, metabolism, interaction and/or efficacy can be determined.

Claims

1. A method for determining metabolism of a test agent in a tissue comprising:

A) incubating a test agent and an enzyme within a three-dimensional tissue engineered system, such that an enzyme-substrate complex is formed between the enzyme and the test agent; and

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

2. The method of claim 1, wherein the tissue-engineered system 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 such as antihistamines and non-steroidal anti-inflammatory drugs (NSAIDs), diuretics such as carbonic anhydrase inhibitors, loop diuretics, high-ceiling diuretics, thiazide and thiazide-like agents, and potassium-sparing diuretics, agents that impinge on the renal and cardiovascular systems such as angiotensin converting enzyme inhibitors, cardiac drugs such as organic nitrates, calcium channel blockers, sympatholytic agents, vasodilators, β

-adrenergic receptor agonists and antagonists, α

-adrenergic receptor agonists and antagonists, cardiac glycosides, anti-arrhythmic drugs, agents that affect hyperlipoproteinemias such as 3-hydroxymethylglutaryl-coenzyme A (HMG-CoA) inhibitors, anti-neoplastic agents such as alkylating agents, antimetabolites, natural products, antibiotics, and other drugs, immunomodulators, anti-diabetic agents, and anti-microbial agents such as antibacterial agents, antiviral agents, antifungal agents, antiprotozoal agents, and antihelminthic 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.

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. A method of determining toxicity of a test agent in a tissue comprising:

A) incubating a test agent within a three-dimensional tissue engineered system; and

B) detecting an undesired effect.

12. The method of claim 11, wherein the tissue-engineered system comprises liver tissue, kidney tissue, cardiac tissue, cartilage tissue or bone marrow tissue, and combinations thereof.

13. The method of claim 11, wherein the test agent is provided for at least 24 hours.

14. The method of claim 11, wherein the test agent is provided for more than 90 days.

15. The method of claim 11, wherein the test agent comprises one or more compounds.

16. The method of claim 11, wherein the undesired effect comprises carcinogenicity, cell death, changes in gene expression, changes in protein expression or irregular metabolism and combinations thereof.

17. The method of claim 16, wherein carcinogenicity is detected by changes in gene expression, changes in protein levels, abnormal cell proliferation, or changes in expression of antigenic determinants and combinations thereof.

18. The method of claim 16, wherein cell death is detected by vital dyes, lactate dehydrogenase release, caspase activity, annexin V staining, phosphatidylserine staining or TUNEL assay.

19. The method of claim 16, wherein changes in gene expression are detected by microchip analysis, RT-PCR, in situ hybridization, fluorescence in situ hybridization or Northern analysis.

20. The method of claim 16, wherein changes in protein expression are detected by quantitative Western blot, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, amino acid sequence analysis, fluorescence activated cell sorting or protein concentration assays.

21. The method of claim 16, wherein irregular metabolism is indicated by detecting abnormal enzyme function.

22. The method of claim 21, 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.

23. A method for determining efficacy of a test agent, wherein efficacy comprises activity sufficient to decrease or eliminate hepatitis C virus in liver tissue comprising:

A) incubating a test agent within a three-dimensional liver tissue engineered system; and

B) measuring levels of hepatitis C virus.

24. A method for determining efficacy of a test agent, wherein efficacy comprises activity sufficient to decrease or eliminate hepatitis C virus in liver tissue comprising:

A) incubating a test agent within a three-dimensional liver tissue engineered system; and

B) detecting improved liver function.

25. The method of claim 24, wherein improved liver function is indicated by detecting normal enzyme levels, histology or protein production and combinations thereof.

26. The method of claim 24, wherein the enzyme is SGOT, ALT or LDH.