Claims for Patent: 7,811,782

Summary for Patent: 7,811,782

Title:	Use of an in vitro hemodynamic endothelial/smooth muscle cell co-culture model to identify new therapeutic targets for vascular disease
Abstract:	Methods and devices for applying hemodynamic patterns to human/animal cells in culture are described. Hemodynamic flow patterns are measured directly from the human circulation and translated to a motor that controls the rotation of a cone. The cone is submerged in fluid (i.e., cell culture media) and brought into close proximity to the cells. Rotation of the cone creates time-varying shear stresses. This model closely mimics the physiological hemodynamic forces imparted on endothelial cells in vivo. A TRANSWELL coculture dish (i.e., a coculture dish comprising an artificial porous membrane) may be incorporated, permitting two, three, or more different cell types to be physically separated within the culture dish environment. In-flow and out-flow tubing may be used to supply media, drugs, etc. separately and independently to both the inner and outer chambers. The physical separation of the cell types permits each cell type to be separately isolated for analysis.
Inventor(s):	Blackman; Brett R. (Charlottesville, VA), Wamhoff; Brian R. (Charlottesville, VA)
Assignee:	Hemoshear, LLC (Charlottesville, VA)
Application Number:	12/007,483
Patent Claims:	1. A method of testing a drug or a compound for an effect on the vascular system, said method comprising: adding a culture media to a Petri dish; adding a drug or a compound to the culture media; plating a first cell type on a first side of a porous membrane, plating a second cell type on a second side of the porous membrane, wherein said porous membrane is suspended in the Petri dish such that the first side is proximal and in spaced relation to a surface of the Petri dish, thereby defining within the Petri dish a lower volume comprising the first cell type and an upper volume comprising the second cell type, the porous membrane being adapted to permit fluid communication of the culture media and physical interaction and communication between cells of the first cell type and cells of the second cell type, and all of the cell types are within the culture media; perfusing culture media into and out of the upper volume; perfusing culture media into and out of the lower volume; applying a shear force upon the plated second cell type, said shear force resulting from flow of the culture media induced by a hemodynamic flow device, said flow mimicking hemodynamic flow; and comparing at least one of the first cell type and the second cell type after applying the shear force for a period of time to at least one of the first cell type and the second cell type after applying the shear force for the period of time wherein the media does not include the drug or compound, to determine the effect of the drug or compound on at least one of the first cell type and the second cell type. 2. The method of claim 1, where the drug or the compound is added to the culture media while applying the shear force. 3. The method of claim 1, where the drug or the compound is added to the culture media before applying the shear force. 4. The method of claim 1, wherein the drug is a cyclooxygenase inhibitor; a taxane; a tyrosine kinase inhibitor; a low molecular weight heparin; an anti-thrombogenic agent; a calcium channel blocker; an anti-platelet agent; an anticlotting agent; a chelating agent; an anti-inflammatory agent; a rho kinase inhibitor; a PDGF inhibitor, a cholesterol lowering agent; an anti-restenosis agent; an antibiotic; an anti-neoplastic agent; an anti-hypertensive agent; a synthetic polysaccharide; an agent that raises HDL; or a combination thereof. 5. The method of claim 4, wherein the cyclooxygenase inhibitor is celecoxib; the taxane is paclitaxel; the tyrosine kinase inhibitor is imatinib; the low molecular weight heparin is enoxaparin; the anti-thrombogenic agent is bivalirudin, dipyridamole, urokinase, r-urokinase, r-prourokinase, reteplase, alteplase, streptokinase, rt-PA, TNK-rt-PA, monteplase, staphylokinase, pamiteplase, unfractionated heparin, or APSAC; the calcium channel blocker is amlodipine or nifedipine; the anti-platelet agent is clopidogrel, abciximab, tirofiban, orbofiban, xemilofiban, sibrafiban, roxifiban or ticlopinin; the anticlotting agent is fondaparinux; the chelating agent is penicillamine, triethylene tetramine dihydrochloride, EDTA, DMSA, deferoxamine mesylate or batimastat; the anti-inflammatory agent is rofecoxib; the rho kinase inhibitor is Y27632; the PDGF inhibitor is AG1295; the cholesterol lowering agent is a statin; the antibiotic is actinomycin-D; the anti-neoplastic agent is c-myc antisense or dexamethasone; or the anti-hypertensive agent is an ACE inhibitor. 6. The method of claim 1 wherein the drug is atorvastatin, sirolimus, tacrolimus, everolimus, wortmannin, or a combination thereof. 7. The method of claim 1, wherein the drug or the compound is a radiocontrast agent, a radio-isotope, a prodrug, an antibody fragment, an antibody, a live cell, a therapeutic drug delivery microsphere or microbead, or a combination thereof. 8. The method of claim 1 wherein the compound is capable of inhibiting, activating or altering the function of proteins or genes in said cell types. 9. The method of claim 1, further comprising the step of culturing all the cell types. 10. The method of claim 1, wherein said at least one of the first cell type and the second cell type is analyzed for toxicity, inflammation, permeability, compatibility, cellular adhesion or phenotypic modulation resulting from the drug or the compound. 11. The method of claim 1, wherein at least one of the first cell type and the second cell type are vascular or organ cells from one or more patients with an identified genotype linked to drug toxicity or a pathophysiological endpoint. 12. The method of claim 11, wherein said one or more patients have a single nucleotide polymorphism linked to drug toxicity or a pathophysiological endpoint. 13. The method of claim 1, wherein said first cell type is renal cells, cells of the airways, or cells of the blood-brain-barrier blood-brain barrier, and said second cell type is vascular cells. 14. The method of claim 1, wherein the hemodynamic flow is derived from a previously measured hemodynamic pattern. 15. The method of claim 14, wherein the previously measured hemodynamic pattern is human derived. 16. The method of claim 15, wherein said pattern is derived from a patient having a pathological condition. 17. The method of claim 1, wherein said hemodynamic flow is time-variant. 18. The method of claim 1, wherein the first cell type is smooth muscle cells, glial cells, astrocytes, neurons, or epithelial podocytes. 19. The method of claim 18, wherein the second cell type is endothelial cells. 20. The method of claim 1 further comprising: modeling a hemodynamic pattern into a set of electronic instructions; and applying the shear force upon the plated second cell type based on the set of electronic instructions. 21. The method of claim 20, wherein said hemodynamic pattern is derived from analysis of ultrasound data. 22. The method of claim 20, wherein said hemodynamic pattern is derived from analysis of magnetic resonance imaging (MRI) data. 23. The method of claim 20, wherein said hemodynamic pattern is time-variant. 24. The method of claim 20, wherein the set of electronic instructions is accepted by an electronic controller of a hemodynamic flow device, the electronic controller operating a motor of the device, and the motor causing rotation of a cone connected to the motor, wherein said rotation of the cone results in said flow of the culture media. 25. The method of claim 1, wherein the shear force is applied by a device for mimicking hemodynamic flow during cell culture, said device comprising: an electronic controller for receiving a set of electronic instructions; a motor operated by the electronic controller; and a shear force applicator operatively connected to the motor for being driven by the motor. 26. The method of claim 25, wherein the shear force applicator comprises a cone attached to the motor. 27. The method of claim 25, wherein the device further comprises inlets and outlets within the portions of the Petri dish defining the upper and lower volumes. 28. The method of claim 1, wherein the compound is a vascular stent material and the method further comprises testing at least one of the cell types for compatibility with, cellular adhesion to, or phenotypic modulation by the vascular stent material. 29. The method of claim 28, wherein the vascular stent material comprises a nanoporous metal, a polymer, or a carbon material. 30. The method of claim 28, wherein the drug or compound is eluted from a vascular stent material adjacent to the second cell type. 31. The method of claim 1, wherein the method further comprises perfusing the drug or compound into at least one of the upper volume and the lower volume. 32. The method of claim 1, further comprising either plating a third cell type on the surface of the Petri dish, or suspending a third cell type in the culture media in the lower volume. 33. The method of claim 32, further comprising the step of culturing all of the cell types. 34. The method of claim 32, further comprising the step of comparing at least one of the first cell type, the second cell type and the third cell type after applying the shear force for a period of time to at least one of the first cell type, the second cell type and the third cell type after applying the shear force for the period of time wherein the media does not include the drug or compound, to determine the effect of the drug or compound on at least one of the first cell type, the second cell type and the third cell type. 35. The method of claim 32, wherein the hemodynamic flow is derived from a previously measured hemodynamic pattern. 36. The method of claim 35, wherein the previously measured hemodynamic pattern is human derived. 37. The method of claim 32, wherein the first cell type is smooth muscle cells, glial cells, astrocytes, neurons, or epithelial podocytes. 38. The method of claim 21, wherein the second cell type is endothelial cells. 39. The method of claim 32, wherein the third cell type is smooth muscle cells, glial cells, astrocytes, neurons, macrophages, or leukocytes. 40. The method of claim 32, wherein said first cell type and said third cell type are, renal cells, cells of the airways, or cells of the blood-brain-barrier, and wherein said second cell type is vascular cells. 41. The method of claim 26, wherein the hemodynamic pattern is from an artery, a vein or an organ. 42. The method of claim 35, wherein said hemodynamic flow is time-variant. 43. The method of claim 1, further comprising analyzing said culture media for cytokine or humoral factor secretion.

Details for Patent 7,811,782

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Microbix Biosystems Inc.	KINLYTIC	urokinase	For Injection	021846	01/16/1978		2027-01-10
Genentech, Inc.	ACTIVASE	alteplase	For Injection	103172	11/13/1987		2027-01-10
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