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US5260210A
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Blood-brain barrier model
Assignee: YEDNICK THEODORE A
•
Filing: 1990-09-04
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Granted: 1993-11-09
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Expires: 2010-09-04
Abstract
Abstract An in vitro model of a blood-brain barrier comprising a porous solid support upon which is disposed an essentially confluent monolayer of brain microvascular endothelial cells in contact with agents that elevate effective cyclic AMP concentrations in endothelial cells, with or without astrocyte-derived or endothelial cell-derived conditioned medium or the equivalent so that high electrical resistance tight junctions are formed between endothelial cells, and endothelial cells exhibit peripheral phalloidin staining and E-cadherin. Also disclosed is the use of agents that reduce effective cyclic AMP concentrations or interfere with the functioning of cyclic AMP or increase effective cyclic GMP concentrations to open up blood-brain barriers in vitro and in vivo, so that drugs normally excluded by such barriers may substantially penetrate such barriers. Also disclosed are uses of the model to screen for reagents with clinical utility in disorders involving brain endothelial cells.
Inventors
- 👤 Lee L. Rubin
- 👤 Seth Porter
- 👤 Heidi C. Horner
- 👤 Theodore A. Yednick
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 16:28
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/06; C12N5/0602; C12N5/069; A; A61; A61K; A61K47/00; A61K47/50; A61K47/51; A61K47/68; A61K47/6835; A61K47/6849; A61K47/69; A61K47/6921; A61P; A61P25/00; A61P25/28; A61P29/00; C07; C07K; C07K16/00; C07K16/18; C07K16/28; C07K16/2803; C07K16/2839; C07K16/2842; C12M; C12M25/00; C12M25/02; C12M35/00; C12M35/08; C12N5/0697; A61K39/00; A61K2039/505; C07K2317/00; C07K2317/70; C07K2317/76; C12N2502/00; C12N2502/08; C12N2502/28; C12N2503/00; C12N2533/00; C12N2533/30; C12N2533/50; C12N2533/52; C12N2533/54
Claims Analysis
Total Claims: 87
Claim 1
Independent Claim
49 words
An in vitro model of a blood-brain barrier, comprising a porous solid support upon which is disposed an essentially confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells such that peripheral phalloidin staining is substantially present.
Claim 2
Dependent Claim
49 words
An in vitro model of a blood-brain barrier, comprising a porous solid support upon which is disposed an essentially confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells such that peripheral phalloidin staining is substantially present.
Claim 3
Dependent Claim
15 words
A model as recited in claim 1, wherein said endothelial cells are mixed endothelial cells.
Claim 4
Dependent Claim
33 words
A model as recited in claim 1, wherein said porous solid support is a porous filter or membrane of a material selected from the group consisting of polycarbonate, nitrocellulose, cellulose, collagen and fiberglass.
Claim 5
Dependent Claim
29 words
A model as recited in claim 1, further comprising an extracellular matrix material disposed upon said porous solid support, interposed between said endothelial cells and said porous solid support.
Claim 6
Dependent Claim
28 words
A model as recited in claim 4, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel™.
Claim 7
Dependent Claim
52 words
A model as recited in claim 1, wherein said agent is selected from the group consisting of agents that increase cell adenylate cyclase activity, agents that inhibit the degradation of intracellular cyclic AMP, and derivatives of cyclic AMP to which endothelial cells are permeable and which act physiologically as does cyclic AMP.
Claim 8
Dependent Claim
41 words
An in vitro model of a blood-brain barrier as recited in claim 1, further comprising an essentially confluent monolayer of brain astrocytes disposed on a side of said porous solid support opposite the side on which are disposed said endothelial cells.
Claim 9
Dependent Claim
56 words
An in vitro model of a blood-brain barrier as recited in claim 1, further comprising an essentially confluent monolayer of brain astrocytes disposed on a second surface other than the porous solid support upon which is disposed a monolayer of endothelial cells, in contact with said growth medium to which the endothelial cells are also exposed.
Claim 10
Dependent Claim
15 words
A model as recited in claim 1, wherein said porous solid support comprises microcarrier beads.
Claim 11
Dependent Claim
25 words
A model as recited in claim 9, further comprising an extracellular matrix material disposed on said beads, interposed between said beads and said endothelial cells.
Claim 12
Dependent Claim
28 words
A model as recited in claim 10, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 13
Dependent Claim
17 words
A model as recited in claim 1, wherein said porous solid support comprises a tubular hollow fiber.
Claim 14
Dependent Claim
27 words
A model as recited in claim 12, further comprising an extracellular matrix material disposed on said hollow fiber, interposed between said hollow fiber and said endothelial cells.
Claim 15
Dependent Claim
28 words
A model as recited in claim 13, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 16
Dependent Claim
15 words
A model as recited in claim 1, wherein E-cadherin is substantially present between endothelial cells.
Claim 17
Dependent Claim
53 words
An in vitro model of a blood-brain barrier, comprising a porous solid support upon which is disposed a confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells, such that the transmonolayer resistance is at least about 200 ohm-cm 2 .
Claim 18
Dependent Claim
53 words
An in vitro model of a blood-brain barrier, comprising a porous solid support upon which is disposed a confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells, such that the transmonolayer resistance is at least about 200 ohm-cm 2 .
Claim 19
Dependent Claim
17 words
A model as recited in claim 16, wherein said endothelial cells are mixed or cloned endothelial cells.
Claim 20
Dependent Claim
33 words
A model as recited in claim 16, wherein said porous solid support is a porous filter or membrane of a material selected from the group consisting of polycarbonate, nitrocellulose, cellulose, collagen and fiberglass.
Claim 21
Dependent Claim
29 words
A model as recited in claim 16, further comprising an extracellular matrix material disposed upon said porous solid support interposed between said endothelial cells and said porous solid support.
Claim 22
Dependent Claim
28 words
A model as recited in claim 19, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 23
Dependent Claim
52 words
A model as recited in claim 16, wherein said agent is selected from the group consisting of agents that increase cell adenylate cyclase activity, agents that inhibit the degradation of intracellular cyclic AMP, and derivatives of cyclic AMP to which endothelial cells are permeable and which act physiologically as does cyclic AMP.
Claim 24
Dependent Claim
41 words
An in vitro model of a blood-brain barrier as recited in claim 16, further comprising an essentially confluent monolayer of brain astrocytes disposed on a side of said porous solid support opposite the side on which are disposed said endothelial cells.
Claim 25
Dependent Claim
57 words
An in vitro model of a blood-brain barrier as recited in claim 16 , further comprising an essentially confluent monolayer of brain astrocytes disposed on a second surface other than the porous solid support upon which is disposed a monolayer of endothelial cells, in contact with said growth medium to which the endothelial cells are also exposed.
Claim 26
Dependent Claim
17 words
A model as recited in claim 16, wherein said porous solid support comprises a tubular hollow fiber.
Claim 27
Dependent Claim
27 words
A model as recited in claim 24, further comprising an extracellular matrix material disposed on said hollow fiber, interposed between said hollow fiber and said endothelial cells.
Claim 28
Dependent Claim
28 words
A model as recited in claim 25, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 29
Dependent Claim
15 words
A model as recited in claim 16, wherein E-cadherin is substantially present between endothelial cells.
Claim 30
Dependent Claim
73 words
An in vitro model of a blood-brain barrier comprising a porous solid support upon which is disposed an essentially confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells, wherein said growth medium is a cell-derived conditioned medium, a medium supplemented with a cell extract or a medium supplemented with a tissue extract, such that peripheral phalloidin staining is substantially present.
Claim 31
Dependent Claim
73 words
An in vitro model of a blood-brain barrier comprising a porous solid support upon which is disposed an essentially confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells, wherein said growth medium is a cell-derived conditioned medium, a medium supplemented with a cell extract or a medium supplemented with a tissue extract, such that peripheral phalloidin staining is substantially present.
Claim 32
Dependent Claim
17 words
A model as recited in claim 28, wherein said endothelial cells are mixed or cloned endothelial cells.
Claim 33
Dependent Claim
34 words
A model as recited in claim 28, wherein said porous solid support is a porous filter or membrane of a material selected from among the group consisting of polycarbonate, nitrocellulose, cellulose, collagen and fiberglass.
Claim 34
Dependent Claim
29 words
A model as recited in claim 28, further comprising an extracellular matrix material disposed upon said porous solid support interposed between said endothelial cells and said porous solid support.
Claim 35
Dependent Claim
28 words
A model as recited in claim 31, wherein said extracellular matrix is selected from among the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 36
Dependent Claim
52 words
A model as recited in claim 28, wherein said agent is selected from the group consisting of agents that increase cell adenylate cyclase activity, agents that inhibit the degradation of intracellular cyclic AMP, and derivatives of cyclic AMP to which endothelial cells are permeable and which act physiologically as does cyclic AMP.
Claim 37
Dependent Claim
21 words
A model as recited in claim 28 wherein said cell-derived conditioned medium is endothelial cell-derived conditioned medium or astrocyte-derived conditioned medium.
Claim 38
Dependent Claim
41 words
An in vitro model of a blood-brain barrier as recited in claim 28, further comprising an essentially confluent monolayer of brain astrocytes disposed on a side of said porous solid support opposite the side on which are disposed said endothelial cells.
Claim 39
Dependent Claim
56 words
An in vitro model of a blood-brain barrier as recited in claim 28, further comprising an essentially confluent monolayer of brain astrocytes disposed on a second surface other than the porous solid support upon which is disposed a monolayer of endothelial cells, in contact with said growth medium to which the endothelial cells are also exposed.
Claim 40
Dependent Claim
15 words
A model as recited in claim 28, wherein said porous solid support comprises microcarrier beads.
Claim 41
Dependent Claim
25 words
A model as recited in claim 37, further comprising an extracellular matrix material disposed on said beads, interposed between said beads and said endothelial cells.
Claim 42
Dependent Claim
28 words
A model as recited in claim 38, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fribronectin, collagen, and Matrigel®.
Claim 43
Dependent Claim
17 words
A model as recited in claim 28, wherein said porous solid support comprises a tubular hollow fiber.
Claim 44
Dependent Claim
29 words
A model as recited in claim 40, further comprising a of an extracellular matrix material disposed on said hollow fiber, interposed between said hollow fiber and said endothelial cells.
Claim 45
Dependent Claim
28 words
A model as recited in claim 41, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 46
Dependent Claim
15 words
A model as recited in claim 28, wherein E-cadherin is substantially present between endothelial cells.
Claim 47
Dependent Claim
87 words
An in vitro model of a blood-brain barrier comprising a porous solid support upon which is disposed a confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells, wherein said growth medium is a cell-derived conditioned medium, a medium supplemented with a cell extract or a medium supplemented with a tissue extract, such that peripheral phalloidin staining is substantially present, and said monolayer has a transmonolayer resistance of at least about 200 ohm-cm 2 .
Claim 48
Dependent Claim
87 words
An in vitro model of a blood-brain barrier comprising a porous solid support upon which is disposed a confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells, wherein said growth medium is a cell-derived conditioned medium, a medium supplemented with a cell extract or a medium supplemented with a tissue extract, such that peripheral phalloidin staining is substantially present, and said monolayer has a transmonolayer resistance of at least about 200 ohm-cm 2 .
Claim 49
Dependent Claim
17 words
A model as recited in claim 44, wherein said endothelial cells are mixed or cloned endothelial cells.
Claim 50
Dependent Claim
34 words
A model as recited in claim 44, wherein said porous solid support is a porous filter or membrane of a material selected from among the group consisting of polycarbonate, nitrocellulose, cellulose, collagen and fiberglass.
Claim 51
Dependent Claim
29 words
A model as recited in claim 44, further comprising an extracellular matrix material disposed upon said porous solid support, interposed between said endothelial cells and said porous solid support.
Claim 52
Dependent Claim
29 words
A model as recited in claim 47, wherein said extracellular matrix material is selected from among the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 53
Dependent Claim
52 words
A model as recited in claim 44, wherein said agent is selected from the group consisting of agents that increase cell adenylate cyclase activity, agents that inhibit the degradation of intracellular cyclic AMP, and derivatives of cyclic AMP to which endothelial cells are permeable and which act physiologically as does cyclic AMP.
Claim 54
Dependent Claim
19 words
A model as recited in claim 44 wherein said cell-derived conditioned medium is endothelial cell-derived or astrocyte-derived conditioned medium.
Claim 55
Dependent Claim
41 words
An in vitro model of a blood-brain barrier as recited in claim 44, further comprising an essentially confluent monolayer of brain astrocytes disposed on a side of said porous solid support opposite the side on which are disposed said endothelial cells.
Claim 56
Dependent Claim
56 words
An in vitro model of a blood-brain barrier as recited in claim 44, further comprising an essentially confluent monolayer of brain astrocytes disposed on a second surface other than the porous solid support upon which is disposed a monolayer of endothelial cells, in contact with said growth medium to which the endothelial cells are also exposed.
Claim 57
Dependent Claim
17 words
A model as recited in claim 44, wherein said porous solid support comprises a tubular hollow fiber.
Claim 58
Dependent Claim
27 words
A model as recited in claim 53, further comprising an extracellular matrix material disposed on said hollow fiber, interposed between said hollow fiber and said endothelial cells.
Claim 59
Dependent Claim
28 words
A model as recited in claim 54, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 60
Dependent Claim
15 words
A model as recited in claim 44, wherein E-cadherin is substantially present between endothelial cells.
Claim 61
Dependent Claim
64 words
An in vitro model of a blood-brain barrier, comprising a porous solid support upon which is disposed a confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells such that peripheral phalloidin staining is substantially present, and said monolayer has a transmonolayer electric resistance of at least about 200 ohm-cm 2 .
Claim 62
Dependent Claim
64 words
An in vitro model of a blood-brain barrier, comprising a porous solid support upon which is disposed a confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said cells such that peripheral phalloidin staining is substantially present, and said monolayer has a transmonolayer electric resistance of at least about 200 ohm-cm 2 .
Claim 63
Dependent Claim
15 words
A model as recited in claim 57, wherein said endothelial cells are mixed endothelial cells.
Claim 64
Dependent Claim
32 words
A model as recited in claim 57, wherein said solid support is a porous filter or membrane of a material selected from the group consisting of polycarbonate, nitrocellulose, cellulose, collagen and fiberglass.
Claim 65
Dependent Claim
29 words
A model as recited in claim 57, further comprising an extracellular matrix material disposed upon said porous solid support interposed between said endothelial cells and said porous solid support.
Claim 66
Dependent Claim
28 words
A model as recited in claim 60, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 67
Dependent Claim
52 words
A model as recited in claim 57, wherein said agent is selected from the group consisting of agents that increase cell adenylate cyclase activity, agents that inhibit the degradation of intracellular cyclic AMP, and derivatives of cyclic AMP to which endothelial cells are permeable and which act physiologically as does cyclic AMP.
Claim 68
Dependent Claim
41 words
An in vitro model of a blood-brain barrier as recited in claim 57, further comprising an essentially confluent monolayer of brain astrocytes disposed on a side of said porous solid support opposite the side on which are disposed said endothelial cells.
Claim 69
Dependent Claim
56 words
An in vitro model of a blood-brain barrier as recited in claim 57, further comprising an essentially confluent monolayer of brain astrocytes disposed on a second surface other than the porous solid support upon which is disposed a monolayer of endothelial cells, in contact with said growth medium to which the endothelial cells, are also exposed.
Claim 70
Dependent Claim
17 words
A model as recited in claim 57, wherein said porous solid support comprises a tubular hollow fiber.
Claim 71
Dependent Claim
27 words
A model as recited in claim 65, further comprising an extracellular matrix material disposed on said hollow fiber, interposed between said hollow fiber and said endothelial cells.
Claim 72
Dependent Claim
28 words
A model as recited in claim 66, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 73
Dependent Claim
15 words
A model as recited in claim 57, wherein E-cadherin is substantially present between endothelial cells.
Claim 74
Dependent Claim
79 words
An in vitro model of a blood-brain barrier, comprising a porous solid support upon which is disposed a confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said endothelial cells, wherein said growth medium is a cell-derived conditioned medium, a medium supplemented with a cell extract or a medium supplemented with a tissue extract, such that the transmonolayer electrical resistance is at least about 200 ohm-cm 2 .
Claim 75
Dependent Claim
79 words
An in vitro model of a blood-brain barrier, comprising a porous solid support upon which is disposed a confluent monolayer of brain microvascular endothelial cells, a growth medium and an agent that effectively increases cyclic AMP concentrations in said endothelial cells, wherein said growth medium is a cell-derived conditioned medium, a medium supplemented with a cell extract or a medium supplemented with a tissue extract, such that the transmonolayer electrical resistance is at least about 200 ohm-cm 2 .
Claim 76
Dependent Claim
17 words
A model as recited in claim 69, wherein said endothelial cells are mixed or cloned endothelial cells.
Claim 77
Dependent Claim
33 words
A model as recited in claim 69, wherein said porous solid support is a porous filter or membrane of a material selected from the group consisting of polycarbonate, nitrocellulose, cellulose, collagen and fiberglass.
Claim 78
Dependent Claim
29 words
A model as recited in claim 69, further comprising an extracellular matrix material disposed upon said porous solid support interposed between said endothelial cells and said porous solid support.
Claim 79
Dependent Claim
28 words
A model as recited in claim 72, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagen, and Matrigel®.
Claim 80
Dependent Claim
52 words
A model as recited in claim 69, wherein said agent is selected from the group consisting of agents that increase cell adenylate cyclase activity, agents that inhibit the degradation of intracellular cyclic AMP, and derivatives of cyclic AMP to which endothelial cells are permeable and which act physiologically as does cyclic AMP.
Claim 81
Dependent Claim
19 words
A model as recited in claim 69, wherein said cell-derived conditioned medium is endothelial cell-derived or astrocyte-derived conditioned medium.
Claim 82
Dependent Claim
41 words
An in vitro model of a blood-brain barrier as recited in claim 69, further comprising an essentially confluent monolayer of brain astrocytes disposed on a side of said porous solid support opposite the side on which are disposed said endothelial cells.
Claim 83
Dependent Claim
56 words
An in vitro model of a blood-brain barrier as recited in claim 69, further comprising an essentially confluent monolayer of brain astrocytes disposed on a second surface other than the porous solid support upon which is disposed a monolayer of endothelial cells, in contact with said growth medium to which the endothelial cells are also exposed.
Claim 84
Dependent Claim
17 words
A model as recited in claim 69, wherein said porous solid support comprises a tubular hollow fiber.
Claim 85
Dependent Claim
27 words
A model as recited in claim 78, further comprising an extracellular matrix material disposed on said hollow fiber, interposed between said hollow fiber and said endothelial cells.
Claim 86
Dependent Claim
28 words
A model as recited in claim 79, wherein said extracellular matrix material is selected from the group consisting of astrocyte extracellular matrix, laminin, vitronectin, fibronectin, collagent, and Matrigel®.
Claim 87
Dependent Claim
15 words
A model as recited in claim 69, wherein E-cadherin is substantially present between endothelial cells.
References Cited (Backward Citations)
US4208406A
Cited
US4764504A
Cited
USPTO Annuity & Maintenance Windows
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| 3.5 Years (Year 4) | 1996-11-08 | 1997-05-10 | 1997-11-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2000-11-08 | 2001-05-10 | 2001-11-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2004-11-08 | 2005-05-10 | 2005-11-09 | $7700.00 | Window Closed / Lapsed |