U.S. patent application number 17/675887 was filed with the patent office on 2022-06-09 for compositions and methods for preventing the proliferation and epithelial-mesenchymal transition of epithelial cells.
The applicant listed for this patent is TissueTech, Inc.. Invention is credited to Hua HE, Ek Kia TAN, Scheffer TSENG.
Application Number | 20220175849 17/675887 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220175849 |
Kind Code |
A1 |
TSENG; Scheffer ; et
al. |
June 9, 2022 |
COMPOSITIONS AND METHODS FOR PREVENTING THE PROLIFERATION AND
EPITHELIAL-MESENCHYMAL TRANSITION OF EPITHELIAL CELLS
Abstract
Compositions and preparations of fetal support tissue that
prevent or reduce the proliferation and epithelial-mesenchymal
transition (EMT) of epithelial cells, wherein the epithelial cells
may be human epithelial cells and the human epithelial cells may be
conjunctival, retinal, corneal, limbal, or renal epithelial cells.
Methods of preventing or reducing the proliferation, cell
migration, and EMT of epithelial cells in an individual in need
thereof, wherein the epithelial cells may be human epithelial cells
and the human epithelial cells may be conjunctival, retinal,
corneal, limbal, or renal epithelial cells. Methods of preventing
or treating proliferative vitreoretinopathy in an individual in
need thereof.
Inventors: |
TSENG; Scheffer; (Pinecrest,
FL) ; TAN; Ek Kia; (Miami, FL) ; HE; Hua;
(Miami, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TissueTech, Inc. |
Miami |
FL |
US |
|
|
Appl. No.: |
17/675887 |
Filed: |
February 18, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16421191 |
May 23, 2019 |
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17675887 |
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15160487 |
May 20, 2016 |
10342831 |
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16421191 |
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62164281 |
May 20, 2015 |
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International
Class: |
A61K 35/50 20060101
A61K035/50; A61K 9/00 20060101 A61K009/00; A61K 35/51 20060101
A61K035/51; A61K 47/36 20060101 A61K047/36; A61P 27/02 20060101
A61P027/02 |
Claims
1. An composition for preventing or reducing proliferation, cell
migration, or epithelial-mesenchymal transition (EMT) of epithelial
cells, comprising: (a) a preparation of fetal support tissue; and
(b) a pharmaceutically acceptable diluent, excipient, vehicle, or
carrier.
2. The composition according to claim 1, wherein the fetal support
tissue is selected from placenta, placental amniotic membrane,
umbilical cord, umbilical cord amniotic membrane, chorion,
amnion-chorion, amniotic stroma, amniotic jelly, or a combination
thereof.
3. The composition of claim 1, wherein the fetal support tissue is
frozen or previously frozen.
4. The composition according to claim 1, wherein the epithelial
cells are selected from retinal pigment epithelial cells (RPE),
conjunctival epithelial cells, corneal epithelial cells, limbal
epithelial cells, and renal epithelial cells.
5. The composition according to claim 1, wherein the epithelial
cells are human epithelial cells.
6. The composition of claim 1, wherein the preparation of fetal
support tissue is an extract of fetal support tissue, a homogenate,
a powder, morselized fetal support tissue, pulverized fetal support
tissue, ground fetal support tissue, purified HC-HA/PTX3, or a
combination thereof.
7. The composition according to claim 1, wherein the composition is
a gel, a solution, or a suspension.
8. The composition according to claim 1, wherein the preparation
comprises HC-HA/PTX3.
9. The composition according to claim 1, wherein the composition is
for local administration.
10. The composition according to claim 1, wherein the composition
is formulated for intraocular injection, subretinal injection,
intravitreal injection, periocular injection, subconjunctival
injection, retrobulbar injection, intracameral injection, or
sub-Tenon's injection.
11. An injectable composition for treating or preventing
Proliferative Vitreoretinopathy (PVR) consisting essentially of:
(a) substantially isolated HC-HA/PTX3, reconstituted HC-HA/PTX3, or
a combination thereof; and (b) a pharmaceutically acceptable
diluent, excipient, vehicle, or carrier; wherein the composition is
suitable for injection.
12. The composition according to claim 11, wherein the HC-HA/PTX3
is isolated from a fetal support tissue, wherein the fetal support
tissue is placenta, placental amniotic membrane, umbilical cord,
umbilical cord amniotic membrane, chorion, amnion-chorion, amniotic
stroma, amniotic jelly, or a combination thereof.
13. The composition of claim 12, wherein the fetal support tissue
is frozen or previously frozen.
14. The composition of claim 11, wherein the composition is in an
amount effective for preventing or reducing the proliferation, cell
migration or EMT of epithelial cells.
15. The composition of claim 14, wherein the epithelial cells are
retinal pigment epithelial cells (RPE).
16. The composition of claim 14, wherein the epithelial cells are
human epithelial cells.
17. The composition of claim 11, wherein the substantially isolated
HC-HA/PTX3 is isolated from fetal support tissue by
ultracentrifugation.
18. The composition according to claim 11, wherein the composition
is a gel, a solution, or a suspension.
19. The composition according to claim 11, wherein the composition
is formulated for intraocular injection, subretinal injection,
intravitreal injection, periocular injection, subconjunctival
injection, retrobulbar injection, intracameral injection or
sub-Tenon's injection.
20. An injectable composition for treating or preventing
Proliferative Vitreoretinopathy (PVR) consisting essentially of:
(a) substantially isolated HC-HA/PTX3, reconstituted HC-HA/PTX3, or
a combination thereof; (b) an additional therapeutic agent; and (c)
a pharmaceutically acceptable diluent, excipient, vehicle, or
carrier; wherein the composition is suitable for injection.
21. An injectable composition for treating or preventing
Proliferative Vitreoretinopathy (PVR) comprising: (a) a preparation
of fetal support tissue comprising HC-HA/PTX3 and at least one
other component of fetal support tissue; and (b) a pharmaceutically
acceptable diluent, excipient, vehicle, or carrier; wherein the
composition is suitable for injection.
22. The composition according to claim 21, wherein the fetal
support tissue is placenta, placental amniotic membrane, umbilical
cord, umbilical cord amniotic membrane, chorion, amnion-chorion,
amniotic stroma, amniotic jelly, amniotic fluid, or a combination
thereof.
23. The composition of claim 21, wherein the fetal support tissue
is frozen or previously frozen.
24. The composition of claim 21, wherein the composition is in an
amount effective for preventing or reducing the proliferation, cell
migration or EMT of epithelial cells.
25. The composition according to claim 24, wherein the epithelial
cells are retinal pigment epithelial (RPE) cells.
26. The composition of claim 21, wherein the preparation of fetal
support tissue is an extract of fetal support tissue, micronized
fetal support tissue, a homogenate, a powder, morselized fetal
support tissue, pulverized fetal support tissue, ground fetal
support tissue, purified HC-HA/PTX3, or a combination thereof.
27. The composition according to claim 21, wherein the composition
is a gel, a solution, or a suspension.
28. The composition according to claim 21, wherein the fetal
support tissue is human, non-human primate, bovine, or porcine.
29. The composition according to claim 21, wherein the composition
is formulated for intraocular injection, subretinal injection,
intravitreal injection, periocular injection, subconjunctival
injection, retrobulbar injection, intracameral injection or
sub-Tenon's injection.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 16/421,191, filed May 23, 2019, which is a continuation of U.S.
application Ser. No. 15/160,487, filed May 20, 2016, which claims
the benefit of and right of priority to U.S. Provisional
Application No. 62/164,281 filed May 20, 2015, which is
incorporated herein by reference in its entirety.
SUMMARY OF THE INVENTION
[0002] Disclosed herein, in certain embodiments, are methods for
preventing or reducing proliferation, cell migration, or
epithelial-mesenchymal transition (EMT) of epithelial cells in an
individual in need thereof, comprising: administering to the
individual a therapeutically effective amount of a composition,
comprising: (a) a preparation of fetal support tissue; and (b) a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier, thereby preventing or reducing the proliferation, cell
migration, or EMT of epithelial cells, wherein the epithelial cells
are not retinal pigment epithelial cells. In some embodiments, the
EMT is associated with a disease or disorder other than
proliferative vitreoretinopathy (PVR). In some embodiments, the EMT
is associated with a disease or disorder selected from cancer,
proliferative diabetic retinopathy, fibrotic lesion, and
Retro-corneal membrane. In some embodiments, the fetal support
tissue is selected from the group consisting of: placenta,
placental amniotic membrane, umbilical cord, umbilical cord
amniotic membrane, chorion, amnion-chorion, amniotic stroma,
amniotic jelly, amniotic fluid, and a combination thereof. In some
embodiments, the fetal support tissue is frozen or previously
frozen. In some embodiments, the epithelial cells are selected from
conjunctival epithelial cells, corneal epithelial cells, limbal
epithelial cells, and renal epithelial cells. In some embodiments,
the epithelial cells are human epithelial cells. In some
embodiments, the human epithelial cells are retinal pigment
epithelial cells (RPE). In some embodiments, the human epithelial
cells are conjunctival epithelial cells. In some embodiments, the
human epithelial cells are corneal epithelial cells. In some
embodiments, the human epithelial cells are limbal epithelial
cells. In some embodiments, the human epithelial cells are renal
epithelial cells. In some embodiments, the preparation of fetal
support tissue is an extract of fetal support tissue, a homogenate,
a powder, morselized fetal support tissue, pulverized fetal support
tissue, ground fetal support tissue, purified HC-HA/PTX3, or a
combination thereof. In some embodiments, the composition is a gel,
a solution, or a suspension. In some embodiments, the composition
is in an injectable form. In some embodiments, the preparation of
fetal support tissue comprises substantially isolated HC-HA/PTX3.
In some embodiments, the preparation of fetal support tissue
consists of substantially isolated HC-HA/PTX3. In some embodiments,
the preparation of fetal support tissue comprises reconstituted
HC-HA/PTX3. In some embodiments, the preparation of fetal support
tissue comprises high molecular weight hyaluronan (HA) that is
cross-linked by a covalent bond to the heavy chain of
inter-.alpha.-trypsin inhibitor (I.alpha.I), the high molecular
weight HA having a molecular weight greater than 1000 kDa. In some
embodiments, the preparation of fetal support tissue comprises
pentraxin 3 (PTX-3). In some embodiments, the preparation of fetal
support tissue comprises tumor necrosis factor-stimulated gene 6
protein (TSG-6). In some embodiments, the preparation of fetal
support tissue comprises thrombospondin-1 (TSP-1). In some
embodiments, the ratio of total protein to HA in the composition is
between 500 parts protein:1 part HA and 500 parts HA:1 parts
protein. In some embodiments, the composition prevents the
proliferation and EMT of epithelial cells by counteracting the
actions of growth factors and cytokines. In some embodiments, the
growth factors and cytokines are selected from the group consisting
of: EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C, TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, CTGF, HGF, IGF-1, G-CSF, IL-6, MCP-1,
TNF-.alpha., VEGF, and IFN-.gamma.. In some embodiments, the
composition further comprises an aqueous adjuvant. In some
embodiments, the composition is for local administration. In some
embodiments the composition if formulated for injection. In some
embodiments, the composition is formulated for intraocular
injection, subretinal injection, intravitreal injection, periocular
injection, subconjunctival injection, retrobulbar injection,
intracameral injection, or sub-Tenon's injection.
[0003] Disclosed herein, in certain embodiments, are methods method
for treating or preventing Proliferative Vitreoretinopathy (PVR) in
an individual in need thereof, comprising administering to the
individual a therapeutically effective amount of an injectable
composition, consisting essentially of: (a) substantially isolated
HC-HA/PTX3, reconstituted HC-HA/PTX3, or a combination thereof; and
(b) a pharmaceutically acceptable diluent, excipient, vehicle, or
carrier, thereby treating or preventing PVR. In some embodiments,
the composition consists of: (a) substantially isolated HC-HA/PTX3,
reconstituted HC-HA/PTX3, or a combination thereof; and (b) a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the composition consists of
reconstituted HC-HA/PTX3 and a pharmaceutically acceptable diluent,
excipient, vehicle, or carrier. In some embodiments, the
composition consists of substantially isolated HC-HA/PTX3 and a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the substantially isolated HC-HA/PTX3
is isolated from fetal support tissue is selected from the group
consisting of: placenta, placental amniotic membrane, umbilical
cord, umbilical cord amniotic membrane, chorion, amnion-chorion,
amniotic stroma, amniotic jelly, amniotic fluid, and a combination
thereof. In some embodiments, the fetal support tissue is frozen or
previously frozen. In some embodiments, the fetal support tissue is
human, non-human primate, bovine, or porcine. In some embodiments,
the fetal support tissue is human. In some embodiments, the
substantially isolated HC-HA/PTX3 is isolated from fetal support
tissue by ultracentrifugation. In some embodiments, the
therapeutically effective amount is effective for preventing or
reducing the proliferation, cell migration or EMT of epithelial
cells. In some embodiments, the epithelial cells are retinal
pigment epithelial cells (RPE). In some embodiments, the epithelial
cells are human epithelial cells. In some embodiments, the human
epithelial cells are retinal epithelial cells. In some embodiments,
the injectable composition is a gel, a solution, or a suspension.
In some embodiments, the composition comprises high molecular
weight hyaluronan (HA) that is cross-linked by a covalent bond to
the heavy chain of inter-.alpha.-trypsin inhibitor (I.alpha.I), the
high molecular weight HA having a molecular weight greater than
1000 kDa. In some embodiments, the composition comprises pentraxin
3 (PTX-3). In some embodiments, the composition comprises tumor
necrosis factor-stimulated gene 6 protein (TSG-6). In some
embodiments, the ratio of total protein to HA in the injectable
composition is between 500 parts protein:1 part HA and 500 parts
HA:1 parts protein. In some embodiments, the injectable composition
prevents the proliferation and EMT of epithelial cells by
inhibiting or suppressing the activity of one or more growth
factors or cytokines. In some embodiments, the growth factors and
cytokines are selected from the group consisting of: EGF, FGF-2,
PDGF-A, PDGF-AB, PDGF-B, PDGF-C, TGF.beta.1, TGF-.beta.2,
TGF-.beta.3, CTGF, HGF, IGF-1, G-CSF, IL-6, MCP-1, TNF-.alpha.,
VEGF, and IFN-.gamma.. In some embodiments, the injectable
composition further comprises an aqueous adjuvant. In some
embodiments, the injectable composition is for local
administration. In some embodiments, the injectable composition is
formulated for intraocular injection, subretinal injection,
intravitreal injection, periocular injection, subconjunctival
injection, retrobulbar injection, intracameral injection or
sub-Tenon's injection. In some embodiments, the composition is
formulated for intravitreal injection.
[0004] Disclosed herein, in certain embodiments, are methods method
for treating or preventing Proliferative Vitreoretinopathy (PVR) in
an individual in need thereof, comprising administering to the
individual a therapeutically effective amount of an injectable
composition, consisting essentially of: (a) substantially isolated
HC-HA/PTX3, reconstituted HC-HA/PTX3, or a combination thereof (b)
an additional therapeutic agent; and (c) a pharmaceutically
acceptable diluent, excipient, vehicle, or carrier, thereby
treating or preventing PVR. In some embodiments, the composition
consists of: (a) substantially isolated HC-HA/PTX3, reconstituted
HC-HA/PTX3, or a combination thereof, (b) an additional therapeutic
agent; and (c) a pharmaceutically acceptable diluent, excipient,
vehicle, or carrier. In some embodiments, the additional
therapeutic agent is an additional agent for treating PVR. In some
embodiments, the additional therapeutic agent is selected from the
group consisting of: oral Accutane, intravitreal triamcinolone
acetonide, ranibizumab, bevacizumab, dasatinib, pegaptanib sodium,
N-acetyl-cysteine (NAC), pioglitazone, glucosamine, genistin,
geldanamycin, fausdil, resveratrol, hepatocyte growth factor (HGF),
BMP-7, LY-364947, diosgenin, emodin, pentoxyfilline, dipyridamole,
a peroxisome proliferative-activated receptor-gamma (PPAR.gamma.)
agonist, a female sex hormone, and an antioxidant. In some
embodiments, the female sex hormone comprises estradiol or
progesterone. In some embodiments, the antioxidant comprises beta
carotene, vitamin C, vitamin E, lutein, zeaxanthin, and omega-3
fatty acids. In some embodiments the additional therapeutic agent
is an additional agent for treating inflammation. In some
embodiments, the composition consists of: (a) substantially
isolated HC-HA/PTX3, reconstituted HC-HA/PTX3, or a combination
thereof; (b) an additional therapeutic agent; and (c) a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the composition consists of
reconstituted HC-HA/PTX3, an additional therapeutic agent, and a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the composition consists of
substantially isolated HC-HA/PTX3, an additional therapeutic agent,
and a pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the substantially isolated HC-HA/PTX3
is isolated from fetal support tissue is selected from the group
consisting of: placenta, placental amniotic membrane, umbilical
cord, umbilical cord amniotic membrane, chorion, amnion-chorion,
amniotic stroma, amniotic jelly, amniotic fluid, and a combination
thereof. In some embodiments, the fetal support tissue is frozen or
previously frozen. In some embodiments, the fetal support tissue is
human, non-human primate, bovine, or porcine. In some embodiments,
the fetal support tissue is human. In some embodiments, the
substantially isolated HC-HA/PTX3 is isolated from fetal support
tissue by ultracentrifugation. In some embodiments, the composition
further comprises an additional therapeutic agent. In some
embodiments, the therapeutically effective amount is effective for
preventing or reducing the proliferation, cell migration or EMT of
epithelial cells. In some embodiments, the epithelial cells are
retinal pigment epithelial cells (RPE). In some embodiments, the
epithelial cells are human epithelial cells. In some embodiments,
the human epithelial cells are retinal epithelial cells. In some
embodiments, the injectable composition is a gel, a solution, or a
suspension. In some embodiments, the composition comprises high
molecular weight hyaluronan (HA) that is cross-linked by a covalent
bond to the heavy chain of inter-.alpha.-trypsin inhibitor
(I.alpha.I), the high molecular weight HA having a molecular weight
greater than 1000 kDa. In some embodiments, the composition
comprises pentraxin 3 (PTX-3). In some embodiments, the composition
comprises tumor necrosis factor-stimulated gene 6 protein (TSG-6).
In some embodiments, the preparation of fetal support tissue
comprises thrombospondin-1 (TSP-1). In some embodiments, the ratio
of total protein to HA in the injectable composition is between 500
parts protein:1 part HA and 500 parts HA:1 parts protein. In some
embodiments, the injectable composition prevents the proliferation
and EMT of epithelial cells by inhibiting or suppressing the
activity of one or more growth factors or cytokines. In some
embodiments, the growth factors and cytokines are selected from the
group consisting of: EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C,
TGF.beta.1, TGF-.beta.2, TGF-.beta.3, CTGF, HGF, IGF-1, G-CSF,
IL-6, MCP-1, TNF-.alpha., VEGF, and IFN-.gamma.. In some
embodiments, the injectable composition further comprises an
aqueous adjuvant. In some embodiments, the injectable composition
is for local administration. In some embodiments, the injectable
composition is formulated for intraocular injection, subretinal
injection, intravitreal injection, periocular injection,
subconjunctival injection, retrobulbar injection, intracameral
injection or sub-Tenon's injection. In some embodiments, the
composition is formulated for intravitreal injection.
[0005] Disclosed herein, in certain embodiments, are methods for
treating or preventing Proliferative Vitreoretinopathy (PVR) in an
individual in need thereof, comprising administering to the
individual a therapeutically effective amount of an injectable
composition, comprising: a preparation of fetal support tissue
comprising HC-HA/PTX3 and at least one other component of fetal
support tissue; and a pharmaceutically acceptable diluent,
excipient, vehicle, or carrier, thereby treating or preventing PVR.
In some embodiments, the fetal support tissue is placenta,
placental amniotic membrane, umbilical cord, umbilical cord
amniotic membrane, chorion, amnion-chorion, amniotic stroma,
amniotic jelly, amniotic fluid, or a combination thereof. In some
embodiments, the fetal support tissue is frozen or previously
frozen. In some embodiments, the fetal support tissue is human,
non-human primate, bovine, or porcine. In some embodiments, the
fetal support tissue is human. In some embodiments, the
therapeutically effective amount is an amount effective for
preventing or reducing the proliferation, cell migration or EMT of
epithelial cells. In some embodiments, the epithelial cells are
retinal pigment epithelial (RPE) cells. In some embodiments, the
preparation of fetal support tissue is an extract of fetal support
tissue, micronized fetal support tissue, a homogenate, a powder,
morselized fetal support tissue, pulverized fetal support tissue,
ground fetal support tissue, purified HC-HA/PTX3, or a combination
thereof. In some embodiments, the composition is a gel, a solution,
or a suspension. In some embodiments, the composition is formulated
for intraocular injection, subretinal injection, intravitreal
injection, periocular injection, subconjunctival injection,
retrobulbar injection, intracameral injection or sub-Tenon's
injection.
[0006] Disclosed herein, in certain embodiments, are compositions
for preventing or reducing proliferation, cell migration, and/or
epithelial-mesenchymal transition (EMT) of epithelial cells,
comprising: (a) a preparation of fetal support tissue; and (b) a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier, wherein the epithelial cells are not retinal pigment
epithelial cells. In some embodiments, the EMT is associated with a
disease or disorder other than proliferative vitreoretinopathy. In
some embodiments, the EMT is associated with a disease or disorder
selected from cancer, proliferative diabetic retinopathy, fibrotic
lesion, and Retro-corneal membrane. In some embodiments, the fetal
support tissue is selected from the group consisting of: placenta,
placental amniotic membrane, umbilical cord, umbilical cord
amniotic membrane, chorion, amnion-chorion, amniotic stroma,
amniotic jelly, amniotic fluid, and a combination thereof. In some
embodiments, the fetal support tissue is frozen or previously
frozen. In some embodiments, the fetal support tissue is human,
non-human primate, bovine, or porcine. In some embodiments, the
fetal support tissue is human. In some embodiments, the composition
is in a therapeutically effective amount for preventing or reducing
the proliferation, cell migration or EMT of epithelial cells. In
some embodiments, the epithelial cells are selected from
conjunctival epithelial cells, corneal epithelial cells, limbal
epithelial cells, and renal epithelial cells. In some embodiments,
the epithelial cells are human epithelial cells. In some
embodiments, the human epithelial cells are retinal pigment
epithelial cells (RPE). In some embodiments, the human epithelial
cells are conjunctival epithelial cells. In some embodiments, the
human epithelial cells are corneal epithelial cells. In some
embodiments, the human epithelial cells are limbal epithelial
cells. In some embodiments, the human epithelial cells are renal
epithelial cells. In some embodiments, the preparation of fetal
support tissue is an extract of fetal support tissue, micronized
fetal support tissue, a homogenate, a powder, morselized fetal
support tissue, pulverized fetal support tissue, ground fetal
support tissue, or purified HC-HA/PTX3. In some embodiments, the
composition is a gel, a solution, or a suspension. In some
embodiments, the preparation of fetal support tissue comprises
HC-HA/PTX3. In some embodiments, the preparation of fetal support
tissue comprises substantially isolated HC-HA/PTX3. In some
embodiments, the preparation of fetal support tissue consists of
substantially isolated HC-HA/PTX3. In some embodiments, the
preparation of fetal support tissue comprises reconstituted
HC-HA/PTX3. In some embodiments, the preparation of fetal support
tissue comprises high molecular weight hyaluronan (HA) that is
cross-linked by a covalent bond to the heavy chain of
inter-.alpha.-trypsin inhibitor (I.alpha.I), the high molecular
weight HA having a molecular weight greater than 1000 kDa. In some
embodiments, the preparation of fetal support tissue comprises
pentraxin 3 (PTX-3). In some embodiments, the preparation of fetal
support tissue comprises tumor necrosis factor-stimulated gene 6
protein (TSG-6). In some embodiments, the preparation of fetal
support tissue comprises thrombospondin-1 (TSP-1). In some
embodiments, the ratio of total protein to HA in the injectable
composition is between 500 parts protein:1 part HA and 500 parts
HA:1 parts protein. In some embodiments, the injectable composition
prevents the proliferation and EMT of epithelial cells by
inhibiting the actions of growth factors and cytokines. In some
embodiments, the growth factors and cytokines are selected from the
group consisting of: EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C,
TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, CTGF, HGF, IGF-1, G-CSF,
IL-6, MCP-1, TNF-.alpha., VEGF and IFN-.gamma.. In some
embodiments, the injectable composition further comprises an
aqueous adjuvant. In some embodiments, the injectable composition
is for local administration. In some embodiments, the composition
is formulated for injection. In some embodiments, the injectable
composition is formulated for intraocular injection, subretinal
injection, intravitreal injection, periocular injection,
subconjunctival injection, retrobulbar injection, intracameral
injection, or sub-Tenon's injection.
[0007] Disclosed herein, in certain embodiments, are injectable
compositions for treating or preventing Proliferative
Vitreoretinopathy (PVR), comprising: (a) substantially isolated
HC-HA/PTX3, reconstituted HC-HA/PTX3, or a combination thereof; and
(b) a pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the composition consists of: (a)
substantially isolated HC-HA/PTX3, reconstituted HC-HA/PTX3, or a
combination thereof; and (b) a pharmaceutically acceptable diluent,
excipient, vehicle, or carrier. In some embodiments, the
composition consists of reconstituted HC-HA/PTX3 and a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the composition consists of
substantially isolated HC-HA/PTX3 and a pharmaceutically acceptable
diluent, excipient, vehicle, or carrier. In some embodiments, the
substantially isolated HC-HA/PTX3 is isolated from fetal support
tissue is selected from the group consisting of: placenta,
placental amniotic membrane, umbilical cord, umbilical cord
amniotic membrane, chorion, amnion-chorion, amniotic stroma,
amniotic jelly, amniotic fluid, and a combination thereof. In some
embodiments, the fetal support tissue is frozen or previously
frozen. In some embodiments, the fetal support tissue is human,
non-human primate, bovine, or porcine. In some embodiments, the
fetal support tissue is human. In some embodiments, the
substantially isolated HC-HA/PTX3 is isolated from fetal support
tissue by ultracentrifugation. In some embodiments, the injectable
composition is in a therapeutically effective amount for preventing
or reducing the proliferation, cell migration or EMT of epithelial
cells. In some embodiments, the epithelial cells are retinal
pigment epithelial cells (RPE). In some embodiments, the epithelial
cells are human epithelial cells. In some embodiments, the human
epithelial cells are retinal epithelial cells. In some embodiments,
the injectable composition is a gel, a solution, or a suspension.
In some embodiments, the preparation of fetal support tissue
comprises high molecular weight hyaluronan (HA) that is
cross-linked by a covalent bond to the heavy chain of
inter-.alpha.-trypsin inhibitor (I.alpha.I), the high molecular
weight HA having a molecular weight greater than 1000 kDa. In some
embodiments, the preparation of fetal support tissue comprises
pentraxin 3 (PTX-3). In some embodiments, the preparation of fetal
support tissue comprises tumor necrosis factor-stimulated gene 6
protein (TSG-6). In some embodiments, the ratio of total protein to
HA in the injectable composition is between 500 parts protein:1
part HA and 500 parts HA:1 parts protein. In some embodiments, the
injectable composition prevents the proliferation and EMT of
epithelial cells by inhibiting or suppressing the activity of
growth factors and/or cytokines. In some embodiments, the growth
factors and cytokines are selected from the group consisting of:
EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C, TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, CTGF, HGF, IGF-1, G-CSF, IL-6, MCP-1,
TNF-.alpha., VEGF and IFN-.gamma.. In some embodiments, the
injectable composition further comprises an aqueous adjuvant. In
some embodiments, the injectable composition is for local
administration. In some embodiments, the injectable composition is
formulated for intraocular injection, subretinal injection,
intravitreal injection, periocular injection, subconjunctival
injection, retrobulbar injection, intracameral injection, or
sub-Tenon's injection.
[0008] Disclosed herein, in certain embodiments, are injectable
compositions for treating or preventing Proliferative
Vitreoretinopathy (PVR), consisting essentially of: (a)
substantially isolated HC-HA/PTX3, reconstituted HC-HA/PTX3, or a
combination thereof; (b) an additional therapeutic agent; and (c) a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the composition consists of: (a)
substantially isolated HC-HA/PTX3, reconstituted HC-HA/PTX3, or a
combination thereof; (b) an additional therapeutic agent; and (c) a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the composition consists of
reconstituted HC-HA/PTX3, an additional therapeutic agent, and a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the composition consists of
substantially isolated HC-HA/PTX3, an additional therapeutic agent,
and a pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the substantially isolated HC-HA/PTX3
is isolated from fetal support tissue is selected from the group
consisting of: placenta, placental amniotic membrane, umbilical
cord, umbilical cord amniotic membrane, chorion, amnion-chorion,
amniotic stroma, amniotic jelly, amniotic fluid, and a combination
thereof. In some embodiments, the fetal support tissue is frozen or
previously frozen. In some embodiments, the fetal support tissue is
human, non-human primate, bovine, or porcine. In some embodiments,
the fetal support tissue is human. In some embodiments, the
substantially isolated HC-HA/PTX3 is isolated from fetal support
tissue by ultracentrifugation. In some embodiments, the additional
therapeutic agent is selected from the group consisting of: oral
Accutane, intravitreal triamcinolone acetonide, ranibizumab,
bevacizumab, dasatinib, pegaptanib sodium, N-acetyl-cysteine (NAC),
pioglitazone, glucosamine, genistin, geldanamycin, fausdil,
resveratrol, hepatocyte growth factor (HGF), BMP-7, LY-364947,
diosgenin, emodin, pentoxyfilline, dipyridamole, a peroxisome
proliferative-activated receptor-gamma (PPAR.gamma.) agonist, a
female sex hormone, and an antioxidant. In some embodiments, the
female sex hormone comprises estradiol or progesterone. In some
embodiments, the antioxidant comprises beta carotene, vitamin C,
vitamin E, lutein, zeaxanthin, and omega-3 fatty acids. In some
embodiments, the injectable composition is in a therapeutically
effective amount for preventing or reducing the proliferation, cell
migration or EMT of epithelial cells. In some embodiments, the
epithelial cells are retinal pigment epithelial cells (RPE). In
some embodiments, the epithelial cells are human epithelial cells.
In some embodiments, the human epithelial cells are retinal
epithelial cells. In some embodiments, the injectable composition
is a gel, a solution, or a suspension. In some embodiments, the
preparation of fetal support tissue comprises high molecular weight
hyaluronan (HA) that is cross-linked by a covalent bond to the
heavy chain of inter-.alpha.-trypsin inhibitor (I.alpha.I), the
high molecular weight HA having a molecular weight greater than
1000 kDa. In some embodiments, the preparation of fetal support
tissue comprises pentraxin 3 (PTX-3). In some embodiments, the
preparation of fetal support tissue comprises tumor necrosis
factor-stimulated gene 6 protein (TSG-6). In some embodiments, the
ratio of total protein to HA in the injectable composition is
between 500 parts protein:1 part HA and 500 parts HA:1 parts
protein. In some embodiments, the injectable composition prevents
the proliferation and EMT of epithelial cells by inhibiting or
suppressing the activity of growth factors and/or cytokines. In
some embodiments, the growth factors and cytokines are selected
from the group consisting of: EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B,
PDGF-C, TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, CTGF, HGF, IGF-1,
G-CSF, IL-6, MCP-1, TNF-.alpha., VEGF and IFN-.gamma.. In some
embodiments, the injectable composition further comprises an
aqueous adjuvant. In some embodiments, the injectable composition
is for local administration. In some embodiments, the injectable
composition is formulated for intraocular injection, subretinal
injection, intravitreal injection, periocular injection,
subconjunctival injection, retrobulbar injection, intracameral
injection, or sub-Tenon's injection.
[0009] Disclosed herein, in certain embodiments, are injectable
compositions for treating or preventing Proliferative
Vitreoretinopathy (PVR) comprising: a preparation of fetal support
tissue comprising HC-HA/PTX3 and at least one other component of
fetal support tissue; and a pharmaceutically acceptable diluent,
excipient, vehicle, or carrier; wherein the composition is suitable
for injection. In some embodiments, the fetal support tissue is
placenta, placental amniotic membrane, umbilical cord, umbilical
cord amniotic membrane, chorion, amnion-chorion, amniotic stroma,
amniotic jelly, amniotic fluid, or a combination thereof. In some
embodiments, the fetal support tissue is frozen or previously
frozen. In some embodiments, the fetal support tissue is human,
non-human primate, bovine, or porcine. In some embodiments, the
fetal support tissue is human. In some embodiments, the composition
is in an amount effective for preventing or reducing the
proliferation, cell migration or EMT of epithelial cells. In some
embodiments, the epithelial cells are retinal pigment epithelial
(RPE) cells. In some embodiments, the preparation of fetal support
tissue is an extract of fetal support tissue, micronized fetal
support tissue, a homogenate, a powder, morselized fetal support
tissue, pulverized fetal support tissue, ground fetal support
tissue, purified HC-HA/PTX3, or a combination thereof. In some
embodiments, the composition is a gel, a solution, or a suspension.
In some embodiments, the composition is formulated for intraocular
injection, subretinal injection, intravitreal injection, periocular
injection, subconjunctival injection, retrobulbar injection,
intracameral injection or sub-Tenon's injection.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 illustrates the signaling pathways in the regulating
of EMT with or without proliferation by growth factors.
[0011] FIGS. 2A-2D illustrate HC-HA/PTX3 formation and
characterization of HC-HA/PTX3 purified from human AME. FIG. 2A
provides a schematic illustration of HC-HA/PTX3 formation. FIG. 2B
illustrates HC-HA/PTX3 purified from human AME. FIG. 2C illustrates
that the HC-HA/PTX3 purified from AME comprises HC1. FIG. 2D
illustrates that HC-HA/PTX3 purified from AME comprises PTX3.
[0012] FIGS. 3A-3B illustrate canonical but not non-canonical Wnt
signaling is suppressed by immobilized HC-HA/PTX3 in LEPCs/LNCs.
FIG. 3A illustrates that HC-HA/PTX3 downregulates canonical Wnt
signaling in human limbal epithelial progenitor cells (LEPCs) and
niche cells (LNCs). FIG. 3B illustrates immunostaining of
.beta.-catenin and C-JUN seeded either on Matrigel or on
immobilized HC-HA/PTX3.
[0013] FIGS. 4A-4D illustrate expression of TGF-.beta. and
TGF-.beta. receptors in human corneal fibroblasts (HCF). FIG. 4A
illustrates TGF-.beta.1 expression in Human Corneal Fibroblasts
(HCFs) seeded on plastic, HA, or HC-HA/PTX3, both with and without
addition of exogenous TGF-.beta.1. FIG. 4B illustrates TGF-.beta.2
expression in HCFs seeded on plastic, HA, or HC-HA/PTX3, both with
and without addition of exogenous TGF-.beta.1. FIG. 4C illustrates
TGF-.beta.3 expression in HCFs seeded on plastic, HA, or
HC-HA/PTX3, both with and without addition of exogenous
TGF-.beta.1. FIG. 4D exemplifies a Northern blot showing expression
of TGF-.beta.RI, TGF-.beta.RII, and TGF-.beta.III in HCFs seeded on
plastic, HA, or HC-HA/PTX3, both with and without addition of
exogenous TGF-.beta.1. FIG. 4E exemplifies nuclear translocation of
pSmad2/3 cause by addition of exogenous TGF-.beta.1. FIG. 4F
exemplifies positive cytoplasmic expression of .alpha.-SMA caused
by addition of exogenous TGF-1.
[0014] FIGS. 5A-5C illustrate HC-HA/PTX3 inhibits proliferation in
ARPE-19 cells when stimulated with EGF+FGF-2. FIG. 5A illustrates
HC-HA/PTX3 does not affect the viability of normal ARPE-19 cells.
FIG. 5B illustrates proliferation of ARPE-19 cells using
immunostaining. 5C illustrates proliferation of ARE-19 cells.
[0015] FIGS. 6A-6B illustrate HC-HA/PTX3 inhibits nuclear
translocation of pSmad2/3 in APRE-19 cells. FIG. 6A illustrates
nuclear localization of phosphorylated Smad2/3 using
immunostaining. FIG. 6B illustrates nuclear localization of
phosphorylated Smad2/3.
[0016] FIGS. 7A-7D illustrate development of PVR in rabbits. FIG.
7A exemplifies fundus photographs of a normal rabbit eye without
PVR. FIG. 7B exemplifies a rabbit with tractional PVR four weeks
after gas vitrectomy and intravitreal injection of RPE cells. FIG.
7C exemplifies a cross-section of the normal rabbit eye without PVR
after enucleation. FIG. 7D exemplifies a cross-section of the eye
of the rabbit with tractional PVR with retinal detachment after
enucleation.
[0017] FIG. 8 illustrates the effect of the addition of collagen
gel (Col), AM extract AME, or collagen gel mixed with AM extract
(Col+AME) on the suppression of TGF-.beta. promoter activity. BSA
was used as a control.
[0018] FIG. 9 illustrates the effect of treatment with AME, HA, or
HA+AME, compared to a control assay with BSA alone, on the
suppression of TGF-.beta. activity. The promoter activity is
displayed as relative luciferase units (RLU).
[0019] FIG. 10 illustrates the molecular weight ranges of
hyaluronan in AM extracts separated by agarose gel electrophoresis.
Amniotic membrane extracted by buffer A, B, C were treated with or
without hyaluronidase and electrophoretically separated by a 0.5%
agarose gel.
[0020] FIG. 11 illustrates the molecular weight ranges of
hyaluronan in AM extracts separated by agarose gel electrophoresis.
Amniotic membrane extracted by buffer PBS were treated with or
without hyaluronidase (10 units/ml in Tris-HCl, pH 7.5, 150 mM
NaCl) for 2 hr at 37.degree. C. and run through 0.5% agarose gels.
HA: positive hyaluronic acid control; L: AM extract after low speed
centrifugation; H: AM extract after high speed centrifugation.
[0021] FIG. 12 illustrates a western blot demonstrating that the
inter-.alpha.-trypsin inhibitor (I.alpha.I) is present in AM
extracts. I.alpha.I was present in AM extract A and C although the
signal of bikunin was very weak (.about.39 kDa). Prior to transfer
to the western blot, the extract was separated on a 4-15% denatured
acrylamide gel.
[0022] FIG. 13 illustrates an immunoblot demonstrating that the
inter-.alpha.-trypsin inhibitor (I.alpha.I) is present in the AM
extracts even after low (LS) or high speed (HS) centrifugation.
[0023] FIG. 14 illustrates an immunoblot of TSG-6 (Tumor Necrosis
Factor-Stimulated Gene 6), either with (+) or without (-)
hyaluronidase treatment. The samples included total AM extract
without centrifugation (T), AM Extract after extraction in isotonic
low salt buffer (buffer A); high salt buffer (B); or 4 M guanidine
HCl (C); as detailed in Example 2. TSG-6 was present in the total
extract, buffer A extract, and buffer C extract. The addition of
hyaluronidase did not appear to alter the TSG-6 level in the
extracts.
[0024] FIG. 15 illustrates an immunoblot analysis of the
deglycosylation of TSG-6 in AM. AM extract A, B, and C were treated
with (+) or without 20 units/ml PNGase F at 37.degree. C. for 3
hours. Glycosylation of TSG-6 in AM was then analyzed by western
blot. The cell lysate of human corneal fibroblast (HCF) was used as
a positive control.
[0025] FIG. 16 illustrates an immunoblot analysis of potential
TSG-6 complexes in AM by digestion with Chondroitin Sulfate ABC
lyase. AM extract A, B, and C were treated without (-) or with (+)
1 unit/ml ABC lyase at 37.degree. C. for 2 hours. The possible
disruption of TSG-6 complexes was then analyzed by western blot
using an anti-TSG-6 antibody RAH-1:1:1000.
[0026] FIG. 17 illustrates an immunoblot of potential TSG-6
complexes in AM by digestion with Chondroitin Sulfate ABC lyase.
This is the same experiment as shown in FIG. 16 except that a
different TSG-6 antibody was used. Here, the anti-TSG-6 antibody
was obtained from R & D Systems (cat #MAB2104).
[0027] FIG. 18 illustrates an immunoblot demonstrating the presence
of Pentraxin (PTX3) in AM, using a rat monoclonal anti-PTX3
antibody obtained from Alexis Biochemicals. HCF: human corneal
fibroblast, T, A, B, C: AM extract Total, A, B, C, respectively;
HAse: Hyaluronidase.
[0028] FIG. 19 illustrates an immunoblot demonstrating the presence
of TSP-1 in AM. The monomeric TSP-1 (180 kDa) and the putative
trimeric TSP-1 (540 kDa) are indicated. The positive control,
TSP-1, was purified from human platelets (Calbiochem, Cat #605225)
and loaded as 100 ng/lane.
[0029] FIG. 20 illustrates an immunoblot demonstrating the presence
of Smad 7 in AM. AM was extracted with PBS or urea (2M urea in 50
mM Tris-HCl, pH 7.5). 20 .mu.g of total protein was loaded for each
extract. Smad 7 was detected with goat anti-human Smad 7 (AF2029,
1:1000, R & D Systems). Smad 7 migrated as a band of .about.51
kDa.
[0030] FIG. 21 illustrates the number of migrated cells counted
from six random microscopic fields (n=4, * indicates p<0.05 when
compared with PBS+EGF+FGF-2+TGF-.beta.1).
[0031] FIG. 22 illustrates the percentage of gel contraction
compared among groups (n=4, * indicates p<0.05 compared with
PBS+TGF-.beta.1).
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present application describes compositions and methods
for preventing or reducing the proliferation, cell migration,
and/or epithelial-mesenchymal transition (EMT) of epithelial cells,
wherein the epithelial cells are human epithelial cells and the
human epithelial cells are selected from: retinal pigment
epithelial, conjunctival, retinal, corneal, limbal, or renal
epithelial cells. Additionally, the present application describes
compositions and methods for the prevention and treatment of
proliferative vitreoretinopathy in an individual in need
thereof.
[0033] It is known that proliferation, cell migration and EMT occur
when epithelial cells such as, for example, retinal pigment
epithelial, human conjunctival, retinal, corneal, limbal, or renal
epithelial cells are exposed to growth factors and cytokines such
as, for example, EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C,
TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, CTGF, HGF, IGF-1, G-CSF,
IL-6, MCP-1, TNF-.alpha., VEGF or IFN-.gamma. and ethylene glycol
tetraacetic acid (EGTA) either in vitro or in vivo.
[0034] Further, it is known that transplantation of cryopreserved
amniotic membrane (AM) tissue onto the ocular surface provides
anti-proliferative, anti-inflammatory, anti-scarring and
anti-angiogenic actions in both corneal and limbal epithelial cells
to promote wound healing.
[0035] What is needed is a composition that prevents or reduces
proliferation, cell migration and EMT of epithelial cells, can be
administered without the need of surgical transplantation and can
additionally be administered to non-surface epithelial cells such
as, for example, retinal and renal epithelial cells.
[0036] A description of certain embodiments follows. It will be
understood that the particular embodiments of the application are
shown by way of illustration and not as limitations of the
application.
Certain Terminology
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the claimed subject matter belongs.
All patents, patent applications, published, applications and
publications, GENBANK sequences, websites and other published
materials referred to throughout the entire disclosure herein,
unless noted otherwise, are incorporated by reference in their
entirety. In the event that there is a plurality of definitions for
terms herein, those in this section prevail. Where reference is
made to a URL or other such identifier or address, it is understood
that such identifiers can change and particular information on the
internet can come and go, but equivalent information is known and
can be readily accessed, such as by searching the internet and/or
appropriate databases. Reference thereto evidences the availability
and public dissemination of such information.
[0038] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence, "about 5 .mu.g" means "about 5 .mu.g" and also "5
.mu.g." Generally, the term "about" includes an amount that would
be expected to be within experimental error.
[0039] As used herein, the terms "subject", "individual", and
"patient" are used interchangeably. None of the terms are to be
interpreted as requiring the supervision of a medical professional
(e.g., a doctor, nurse, physician's assistant, orderly, hospice
worker). As used herein, the subject is any animal, including
mammals (e.g., a human or non-human animal) and non-mammals. In one
embodiment of the methods and compositions provided herein, the
mammal is a human.
[0040] As used herein, the terms "treat," "treating" or
"treatment," and other grammatical equivalents, include:
alleviating, abating or ameliorating one or more symptoms of a
disease or condition. In some embodiments, treating is alleviating,
abating or ameliorating one or more symptoms of
epithelial-mesenchymal transition. In some embodiments, treating is
alleviating, abating or ameliorating one or more symptoms of
proliferative vitreoretinopathy. In some embodiments, treating is
alleviating, abating or ameliorating one or more symptoms of
inflammation. In some embodiments, treating is preventing or
reducing the appearance, severity or frequency of one or more
additional symptoms of a disease or condition. In some embodiments,
the methods include preventing or reducing the appearance, severity
or frequency of one or more additional symptoms of
epithelial-mesenchymal transition. In some embodiments, the methods
include preventing or reducing the appearance, severity or
frequency of one or more additional symptoms of proliferative
vitreoretinopathy. In some embodiments, the methods include
preventing or reducing the appearance, severity or frequency of one
or more additional symptoms of inflammation. In some embodiments,
the methods include ameliorating or preventing the underlying
metabolic causes of one or more symptoms of a disease or condition,
inhibiting the disease or condition, such as, for example,
arresting the development of the disease or condition, relieving
the disease or condition, causing regression of the disease or
condition, relieving a condition caused by the disease or
condition, or inhibiting the symptoms of the disease or condition
either prophylactically and/or therapeutically.
[0041] As used herein, "fetal support tissue" means tissue used to
support the development of a fetus. Examples of fetal support
tissue include, but are not limited to, (i) placental amniotic
membrane (PAM), or substantially isolated PAM, (ii) umbilical cord
amniotic membrane (UCAM) or substantially isolated UCAM, (iii)
chorion or substantially isolated chorion, (iv) amnion-chorion or
substantially isolated amnion-chorion, (v) amniotic stroma or
substantially isolated amniotic stroma, (vi) placenta or
substantially isolated placenta, (vii) umbilical cord or
substantially isolated umbilical cord, (viii) amniotic fluid, or
(ix) any combinations thereof. Fetal support tissue is also used
interchangeably with "gestational tissue." In some embodiments the
gestational tissue is "mammalian gestational tissue" or "human
gestational tissue ("HGT")." In some embodiments, the fetal support
tissue is obtained from a mammal. In some embodiments, the fetal
support tissue is from human, non-human primate, cow, or pig. In
some embodiments, the fetal support tissue is from human. In some
embodiments, the fetal support tissue is ground, pulverized,
morselized, a graft, a powder, a gel, a homogenate, or an extract.
In some embodiments, the fetal support tissue is aseptically
processed. In some embodiment, the fetal support tissue is
terminally-sterilized.
[0042] As used herein, "placenta" means the organ that connects a
developing fetus to the maternal uterine wall to allow nutrient
uptake, waste elimination, and gas exchange via the maternal blood
supply. The placenta is composed of three layers. The innermost
placental layer surrounding the fetus is called amnion. The
allantois is the middle layer of the placenta (derived from the
embryonic hindgut); blood vessels originating from the umbilicus
traverse this membrane. The outermost layer of the placenta, the
chorion, comes into contact with the endometrium. The chorion and
allantois fuse to form the chorioallantoic membrane.
[0043] As used herein, "chorion" means the membrane formed by
extraembryonic mesoderm and the two layers of trophoblasts. The
chorionic villi emerge from the chorion, invade the endometrium,
and allow transfer of nutrients from the maternal blood to fetal
blood. The chorion consists of two layers: an outer layer formed by
the trophoblast, and an inner layer formed by the somatic mesoderm;
the amnion is contact with the latter. The trophoblast is made up
of an internal layer of cubical or prismatic cells, the
cytotrophoblast or layer of Langhans, and an external layer of
richly nucleated protoplasm devoid of cell boundaries, the
syncytiotrophobast. The avascular amnion is adherent to the inner
layer of the chorion.
[0044] As used herein, "amnion-chorion" means a product comprising
amnion and chorion. In some embodiments, the amnion and the chorion
are not separated (i.e., the amnion is naturally adherent to the
inner layer of the chorion). In some embodiments, the amnion is
initially separated from the chorion and later combined with the
chorion during processing.
[0045] As used herein, "umbilical cord" means the organ that
connects a developing fetus to the placenta. The umbilical cord is
composed of Wharton's jelly, a gelatinous substance made largely
form mucopolysaccharides. It contains one vein, which carries
oxygenated, nutrient-rich blood to the fetus, and two arteries that
carry deoxygenated, nutrient-depleted blood away. In some
embodiments, the blood vessels have been substantially removed from
the umbilical cord tissue. In some embodiments, a portion of the
Wharton's Jelly has been removed. In some embodiments, the blood
vessels and a portion of the Wharton's Jelly have been removed.
[0046] As used herein, "placental amniotic membrane" (PAM) means
amniotic membrane derived from the placenta. In some embodiments,
the PAM is substantially isolated.
[0047] As used herein, "umbilical cord amniotic membrane" (UCAM)
means amniotic membrane derived from the umbilical cord. UCAM is a
translucent membrane. The UCAM has multiple layers: an epithelial
layer; a basement membrane; a compact layer; a fibroblast layer;
and a spongy layer. It lacks blood vessels or a direct blood
supply. In some embodiments, the UCAM is substantially isolated. In
some embodiments, the UCAM comprises all of the Wharton's Jelly. In
some embodiments, the UCAM comprises a portion of the Wharton's
Jelly. In some embodiments, the UCAM comprises blood vessels and/or
arteries. In some embodiments, the UCAM comprises all of the
Wharton's Jelly and blood vessels and/or arteries. In some
embodiments, the UCAM comprises part of the Wharton's Jelly and
blood vessels and/or arteries.
[0048] As used herein, "substantially isolated" or "isolated" means
that the fetal support tissue product has been separate from
undesired materials (e.g., red blood cells, blood vessels, and
arteries) derived from the original source organism. Purity, or
"isolation" may be assayed by standard methods, and will ordinarily
be at least about 10% pure, more ordinarily at least about 20%
pure, generally at least about 30% pure, and more generally at
least about 40% pure; in further embodiments at least about 50%
pure, or more often at least about 60% pure; in still other
embodiments, at least about 95% pure.
[0049] As used herein, "biological activity" means the activity of
polypeptides and polysaccharides. In some embodiments, the activity
of polypeptides and polysaccharides found in umbilical cord (and
substantially isolated umbilical cord), UCAM (and substantially
isolated UCAM), placenta (and substantially isolated placenta), PAM
(and substantially isolated PAM), chorion (and substantially
isolated chorion), or amnion-chorion (and substantially isolated
amnion-chorion). In some embodiments, the biological activity is
anti-scarring activity, anti-inflammation activity, anti-angiogenic
activity, and wound healing. In some embodiments, the biological
activity is anti-inflammation activity. In some embodiments, the
biological activity comprises the biological activity of a fetal
support tissue preparation or composition. In some embodiments, the
biological activity comprises the biological activity of
HC-HA/PTX3.
[0050] As used herein, the substantial preservation of biological
activity or structural integrity means that when compared to the
biological activity and structural integrity of non-processed
tissue, the biological activity and structural integrity of the
fetal support tissue product has only decreased by about 5%, about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 50%, or about 60%.
[0051] As used herein, "freezing" refers to exposing the fetal
support tissue product below about or at 0.degree. C., -5.degree.
C., -10.degree. C., -20.degree. C., -40.degree. C., -50.degree. C.,
-60.degree. C., -70.degree. C., -80.degree. C., -90.degree. C., or
-100.degree. C. for a period of time of about 1 hour, 2 hours, 3
hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10
hours, 11 hours, 12 hours, 18 hours, 24 hours, or longer.
[0052] As used herein, "powder" means matter in the form of fine
dry particles or matrix. In some embodiments, the particles are not
uniform in size. In some embodiments, the particles are
substantially uniform in size.
[0053] As used herein, "grinding" means any method of reducing
fetal support tissue to small particles or a powder. The term
grinding includes micronizing, pulverizing, homogenizing, filing,
milling, grating, pounding, and crushing.
[0054] The terms "effective amount" or "therapeutically effective
amount," as used herein, refer to a sufficient amount of an agent
or a compound being administered which will relieve to some extent
one or more of the symptoms of the disease or condition being
treated. The result can be reduction and/or alleviation of the
signs, symptoms, or causes of a disease, or any other desired
alteration of a biological system. For example, an "effective
amount" for therapeutic uses is the amount of the composition
including a compound as disclosed herein required to provide a
clinically significant decrease in disease symptoms without undue
adverse side effects. An appropriate "effective amount" in any
individual case may be determined using techniques, such as a dose
escalation study. The term "therapeutically effective amount"
includes, for example, a prophylactically effective amount. An
"effective amount" of a compound disclosed herein, is an amount
effective to achieve a desired effect or therapeutic improvement
without undue adverse side effects. It is understood that "an
effective amount" or "a therapeutically effective amount" can vary
from individual to individual, due to variation in metabolism of
the injectable composition, age, weight, general condition of the
individual, the condition being treated, the severity of the
condition being treated, and the judgment of the prescribing
physician. In some embodiments, an effective amount is an amount
that prevents or reduces the symptoms of PVR. In some embodiments,
an effective amount is an amount that reduces, inhibits or prevents
cell migration, cell proliferation and/or EMT of epithelial
cells.
[0055] Epithelial-mesenchymal transition (EMT) is a process by
which epithelial cells lose their cell polarity and cell-cell
adhesion, and gain migratory and invasive properties. EMT occurs in
processes such as mesoderm formation, neural tube formation, wound
healing, as well as the initiation of metastasis for cancer
progression. EMT can be induced through several signal signaling
pathways, including TGF-.beta., FGF, EGF, HGF, Wnt/beta-catenin,
and Notch.
[0056] Proliferative vitreoretinopathy (PVR) is a disease that
develops as a complication of rhegmatogenous retinal detachment.
When fluid from the vitreous humor enters a hole in the retina and
accumulates in the subretinal space, the tractional force of the
vitreous on the retina is what results in rhegmatogenous retinal
detachment. During this process the retinal cell layers come in
contact with vitreous cytokines, which can trigger the retinal
pigmented epithelium (RPE) to proliferate and migrate. The RPE
cells undergo epithelial-mesenchymal transition (EMT) and develop
the ability to migrate out into the vitreous. During migration of
the RPE, these cells lay down fibrotic membranes which contract and
pull at the retina, and can lead to secondary retinal detachment
after primary retinal detachment surgery.
Compositions
[0057] Disclosed herein, in certain embodiments, are compositions
for preventing or reducing proliferation, cell migration, and/or
epithelial-mesenchymal transition (EMT) of epithelial cells,
comprising: a preparation of fetal support tissue; and a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. Further disclosed herein, in certain embodiments, are
injectable compositions for preventing or reducing proliferative
venous retinopathy (PVR) in an individual in need thereof,
comprising: a preparation of fetal support tissue; and a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. Further disclosed herein, in certain embodiments, are
injectable compositions for preventing or reducing proliferative
venous retinopathy (PVR) in an individual in need thereof,
consisting essentially of: substantially isolated HC-HA/PTX3,
reconstituted HC-HA/PTX3 (rcHC-HA/PTX3); and a pharmaceutically
acceptable diluent, excipient, vehicle, or carrier. Further
disclosed herein, in certain embodiments, are injectable
compositions for preventing or reducing proliferative venous
retinopathy (PVR) in an individual in need thereof, consisting
essentially of: substantially isolated HC-HA/PTX3, reconstituted
HC-HA/PTX3 (rcHC-HA/PTX3); an additional therapeutic agent; and a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier.
[0058] In some embodiments, the preparation of fetal support tissue
comprises HC-HA/PTX3. In some embodiments, the preparation of fetal
support tissue comprises: high molecular weight hyaluronan (HA)
that is cross-linked by a covalent bond to the heavy chain of
inter-.alpha.-trypsin inhibitor (I.alpha.I), the high molecular
weight HA having a molecular weight greater than 1000 kDa. In some
embodiments, the preparation of fetal support tissue comprises:
pentraxin 3 (PTX-3, PTX3). In some embodiments, the preparation of
fetal support tissue comprises: tumor necrosis factor-stimulated
gene 6 protein (TSG-6). In some embodiments, the preparation of
fetal support tissue comprises: thrombospondin-1 (TSP-1). In some
embodiments, the ratio of total protein to HA in the composition is
less than 500 parts protein:1 part HA. In some embodiments, the
ratio of HA to total protein in the composition is less than 500
parts HA:1 part protein. In some embodiments, the preparation of
fetal support tissue comprises HC-HA/PTX3 complex. In some
embodiments, the preparation of fetal support tissue comprises
substantially purified HC-HA/PTX3 complex.
[0059] In some embodiments, the epithelial cells are human
epithelial cells. In some embodiments, the human epithelial cells
are retinal pigment epithelial cells (RPE). In some embodiments,
the human epithelial cells are corneal epithelial cells. In some
embodiments, the human epithelial cells are limbal epithelial
cells. In some embodiments, the human epithelial cells are
conjunctival epithelial cells. In some embodiments, the human
epithelial cells are renal epithelial cells.
[0060] In some embodiments, the composition prevents the
proliferation and EMT of epithelial cells by suppressing the
activity of growth factors and cytokines. In some embodiments, the
growth factors and cytokines are selected from the group consisting
of: EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C, TGF-.beta.1,
TGF-.beta.2, TGF-.beta.3, CTGF, HGF, IGF-1, G-CSF, IL-6, MCP-1,
TNF-.alpha., VEGF and IFN-.gamma.. In some embodiments, the
composition inhibits signaling pathways in epithelial cells to
inhibit proliferation and EMT. In some embodiments, the signaling
pathways are canonical Wnt signaling and TGF-.beta.-induced
Smad/ZEB signaling.
[0061] In some embodiments, the composition comprises the
preparation of fetal support tissue and a pharmaceutically
acceptable diluent, excipient, or carrier. In some embodiments, the
composition further comprises an aqueous adjuvant. In some
embodiments, the composition is for local administration. In some
embodiments, the composition is formulated for injection. In some
embodiments, the composition is formulated for intraocular
injection, subretinal injection, intravitreal injection, periocular
injection, subconjunctival injection, retrobulbar injection,
intracameral injection or sub-Tenon's injection.
[0062] Preparations of Fetal Support Tissue
[0063] In some embodiments, the preparation of fetal support tissue
comprises placental tissue, umbilical cord tissue, placental
amniotic membrane tissue, chorion tissue, amniotic stroma,
amnion-chorion tissue, UCAM tissue, amniotic fluid, or combinations
thereof. In some embodiments, the preparation of fetal support
tissue is an extract of fetal support tissue, micronized fetal
support tissue, a homogenate of fetal support tissue, a powder of
fetal support tissue, morselized fetal support tissue, pulverized
fetal support tissue, ground fetal support tissue, purified
HC-HA/PTX3, or a combination thereof. In some embodiments, the
preparation of fetal support tissue is prepared from fresh, frozen
or previously frozen fetal support tissue. In some embodiments, the
preparation of fetal support tissue is prepared from frozen or
previously frozen of fetal support tissue. In some embodiments, the
preparation of fetal support tissue comprises HA, I.alpha.I, TSG-6,
PTX-3, TSP-1, or a combination thereof. In some embodiments, the
preparation of fetal support tissue comprises HC-HA/PTX3 complex.
In some embodiments, the preparation of fetal support tissue
comprises purified HC-HA/PTX3. In some embodiments, the preparation
of fetal support tissue comprises ultracentrifuged HC-HA/PTX3. In
some embodiments, the preparation of fetal support tissue consists
of purified HC-HA/PTX3. In some embodiments, the preparation of
fetal support tissue comprises reconstituted HC-HA/PTX3.
[0064] In some embodiments, the preparation of fetal support tissue
suppresses TGF-.beta. promoter activity; increases apoptosis in
macrophages; decreases proliferation, decreases migration, and
increases apoptosis of human vascular endothelial cells; decreases
viability of human fibroblasts; decreases inflammation; and
prevents apoptosis of epithelial cells exposed to storage and
injury. In some embodiments, the preparations of fetal support
tissue and injectable compositions described herein are used to
treat diseases related to TGF-.beta. upregulation, such as
angiogenesis, wound healing, and tissue inflammation.
[0065] TGF-.beta. is the prototypic cytokine that is involved in
tissue inflammation, in addition to wound healing and scar
formation. Mammalian cells express three different TGF-.beta.s:
TGF-.beta.1, TGF-.beta.2, and TGF.beta.3. TGF-.beta. is the most
potent cytokine promoting myofibroblast differentiation by
up-regulating expression of .alpha.-SMA, integrin .alpha.5.beta.1,
and EDA domain-containing fibronectin (Fn) in a number of cell
types, including fibroblasts. TGF-.beta. also up-regulates the
expression of such matrix components as collagens and
proteoglycans, down-regulates proteinase and matrix
metalloproteinases, and up-regulates their inhibitors.
Collectively, these actions result in increased cell-matrix
interactions and adhesiveness, as well as deposition and formation
of scar tissue.
[0066] TGF-.beta.s exert their actions via binding with TGF-.beta.
receptors (TGF-.beta.Rs) on the cell membrane. In human cells,
there are three TGF-.beta.Rs, namely TGF-.beta.R type I
(TGF-.beta.RI), type II (TGF-.beta.RII), and type III
(TGF-.beta.RIII). TGF-.beta.s, serving as ligands, bind with a
serine, threonine kinase receptor complex made of TGF-.beta.RI and
TGF-.beta.RII; such a binding is facilitated by TGF-.beta.RIII,
which is not a serine, threonine kinase receptor. Binding with
TGF-.beta.RII activates TGF-.beta.RI, which is responsible for
direct phosphorylation of a family of effector proteins known as
Smads, which modulate transcription of a number of target genes,
including those described herein, participating in scar
formation.
[0067] Suppression of TGF-.beta. can be achieved by neutralizing
antibodies to TGF-.beta. and agents that intercede the signaling
mediated by TGF-.beta. such as decorin. Most of the literature has
shown suppression of TGF-.beta. being achieved at the level of
modulating the TGF-.beta. activation, binding with its receptor, or
its signal transduction. It has been shown that amniotic membrane
can achieve such an inhibition at the level of transcription, i.e.,
to turn off transcription of TGF-.beta.1 genes. In particular,
amniotic membrane has been shown to suppress TGF-.beta. signaling
in human corneal and limbal fibroblasts, and human conjunctival and
pterygium body fibroblasts.
[0068] Hyaluronic acid (HA) is a natural sugar found in the
synovial joint fluid, the vitreous humor of the eye, the cartilage,
blood vessels, extra-cellular matrix, skin, and umbilical cord. In
some embodiments, the cross-linking of HA is through a covalent
bond to another molecule, such as a protein. In some embodiments,
HA is covalently bound to the heavy chain of inter-.alpha.-trypsin
inhibitor (I.alpha.I). In some embodiments, the ratio of protein to
HA in the preparation of fetal support tissue is less than about
500:1, less than about 200:1, less than about 100:1, less than
about 50:1, or less than about 10:1 protein:HA. In some
embodiments, the ratio of HA to protein in the preparation of fetal
support tissue is less than about 500:1, less than about 200:1,
less than about 100:1, less than about 50:1, or less than about
10:1 HA:protein.
[0069] TSG-6 is a hyaluronan binding protein that plays a role in
extracellular matrix remodeling, cell proliferation, and leucocyte
migration. TSG-6 can form a complex with the serine protease
inhibitor inter-.alpha.-inhibitor (I.alpha.I) and catalyze the
transfer of a heavy chain from I.alpha.I to HA. PTX-3 is Ca.sup.2+
dependent ligand binding protein that has a pentameric discoid
structure and are present in plasma. TSP-1 (Thrombospondin 1) is a
homotrimeric glycoprotein having a potent anti-angiogenic and other
biological activities. TSP-1 is secreted into the extracellular
matrix by a variety of cell types.
[0070] In some embodiments, the preparation of fetal support tissue
comprises a purified component selected from HA, I.alpha.I, TSG-6,
PTX-3, TSP-1, HC-HA/PTX3, or a combination thereof. In some
embodiments the preparation of fetal support tissue comprises
reconstituted HC-HA/PTX3. In some embodiments, the preparation of
fetal support tissue comprises purified HC-HA/PTX3. In some
embodiments, the preparation of fetal support tissue consists of
HC-HA/PTX3. In some embodiments, the preparation of fetal support
tissue comprises purified HC-HA/PTX3 at a high concentration. In
some embodiments, the HC-HA/PTX3 is at a concentration of 25 to 750
.mu.g/ml, 50 to 500 .mu.g/ml, 50 to 250 .mu.g/ml, or about 250
ug/ml, about 500 ug/ml, or about 750 ug/ml. In some embodiments,
the purified component is obtained from any suitable source. In
some embodiments, the purified component is obtained from a fetal
support tissue. In some embodiments, the purified component of
fetal support tissue is obtained from a commercial source. In some
embodiments, the purified component of fetal support tissue is
isolated from a transgenic organism. In some embodiments, a protein
sequence of the purified component of fetal support tissue has a
similarity of at least 90%, 93%, 95%, 97%, 99% or 99.5% to a human
protein sequence. In some embodiments, the purified component of
fetal support tissue is purified, substantially purified, partially
purified, or are present in crude extracts. In some embodiments,
the purified component of fetal support tissue is HC-HA/PTX3. In
some embodiments, the purified component of fetal support tissue is
isolated from the preparation of fetal support tissue at any time
during the process.
[0071] In some embodiments, the preparation of fetal support tissue
comprises Smad7. In some embodiments, Smad7 is obtained from any
suitable source, such as from amniotic membrane, from a commercial
source or isolated from a transgenic organism. In some embodiments,
Smad7 is purified, substantially purified, partially purified, or
is present in a crude extract.
[0072] In some embodiments, HA, I.alpha.I, TSG-6, PTX-3, TSP-1, and
optionally Smad7 are obtained from the preparation of fetal support
tissue. In some embodiments, the preparation of fetal support
tissue containing the combination of HA, I.alpha.I, TSG-6, PTX-3,
TSP-1 and optionally Smad7 is prepared.
[0073] In some embodiments, after homogenization of the fetal
support tissue, is centrifuged to remove the insoluble material. In
some embodiments, after homogenization of the fetal support tissue,
the insoluble material is left in the preparation of fetal support
tissue. In some embodiments, the preparation of fetal support
tissue is dried. In some embodiments, a preparation of fetal
support tissue is prepared according to a method described in
Example 1.
[0074] In some embodiments, the fetal support tissue is obtained
from sources such as Bio-Tissue, Inc. (Miami, Fla.) and Baptist
Hospital (Miami, Fla.) (under IRB approval). In some embodiments,
the fetal support tissue is obtained in either a fresh, frozen, or
previously frozen state. In some embodiments, the fetal support
tissue is washed to remove excess storage buffer, blood, or
contaminants. In some embodiments, the excess liquid is removed
using a brief centrifugation step, or by other means. In some
embodiments, the fetal support tissue is frozen using liquid
nitrogen or other cooling means to facilitate the subsequent
homogenization. In some embodiments, the source of the fetal
support tissue is a mammal. In some embodiments, the source of the
fetal support tissue is a human. In some embodiments, other sources
of fetal support tissue, such as non-human primate, bovine or
porcine, are used.
[0075] In some embodiments, the preparation of fetal support tissue
is obtained from AM jelly. In some embodiments, the AM jelly is
obtained from fresh AM tissue. In some embodiments, AM jelly is
obtained before freezing the fresh AM tissue. In some embodiments,
AM jelly is obtained after freezing the fresh AM tissue. In some
embodiments, AM jelly is obtained from frozen or previously frozen
AM tissue. In some embodiments, the AM jelly is frozen. In some
embodiments, the AM jelly is freeze-ground following the procedure
for AM preparations as described herein. In some embodiments, the
AM jelly is centrifuged. In some embodiments, the AM jelly is
lyophilized.
[0076] In some embodiments, the preparation of fetal support tissue
is made from a stroma of the AM. In some embodiments, the stroma is
separated from a layer of fresh, frozen, thawed, or otherwise
treated AM membrane. In some embodiments, the stroma removal occurs
by enzymatic methods, mechanical methods, or by any other suitable
means. In some embodiments, the stroma is fresh, frozen, or
previously frozen. In some embodiments, the stroma is ground or
freeze-ground following the procedure for generating the
preparation of fetal support tissue from AM as described herein. In
some embodiments, the stroma is centrifuged. In some embodiments,
the stroma is lyophilized.
[0077] In some embodiment, the preparation is ground fetal support
tissue. In some embodiments, the fetal support tissue is frozen
prior to the grinding process. In some embodiments, the freezing
step occurs by any suitable cooling process. In some embodiments,
the fetal support tissue is flash-frozen using liquid nitrogen. In
some embodiments, the fetal support tissue is placed in an
isopropanol/dry ice bath or is flash-frozen in other coolants. In
some embodiments, a commercially available quick freezing process
is used. In some embodiments, the fetal support tissue is placed in
a freezer and allowed to equilibrate to the storage temperature
more slowly, rather than being flash-frozen. In some embodiments,
the fetal support tissue is stored at any desired temperature. In
some embodiments, the fetal support tissue is stored at -20.degree.
C. or -80.degree. C.
[0078] In some embodiment, the preparation is pulverized fetal
support tissue. In some embodiments, the fetal support tissue is
pulverized while frozen. In some embodiments, fresh, partially
thawed, or thawed fetal support tissue is used in the grinding
step. In some embodiments, the fetal support tissue (fresh, frozen,
or thawed) is sliced into pieces of a desired size with a suitable
device, such as a scalpel, then ground to fine particles using a
BioPulverizer (Biospec Products, Inc., Bartlesville, Okla.) or
other suitable devices, and homogenized with a homogenization
device such as a Tissue Tearor (Biospec Products, Inc., Dremel,
Wis.), in a suitable solution, forming a homogenate. Non-limiting
examples of solutions include, but are not limited to, phosphate
buffered saline (PBS), DMEM, NaCl solution, and water. In some
embodiments, the pH of the solution is adjusted as needed. In some
embodiments, the pH range is from about 5.5 or 6.0 to about 8.5. In
some embodiments, the frozen tissue is ground in a solution having
a pH of between about 6.3 and about 7.8.
[0079] In some embodiment, the preparation is a homogenate of fetal
support tissue. In some embodiments, the homogenate is mixed at any
suitable speed, temperature, or other parameters. In some
embodiments, the mixing occurs at a temperature range of from about
1.degree. C., or 3.degree. C., to about 6.degree. C., 10.degree.
C., 15.degree. C., or 20.degree. C. In some embodiments, the mixing
occurs at about 4.degree. C. In some embodiments, the homogenate is
mixed, for example, from less than about 1 minute, 10 minutes, or
20 minutes to about 1, 2, 3 or more hours.
[0080] In some embodiments, the homogenate is centrifuged to remove
any remaining insoluble material and/or cellular debris. In some
embodiments, the centrifugation is performed using any suitable
range of time, temperature, protein concentration, buffers, and
speed. In some embodiments, the centrifugation occurs at a range of
about 1,000, 5,000, or 10,000.times.g to about 20,000.times.g. In
some embodiments, the centrifugation occurs at about
15,000.times.g. In some embodiments, the centrifugation occurs for
a duration of from less than 1 minute, 5 minutes, 10 minutes, 20
minutes, to about 40 minutes, 60 minutes, 1.5 hours, or more. In
some embodiments, the supernatant is collected and stored in
aliquots at -80.degree. C. In some embodiments, the total protein
is quantitated using any suitable commercial protein analysis kit,
such as a BCA assay (Pierce, Rockford, Ill.). Example 2, Table 1
and FIG. 13 describe the analysis of AM preparations after low
speed or high speed centrifugation.
[0081] In some embodiments, for biochemical characterization and
purification, the above solutions are supplemented with protease
inhibitors. An exemplary mixture of protease inhibitors is the
following: 1 .mu.g/ml aprotinin, 1 .mu.g/ml leupeptin, 1 .mu.g/ml
pepstatin A, and 1 mM PMSF. In some embodiments, a protease
inhibitor is not added to the preparation of fetal support tissue
if the preparation of fetal support tissue is to be added to live
cells or tissues.
[0082] In some embodiment, the preparation is an extract fetal
support tissue. In some embodiments, any suitable buffer or liquid
is used to prepare an extract of fetal support tissue. Example 2
examines the use of various extraction buffers (high salt, low
salt, PBS, etc.) on total protein content and HA in the extract of
fetal support tissue (Table 1). Example 2 examined the levels of
the specific proteins TSG-6 (FIG. 14), PTX-3 (FIG. 18), TSP-1 (FIG.
19), and Smad7 (FIG. 20) using several extraction methods.
[0083] In some embodiments, the preparation of fetal support tissue
is tested to confirm the presence of specific components or
proteins. In some embodiments, the preparation of fetal support
tissue is tested for the presence of molecules including, but not
limited to, HA, I.alpha.I, TSG-6, PTX-3, TSP-1, and Smad7. In some
embodiments, the preparation of fetal support tissue is tested to
confirm the absence of pathogens at any point during the
preparation process.
[0084] In some embodiments, the preparation of fetal support tissue
is a dry powder. In some embodiments, the dry powder does not
require refrigeration or freezing during storage to keep the dry
powder from degrading over time. In some embodiments, the dry
powder is stored and reconstituted prior to use. In some
embodiments, the dry powder is prepared by preparing the
freeze-ground fetal support tissue as described herein, then
removing at least a portion of the water in the preparation of
fetal support tissue. In some embodiments, the excess water is
removed from the preparation of fetal support tissue by any
suitable means. In some embodiments, is the excess water is removed
by use of lyophilization. In some embodiments, lyophilizing the
preparation of fetal support tissue comprises using a commercially
available lyophilizer or freeze-dryer. In some embodiments,
suitable equipment is found, for example, through Virtis (Gardiner,
N.Y.); FTS Systems (Stone Ridge, N.Y.); and SpeedVac (Savant
Instruments Inc., Farmingdale, N.Y.). In some embodiments, the
amount of water that is removed is from about 5%, 10%, 20%, 30% to
about 60, 70, 80, 90, 95 or 99% or more. In some embodiments,
substantially all of the excess water is removed from the
preparation of fetal support tissue. In some embodiments, the dry
powder is stored. In some embodiments, the storage temperature
varies from less than about -196.degree. C., -80.degree. C.,
-50.degree. C., or -20.degree. C. to more than about 23.degree. C.
In some embodiments, the dry powder is characterized (weight,
protein content, etc.) prior to storage.
[0085] In some embodiments, the dry powder is reconstituted in a
suitable solution or buffer prior to use. Non-limiting examples of
solutions include, but are not limited to, PBS, DMEM, and BSS. In
some embodiments, the pH of the solution is adjusted as needed. In
some embodiments, the dry powder is reconstituted with a sufficient
volume of solution to produce a high concentration of the fetal
support tissue reconstituted composition. In some embodiments, the
dry powder is reconstituted with a sufficient volume of solution to
produce a low concentration of the fetal support tissue
reconstituted composition.
[0086] In some embodiments, the dry powder is reconstituted in a
cream, ointment, gel, foam or lotion].
[0087] In some embodiments, the preparation of fetal support tissue
is used to produce a phenotypic reversal of AMSCs from
myofibroblasts to fibroblasts. In some embodiments, the preparation
of fetal support tissue is used to prevent or slow differentiation
of various cell types. In some embodiments, many types of cells are
treated with the preparation of fetal support tissue.
[0088] Isolated nHC-HA/PTX3 Complexes
[0089] In some embodiments, the compositions include isolated
native HC-HA/PTX3 complexes (nHC-HA/PTX3).
[0090] In some embodiments, the nHC-HA/PTX3 complexes are isolated
from an isolated cell. In some embodiments, the nHC-HA/PTX3
complexes are isolated from a cultured cell. In some embodiments,
the nHC-HA/PTX3 complexes are isolated from a stem cell. In some
embodiments, the nHC-HA/PTX3 complexes are isolated from a water
soluble fraction of an extract prepared from a tissue, such as
umbilical cord or amniotic membrane. In some embodiments, the water
soluble fraction is extracted with an isotonic salt solution. In
some embodiments, the nHC-HA/PTX3 complexes are isolated from a
water insoluble fraction of an extract prepared from a tissue, such
as umbilical cord or amniotic membrane. In some embodiments, the
insoluble fraction is extracted with GnHCl.
[0091] In some embodiments, the isolated nHC-HA/PTX3 complex is
isolated from an amniotic tissue. In some embodiments, the isolated
nHC-HA/PTX3 complex is isolated from an amniotic membrane or an
umbilical cord. In some embodiments, the isolated nHC-HA/PTX3
complex is isolated from fresh, frozen or previously frozen
placental amniotic membrane (PAM), fresh, frozen or previously
frozen umbilical cord amniotic membrane (UCAM), fresh, frozen or
previously frozen placenta, fresh, frozen or previously frozen
umbilical cord, fresh, frozen or previously frozen chorion, fresh,
frozen or previously frozen amnion-chorion, or any combinations
thereof. Such tissues can be obtained from any mammal, such as, for
example, but not limited to a human, non-human primate, cow or
pig.
[0092] In some embodiments, the nHC-HA/PTX3 is purified by any
suitable method. In some embodiments, the nHC-HA/PTX3 complex is
purified by centrifugation (e.g., ultracentrifugation, gradient
centrifugation), chromatography (e.g., ion exchange, affinity, size
exclusion, and hydroxyapatite chromatography), gel filtration, or
differential solubility, ethanol precipitation or by any other
available technique for the purification of proteins (See, e.g.,
Scopes, Protein Purification Principles and Practice 2nd Edition,
Springer-Verlag, New York, 1987; Higgins, S. J. and Hames, B. D.
(eds.), Protein Expression: A Practical Approach, Oxford Univ
Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J. N.
(eds.), Guide to Protein Purification: Methods in Enzymology
(Methods in Enzymology Series, Vol 182), Academic Press, 1997, all
incorporated herein by reference).
[0093] In some embodiments, the nHC-HA/PTX3 is isolated from an
extract. In some embodiments, the extract is prepared from an
amniotic membrane extract. In some embodiments, the extract is
prepared from an umbilical cord extract. In some embodiments, the
umbilical cord extract comprises umbilical cord stroma and/or
Wharton's jelly. In some embodiments, the nHC-HA/PTX3 complex is
contained in an extract that is prepared by ultracentrifugation. In
some embodiments, the nHC-HA/PTX3 complex is contained in an
extract that is prepared by ultracentrifugation using a CsCl/4-6M
guanidine HCl gradient. In some embodiments, the extract is
prepared by at least 2 rounds of ultracentrifugation. In some
embodiments, the extract is prepared by more than 2 rounds of
ultracentrifugation (i.e. nHC-HA/PTX3 2nd). In some embodiments,
the extract is prepared by at least 4 rounds of ultracentrifugation
(i.e. nHC-HA/PTX3 4th). In some embodiments, the nHC-HA/PTX3
complex comprises a small leucine-rich proteoglycan. In some
embodiments, the nHC-HA/PTX3 complex comprises HC1, HA, PTX3 and/or
a small leucine-rich proteoglycan.
[0094] In some embodiments, ultracentrifugation is performed on an
extract prepared by extraction in an isotonic solution. In some
embodiments, the isotonic solution is PBS. For example, in some
embodiments the tissue is homogenized in PBS to produce a
homogenized sample. The homogenized sample is then separated into a
soluble portion and insoluble portion by centrifugation. In some
embodiments, ultracentrifugation is performed on the soluble
portion of the PBS-extracted tissue. In such embodiments, the
nHC-HA/PTX3 purified by ultracentrifugation of the PBS-extracted
tissue called an nHC-HA/PTX3 soluble complex. In some embodiments,
the nHC-HA soluble complex comprises a small leucine-rich
proteoglycan. In some embodiments, the nHC-HA/PTX3 soluble complex
comprises HC1, HA, PTX3 and/or a small leucine-rich
proteoglycan.
[0095] In some embodiments, ultracentrifugation is performed on an
extract prepared by direct guanidine HCl extraction (e.g. 4-6 M
GnHCl) of the amniotic membrane and/or umbilical cord tissue. In
some embodiments, the GnHCl extract tissues is then centrifuged to
produce GnHCl soluble and GnHCl insoluble portions. In some
embodiments, ultracentrifugation is performed on the GnHCl soluble
portion. In such embodiments, the nHC-HA/PTX3 purified by
ultracentrifugation of the guanidine HCl-extracted tissue is called
an nHC-HA/PTX3 insoluble complex. In some embodiments, the nHC-HA
insoluble complex comprises a small leucine-rich proteoglycan. In
some embodiments, the nHC-HA/PTX3 insoluble complex comprises HC1,
HA, PTX3 and/or a small leucine-rich proteoglycan.
[0096] In some embodiments, ultracentrifugation is performed on an
extract prepared by further guanidine HCl extraction of the
insoluble portion of the PBS-extracted tissue. For example, in some
embodiments the tissue is homogenized in PBS to produce a
homogenized sample. The homogenized sample is then separated into a
soluble portion and insoluble portion by centrifugation. The
insoluble portion is then further extracted in guanidine HCl (e.g.
4-6 M GnHCl) and centrifuged to produce a guanidine HCl soluble and
insoluble portions. In some embodiments, ultracentrifugation is
performed on the guanidine HCl soluble portion. In such
embodiments, the nHC-HA/PTX3 purified by ultracentrifugation of the
guanidine HCl-extracted tissue is called an nHC-HA/PTX3 insoluble
complex. In some embodiments, the nHC-HA insoluble complex
comprises a small leucine-rich proteoglycan. In some embodiments,
the nHC-HA/PTX3 insoluble complex comprises HC1, HA, PTX3 and/or a
small leucine-rich proteoglycan.
[0097] In some embodiments, the method of purifying the isolated
nHC-HA/PTX3 extract comprises: (a) dissolving the isolated extract
(e.g. prepared by the soluble or insoluble method described herein)
in CsCl/4-6M guanidine HCl at the initial density of 1.35 g/ml, to
generate a CsCl mixture, (b) centrifuging the CsCl mixture at
125,000.times.g for 48 h at 15.degree. C., to generate a first
purified extract, (c) extracting the first purified extract and
dialyzing it against distilled water to remove CsCl and guanidine
HCl, to generate a dialysate. In some embodiments, the method of
purifying the isolated extract further comprises (d) mixing the
dialysate with 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v)
potassium acetate at 0.degree. C. for 1 h, to generate a first
dialysate/ethanol mixture, (e) centrifuging the first
dialysate/ethanol mixture at 15,000.times.g, to generate a second
purified extract, and (f) extracting the second purified extract.
In some embodiments, the method of purifying the isolated extract
further comprises: (g) washing the second purified extract with
ethanol (e.g., 70% ethanol), to generate a second purified
extract/ethanol mixture; (h) centrifuging the second purified
extract/ethanol mixture, to generate a third purified extract; and
(i) extracting the third purified extract. In some embodiments, the
method of purifying the isolated extract further comprises: (j)
washing the third purified extract with ethanol (e.g., 70%
ethanol), to generate a third purified extract/ethanol mixture; (k)
centrifuging the third purified extract/ethanol mixture, to
generate a forth purified extract; and (l) extracting the forth
purified extract. In some embodiments, the purified extract
comprises an nHC-HA/PTX3 complex.
[0098] In some embodiments, the nHC-HA/PTX3 complex is purified by
immunoaffinity chromatography. In some embodiments, anti HC1
antibodies, anti-HC2 antibodies, or both are generated and affixed
to a stationary support. In some embodiments, the unpurified HC-HA
complex (i.e., the mobile phase) is passed over the support. In
certain instances, the HC-HA complex binds to the antibodies (e.g.,
via interaction of (a) an anti-HC1 antibody and HC1, (b) an
anti-HC2 antibody and HC2, (c) an anti-PTX antibody and PTX3, (d)
an anti-SLRP antibody and the SLRP, or (e) any combination
thereof). In some embodiments the support is washed (e.g., with
PBS) to remove any unbound or loosely bound molecules. In some
embodiments, the support is then washed with a solution that
enables elution of the nHC-HA/PTX3 complex from the support (e.g.,
1% SDS, 6M guanidine-HCl, or 8M urea).
[0099] In some embodiments, the nHC-HA/PTX3 complex is purified by
affinity chromatography. In some embodiments, HABP is generated and
affixed to a stationary support. In some embodiments, the
unpurified nHC-HA/PTX3 complex (i.e., the mobile phase) is passed
over the support. In certain instances, the nHC-HA/PTX3 complex
binds to the HABP. In some embodiments the support is washed (e.g.,
with PBS) to remove any unbound or loosely bound molecules. In some
embodiments, the support is then washed with a solution that
enables elution of the HC-HA complex from the support.
[0100] In some embodiments, the nHC-HA/PTX3 complex is purified by
a combination of HABP affinity chromatography, and immunoaffinity
chromatography using anti HC1 antibodies, anti-HC2 antibodies,
anti-PTX3 antibodies, antibodies against a SLRP or a combination of
SLRPs, or any combination of antibodies thereof.
[0101] In some embodiments, the nHC-HA/PTX3 complex is purified
from the insoluble fraction as described herein using one or more
antibodies. In some embodiments, the nHC-HA/PTX3 complex is
purified from the insoluble fraction as described herein using
anti-SLRP antibodies.
[0102] In some embodiments, the nHC-HA/PTX3 complex is purified
from the soluble fraction as described herein. In some embodiments,
the nHC-HA/PTX3 complex is purified from the soluble fraction as
described herein using anti-PTX3 antibodies.
[0103] In some embodiments, the nHC-HA/PTX3 complex comprises a
small leucine rich proteoglycan (SLRP). In some embodiments, the
nHC-HA/PTX3 complex comprises a class I, class II or class II SLRP.
In some embodiments, the small leucine-rich proteoglycan is
selected from among class I SLRPs, such as decorin and biglycan. In
some embodiments, the small leucine-rich proteoglycan is selected
from among class II SLRPs, such as fibromodulin, lumican, PRELP
(proline arginine rich end leucine-rich protein), keratocan, and
osteoadherin. In some embodiments, the small leucine-rich
proteoglycan is selected from among class III SLRPs, such as
epipycan and osteoglycin. In some embodiments, the small
leucine-rich proteoglycan is selected from among bikunin, decorin,
biglycan, and osteoadherin. In some embodiments, the small
leucine-rich protein comprises a glycosaminoglycan. In some
embodiments, the small leucine-rich proteoglycan comprises keratan
sulfate.
[0104] rcHC-HA/PTX3 Complexes
[0105] In some embodiments, the compositions comprise reconstituted
HC-HA/PTX3 complexes (rcHC-HA/PTX3) with or without SLRPs.
[0106] In some embodiments, a method for generating reconstituted
HC-HA/PTX3 complexes comprises (a) contacting immobilized high
molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) under
suitable conditions to form a PTX3/HA complex, and (b) contacting
the PTX3/HA complex with I.alpha.I and Tumor necrosis
factor-Stimulated Gene-6 (TSG-6). Provided herein are rcHC-HA/PTX3
complexes produced by such method. In some embodiments, TSG-6
catalyzes the transfer of heavy chain 1 (HC1) of
inter-.alpha.-inhibitor (I.alpha.I) to HA. In some embodiments, HC1
of I.alpha.I forms a covalent linkage with HA. In some embodiments,
the steps (a) and (b) of the method are performed sequentially in
order.
[0107] In some embodiments, a method for generating reconstituted
HC-HA/PTX3 complexes comprises contacting a PTX3/HA complex with
I.alpha.I and TSG-6. In some embodiments, TSG-6 catalyzes the
transfer of heavy chain 1 (HC1) of inter-.alpha.-inhibitor
(I.alpha.I) to HA. Provided herein are rcHC-HA/PTX3 complexes
produced by such method. In some embodiments, HC1 of I.alpha.I
forms a covalent linkage with HA.
[0108] In some embodiments, a method for generating a complex of HA
bound to PTX3 comprises contacting immobilized high molecular
weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) under suitable
conditions to form a PTX3/HA complex. Provided herein are PTX3/HA
complexes produced by such method.
[0109] In some embodiments, a method for generating reconstituted
HC-HA/PTX3 complexes comprises (a) contacting immobilized high
molecular weight hyaluronan (HMW HA) with I.alpha.I and TSG-6 to HA
to form an HC-HA complex pre-bound to TSG-6 and (b) contacting the
HC-HA complex with pentraxin 3 (PTX3) under suitable conditions to
form an rcHC-HA/PTX3 complex. Provided herein are rcHC-HA/PTX3
complexes produced by such method. In some embodiments, HC1 of
I.alpha.I forms a covalent linkage with HA. In some embodiments,
the steps (a) and (b) of the method are performed sequentially in
order. In some embodiments, the method comprises contacting an
HC-HA complex pre-bound to TSG-6 with PTX3.
[0110] In some embodiments, the method comprises first contacting
high molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3)
under suitable conditions to form a PTX3/HA complex, then
contacting the PTX3/HA complex with I.alpha.I and TSG-6.
[0111] In some embodiments, the I.alpha.I protein and TSG-6 protein
are contacted to the complex at a molar ratio of about 1:1, 2:1,
3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1
(I.alpha.I:TSG-6). In some embodiments the ratio of I.alpha.I:TSG-6
ranges from about 1:1 to about 20:1, such as about 1:1 to about
10:1, such as about 1:1 to 5 about:1, such as about 1:1 to about
3:1. In some embodiments, the ratio of I.alpha.I:TSG-6 is 3:1 or
higher. In some embodiments, the ratio of I.alpha.I:TSG-6 is
3:1.
[0112] In some embodiments, the steps (a) and (b) of the method are
performed sequentially in order. In some embodiments, the method
comprises contacting a PTX3/HA complex with I.alpha.I and
TSG-6.
[0113] In certain instances, TSG-6 interacts with I.alpha.I and
forms covalent complexes with HC1 and HC2 of I.alpha.I (i.e.
HC1.TSG-6 and HC2.TSG-6). In certain instances, in the presence of
HA, the HCs are transferred to HA to form rcHC-HA. In some
embodiments, a TSG-6.HC1 complex is added to pre-bound PTX3/HA
complex to catalyze the transfer of HC1 to HA. In some embodiments,
the method comprises first contacting immobilized high molecular
weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) under suitable
conditions to form a PTX3/HA complex, then contacting the PTX3/HA
complex with a HC1.TSG-6 complex. In some embodiments, a
combination of HC1.TSG-6 complex and HC2.TSG-6 complex is added to
a PTX3/HA complex.
[0114] In some embodiments, the step of contacting PTX3 to
immobilized HMW HA occurs for at least 10 minutes, at least 30
minutes, at least 1 hour, at least 2 hours, at least 3 hours, at
least 4 hours, at least 5 hours, at least 6 hours, at least 12
hours, or at least 24 hours or longer. In some embodiments, the
step of contacting PTX3 to immobilized HMW HA occurs for at least 2
hours or longer. In some embodiments, the step of contacting PTX3
to immobilized HMW HA occurs for at least 2 hours. In some
embodiments, the step of contacting PTX3 to immobilized HMW HA
occurs at 37.degree. C. In some embodiments, the step of contacting
PTX3 to immobilized HMW HA occurs in 5 mM MgCl.sub.2 in PBS.
[0115] In some embodiments, the step of contacting the PTX3/HA
complex with I.alpha.I and TSG-6 to HA occurs for at least 10
minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at
least 3 hours, at least 4 hours, at least 5 hours, at least 6
hours, at least 12 hours, or at least 24 hours or longer. In some
embodiments the step of contacting the PTX3/HA complex with a
HC1.TSG-6 complex and/or a HC2.TSG-6 complex occurs for at least 10
minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at
least 3 hours, at least 4 hours, at least 5 hours, at least 6
hours, at least 12 hours, or at least 24 hours or longer. In some
embodiments the step of contacting the PTX3/HA complex with a
HC1.TSG-6 complex and/or a HC2.TSG-6 complex occurs for at least 2
hours or longer. In some embodiments the step of contacting the
PTX3/HA complex with a HC1.TSG-6 complex and/or a HC2.TSG-6 complex
occurs for at least 2 hours. In some embodiments the step of
contacting the PTX3/HA complex with a HC1.TSG-6 complex and/or a
HC1.TSG-6 complex occurs at 37.degree. C. In some embodiments the
step of contacting the PTX3/HA complex with a HC1.TSG-6 complex
and/or a HC1.TSG-6 complex occurs in 5 mM MgCl.sub.2 in PBS.
[0116] In some embodiments, the method comprises contacting high
molecular weight hyaluronan (HMW HA) with a pentraxin 3 (PTX3)
protein, inter-.alpha.-inhibitor (I.alpha.I) protein comprising
heavy chain 1 (HC1) and Tumor necrosis factor .alpha.-stimulated
gene 6 (TSG-6) simultaneously under suitable conditions to form a
HC-HA/PTX3 complex. In some embodiments, the contacting the HMW HA
with PTX3, I.alpha.I and TSG-6 occurs for at least 10 minutes, at
least 30 minutes, at least 1 hour, at least 2 hours, at least 3
hours, at least 4 hours, at least 5 hours, at least 6 hours, at
least 12 hours, or at least 24 hours or longer. In some embodiments
the step of contacting the HMW HA, PTX3, I.alpha.I, and TSG-6
occurs at 37.degree. C. In some embodiments the step of contacting
the HMW HA, PTX3, I.alpha.I, and TSG-6 occurs in 5 mM MgCl.sub.2 in
PBS.
[0117] In some embodiments, the method comprises contacting high
molecular weight hyaluronan (HMW HA) with a pentraxin 3 (PTX3)
protein, inter-.alpha.-inhibitor (I.alpha.I) protein comprising
heavy chain 1 (HC1) and Tumor necrosis factor .alpha.-stimulated
gene 6 (TSG-6) sequentially, in any order, under suitable
conditions to form a HC-HA/PTX3 complex. In some embodiments, the
contacting the HMW HA with PTX3, I.alpha.I and TSG-6 occurs for at
least 10 minutes, at least 30 minutes, at least 1 hour, at least 2
hours, at least 3 hours, at least 4 hours, at least 5 hours, at
least 6 hours, at least 12 hours, or at least 24 hours or longer.
In some embodiments the step of contacting the HMW HA, PTX3,
I.alpha.I, and TSG-6 occurs at 37.degree. C. In some embodiments
the step of contacting the HMW HA, PTX3, I.alpha.I, and TSG-6
occurs in 5 mM MgCl.sub.2 in PBS.
[0118] In some embodiments, the methods for production of an
rcHC-HA/PTX3 complex further comprises addition of one or more
small leucine rich proteoglycans (SLRPs). In some embodiments, a
method for generating reconstituted HC-HA/PTX3 complexes comprises
(a) contacting immobilized high molecular weight hyaluronan (HMW
HA) with pentraxin 3 (PTX3) under suitable conditions to form a
PTX3/HA complex, (b) contacting the PTX3/HA complex with I.alpha.I
and Tumor necrosis factor-Stimulated Gene-6 (TSG-6) and (c)
contacting the PTX3/HA complex with one or more SLRPS. Provided
herein are rcHC-HA/PTX3 complexes produced by such method. In some
embodiments, TSG-6 catalyzes the transfer of heavy chain 1 (HC1) of
inter-.alpha.-inhibitor (I.alpha.I) to HA. In some embodiments, HC1
of I.alpha.I forms a covalent linkage with HA. In some embodiments,
the steps (a), (b), and (c) of the method are performed
sequentially in order. In some embodiments, the steps (a), (b), and
(c) of the method are performed simultaneously. In some
embodiments, the step (a) of the method is performed and then steps
(b) and (c) of the method are performed sequentially in order. In
some embodiments, the step (a) of the method is performed and then
steps (b) and (c) of the method are performed simultaneously.
[0119] In some embodiments, a method for generating reconstituted
HC-HA/PTX3 complexes comprises (a) contacting immobilized high
molecular weight hyaluronan (HMW HA) with I.alpha.I and TSG-6 to HA
to form an HC-HA complex pre-bound to TSG-6, (b) contacting the
HC-HA complex with pentraxin 3 (PTX3) and (c) contacting the HC-HA
complex with one or more SLRPS under suitable conditions to form an
rcHC-HA/PTX3 complex. Provided herein are rcHC-HA/PTX3 complexes
produced by such method. In some embodiments, HC1 of I.alpha.I
forms a covalent linkage with HA. In some embodiments, the method
comprises contacting an HC-HA complex pre-bound to TSG-6 with PTX3.
In some embodiments, the steps (a), (b), and (c) of the method are
performed sequentially in order. In some embodiments, the steps
(a), (b), and (c) of the method are performed simultaneously. In
some embodiments, the step (a) of the method is performed and then
steps (b) and (c) of the method are performed sequentially in
order. In some embodiments, the step (a) of the method is performed
and then steps (b) and (c) of the method are performed
simultaneously.
[0120] In some embodiments, the SLRP is selected from among a class
I, class II or class II SLRP. In some embodiments, the SLRP is
selected from among class I SLRPs, such as decorin and biglycan. In
some embodiments, the small leucine-rich proteoglycan is selected
from among class II SLRPs, such as fibromodulin, lumican, PRELP
(proline arginine rich end leucine-rich protein), keratocan, and
osteoadherin. In some embodiments, the small leucine-rich
proteoglycan is selected from among class III SLRPs, such as
epipycan and osteoglycin. In some embodiments, the small
leucine-rich proteoglycan is selected from among bikunin, decorin,
biglycan, and osteoadherin. In some embodiments, the small
leucine-rich protein comprises a glycosaminoglycan. In some
embodiments, the small leucine-rich proteoglycan comprises keratan
sulfate.
[0121] PTX3
[0122] In some embodiments, PTX3 for use in the methods is isolated
from a cell or a plurality of cells (e.g., a tissue extract).
Exemplary cells suitable for the expression of PTX3 include, but
are not limited to, animal cells including, but not limited to,
mammalian cells, primate cells, human cells, rodent cells, insect
cells, bacteria, and yeast, and plant cells, including, but not
limited to, algae, angiosperms, gymnosperms, pteridophytes and
bryophytes. In some embodiments, PTX3 for use in the methods is
isolated from a human cell. In some embodiments, PTX3 for use in
the methods is isolated from a cell that is stimulated with one or
more proinflammatory cytokines to upregulate PTX3 expression. In
some embodiments, the proinflammatory cytokine is IL-1 or
TNF-.alpha..
[0123] In some embodiments, PTX3 for use in the methods is isolated
from an amniotic membrane cell. In some embodiments, PTX3 for use
in the methods is isolated from an amniotic membrane cell from an
umbilical cord. In some embodiments, the amniotic membrane cell is
stimulated with or more proinflammatory cytokines to upregulate
PTX3 expression. In some embodiments, the proinflammatory cytokine
is IL-1 or TNF-.alpha..
[0124] In some embodiments, PTX3 for use in the methods is isolated
from an umbilical cord cell. In some embodiments, the umbilical
cord cell is stimulated with or more proinflammatory cytokines to
upregulate PTX3 expression. In some embodiments, the
proinflammatory cytokine is IL-1 or TNF-.alpha..
[0125] In some embodiments, PTX3 for use in the methods is isolated
from an amniotic epithelial cell. In some embodiments, PTX3 for use
in the methods is isolated from an umbilical cord epithelial cell.
In some embodiments, the amniotic epithelial cell or umbilical cord
epithelial cell is stimulated with or more proinflammatory
cytokines to upregulate PTX3 expression. In some embodiments, the
proinflammatory cytokine is IL-1 or TNF-.alpha..
[0126] In some embodiments, PTX3 for use in the methods is isolated
from an amniotic stromal cell. In some embodiments, PTX3 for use in
the methods is isolated from an umbilical cord stromal cell. In
some embodiments, the amniotic stromal cell or umbilical cord
stromal cell is stimulated with or more proinflammatory cytokines
to upregulate PTX3 expression. In some embodiments, the
proinflammatory cytokine is IL-1 or TNF-.alpha..
[0127] In some embodiments, PTX3 for use in the methods is a native
PTX3 protein isolated from a cell. In some embodiments, the cell is
stimulated with or more proinflammatory cytokines to upregulate
PTX3 expression. In some embodiments, the proinflammatory cytokine
is IL-1 or TNF-.alpha..
[0128] In some embodiments, PTX3 is prepared by recombinant
technology. In some embodiments, PTX3 is expressed from a
recombinant expression vector. In some embodiments, nucleic acid
encoding PTX3 is operably linked to a constitutive promoter. In
some embodiments, nucleic acid encoding PTX3 is operably linked to
an inducible promoter. In some embodiments, PTX3 is expressed in a
transgenic animal. In some embodiments, PTX3 is a recombinant
protein. In some embodiments, PTX3 is a recombinant protein
isolated from a cell. In some embodiments, PTX3 is a recombinant
protein produced in a cell-free extract.
[0129] In some embodiments, PTX3 is purified from amniotic
membrane, umbilical cord, umbilical cord amniotic membrane,
chorionic membrane, amniotic fluid, or a combination thereof. In
some embodiments, PTX3 is purified from amniotic membrane cells. In
some embodiments, the amniotic membrane cell is an amniotic
epithelial cell. In some embodiments, the amniotic membrane cell is
an umbilical cord epithelial cell. In some embodiments, the
amniotic membrane cell is an amniotic stromal cell. In some
embodiments, the amniotic membrane cell is an umbilical cord
stromal cell. In some embodiments, the amniotic membrane cell is
stimulated with or more proinflammatory cytokines to upregulate
PTX3 expression. In some embodiments, the proinflammatory cytokine
is IL-1 or TNF-.alpha..
[0130] In some embodiments, PTX3 is not isolated from a cell or a
plurality of cells (e.g., a tissue extract).
[0131] In some embodiments, PTX3 comprises a polypeptide having the
sequence set forth in SEQ ID NO: 33 or a variant thereof having at
least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
sequence amino acid identity to the polypeptide having the sequence
set forth in SEQ ID NO: 33. Exemplary variants include, for
example, species variants, allelic variants and variants that
contain conservative and non-conservative amino acid mutations. In
some embodiments, PTX3 comprises a fragment of PTX3 sufficient to
bind to HA and facilitate the formation of rcHC-HA/PTX3 complex. In
some embodiments, PTX3 comprises Glu18 to Ser277 of human PTX3.
Variants of PTX3 for use in the provided methods include variants
with an amino acid modification that is an amino acid replacement
(substitution), deletion or insertion. In some embodiments, such
modification improves one or more properties of the PTX3
polypeptides such as improving the one or more therapeutic
properties of the rcHC-HA/PTX3 complex (e.g., anti-inflammatory,
anti-immune, anti-angiogenic, anti-scarring, anti-adhesion,
regeneration or other therapeutic activities as described
herein).
[0132] In some embodiments PTX3 protein is obtained from a
commercial source. An exemplary commercial source for PTX3 is, but
is not limited to, PTX3 (Catalog No. 1826-TS; R&D Systems,
Minneapolis, Minn.).
[0133] In some embodiments, the PTX3 protein used in the methods is
a multimeric protein. In some embodiments, the PTX3 protein used in
the methods is a homomultimer. In some embodiments, the
homomultimer is a dimer, trimer, tetramer, hexamer, pentamer, or
octamer. In some embodiments, the PTX3 homomultimer is a trimer,
tetramer, or octamer. In particular embodiments, the PTX3
homomultimer is an octamer. In some embodiments, the
multimerization domain is modified to improve multimerization of
the PTX3 protein. In some embodiments, the multimerization domain
is replaced with a heterogeneous multimerization domain (e.g., an
Fc multimerization domain or leucine zipper) that when fused to
PTX3 improves the multimerization of PTX3.
[0134] TSG-6
[0135] In some embodiments, TSG-6 for use in the methods is
isolated from a cell or a plurality of cells (e.g., a tissue
extract). Exemplary cells suitable for the expression of TSG-6
include, but are not limited to, animal cells including, but not
limited to, mammalian cells, primate cells, human cells, rodent
cells, insect cells, bacteria, and yeast, and plant cells,
including, but not limited to, algae, angiosperms, gymnosperms,
pteridophytes and bryophytes. In some embodiments, TSG-6 for use in
the methods is isolated from a human cell. In some embodiments,
TSG-6 for use in the methods is isolated from a cell that is
stimulated with one or more proinflammatory cytokines to upregulate
TSG-6 expression. In some embodiments, the proinflammatory cytokine
is IL-1 or TNF-.alpha..
[0136] In some embodiments, TSG-6 for use in the methods is
isolated from an amniotic membrane cell. In some embodiments, TSG-6
for use in the methods is isolated from an amniotic membrane cell
from an umbilical cord. In some embodiments, TSG-6 for use in the
methods is isolated from an amniotic membrane cell that is
stimulated with one or more proinflammatory cytokines to upregulate
TSG-6 expression. In some embodiments, the proinflammatory cytokine
is IL-1 or TNF-.alpha..
[0137] In some embodiments, TSG-6 for use in the methods is
isolated from an umbilical cord cell. In some embodiments, TSG-6
for use in the methods is isolated from an umbilical cord cell that
is stimulated with one or more proinflammatory cytokines to
upregulate TSG-6 expression. In some embodiments, the
proinflammatory cytokine is IL-1 or TNF-.alpha..
[0138] In some embodiments, TSG-6 for use in the methods is
isolated from an amniotic epithelial cell. In some embodiments,
TSG-6 for use in the methods is isolated from an umbilical cord
epithelial cell. In some embodiments, TSG-6 for use in the methods
is isolated from an amniotic epithelial cell or an umbilical cord
epithelial cell that is stimulated with one or more proinflammatory
cytokines to upregulate TSG-6 expression. In some embodiments, the
proinflammatory cytokine is IL-1 or TNF-.alpha..
[0139] In some embodiments, TSG-6 for use in the methods is
isolated from an amniotic stromal cell. In some embodiments TSG-6
for use in the methods is isolated from an umbilical cord stromal
cell. In some embodiments, TSG-6 for use in the methods is isolated
from an amniotic stromal cell or an umbilical cord stromal cell
that is stimulated with one or more proinflammatory cytokines to
upregulate TSG-6 expression. In some embodiments, the
proinflammatory cytokine is IL-1 or TNF-.alpha..
[0140] In some embodiments, TSG-6 for use in the methods is a
native TSG-6 protein isolated from a cell. In some embodiments, the
cell is stimulated with or more proinflammatory cytokines to
upregulate TSG-6 expression. In some embodiments, the
proinflammatory cytokine is IL-1 or TNF-.alpha..
[0141] In some embodiments, TSG-6 is prepared by recombinant
technology. In some embodiments, TSG-6 is expressed from a
recombinant expression vector. In some embodiments, nucleic acid
encoding TSG-6 is operably linked to a constitutive promoter. In
some embodiments, nucleic acid encoding TSG-6 is operably linked to
an inducible promoter. In some embodiments, TSG-6 is expressed in a
transgenic animal. In some embodiments, TSG-6 is a recombinant
protein. In some embodiments, TSG-6 is a recombinant protein
isolated from a cell. In some embodiments, TSG-6 is a recombinant
protein produced in a cell-free extract.
[0142] In some embodiments, TSG-6 is purified from amniotic
membrane, amniotic membrane, chorionic membrane, amniotic fluid, or
a combination thereof. In some embodiments, PTX3 is purified from
amniotic membrane cells. In some embodiments, the amniotic membrane
cell is an amniotic epithelial cell. In some embodiments, the
amniotic epithelial cell is an umbilical cord epithelial cell. In
some embodiments, the amniotic membrane cell is an amniotic stromal
cell. In some embodiments, the amniotic membrane cell is an
umbilical cord stromal cell. In some embodiments, the amniotic
membrane cell is stimulated with or more proinflammatory cytokines
to upregulate TSG-6 expression. In some embodiments, the
proinflammatory cytokine is IL-1 or TNF-.alpha..
[0143] In some embodiments, TSG-6 is not isolated from a cell or a
plurality of cells (e.g., a tissue extract).
[0144] In some embodiments, TSG-6 comprises a fragment of TSG-6
that is sufficient to facilitate or catalyze the transfer HC1 of
I.alpha.I to HA. In some embodiments, TSG-6 comprises the link
module of TSG-6. In some embodiments, TSG-6 comprises amino acids
Trp18 through Leu277 of TSG-6. In some embodiments, TSG-6 comprises
a polypeptide having the sequence set forth in SEQ ID NO: 2 or a
variant thereof having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% sequence amino acid identity to the
polypeptide having the sequence set forth in SEQ ID NO: 2.
Exemplary variants include, for example, species variants, allelic
variants and variants that contain conservative and
non-conservative amino acid mutations. Natural allelic variants of
human TSG-6 include, for example, TSG-6 containing the amino acid
replacement Q144R. Variants of TSG-6 or HA binding fragments
thereof for use in the provided methods include variants with an
amino acid modification that is an amino acid replacement
(substitution), deletion or insertion. In some embodiments, such
modification improve one or more properties of the TSG-6
polypeptides such as improved transfer of HC1 of I.alpha.I to HA or
improved release of the TSG-6 polypeptide from the rcHC-HA/PTX3
complex following transfer of HC1 of I.alpha.I to HA.
[0145] In some embodiments, TSG-6 comprises an affinity tag.
Exemplary affinity tags include but are not limited to a
hemagglutinin tag, a poly-histidine tag, a myc tag, a FLAG tag, a
glutathione-S-transferase (GST) tag. Such affinity tags are well
known in the art for use in purification. In some embodiments, such
an affinity tag incorporated into the TSG-6 polypeptide as a fusion
protein or via a chemical linker. In some embodiments, TSG-6
comprises an affinity tag and the unbound TSG-6 is removed from the
rcHC-HA/PTX3 complex by affinity purification.
[0146] In some embodiments TSG-6 protein is obtained from a
commercial source. An exemplary commercial source for TSG-6 is, but
is not limited to, TSG-6 (Catalog No. 2104-TS R&D Systems,
Minneapolis, Minn.).
[0147] I.alpha.I
[0148] In some embodiments, the I.alpha.I comprises an HC1 chain.
In some embodiments, the I.alpha.I comprises an HC1 and an HC2
chain. In some embodiments, the I.alpha.I comprises an HC1 and
bikunin. In some embodiments, the I.alpha.I comprises an HC1, and
HC2 chain and bikunin. In some embodiments, the I.alpha.I comprises
an HC1, and HC2 chain and bikunin linked by a chondroitin sulfate
chain.
[0149] In some embodiments, I.alpha.I is isolated from a biological
sample. In some embodiments the biological sample is a biological
sample from a mammal. In some embodiments, the mammal is a human.
In some embodiments, the biological sample is a blood, serum,
plasma, liver, amniotic membrane, chorionic membrane or amniotic
fluid sample. In some embodiments, the biological sample is a
blood, serum, or plasma sample. In some embodiments, the biological
sample is a blood sample. In some embodiments, the biological
sample is a serum sample. In some embodiments, the biological
sample is a plasma sample. In some embodiments, the I.alpha.I is
purified from human blood, plasma or serum. In some embodiments,
I.alpha.I is isolated from human serum. In some embodiments,
I.alpha.I is not isolated from serum. In some embodiments,
I.alpha.I for use in the methods is produced in an amniotic
membrane cell. In some embodiments, I.alpha.I for use in the
methods is produced in an umbilical cord cell. In some embodiments,
I.alpha.I for use in the methods is produced in an amniotic
membrane cell from an umbilical cord. In some embodiments,
I.alpha.I for use in the methods is produced in an amniotic
epithelial cell. In some embodiments, I.alpha.I for use in the
methods is produced in an umbilical cord epithelial cell. In some
embodiments, I.alpha.I for use in the methods is produced in an
amniotic stromal cell. In some embodiments, I.alpha.I for use in
the methods is produced in an umbilical cord stromal cell. In some
embodiments, I.alpha.I for use in the methods is produced in a
hepatic cell. In some embodiments, I.alpha.I is prepared by
recombinant technology.
[0150] In some embodiments, HC1 of I.alpha.I is isolated from a
biological sample. In some embodiments the biological sample is a
biological sample from a mammal. In some embodiments, the mammal is
a human. In some embodiments, the biological sample is a blood,
serum, plasma, liver, amniotic membrane, chorionic membrane or
amniotic fluid sample. In some embodiments, the biological sample
is a blood, serum, or plasma sample. In some embodiments, the
biological sample is a blood sample. In some embodiments, the
biological sample is a serum sample. In some embodiments, the
biological sample is a plasma sample. In some embodiments, the HC1
of I.alpha.I is purified from human blood, plasma or serum. In some
embodiments, I.alpha.I is isolated from human serum. In some
embodiments, HC1 of I.alpha.I is not purified from serum. In some
embodiments, HC1 of I.alpha.I is prepared by recombinant
technology. In some embodiments, HC1 of I.alpha.I is purified from
hepatic cells. In some embodiments, HC1 of I.alpha.I is purified
from amniotic membrane cells. In some embodiments, HC1 of I.alpha.I
is purified from amniotic epithelial cells or umbilical cord
epithelial cells. In some embodiments, HC1 of I.alpha.I is purified
from amniotic stromal cells or umbilical cord stromal cells.
[0151] In some embodiments, HC1 comprises a polypeptide having the
sequence set forth in SEQ ID NO: 47 or a polypeptide having at
least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino
acid identity to the polypeptide having the sequence set forth in
SEQ ID NO: 47.
[0152] In some embodiments, HC2 of I.alpha.I is isolated from a
biological sample. In some embodiments the biological sample is a
biological sample from a mammal. In some embodiments, the mammal is
a human. In some embodiments, the biological sample is a blood,
serum, plasma, liver, amniotic membrane, chorionic membrane or
amniotic fluid sample. In some embodiments, the biological sample
is a blood, serum, or plasma sample. In some embodiments, the
biological sample is a blood sample. In some embodiments, the
biological sample is a serum sample. In some embodiments, the
biological sample is a plasma sample. In some embodiments, the HC2
of I.alpha.I is purified from human blood, plasma or serum. In some
embodiments, HC2 of I.alpha.I is isolated from human serum. In some
embodiments, HC2 of I.alpha.I is isolated from human serum. In some
embodiments, HC2 of I.alpha.I is not isolated from blood serum. In
some embodiments, HC2 of I.alpha.I is prepared by recombinant
technology. In some embodiments, HC2 of I.alpha.I is purified from
hepatic cells. In some embodiments, HC2 of I.alpha.I is purified
from amniotic membrane cells. In some embodiments, HC2 of I.alpha.I
is purified from amniotic epithelial cells or umbilical cord
epithelial cells. In some embodiments, HC2 of I.alpha.I is purified
from amniotic stromal cells or umbilical cord stromal cells.
[0153] In some embodiments, HC2 comprises a polypeptide having the
sequence set forth in SEQ ID NO: 49 or a polypeptide having at
least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino
acid identity to the polypeptide having the sequence set forth in
SEQ ID NO: 49.
[0154] In some embodiments, I.alpha.I comprises bikunin. In some
embodiments, bikunin comprises a polypeptide having the sequence
set forth in SEQ ID NO: 53 or a polypeptide having at least 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid
identity to the polypeptide having the sequence set forth in SEQ ID
NO: 53. In some embodiments, I.alpha.I comprises a chondroitin
sulfate chain.
[0155] HA
[0156] In some embodiments, HA is purified from a cell, tissue or a
fluid sample. In some embodiments, HA is obtained from a commercial
supplier (e.g., Sigma Aldrich or Advanced Medical Optics, Irvine,
Calif. (e.g., Healon)). In some embodiments, HA is obtained from a
commercial supplier as a powder. In some embodiments, HA is
expressed in a cell. Exemplary cells suitable for the expression of
HA include, but are not limited to, animal cells including, but not
limited to, mammalian cells, primate cells, human cells, rodent
cells, insect cells, bacteria, and yeast, and plant cells,
including, but not limited to, algae, angiosperms, gymnosperms,
pteridophytes and bryophytes. In some embodiments, HA is expressed
in a human cell. In some embodiments, HA is expressed in a
transgenic animal. In some embodiments, HA is obtained from a cell
that expresses a hyaluronan synthase (e.g., HAS1, HAS2, and HAS3).
In some embodiments, the cell contains a recombinant expression
vector that expresses an HA synthase. In certain instances, an HA
synthase lengthens hyaluronan by repeatedly adding glucuronic acid
and N-acetylglucosamine to the nascent polysaccharide as it is
extruded through the cell membrane into the extracellular
space.
[0157] HA for use in the methods is typically high molecular weight
(HMW) HA. In some embodiments, the weight average molecular weight
of HMW HA is greater than about 500 kilodaltons (kDa), such as, for
example, between about 500 kDa and about 10,000 kDa, between about
800 kDa and about 8,500 kDa, between about 1100 kDa and about 5,000
kDa, or between about 1400 kDa and about 3,500 kDa. In some
embodiments, the weight average molecular weight of HMW HA is about
3000 kDa.
[0158] Additional Components
[0159] In some embodiments, one or more additional components are
added to generate an rcHC-HA/PTX3 complex. In some embodiments, a
small leucine rich proteoglycan (SLRP) is added to generate an
rcHC-HA/PTX3 complex. In some embodiments, the SLRP is a class I,
class II or class II SLRP. In some embodiments, the SLRP is
selected from among class I SLRPs, such as decorin and biglycan. In
some embodiments, the SLRP is selected from among class II SLRPs,
such as fibromodulin, lumican, PRELP (proline arginine rich end
leucine-rich protein), keratocan, and osteoadherin. In some
embodiments, the SLRP is selected from among class III SLRPs, such
as epipycan and osteoglycin. In some embodiments, the SLRP is
selected from among bikunin, decorin, biglycan, and osteoadherin.
In some embodiments, the SLRP comprises a glycosaminoglycan. In
some embodiments, the SLRP comprises keratan sulfate.
[0160] HA Immobilization
[0161] In some embodiments, HMW HA is immobilized by any suitable
method. In some embodiments, HMW HA is immobilized to a solid
support, such as culture dish, bead, a column or other suitable
surfaces, such as, for example, a surface of an implantable medical
device or a portion thereof or on a surface that is subsequently
connected to or combined with an implantable medical device as
described herein. In some embodiments, HMW HA is immobilized
directly to the solid support, such a by chemical linkage. In some
embodiments, HMW HA is attached indirectly to the solid support via
a linker or an intermediary protein. Numerous heterobifunctional
cross-linking reagents that are used to form covalent bonds between
amino groups and thiol groups and to introduce thiol groups into
proteins, are known to those of skill in this art. In some
embodiments, HMW HA is immobilized directly to the solid support
via crosslinking to the solid support. In some embodiments, HMW HA
is immobilized directly to the solid support without crosslinking
to the solid support. In some embodiments, HMW HA is immobilized
directly to the solid support as a coating. In some embodiments,
HMW HA is immobilized to a Covalink.TM.-NH surface. In some
embodiments, HMW HA is immobilized directly to the solid support as
a coating. In some embodiments, HMW HA is immobilized to a
Covalink.TM.-NH surface for about 16 h at 4.degree. C.
[0162] In some embodiments, the method comprises immobilizing HMW
HA to a solid surface via direct linkage to a solid support (i.e.
without an intermediary protein). In some embodiments, the solid
support is washed to remove unbound HMW HA prior to contacting the
immobilized HA with PTX3. In some embodiments, the solid support is
washed with washes of 8M GnHCl and PBS to remove unbound HMW HA
prior to contacting the immobilized HA with PTX3.
[0163] In some embodiments, the method comprises immobilizing HA to
a solid surface via an intermediary protein or a linker. In some
embodiments, the linker is a peptide linker. In some embodiments,
the intermediary protein is an HA binding protein (HABP). In some
embodiments, HABP is first attached to a solid support (e.g., by
cross-linking, chemical linkage or via a chemical linker). In some
embodiments, the solid support comprising HABP is then contacted
with HA (e.g., HMW HA) to immobilize HA to the solid support via
binding of the HABP to HA. In some embodiments, the solid support
is washed to remove unbound HMW HA prior to contacting the
immobilized HMW HA with PTX3. In some embodiments, the solid
support is washed with washes of 8M GnHCl and PBS to remove unbound
HMW HA prior to contacting the immobilized HA with PTX3.
[0164] In some embodiments, the method comprises immobilizing HA to
a solid surface via attachment of a peptide linker to the solid
support and attachment HA to the peptide linker. In some
embodiments, the peptide linker comprises a protease cleavage
site.
[0165] In some embodiments, the method comprises immobilizing HA to
a solid surface via attachment of a cleavable chemical linker, such
as, but not limited to a disulfide chemical linker.
[0166] In some embodiments, the HABP selected for use in the
methods is an HABP that is dissociated from HA following formation
of the rcHC-HA/PTX3 complex. In some embodiments, the HABP
non-covalently binds to HA. In some embodiments, the method further
comprises dissociating the rcHC-HA/PTX3 complex from HABP using one
or more dissociating agents. Dissociating agents for the disruption
of non covalent interactions (e.g., guanidine hydrochloride, urea
and various detergents, e.g., SDS) are known in the art. In some
embodiments the dissociating agent is urea. In some embodiments the
dissociating agent is guanidine hydrochloride. In some embodiments,
the dissociation agent is about 4M to about 8M guanidine-HCl. In
some embodiments, the dissociation agent is about 4M, about 5M,
about 6M, about 7M, about 8M guanidine-HCl. In some embodiments,
the dissociation agent is about 4M to about 8M guanidine-HCl in PBS
at pH 7.5.
[0167] In some embodiments, such dissociating agents are employed
to dissociate the rcHC-HA/PTX3 complex from an intermediary HABP.
An HABP for use in the methods typically is selected such that the
binding affinity for HA is strong enough to permit assembly of the
rcHC-HA/PTX3 complex but is dissociated from the rcHC-HA/PTX3
complex with a suitable dissociation agent. In some embodiments the
dissociating agent is guanidine hydrochloride.
[0168] Exemplary HABPs for use with the methods provided herein
include, but are not limited to, HAPLN1, HAPLN2, HAPLN3, HAPLN4,
aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44,
stabilin-1, stabilin-2, or portions thereof (e.g., link modules
thereof) sufficient to bind HA. In some embodiments, the HABP
comprises a polypeptide having the sequence set forth in any of SEQ
ID NOS: 54-99 or a polypeptide having at least 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% sequence amino acid identity to the
polypeptide having the sequence set forth in any of SEQ ID NOS:
54-99. In some embodiments, the HABP is versican. In some
embodiments, the HABP is a recombinant protein. In some
embodiments, the HABP is a recombinant mammalian protein. In some
embodiments, the HABP is a recombinant human protein. In some
embodiments, the HABP is a recombinant versican protein or a
portion thereof sufficient to bind to HA. In some embodiments, the
HABP is a recombinant aggrecan protein or a portion thereof
sufficient to bind to HA. In some embodiments, the HABP is a native
HABP or a portion thereof sufficient to bind to HA. In some
embodiments, the native HABP is isolated from mammalian tissue or
cells. In some embodiments, the HABP is isolated from bovine nasal
cartilage (e.g. HABP from Seikagaku which contains the HA binding
domains of aggrecan and link protein).
[0169] In some embodiments, the HABP comprises a link module of
HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan,
brevican, phosphacan, TSG-6, CD44, stabilin-1, or stabilin-2. In
some embodiments, the HABP comprising a link module comprises a
polypeptide having the sequence set forth in any of link domains of
SEQ ID NOS: 54-99 or a polypeptide having at least 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to the
polypeptide having the sequence set forth in any of link domains of
SEQ ID NOS: 54-99. In some embodiments, the HABP comprises a link
module of versican. In some embodiments, the HABP comprising a link
module is a recombinant protein. In some embodiments, the HABP
comprising a link module of versican is a recombinant protein.
[0170] In some embodiments, the or intermediary protein, such as an
HABP, contains a proteolytic cleavage sequence that is recognized
by and is hydrolyzed by a site specific protease, such as furin, 3C
protease, caspase, matrix metalloproteinase or TEV protease. In
such embodiments, assembled rcHC-HA/PTX3 complexes are released
from the solid support by contacting the immobilized complexes with
a protease that cleaves the specific cleavage sequence.
[0171] In some embodiments, the rcHC-HA/PTX3 complex is purified.
In some embodiments, the rcHC-HA/PTX3 complex is purified by any
suitable method or combination of methods. The embodiments
described below are not intended to be exclusive, only
exemplary.
[0172] In some embodiments, the rcHC-HA/PTX3 complex is purified by
chromatography (e.g., ion exchange, affinity, size exclusion, and
hydroxyapatite chromatography), gel filtration, centrifugation
(e.g., gradient centrifugation), or differential solubility,
ethanol precipitation or by any other available technique for the
purification of proteins.
[0173] In some embodiments, the rcHC-HA/PTX3 complex is purified by
immunoaffinity chromatography. In some embodiments antibodies are
generated against a component of the rcHC-HA/PTX3 complex (e.g.,
anti-HC1, anti-PTX, an antibody against one or more SLRPs of the
rcHC-HA/PTX3 complex, e.g., anti-bikunin, anti-decorin,
anti-biglycan, or anti-osteoadherin) and affixed to a solid
support. In some embodiments, the unpurified rcHC-HA/PTX3 complex
(i.e., the mobile phase) is passed over the support. In certain
instances, the rcHC-HA/PTX3 complex binds to the antibodies. In
some embodiments, the support is washed (e.g., with PBS) to remove
any unbound or loosely bound molecules. In some embodiments, the
support is then washed with a solution that enables elution of the
rcHC-HA/PTX3 complex from the support (e.g., 1% SDS, 6M
guanidine-HCl, or 8M urea). In some embodiments, the dissociating
agent is removed from the dissociated rcHC-HA/PTX3 complex. In some
embodiments, the dissociating agent is removed from the dissociated
rcHC-HA/PTX3 complex by a method including, but not limited to,
ion-exchange chromatography, dialysis, gel filtration
chromatography, ultrafiltration, or diafiltration.
[0174] In some embodiments, the rcHC-HA/PTX3 complex is purified by
affinity chromatography. In some embodiments, an HABP is employed
to bind to the rcHC-HA/PTX3 complex for purification of the complex
and affixed to a stationary support. In some embodiments, the
unpurified rcHC-HA/PTX3 complex (i.e., the mobile phase) is passed
over the support. In certain instances, the rcHC-HA/PTX3 complex
binds to the HABP. In some embodiments the support is washed (e.g.,
with PBS) to remove any unbound or loosely bound molecules. In some
embodiments, the support is then washed with a solution (e.g., a
dissociating agent) that enables elution of the rcHC-HA/PTX3
complex from the support. In some embodiments, the dissociating
agent is removed from the dissociated rcHC-HA/PTX3 complex by a
method including, but not limited to, ion-exchange chromatography,
dialysis, gel filtration chromatography, ultrafiltration, or
diafiltration.
[0175] In some embodiments, the rcHC-HA/PTX3 complex is purified by
a combination of HABP affinity chromatography, and immunoaffinity
chromatography using antibodies against one or more components of
the rcHC-HA/PTX3 complex.
[0176] In some embodiments, one or more components of the
rcHC-HA/PTX3 complex disclosed herein comprise an affinity tag
(e.g., a fusion protein of PTX3 or HC1 with an affinity tag).
Exemplary affinity tags that are incorporated into one or more
components of the rcHC-HA/PTX3 complex in some embodiments include,
but are not limited to, a hemagglutinin tag, poly-histidine, a myc
tag, a FLAG tag, or glutathione-S-transferase sequence. In some
embodiments, the ligand for the affinity tag is affixed to the
solid support. In some embodiments, the unpurified rcHC-HA/PTX3
complex is passed over the support. In certain instances, the
rcHC-HA/PTX3 complex binds to the ligand. In some embodiments the
support is washed (e.g., with PBS) to remove any unbound or loosely
bound molecules. In some embodiments, the support is then washed
with a solution that enables elution of an rcHC-HA/PTX3 complex
disclosed herein from the support. In some embodiments, the elution
agent is removed from the dissociated rcHC-HA/PTX3 complex by a
method including, but not limited to, ion-exchange chromatography,
dialysis, gel filtration chromatography, ultrafiltration, or
diafiltration.
[0177] In some embodiments, the PTX3, TSG-6, and/or HC1 are
conjugated to a label. A "label" refers to a detectable compound or
composition which is conjugated directly or indirectly to a
polypeptide so as to generate a labeled polypeptide. In some
embodiments, the label is detectable by itself (e.g., radioisotope
labels or fluorescent labels) or, in the case of an enzymatic
label, catalyzes chemical alteration of a substrate compound
composition which is detectable. Non-limiting examples of labels
include fluorogenic moieties, dyes, fluorescent tags, green
fluorescent protein, or luciferase.
[0178] Excipients
[0179] In some embodiments, the compositions comprise excipients.
In some embodiments, the excipient is chosen from the group
comprising pH modifiers, buffers, collagen, HA, antibiotics,
surfactants, stabilizers, proteins, and combinations thereof. In
some embodiments, excipient comprises an extracellular matrix (ECM)
component. In some embodiments, the ECM component comprises
collagen, fibrin, HA, or a combination thereof.
[0180] Collagen is a major structural protein found in the body. It
provides support for tissues, connects tissue to bone, and provides
the structure of the body. When the body is in the healing process,
collagen plays a role in helping to build a cellular structure.
Hyaluronic acid is a natural sugar found in the synovial joint
fluid, the vitreous humor of the eye, the cartilage, blood vessels,
extra-cellular matrix, skin, and umbilical cord. Fibrin is a
protein involved in the clotting of blood.
[0181] In some embodiments, the preparation of fetal support tissue
is mixed with collagen, fibrin or HA. Collagen, fibrin and HA can
be suitable delivery vehicles, as AM preparations mixed with
collagen or HA were shown to exert a suppressive effect upon TGF 13
promoter activity. Although the preparations of fetal support
tissue were mixed with collagen gel and HA gel in the experiments
described herein, in some embodiments, any soluble form (e.g.,
liquid) of collagen and HA or other ECM components (e.g., fibrin)
is used. In some embodiments, the collagen, fibrin or HA is derived
from any suitable source. In some embodiments, the ratio of AM to
collagen, fibrin or HA is varied. In some embodiments, the ratio of
AM to collagen, fibrin, or HA is less than about 0.001:1, 0.01:1,
0.05:1, or 0.1:1, to about 1:1, 1.5:1, 2:1, 5:1, 10:1, 100:1 or
1000:1 or more is used.
[0182] In some embodiments, collagen gel is prepared by diluting
the stock solution (4 mg/ml) with 0.1 N acetic acid and by mixing
it with appropriate volume ratios of 20.times. of DMEM or suitable
buffer, and 1 N NaOH, as described in Example 1. In some
embodiments, the collagen in the composition is present at a range
of from less than about 2 mg/ml to more than about 4 mg/ml.
[0183] In some embodiments, the HA is a high molecular weight (MW)
HA. In some embodiments, various dilutions of high MW HA are
prepared by diluting commercially prepared HA (Healon.TM. (10 mg
HA/nil) (Pharmacia, LaJolla, Calif.) in DMEM or suitable buffer. In
some embodiments, dry powder and water-soluble forms of the
preparation of fetal support tissue are diluted in a solution such
as PBS, DMEM, or other solutions into the desired collagen
concentration. In some embodiments, the HA in the preparation of
fetal support tissue is present at a range of from less than about
2 .mu.g/ml to more than about 129 .mu.g/ml.
[0184] Illustrative Preparations
[0185] Examples 8 through 15 represent illustrative methods for
preparing the preparations of fetal support tissue described and
used herein.
[0186] Compositions
[0187] In some embodiments, the composition comprising the
preparation of fetal support tissue is formulated for
administration purposes as a non-solid dosage form. In some
embodiments, the non-solid dosage form comprises combining the
preparation with a delivery vehicle to create a composition such as
a solution, drop, suspension, paste, spray, ointment, oil,
emulsion, aerosol, coated bandage, patch, cream, lotion, gel, and
the like. The formulation used will depend upon the particular
application. Gels are useful for administering the composition
because they allow better retention of the active ingredient at the
site of introduction, allowing the active ingredient to exert its
effect for a longer period of time before clearance of the active
ingredient. In some embodiments, the composition is formulated as
extended-release solid dosage forms (including oral dosage
forms).
[0188] In some embodiments, the composition is formulated in a
conventional manner using one or more physiologically acceptable
carriers including excipients and auxiliaries which facilitate
processing of preparation of fetal support tissue into compositions
which are used pharmaceutically. Proper formulation is dependent
upon the route of administration chosen. Any of the well-known
techniques, carriers, and excipients may be used as suitable and as
understood in the art.
[0189] In some embodiments, the composition of fetal support tissue
is in a liquid, suspension, a gel, or lyophilized powder, or other
forms. In some embodiments, the composition is injectable. In some
embodiments, the composition of fetal support tissue comprises an
antimicrobial agent. In some embodiments, the antimicrobial agent
is an antibiotic or anti-fungal agent. In some embodiments, the
composition of fetal support tissue comprises an additional
substance to stabilize and/or preserve the composition of fetal
support tissue. In some embodiments, the composition of fetal
support tissue is packaged and stored at room temperature,
-20.degree. C. or -80.degree. C. prior to use.
[0190] In certain embodiments, the composition comprises a
pharmaceutically acceptable diluent, excipient, or carrier. In some
embodiments, the composition further comprises other active
ingredients, as in combination therapy. In some embodiments, the
composition comprises other medicinal or pharmaceutical agents,
carriers, adjuvants, such as preserving, stabilizing, wetting or
emulsifying agents, solution promoters, and salts for regulating
the osmotic pressure, buffers, or a combination thereof. In some
embodiments, the composition comprises an additional therapeutic
agent.
[0191] In some embodiments, the composition further comprises a
chemical component, such as a carrier, stabilizer, diluent,
dispersing agent, suspending agent, thickening agent, excipient, or
a combination thereof. In some embodiments, the composition
facilitates administration of the preparation to the individual. In
some embodiments, a therapeutically effective amount of the
composition of fetal support tissue is administered as an
injectable composition to an individual having a disease, disorder,
or condition to be treated. In some embodiments, the individual is
a mammal. In some embodiments, the mammal is a human. In some
embodiments, the therapeutically effective amount varies depending
on the severity of the disease, the age and relative health of the
individual, the potency of the composition used and other factors.
In some embodiments, the composition is used singly or in
combination with one or more therapeutic agents as components of
mixtures.
[0192] Ophthalmic Compositions:
[0193] In some embodiments, the ophthalmic compositions comprise a
preparation of a fetal support tissue; and a pharmaceutically
acceptable diluent, excipient, vehicle, or carrier. In some
embodiments, the ophthalmic compositions consist essentially of
substantially isolated HC-HA/PTX3, reconstituted HC-HA/PTX3, or a
combination thereof and a pharmaceutically acceptable diluent,
excipient, vehicle, or carrier. In some embodiments, the
composition is prepared for local delivery to the eye. In some
embodiments, the composition is administered systemically, such as
intravenously. In some embodiments, the composition is administered
topically to the eye. In some embodiments, the composition is
formulated into a variety of topically administrable ophthalmic
compositions. In some embodiments, the topically administrable
ophthalmic composition comprises a solution, suspension, gel or
ointment. In some embodiments, the composition is formulated for
injection into the eye. In some embodiments, the composition is
administered by intravitreal injection into the eye. In some
embodiments, the composition is administered by intraocular
injection, subretinal injection, intravitreal injection, periocular
administration, subconjunctival injections, retrobulbar injections,
intracameral injections (including into the anterior or vitreous
chamber), or sub-Tenon's injections. In some embodiments, the
composition is administered by implants, ophthalmic solutions,
ophthalmic suspensions, ophthalmic ointments, ocular implants and
ocular inserts, intraocular solutions, use of iontophoresis,
incorporation in surgical irrigating solutions, and packs (by way
of example only, a saturated cotton pledget inserted in the
fornix).
[0194] In some embodiments, the composition is a liquid composition
where the preparation of fetal support tissue is present in
solution, in suspension or both. In some embodiments, the
composition includes a gel formulation. In other embodiments, the
liquid composition is aqueous. In some embodiments, the composition
is an ointment.
[0195] In some embodiments, the composition is an aqueous
composition. In some embodiments, the aqueous composition is an
aqueous solution, suspension or solution/suspension. In some
embodiments, the aqueous composition is presented in the form of
eye drops. In some embodiments, a desired dosage is administered
via a known number of drops into the eye. For example, for a drop
volume of 25 .mu.l, administration of 1-6 drops will deliver 25-150
.mu.l of the composition. In some embodiments, the aqueous
composition comprises from about 0.01% to about 50% weight/volume
of the preparation of fetal support tissue or purified component.
In some embodiments, the aqueous composition comprises from about
0.1% to about 20% weight/volume of the preparation of fetal support
tissue or purified component. In some embodiments, the aqueous
composition comprises from about 0.2% to about 10% weight/volume of
the preparation of fetal support tissue or purified component. In
some embodiments, the aqueous composition comprises from about 0.5%
to about 5%, weight/volume of the preparation of fetal support
tissue or purified component. In some embodiments, the aqueous
composition has an ophthalmically acceptable pH and osmolality.
"Ophthalmically acceptable" with respect to a formulation,
composition or ingredient typically means having no persistent
detrimental effect on the treated eye or the functioning thereof,
or on the general health of the subject being treated. Transient
effects such as minor irritation or a "stinging" sensation are
common with topical ophthalmic administration of agents and
consistent with the formulation, composition or ingredient in
question being "ophthalmically acceptable."
[0196] In some embodiments, the composition is an aqueous
composition and comprises a polymer as a suspending agent. In some
embodiments, the aqueous composition comprises more than one
polymer as the suspending agent. In some embodiments, the polymer
comprises a water-soluble polymer, a water-insoluble polymer, or a
combination thereof. In some embodiments, the water-soluble polymer
comprises a cellulosic polymer. In some embodiments, the cellulosic
polymer comprises hydroxypropyl methylcellulose. In some
embodiments, the water-insoluble polymer comprises a cross-linked
carboxyl-containing polymer. In some embodiments, the aqueous
composition comprises an ophthalmically acceptable mucoadhesive
polymer. In some embodiments, the mucoadhesive polymer comprises
carboxymethylcellulose, carbomer (acrylic acid polymer), poly
(methylmethacrylate), polyacrylamide, polycarbophil, acrylic
acid/butyl acrylate copolymer, sodium alginate, dextran, or a
combination thereof.
[0197] In some embodiments, the composition comprises an
ophthalmically acceptable solubilizing agent to aid in the
solubility of the preparation of fetal support tissue in the
composition. In some embodiments, the composition comprises an
ophthalmically acceptable solubilizing agent to aid in the
solubility of purified HC-HA/PTX3 in the composition. The term
"solubilizing agent" generally includes agents that result in
formation of a micellar solution or a true solution of the agent.
In some embodiments, the ophthalmically acceptable solubilizing
agent is a nonionic surfactants. In some embodiments, the nonionic
surfactant comprises polysorbate 80, glycol, polyglycol,
polyethylene glycol 400, glycol ethers, derivatives thereof, or any
combination thereof.
[0198] In some embodiments, the composition comprises one or more
ophthalmically acceptable pH adjusting agents or buffering agents.
In some embodiments, the pH adjusting agent comprises an acid. In
some embodiments, the acid is chosen from a list comprising:
acetic, boric, citric, lactic, phosphoric acid, and hydrochloric
acid. In some embodiments, the pH adjusting agent comprises a base.
In some embodiments, the base is chosen from a list comprising:
sodium hydroxide, sodium phosphate, sodium borate, sodium citrate,
sodium acetate, sodium lactate and tris-hydroxymethylaminomethane.
In some embodiments, the buffering agent is chosen from a list
comprising: citrate/dextrose, sodium bicarbonate, and ammonium
chloride. In some embodiments, the acid, the base or the buffers
are included in an amount required to maintain pH of the
composition in an ophthalmically acceptable range.
[0199] In some embodiments, the composition comprises an
ophthalmically acceptable salt in an amount required to bring
osmolality of the composition into an ophthalmically acceptable
range. In some embodiments, the salt comprises sodium, potassium or
ammonium cations and chloride, citrate, ascorbate, borate,
phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions.
In some embodiments, the salt is chosen from a list comprising:
sodium chloride, potassium chloride, sodium thiosulfate, sodium
bisulfite, ammonium sulfate, or a combination thereof.
[0200] In some embodiments, the composition comprises an
ophthalmically acceptable preservative to inhibit microbial
activity. In some embodiments, the preservative comprises a
mercury-containing substance, stabilized chlorine dioxide, a
quaternary ammonium compound, or a combination thereof. In some
embodiments, the mercury-containing substance comprises merfen,
thiomersal, or a combination thereof. In some embodiments, the
quaternary ammonium compound comprises benzalkonium chloride,
cetyltrimethylammonium bromide, cetylpyridinium chloride, or a
combination thereof.
[0201] In some embodiments, the composition comprises one or more
ophthalmically acceptable surfactants to enhance physical stability
or for other purposes. In some embodiments, the surfactant
comprises a nonionic surfactant. In some embodiments, the nonionic
surfactant is chosen from a list comprising: polyoxyethylene fatty
acid glycerides and vegetable oils, e.g., polyoxyethylene (60)
hydrogenated castor oil; and polyoxyethylene alkylethers and
alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.
[0202] In some embodiments, the composition comprises one or more
antioxidants to enhance chemical stability where required. In some
embodiments, the antioxidant comprises ascorbic acid, sodium
metabisulfite, or a combination thereof.
[0203] In some embodiments, the composition is packaged in
single-dose non-reclosable containers. In some embodiments, the
composition is packaged in a multiple-dose reclosable container. In
some embodiments, the composition further comprises a preservative
when packaged in the multiple-dose reclosable container.
[0204] In some embodiments, the composition is in the form of a
solid article that is inserted between the eye and eyelid or in the
conjunctival sac, where it releases the preparation. In some
embodiments, the preparation is released to the lacrimal fluid that
bathes the surface of the cornea, or directly to the cornea itself,
with which the solid article is generally in intimate contact. In
some embodiments, the solid article suitable for implantation in
the eye comprises polymers. In some embodiments, the solid article
suitable for implantation in the eye is biodegradable or
non-biodegradable.
[0205] Injectable Compositions:
[0206] In some embodiments, the composition is an injectable
composition. In some embodiments, the injectable compositions
comprise a preparation of a fetal support tissue; and a
pharmaceutically acceptable diluent, excipient, vehicle, or
carrier. In some embodiments, the injectable compositions consist
essentially of substantially isolated HC-HA/PTX3, reconstituted
HC-HA/PTX3, or a combination thereof; and a pharmaceutically
acceptable diluent, excipient, vehicle, or carrier. In some
embodiments, the injectable composition is suitable for
intraocular, intramuscular, subcutaneous, or intravenous injection.
In some embodiments, the injectable composition comprises
physiologically acceptable sterile aqueous or non-aqueous
solutions, dispersions, suspensions or emulsions, and sterile
powders for reconstitution into sterile injectable solutions or
dispersions. Non-limiting examples of suitable aqueous and
non-aqueous carriers, diluents, solvents, or vehicles including
water, ethanol, polyols (propyleneglycol, polyethylene-glycol,
glycerol, cremophor and the like), suitable mixtures thereof,
vegetable oils (such as olive oil) and injectable organic esters
such as ethyl oleate. In some embodiments, proper fluidity is
maintained by the use of a coating, a surfactant, or a combination
thereof. In some embodiments, the coating is lecithin. In some
embodiments, the injectable composition comprises an additive. In
some embodiments, the additive is chosen from the list comprising:
a preserving agent, a wetting agent, an emulsifying agent, a
dispensing agent, or a combination thereof. In some embodiments,
the injectable composition comprises an antibacterial or antifungal
agent. In some embodiments, the antibacterial or antifungal agent
is comprises a paraben, chlorobutanol, phenol, sorbic acid, or a
combination thereof. In some embodiments, the injectable
composition comprises an isotonic agent. In some embodiments, the
isotonic agent comprises sugar, sodium chloride, or a combination
thereof. In some embodiments, the injectable composition comprises
an absorption delaying agent. In some embodiments, the absorption
delaying agent comprises aluminum monostearate gelatin, or a
combination thereof.
[0207] In some embodiments, the injectable composition is
administered intravenously. In some embodiments, the injectable
composition is formulated in an aqueous solution, in a
physiologically compatible buffer such as Hank's solution, Ringer's
solution, a physiological saline buffer, or another suitable
solution. In some embodiments, for transmucosal administration, a
penetrant appropriate to the barrier to be permeated is used in the
formulation. Such penetrants are generally known in the art. In
some embodiments, for a parenteral injection, an appropriate
formulation includes aqueous or nonaqueous solutions, preferably
with physiologically compatible buffers or excipients. Such
excipients are generally known in the art.
[0208] In some embodiments, parenteral injections involve bolus
injection or continuous infusion. In some embodiments, the
composition is presented in unit dosage form, e.g., in ampoules or
in multi dose containers, with an added preservative. In some
embodiments, the injectable composition is in a formulation
suitable for parenteral injection as a sterile suspensions,
solutions or emulsions in oily or aqueous vehicles. In some
embodiments, the injectable composition comprises a formulary
agent. In some embodiments, the formulary agent is a suspending
agent, stabilizing agent, dispersing agent, or a combination
thereof. In some embodiments, the injectable composition for
parenteral administration comprises the aqueous solution of
preparation of fetal support tissue in water soluble form. In some
embodiments, the suspension of the active compounds is prepared as
an oily injection suspension. In some embodiments, the injectable
composition comprises a lipophilic solvent or vehicle. Non-limiting
examples of lipophilic solvents or vehicles include, but are not
limited to, fatty oils such as sesame oil, or synthetic fatty acid
esters, such as ethyl oleate or triglycerides, or liposomes. In
some embodiments, the injectable injection composition contains a
substance which increases the viscosity of the suspension, such as
sodium carboxymethyl cellulose, sorbitol, or dextran. In some
embodiments, the injectable composition contains suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. In some embodiments, the preparation of fetal support
tissue is in powder form for constitution with a suitable vehicle,
e.g., sterile pyrogen-free water, before use.
[0209] Methods of Dosing and Treatments Regimens:
[0210] In some embodiments, the composition is administered by any
suitable technique. In some embodiments, composition is
administered directly to a target site (e.g., ocular surface,
vitreous, etc.). In some embodiments, the composition is
administered topically. In some embodiments, the composition is
administered parentally (e.g., subcutaneous). In some embodiments,
composition is administered intraocularly.
[0211] In some embodiments, the composition is administered for
prophylactic and/or therapeutic applications. In some embodiments,
the composition is administered to an individual already suffering
from a disease or condition, in an amount sufficient to cure or at
least partially arrest the symptoms of the disease or condition. In
some embodiments, amounts effective for this use depend on the
severity and course of the disease or condition, previous therapy,
the individual's health status, weight, and response to the drugs,
and the judgment of the treating physician.
[0212] In some embodiments, the composition is administered to an
individual susceptible to or otherwise at risk of a particular
disease, disorder or condition. Such an amount is defined to be a
"prophylactically effective amount or dose." In this use, the
precise amounts also depend on the individual's state of health,
weight, and the like. In some embodiments, a dose escalation trial
is used to determine a prophylactically effective amount. In some
embodiments, any suitable method is used to determine the
prophylactically effective amount. In some embodiments, the
prophylactically effective amount depends on the severity and
course of the disease, disorder or condition, previous therapy, the
individual's health status and response to the drugs, and the
judgment of the treating physician.
[0213] In the case wherein the individual's condition does not
improve, upon the doctor's discretion the composition is
administered chronically, that is, for an extended period of time,
including throughout the duration of the individual's life in order
to ameliorate or otherwise control or limit the symptoms of the
individual's disease or condition.
[0214] In the case wherein the individual's status does improve,
upon the doctor's discretion the composition is given continuously
or the dose of drug being administered is temporarily reduced or
temporarily suspended for a certain length of time (i.e., a "drug
holiday"). In some embodiments, the length of the drug holiday
varies between 2 days and 1 year, including by way of example only,
2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days,
15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120
days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,
320 days, 350 days, or 365 days. In some embodiments, the dose
reduction during a drug holiday is from 10%-100%, including, by way
of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
[0215] Once improvement of the individual's conditions has
occurred, a maintenance dose is administered if necessary.
Subsequently, the dosage or the frequency of administration, or
both, is reduced, as a function of the symptoms, to a level at
which the improved disease, disorder or condition is retained. In
some embodiments, the individual requires intermittent treatment on
a long-term basis upon any recurrence of symptoms.
[0216] In some embodiments, the amount of the composition
administered to the individual varies depending upon factors such
as the disease or condition and its severity, the identity (e.g.,
weight, gender, age, overall health) of the individual in need of
treatment. In some embodiments, the amount of composition
administered is determined in a manner known in the art according
to the particular circumstances surrounding the case, including,
e.g., the specific preparation, composition, or formulation being
administered, the route of administration, the condition being
treated, and the individual being treated. In some embodiments, the
amounts or doses employed for adult human treatment are in the
range of 0.02-5000 mg per day, preferably 1-1500 mg per day. In
some embodiments, a desired dose is presented in a single dose or
as divided doses administered simultaneously (or over a short
period of time) or at appropriate intervals, for example as two,
three, four or more sub-doses per day.
[0217] In some embodiments, the composition is in unit dosage forms
suitable for single administration of precise amounts or dosages.
In unit dosage form, the composition is divided into unit doses
containing appropriate amounts or doses of the composition. In some
embodiments, the unit dosage is in the form of a package containing
discrete quantities of the composition. Non-limiting examples are
powders packaged in vials or ampoules. In some embodiments,
compositions are packaged in single-dose non-reclosable containers.
In some embodiments, multiple-dose reclosable containers are used,
in which case it is typical to include a preservative in the
composition. In some embodiments, the composition for parenteral
injection are presented in unit dosage form, which include, but are
not limited to ampoules, or in multi-dose containers, with an added
preservative.
[0218] In some embodiments, the daily dosage appropriate for the
composition is from about 0.01 to 2.5 mg/kg per body weight. An
indicated daily dosage is in the range from about 0.5 mg to about
100 mg, conveniently administered in divided doses, including, but
not limited to, up to four times a day or in extended release form.
The foregoing ranges are merely suggestive, as the number of
variables in regard to an individual treatment regime is large, and
considerable excursions from these recommended values are not
uncommon. In some embodiments, the dosage is altered depending on a
number of variables, not limited to the activity of the
composition, the disease or condition to be treated, the mode of
administration, the requirements of the individual, the severity of
the disease or condition being treated, and the judgment of the
practitioner.
[0219] In some embodiments, the toxicity and therapeutic efficacy
of such therapeutic regimens is determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
including, but not limited to, the determination of the LD.sub.50
(the dose lethal to 50% of the population) and the ED.sub.50 (the
dose therapeutically effective in 50% of the population). In some
embodiments, the dose ratio between the toxic and therapeutic
effects is the therapeutic index and is expressed as the ratio
between LD.sub.50 and ED.sub.50. Compositions exhibiting high
therapeutic indices are preferred. In some embodiments, data
obtained from a cell culture assay or animal study is used in
formulating a range of dosage for use in the individual. In some
embodiments, the dosage of the composition is within a range of
circulating concentrations that include the ED.sub.50 with minimal
toxicity. In some embodiments, the dosage varies within this range
depending upon the dosage form employed and the route of
administration utilized.
[0220] Combination Treatments:
[0221] In some embodiments, the composition is co-administered with
an additional therapeutic compound. In some embodiments, the
additional therapeutic agent is not administered in the same
composition. In some embodiments, the additional therapeutic agent
is administered by a different route than the composition. The
determination of the mode of administration and the advisability of
administration, where possible, in the same composition, is well
within the knowledge of the skilled clinician. In some embodiments,
the initial administration is made according to established
protocols known in the art, and then modified by the skilled
clinician based upon the observed effects, the dosage, modes of
administration and times of administration.
[0222] In some embodiments, the particular choice of the additional
therapeutic compound used depends upon the diagnosis of the
attending physicians and their judgment of the condition of the
individual and the appropriate treatment protocol. In some
embodiments, the additional therapeutic compound is administered
concurrently (e.g., simultaneously, essentially simultaneously or
within the same treatment protocol) or sequentially, depending upon
the nature of the disease, disorder, or condition, the condition of
the individual, and the actual choice of compounds used. The
determination of the order of administration, and the number of
repetitions of administration of each therapeutic agent during a
treatment protocol, is well within the knowledge of the skilled
physician after evaluation of the disease being treated and the
condition of the individual.
[0223] In some embodiments, a therapeutically-effective dosage
varies when the composition is used in a combination treatment. In
some embodiments, any suitable method is used to determine a
therapeutically effective dosage of a drug and other agents for use
in the combination treatment regimens. In some embodiments,
metronomic dosing (i.e., providing more frequent, lower doses in
order to minimize toxic side effects) is used to determine a
therapeutically effective dosage of a drug and other agents for use
in the combination treatment. In some embodiments, the combination
treatment comprises periodic treatments that start and stop at
various times to assist with the clinical management of the
individual.
[0224] In some embodiments, dosage of the additional therapeutic
agent varies depending on the type of co-drug employed, on the
specific drug employed, on the disease or condition being treated
and so forth. In some embodiments, when co-administered with one or
more additional therapeutic agents, the composition is administered
either simultaneously with the additional therapeutic agent, or
sequentially. If administered sequentially, the attending physician
will decide on the appropriate sequence of administering the
composition in combination with the additional therapeutic
agent.
[0225] In some embodiments, multiple additional therapeutic agents
are administered in combination with the composition. In some
embodiments, the multiple additional therapeutic agents are
administered in any order or even simultaneously. If
simultaneously, the multiple additional therapeutic agents are
provided in a single, unified form, or in multiple forms (by way of
example only, either as a single pill or as two separate pills). In
some embodiments, one of the additional therapeutic agents is given
in multiple doses, or both may be given as multiple doses. In some
embodiments, if administration is not simultaneous, the timing
between the multiple doses varies from more than zero weeks to less
than four weeks.
[0226] In some embodiments, the dosage regimen to treat, prevent,
or ameliorate the condition(s) for which relief is sought, is
modified in accordance with a variety of factors. In some
embodiments, the factors comprise: a disorder from which the
individual suffers, as well as the age, weight, sex, diet, and
medical condition of the individual, or a combination thereof. In
some embodiments, the dosage regimen varies widely and deviates
from the dosage regimens set forth herein.
[0227] In some embodiments, the composition and additional
therapeutic agent which make up the combination therapy are a
combined dosage form or in separate dosage forms intended for
substantially simultaneous administration. In some embodiments, the
composition and additional therapeutic agent that make up the
combination therapy are administered sequentially, with either the
composition or the additional therapeutic agent being administered
by a regimen calling for two-step administration. In some
embodiments, the two-step administration regimen calls for
sequential administration of the composition and additional
therapeutic agent or spaced-apart administration of the composition
and additional therapeutic agent. In some embodiments, the time
period between the multiple administration steps ranges from, a few
minutes to several hours, depending upon the properties of each
pharmaceutical agent, such as potency, solubility, bioavailability,
plasma half-life and kinetic profile of the composition or
additional therapeutic agent. In some embodiments, circadian
variation of the composition or therapeutic agent concentration
determines the optimal dose interval.
[0228] In some embodiments, the composition is used in combination
with procedures that may provide additional or synergistic benefit
to the individual. By way of example only, individuals are expected
to find therapeutic and/or prophylactic benefit in the methods
described herein, wherein the composition or the composition in
combination with the additional therapeutic agent is combined with
genetic testing to determine whether that individual is a carrier
of a mutant gene that is known to be correlated with certain
diseases or conditions.
[0229] In some embodiments, the composition and combination
therapies are administered before, during or after the occurrence
of a disease or condition, and the timing of administering the
composition containing a compound varies. In some embodiments, the
composition is used as a prophylactic and administered continuously
to individuals with a propensity to develop conditions or diseases
in order to prevent the occurrence of the disease or condition. In
some embodiments, the composition is administered to the individual
during or as soon as possible after the onset of the symptoms. In
some embodiments, the administration of the composition is
initiated within the first 48 hours of the onset of the symptoms,
preferably within the first 48 hours of the onset of the symptoms,
more preferably within the first 6 hours of the onset of the
symptoms, and most preferably within 3 hours of the onset of the
symptoms. In some embodiments, the initial administration is via
any route practical, such as, for example, an intravenous
injection, a bolus injection, infusion over 5 minutes to about 5
hours, and the like, or combination thereof. In some embodiments,
the composition is administered as soon as is practicable after the
onset of a disease or condition is detected or suspected, and for a
length of time necessary for the treatment of the disease, such as,
for example, from about 1 month to about 3 months. In some
embodiments, the length of treatment varies for each individual,
and the length is determined using the known criteria. In some
embodiments, the composition is administered for at least 2 weeks,
preferably about 1 month to about 5 years, and more preferably from
about 1 month to about 3 years.
Methods of Treatment:
[0230] Disclosed herein, in certain embodiments, are methods for
preventing or reducing proliferation, cell migration, and/or EMT of
epithelial cells in an individual in need thereof, comprising
administering to the individual a therapeutically effective amount
of an injectable composition, comprising: (a) a preparation of a
fetal support tissue; and (b) a pharmaceutically acceptable
diluent, excipient, vehicle, or carrier, thereby preventing or
reducing the proliferation, cell migration, and/or EMT of
epithelial cells. In some embodiments, the EMT is associated with a
disease other than PVR.
[0231] Disclosed herein, in certain embodiments, are methods for
treating or preventing of Proliferative Vitreoretinopathy (PVR) in
an individual in need thereof, comprising administering to the
individual a therapeutically effective amount of an injectable
composition, comprising: (a) a preparation of fetal support tissue;
and (b) a pharmaceutically acceptable diluent, excipient, vehicle,
or carrier, thereby treating or preventing PVR.
[0232] In some embodiments, the preparation of fetal support tissue
comprises HC-HA/PTX3. In some embodiments, the preparation of fetal
support tissue comprises purified HC-HA/PTX3. In some embodiments,
the preparation of fetal support tissue comprises ultracentrifuged
HC-HA/PTX3. In some embodiments, the preparation of fetal support
tissue consists of purified HC-HA/PTX3. In some embodiments, the
preparation of fetal support tissue comprises reconstituted
HC-HA/PTX3. In some embodiments, the preparation of fetal support
tissue comprises: high molecular weight hyaluronan (HA) that is
cross-linked by a covalent bond to the heavy chain of
inter-.alpha.-trypsin inhibitor (I.alpha.I), the high molecular
weight HA having a molecular weight greater than 1000 kDa. In some
embodiments, the preparation comprises: pentraxin 3 (PTX-3). In
some embodiments, the preparation of fetal support tissue
comprises: tumor necrosis factor-stimulated gene 6 protein (TSG-6).
In some embodiments, the preparation of fetal support tissue
comprises: thrombospondin-1 (TSP-1). In some embodiments, the ratio
of total protein to HA in the composition is less than 500 parts
protein:1 part HA. In some embodiments, the ratio of HA to total
protein in the compositions is less than 500 parts HA:1 part
protein.
[0233] In some embodiments, the epithelial cells are human
epithelial cells. In some embodiments, the human epithelial cells
are retinal pigment epithelial cells (RPE). In some embodiments,
the human epithelial cells are renal epithelial cells. In some
embodiments, the human epithelial cells are corneal epithelial
cells. In some embodiments, the human epithelial cells are limbal
epithelial cells. In some embodiments, the human epithelial cells
are conjunctival epithelial cells.
[0234] In some embodiments, the composition prevents the
proliferation and EMT of epithelial cells by inhibiting or
suppressing the activity of growth factors or cytokines. In some
embodiments, the growth factors and cytokines are selected from the
group consisting of: EGF, FGF-2, PDGF-A, PDGF-AB, PDGF-B, PDGF-C,
TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, CTGF, HGF, IGF-1, G-CSF,
IL-6, MCP-1, TNF-.alpha., VEGF and IFN-.gamma.. In some
embodiments, the composition inhibits signaling pathways in
epithelial cells to inhibit proliferation and EMT. In some
embodiments, the signaling pathways are canonical Wnt signaling and
TGF-.beta.-induced Smad/ZEB signaling.
[0235] In some embodiments, the compositions comprise a preparation
of fetal support tissue prepared from placental tissue, umbilical
cord tissue, umbilical cord amniotic membrane tissue, placental
amniotic membrane tissue, amniotic stromal tissue, amnion-chorion
tissue, chorion tissue, amniotic fluid, or combinations thereof. In
some embodiments, the placental tissue, umbilical cord tissue,
amniotic membrane tissue, chorion tissue or combinations thereof is
homogenized, pulverized or ground. In some embodiments, the
placental tissue, umbilical cord tissue, amniotic membrane tissue,
chorion tissue or combinations thereof is fresh, frozen or has been
previously frozen. In some embodiments, a composition comprises the
preparation of fetal support tissue and a pharmaceutically
acceptable diluent, excipient, or carrier. In some embodiments, the
composition further comprises an aqueous adjuvant. In some
embodiments, the composition is for local administration. In some
embodiments, the composition is for injection. In some embodiments,
the composition is formulated for intraocular injection, subretinal
injection, intravitreal injection, periocular injection,
subconjunctival injection, retrobulbar injection, intracameral
injection or sub-Tenon's injection.
[0236] The methods disclosed herein have many uses including
research and clinical applications. In some embodiments, the
methods are applied to tissues or cells to achieve a desired
modulation of physiology. In some embodiments, the methods are used
on cell cultures or tissue cultures to achieve a desired
effect.
[0237] In some embodiments, the methods are used to prevent,
lessen, or treat apoptosis in tissues. In some embodiments, the
methods are used to decrease or prevent apoptosis in a tissue that
has been injured. In some embodiments, the methods are used to
prolong the life of organs being stored prior to transplant. In
some embodiments, the methods are used to treat or prevent damage
during and after surgical procedures.
[0238] In some embodiments, methods are useful for preserving
tissues (e.g., cornea) before transplantation. In some embodiments,
the methods lessen cellular damage due to the storage process. In
some embodiments, the methods are used to decrease the amount of
degradation that occurs in a tissue that is being stored prior to
transplantation or surgical procedures. In some embodiments, the
preparation or composition is added to the storage medium, with or
without collagen and/or HA. Stored tissues such as eyes, organs,
skin, and the like can benefit from the decreased cellular
apoptosis that occurs when the composition is added.
[0239] In some embodiments, the methods further comprise storing a
donor tissue in a storage medium until transplantation after the
donor tissue is harvested. In some embodiments, the composition is
added to the storage medium to prevent cellular apoptosis. In some
embodiments, the composition is added to storage media for
preserving limbal epithelial stem cells. In some embodiments, the
composition is added to cell culture medium or digestion medium to
prevent cellular (e.g., keratocyte) apoptosis. Because studies
described herein show that incubation of composition during dispase
digestion (a treatment which mimics surgical and pathological
insults such as excimer ablation in PRK and recurrent corneal
erosion, respectively) significantly reduced apoptosis of both
epithelial cells and keratocytes. In some embodiments, the
composition is administered to an eye receiving mechanical scraping
or excimer laser photoablation to attempt to reduce keratocyte
apoptosis, and hence reduce corneal haze. In some embodiments, the
methods are used in a surgical condition or disease such as
recurrent corneal erosion or keratoconus where the basement
membrane is dissolved to reduce the keratocyte apoptosis.
[0240] In some embodiments, the method is used to produce a
phenotypic reversal of AMSCs from myofibroblasts to fibroblasts. In
some embodiments, the method is used to prevent or slow
differentiation of various cell types. In some embodiments, many
types of cells are treated with the method. This method is
particularly useful for expanding cell cultures without causing
differentiation of the culture to unwanted cell types.
Kits/Articles of Manufacture:
[0241] For use in the methods described herein, kits and articles
of manufacture are also described herein. In some embodiments, the
kits comprise a carrier, package, or container that is
compartmentalized to receive one or more containers such as vials,
tubes, and the like, each of the container(s) including one of the
separate elements to be used in a method described herein. In some
embodiments, the container is a bottle, vial, syringe, or test
tube. In some embodiments, the container is formed from a variety
of materials such as glass or plastic. In some embodiments, the kit
comprising one or more prefilled syringes comprising a composition
disclosed herein.
[0242] In some embodiments, the article of manufacture contains
packaging materials. Packaging materials for use in packaging
pharmaceutical products are well known to those of skill in the
art. Non-limiting examples of pharmaceutical packaging materials
include, but are not limited to, blister packs, bottles, tubes,
inhalers, pumps, bags, vials, containers, syringes, bottles, and
any packaging material suitable for a selected formulation and
intended mode of administration and treatment. A wide array of
formulations of the preparations and compositions provided herein
are contemplated as are a variety of treatments for any disease,
disorder, or condition.
[0243] In some embodiments, the container includes one or more
preparations of fetal support tissue, optionally in a composition
or in combination with another agent as disclosed herein. In some
embodiments, the container comprises a sterile access port. In some
embodiments, the container is an intravenous solution bag or a
vial. In some embodiments, the sterile access port is a stopper
pierceable by a hypodermic injection needle. In some embodiments,
the kit comprises a composition with an identifying description or
label or instructions relating to its use in the methods described
herein.
[0244] In some embodiments, the kit comprises one or more
additional containers, each with one or more of various materials
(such as reagents, optionally in concentrated form, and/or devices)
desirable from a commercial and user standpoint for use of the
composition comprising fetal support tissue. Non-limiting examples
of such materials include, but not limited to, buffers, diluents,
filters, needles, syringes; carrier, package, container, vial
and/or tube labels listing contents and/or instructions for use,
and package inserts with instructions for use. In some embodiments,
a set of instructions is included.
[0245] In some embodiments, a label is on or associated with the
container. In some embodiments, the label is on a container when
letters, numbers or other characters forming the label are
attached, molded or etched into the container itself. In some
embodiments, the label is associated with a container when it is
present within a receptacle or carrier that also holds the
container, e.g., as a package insert. In some embodiments, the
label is used to indicate that the contents are to be used for a
specific therapeutic application. In some embodiments, the label
indicates directions for use of the contents, such as in the
methods described herein.
[0246] In certain embodiments, the injectable composition is
presented in a pack or dispenser device which contains one or more
unit dosage forms containing the injectable composition provided
herein. In some embodiments, the pack contains metal or plastic
foil, such as a blister pack. In some embodiments, the pack or
dispenser device is accompanied by instructions for administration.
In some embodiments, the pack or dispenser device is accompanied
with a notice associated with the container in form prescribed by a
governmental agency regulating the manufacture, use, or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the drug for human or veterinary
administration. In some embodiments, the notice is the labeling
approved by the U.S. Food and Drug Administration for prescription
drugs, or the approved product insert. In some embodiments, the
injectable composition is prepared, placed in an appropriate
container, and labeled for treatment of an indicated condition.
[0247] The compositions and methods described herein are provided
in further detail in the following examples. These examples are
provided by way of illustration and are not intended to limit the
invention in any way.
EXAMPLES
Example 1: Example Preparation
[0248] An injectable composition is prepared by mixing 10 mg each
of: HA, TSG-6, PTX-3, and TSP-1, each of which is obtained from a
commercial source, with 100 mg of a preparation comprising:
placental tissue, umbilical cord tissue, amniotic membrane tissue,
chorion tissue or combinations thereof; and then mixed with 10 mL
of 0.9% sterile saline. The mixture is incorporated into a dosage
unit form suitable for administration by injection.
Example 2: Characterization of Amniotic Membrane Components
[0249] Material and Methods
[0250] The concentration of proteins in each extract was
quantitated by the BCA Protein Assay Kit (Pierce, Rockford, Ill.).
The concentration of hyaluronic acid (HA) in each extracts was
assayed with Hyaluronic Acid (HA) Quantitative Test Kit (Corgenix,
Westminster, Colo.) based on ELISA using a standard curve provided
by the manufacturer prepared by serial dilution of HA.
[0251] HA Molecular Weight Range Analysis by Hyaluronidase
Digestion
[0252] The HA molecular weight ranges of the extracts were analyzed
by agarose gel electrophoresis according to the method described by
Lee and Cowman (Lee H. G. and Cowman, M. K. An Agarose Gel
Electrophoretic Method for Analysis of Hyaluronan Molecular Weight
Distribution. Analytical Biochemistry, 1994, 219, 278-287). The
samples were subjected to 0.5% agarose gel electrophoresis followed
by staining using 0.005% Stains-All (Sigma, cat #23096-0) in 50%
ethanol. The gel was stained overnight under a light-protective
cover at room temperature (Shorter staining periods of 3-4 hr can
also give acceptable results). HA was visualized as blue bands
after destaining by transferring the gel to H.sub.2O and exposed to
the room light for approximately 6 hr. The molecular weight
standards included .lamda. DNA-BstE II digested restriction
fragments (cat #D9793, Sigma) ranging in MW from 0.9 to
5.7.times.10.sup.6. The authenticity of HA was further verified by
incubation of the extract with or without 10 units/ml hyaluronidase
(Sigma #H1136) in the reaction buffer (50 mM Tris-HCl, pH7.5, 0.1 M
NaCl, 1% Triton X-100, 0.1% BSA supplemented with the above
protease and phosphatase inhibitors) for 2 h at 37.degree. C. using
a positive control of high MW HA (cat #H1876, Sigma) purified from
human umbilical cords.
[0253] Western Blot Analyses
[0254] The above extracts were electrophoresed on 4-15% denatured
acrylamide gels and transferred to the nitrocellulose membrane, and
then immunoblotted with a rabbit antihuman inter-.alpha.-trypsin
inhibitor (rabbit polyclonal antibody (cat #A0301, DAKO at 1:1000),
a rabbit anti-human TSG-6 polyclonal antibody (provided by Dr. Tony
Day at 1:1000 dilution), a rat monoclonal anti-PTX3 antibody
(Alexis Biochemicals, ALX-804-464, 1 .mu.g/ml), an
anti-thrombospondin-1 antibody obtained from Calbiochem (Cat
#BA24), and a goat anti-human Smad 7 antibody (AF2029, 1:1000, R
& D Systems). Imunoreactive protein bands were detected by
Western Lighting.TM. Chemiluminesence Reagent (PerkinElmer).
[0255] Results
[0256] Experiments showed that the observed suppressive effect on
the TGF .beta.1 promoter activity was abolished when water-soluble
AM extracts were pre-heated at 90.degree. C. for 10 minutes,
suggesting that the responsible component(s) most likely contained
protein(s), of which the conformation is important.
[0257] Quantitation of HA and Proteins in AM Extracts
[0258] The results, summarized Table 1, showed that all AM and
jelly extracts contained both HA and proteins. In general, the
weight ratio between proteins and HA was high in the Total Extract
than the supernatant (e.g., L and H for PBS, and A for Buffer A)
after centrifugation for AM, suggesting that most
protein-containing materials were eliminated by centrifugation.
However, this trend was not noted in AM Jelly, suggesting that AM
extracts contained more proteins than Jelly (see T under PBS and T
under A/B/C). The ratio between proteins and HA was also increased
from Extract A to Extracts B and C for both AM and AM jelly,
further supporting that HA was mostly present in the soluble form,
and vice versa proteins were found more in the water-insoluble
components. Furthermore, HA was largely removed from AM Jelly after
centrifugation in A/B/C.
TABLE-US-00001 TABLE 1 Tissue AM Jelly Buffer PBS A/B/C PBS A/B/C
Fraction T L H T A B C T L H T A B C Protein 8645 1370 1467 8645
2731 930 2698 3836 3645 3589 3836 3893 527 1364 (.mu.g/ml) HA 75 62
44 60 74 7 35 80 90 96 129 94 2 7 (.mu.g/ml) Protein/HA 115 22 33
144 37 133 77 48 41 37 30 41 264 195 [Note]: T: Total; L: the
supernatant following the low speed centrifugation of the total
extract; H: the supernatant following the low speed centrifugation
of the total extract; A, B, C: Extracts, see text.
[0259] HA in Different AM Extracts had Molecular Weights Greater
than One Million Daltons
[0260] High molecular weight (>10.sup.6 daltons) of HA was
present in the total extracts and Extract A (FIG. 10). However,
even higher MW of HA was present in Extract B, while HA was found
in a narrow band with even higher MW in Extract C (FIG. 10). All of
the HA-containing components disappeared after hyaluronidase
digestion, confirming that they indeed contained HA. Compared to
the positive control of HA obtained from Sigma (cat #H1136), a
similar high molecular weight (>10.sup.6 daltons) of HA was also
found in both supernatants obtained after low and high speeds of
centrifugation (FIG. 11). Again these HA-containing bands
disappeared after hyaluronidase digestion. A similar result was
obtained for AM jelly.
[0261] Inter-.alpha.-Trypsin Inhibitor (I.alpha.I) was Present in
Different AM Extracts and its Heavy Chains (HCs) were Covalently
Linked with HA
[0262] FIG. 12 showed that before digestion with hyaluronidase,
free heavy chains were present in different complexes, and a small
amount of light chain was also present (UTI or bikunin). However,
in all extracts, i.e., total and Extracts A, B, and C, there was
also a covalently linked complex between HA and heavy chains of
I.alpha.I as the latter was released only after hyaluronidase
digestion. The same result was obtained in Extracts H and L
obtained by two different speeds of centrifugation (FIG. 13).
[0263] Tumor Necrosis Factor-Stimulated Gene 6 (TSG-6) was Also
Present in AM Extracts
[0264] FIG. 14 showed that TSG-6 (about 38 kDa) was present in
Total, Extract A and Extract C. In Extract A, there was a band of
about 38 kDa migrated close to that of the purified TSG-6 (35 kD).
The identity of other bands of about 45 and 55 kDa was unknown.
Total AM extract (without centrifugation) "T" showed two bands
(both above 35 kD), and the higher one (55 kD) that were found in
Extract A (after centrifugation), while the lower one (45 kD) was
found in Extract C. All of these bands were not significantly
altered when samples were treated with hyaluronidase (FIG. 14) or
with F-glycosidase (FIG. 15). However, digestion with chondroitin
sulfate ABC lyase resulted in more noticeable 38 kD band using
antibody RAH-1 (FIG. 16) but not using antibody MAB2104 (FIG.
17).
[0265] Pentraxin-3 (PTX-3) was Exclusively Present in Water-Soluble
AM Extracts and Formed a Complex with HA
[0266] FIG. 18 showed that PTX3 was also present in AM extracts and
was complexed with HA in the water soluble extract A only.
[0267] Thrombospondin-1 (TSP-1) was Present in Different AM
Extracts
[0268] FIG. 19 showed that all AM extracts had a high molecular
weight band of TSP-1 while the total extract (T) and Extract C also
had some bands between 35-120 kDa. Hyaluronidase digestion did not
change the reactive pattern except some bands became a little
stronger or weaker.
[0269] Smad7 was Present in Mostly in Water-Insoluble AM
Extracts
[0270] Smad 7 was found in both PBS extracts and urea extracts of
AM (FIG. 20).
Example 3: Signaling Pathways Control Proliferation and EMT of
Epithelial Cells
[0271] Proliferation and EMT by dysfunctional epithelial cells are
two major pathological processes. During rhegmatogenous retinal
detachments (RRDs), retinal pigment epithelium (RPE) cells are
dispersed into the vitreous, which contains many growth factors and
cytokines (e.g., EGF, FGF, PDGF, TGF-.beta., VEGF and IFN-.gamma.)
necessary for the bioactivity of proliferative vitreoretinopathy
(PVR) as identified recently. To understand how growth factors
might contribute to proliferation and EMT of dispersed RPE cells,
an in vitro culturing model of ARPE-19 cells, which these cells
exhibit contact inhibition after seven days of post-confluence was
used.
[0272] Following perturbation of contact inhibition by EGTA, cell
proliferation (BrdU labeling) and EMT (loss of normal RPE phenotype
markers of N-cadherin, ZO-1, Na, K-ATPase, and RPE65, and express
of mesenchymal phenotype markers of vimentin, S100A4, and
.alpha.-SMA) were only induced in the presence of EGF and/or FGF-2.
This pathological process required the activation of canonical Wnt
signaling, as evidenced by the increased nuclear level and
interaction of .beta.-catenin and LEF, as well as upregulation of
TCF/LEF transcriptional activation. The activation of canonical Wnt
signaling was confirmed by using a Wnt inhibitor XAV939 and
overexpression of constitutive active .beta.-catenin (S33Y) in
blocking or rescuing experiments. Addition of TGF-.beta.1 also lead
to EMT by activating Smad/ZEB1/2 signaling, which suppressed
proliferation and activation of canonical Wnt signaling.
Furthermore, canonical Wnt signaling triggered by EGF+FGF-2 was
sufficient and synergized with TGF-.beta.1 to lead to EMT (FIG. 1).
These findings provided the mechanistic insight for us to target
these two signaling pathways so as to prevent PVR. The in vitro
model using ARPE-19 cell line was further optimized based on a low
cell density instead of by adding EGTA to confluent cells to
initiate proliferation to better mimic PVR. The results showed
HC-HA/PTX3 did not harm the non-stimulated ARPE-19 cells (FIG. 5A),
but significantly suppressed the proliferation (FIGS. 5B and 5C)
and the nuclear localization of phosphorylated Smad2/3 (FIGS. 6A
and 6B) after stimulation with EGF+FGF-2 and EGF+FGF-2+TGF-.beta.1,
respectively. The establishment of such an in vitro model allowed
for the determination of optimal dosing of HC-HA/PTX3 to be used
for in vivo testing, in the rabbit PVR animal model provided herein
(FIGS. 7A-D).
Example 4: Development of an Animal Model of PVR
[0273] PVR was successfully reproduced in rabbits (see FIGS. 7A-7D)
by vitreous detachment by gas compression vitrectomy followed by
intravitreal injection of rabbit RPE cells to mimic human PVR.
Rabbits were chosen because they can develop medullary wing
detachments that simulate retinal detachments in humans and show
PVR-like features.
[0274] NZW rabbits (Female, aged 3-7 months, weighing between 1.5
and 5.0 kg) were subjected to vitrectomy by intravitreal gas
injection by injecting 0.3 ml of 100% C3F8 gas into the vitreous
cavity using a 32 gauge 1/2'' needle 3 mm posterior to the
corneoscleral limbus under direct visualization using indirect
ophthalmoscopy following anterior chamber paracentesis performed to
lower the intraocular pressure and reduce the possibility of ocular
damage caused by an acute increase in pressure. Indirect
ophthalmoscopy was performed to ensure there is normal vascular
flow in the retina. The intraocular pressure was checked using a
Perkins tonometer until the intraocular pressure is below 20 mmHg.
PVR was created by intravitreal injection of 2.0.times.10.sup.5
rabbit RPE cells that had been prepared from tissue cultured
homologous primary rabbit RPE cells in a total of 0.1 ml volume via
a 32 gauge 1/2'' needle, with the bevel facing upward, and injected
into the vitreous cavity, just in front of the optic nerve head
(slowly, to prevent retinal damage). If the treatment of HC-HA/PTX3
was simultaneous with RPE cells, then PBS or two different doses of
HC-HA/PTX3 were injected similarly into the vitreous cavity of the
control rabbits or treated rabbits, respectively. If the treatment
of HC-HA/PTX3 was subsequent with RPE cells, PBS and HC-HA/PTX3 was
injected into the vitreous cavity one week later. In each
condition, the rabbits were immediately placed on their backs for 1
h to allow the cells and reagents to settle over the vascular wings
of the retina.
[0275] Four weeks after injection of the intravitreal HC-HA/PTX3 or
saline the rabbits were sacrificed by euthanasia by anesthetic
overdose with Euthasol (390 mg/mL/kg, intravenous). The eyeball was
enucleated with all conjunctival tissues, and fixed in 10%
formalin. The eyes underwent external examination and then the
superior cap is removed to allow internal examination. The gross
anatomical examination of the enucleated eyes was photographed
using a Nikon D600 camera (FIG. 7C and FIG. 7D).
Example 5: HC-HA/PTX3 is a Unique Matrix Component Responsible for
AM's Therapeutic Actions
[0276] HC-HA/PTX3 complex, first found in the cumulus-oocyte
complex surrounding the ovulated oocyte, plays a critical role in
fertilization. HC-HA/PTX3 is abundantly present in human AM and
this discovery has led to several exciting findings: (1) AM
epithelial and stromal cells express all components (HA, HC1, HC2,
bikunin, TSG-6, and PTX3) necessary for HC-HA/PTX3 biosynthesis
(FIG. 2A); (2) HC-HA/PTX3 purified from AM extract (AME) consists
of HMW HA (>3000 kDa) with covalently linked HC1 of I.alpha.I
and tightly bound PTX3 (FIG. 2B-2D), but not HC2, bikunin, or TSG-6
and (3) HC-HA/PTX3 is responsible for AM's therapeutic actions
which is briefly summarized below.
[0277] To make sure HC-HA/PTX3 prepared from each lot of AM donors
was consistent biochemically and functionally, a manufacturing
process using optimized SOPs under GMP facility was established.
Although the yield of HC-HA/PTX3 from different AM donors varied,
no significant differences in the potency assays were observed,
which were developed based on inhibition of tartrate resistant acid
phosphatase activity in osteoclasts and on promotion of macrophage
M2 polarization in IFN-.gamma./LPS-stimulated macrophages.
Consequently, the reference material to validate the release of
each lot of HC-HA/PTX3 to be used in in vitro and in vivo studies
was established.
[0278] Inflammation involving neutrophils and macrophages plays an
important role in PVR development. Injection of macrophages into
the rabbit vitreous induced epiretinal membranes, posterior
vitreous separation, and retinal detachment. Macrophages can
transdifferentiate into fibroblast-like cells and secrete growth
factors (e.g., PDGF), which contribute to proliferation and EMT of
RPE cells, the two key events in PVR pathogenesis. Soluble
HC-HA/PTX3, but not HA, significantly promoted apoptosis of
activated (by fMLP or LPS) but not resting neutrophils. Similarly,
soluble HC-HA/PTX3, but not HA, dose-dependently promoted apoptosis
of activated (by IFN-.gamma., LPS or IFN-.gamma./LPS) but not
resting macrophages. In addition, soluble and immobilized
HC-HA/PTX3, but not HA, promoted phagocytosis of apoptotic
neutrophils by macrophages. Immobilized HC-HA/PTX3 promoted
anti-inflammatory M2 polarization of LPS- or
IFN-.gamma./LPS-activated macrophages. In addition, such M2
polarization was coupled with notable downregulation of IL-23,
which was produced by activated macrophages and dendritic cells to
activate Th17 cells. Consequently, subconjunctival injection of
HC-HA/PTX3 prolonged survival of allogeneic corneal transplants in
mice. These data support the notion that HC-HA/PTX3 is a novel
complex which can suppress inflammation mediated by both
neutrophils and macrophages.
Example 6: HC-HA/PTX3 Downregulated Canonical Wnt Signaling in
Human Limbal Epithelial Progenitor and Niche Cells
[0279] AM inhibited squamous metaplasia of human conjunctival
epithelium by downregulating the expression, phosphorylation, and
nuclear translocation of .beta.-catenin. Furthermore, HC-HA/PTX3
downregulated canonical Wnt signaling in human limbal epithelial
progenitor cells (LEPCs) and niche cells (LNCs). Specifically,
immobilized HC-HA/PTX3 upregulated transcript expression of
non-canonical but not canonical Wnt ligands (e.g., Wnt 2B, Wnt 3A,
Wnt 5A, Wnt 5B, Wnt7A), Wnt negative regulators, and planer cell
polarity (PCP) factors in LEPCs/LNCs as measured by Wnt Signaling
Pathway RT.sup.2 Profiler PCR Array Plate (FIG. 3A). The
immunostaining data further confirmed that immobilized HC-HA/PTX3
prevented the nuclear translocation of .beta.-catenin as shown in
the positive control cells seeded in 3D Matrigel. In contrast,
transcript expression (FIG. 3A) and nuclear translocation (FIG. 3B)
of C-JUN, a key player of non-canonical Wnt (PCP) signaling, was
noted in LNCs when seeded on immobilized HC-HA/PTX3 but not 3D
Matrigel (FIG. 3A). Note that activation of non-canonical Wnt (PCP)
signaling is known to suppress that of canonical Wnt signaling.
Example 7: HC-HA/PTX3 Downregulates Canonical TGF-.beta.1/Smad
Signaling in Human Corneal Fibroblasts (HCF)
[0280] It has been reported that expression of TGF-.beta.1,2,3 and
TGF-.beta.RII transcripts (using Northern blot) is downregulated in
HCF and human limbal and conjunctival fibroblasts cultured on the
AM stroma. AME induced cell aggregation and prevents expression of
.alpha.-SMA by myofibroblasts. Human and mouse keratocytes seeded
on AM stroma maintained their normal phenotype without eliciting
nuclear translocation of pSmad2/3 even if they were exposed to
serum or TGF-.beta.1. Soluble HC-HA/PTX3 suppressed the TGF-.beta.1
promoter activity of HCF (FIG. 4A). It is known that exogenous
TGF-.beta.1 expectedly upregulates TGF-.beta.1, but not TGF-.beta.2
(FIG. 4B), in HCF seeded on both plastic and immobilized HA.
However, TGF-.beta.1 upregulation was not observed on immobilized
HC-HA/PTX3. Surprisingly, TGF-.beta.3, an anti-scarring isoform,
was upregulated only by HC-HA/PTX3, with or without TGF-.beta.1
(FIG. 4C). Expression of TGF-.beta.RII was reduced to nearly nil on
HC-HA/PTX3 after TGF-.beta.1 challenge (FIG. 4D). As expected,
exogenous TGF-.beta.1 caused the nuclear translocation of pSmad2/3
(FIG. 4E) and positive cytoplasmic expression of .alpha.-SMA (FIG.
4F) in HCF on plastic and HA. However, HC-HA/PTX3 effectively
blocked these TGF-.beta.1-induced changes in HCF. Collectively,
HC-HA/PTX3 downregulated canonical TGF-.beta.1 signaling and
prevented myofibroblast differentiation triggered by exogenous
TGF-.beta.1 in HCF.
Example 8: Preparation of Preserved Human Fetal Support Tissue
[0281] Human placenta was collected at elective cesarean delivery.
The placenta was flattened onto nitrocellulose paper (Hybond N+,
Amersham, England), with the epithelium surface up. The fetal
support tissue samples were stored at -80.degree. C. in
DMEM/glycerol 1:2 (v/v) until use.
Example 9: Amniotic Membrane Extract Preparations
[0282] Fresh and frozen human placentas were obtained from
Bio-tissue, Inc. (Miami, Fla.). The entire procedure for
preparation of total human AM extracts (AME) was carried out
aseptically so as to be used for subsequent cell culture-based
experiments. The AM was sliced into small pieces to fit into the
barrel of a BioPulverizer (Biospec Products, Inc., Bartlesville,
Okla.), frozen in the liquid nitrogen, pulverized into a fine
powder, and weighed. Cold 1.times.PBS buffer, pH 7.4, containing
protease inhibitors (protease inhibitor cocktail, P8340, Sigma, and
supplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM
sodium fluoride and 0.2 mM sodium vanadate) was added to the powder
at 1:1 (ml/g). The mixture was kept on ice and homogenized with a
Tissue Tearor (Biospec Products, Inc., Dremel, Wis.) 5 times, 1
minute each, with a 2 minute cooling interval. These water-soluble
extracts were designated as "Total" AM extracts (AME).
[0283] Total AM extracts were divided into two 50 ml conical
centrifuge tubes. One was centrifuged at high speed (HS,
48,000.times.g) and the other one was centrifuged at a low speed
(LS, 15,000.times.g) at 4 C.degree. Aliquots of the HS and LS
supernatant were transferred to sterile 1.5 ml Eppendorf tubes and
were designated as AM/HS and AM/LS, respectively. Desired AM/HS
samples were frozen at -20 C.degree. for 1 h before lyophilization.
The samples were then placed in the chamber of FreeZone (Labconco,
Kansas City, Mo.) with holes on the cap. Samples were lyophilized
at -50 C.degree. at a vacuum of 0.85 mBar for 5 hours. Before use,
the samples were reconstituted with the sterile distilled H.sub.2O
to the same volume. The same method was also used to prepare
extracts from AM jelly, which was easily scraped from the adherent
material on the AM stroma.
Example 10: Total Soluble Human Amniotic Membrane and Amniotic
Membrane Jelly Extract Preparations
[0284] Frozen human placenta material was obtained from Bio-Tissue,
Bio-tissue, Inc. (Miami, Fla.). The entire procedure for
preparation of total human AM extracts (AME) was carried out
aseptically so as to be used for subsequent cell culture-based
experiments. The AM was sliced into small pieces to fit into the
barrel of a BioPulverizer (Biospec Products, Inc., Bartlesville,
Okla.), frozen in the liquid nitrogen, pulverized into a fine
powder, and weighed. Cold 1.times.PBS buffer, pH 7.4, containing
protease inhibitors (protease inhibitor cocktail, P8340, Sigma, and
supplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM
sodium fluoride and 0.2 mM sodium vanadate) was added to the powder
at 1:1 (ml/g). The mixture was kept on ice and homogenized with a
Tissue Tearor (Biospec Products, Inc., Dremel, Wis.) 5 times, 1
minute each, with a 2 minute cooling interval. These water-soluble
extracts were designated as "Total" AM extracts (AME).
[0285] The total water-soluble extract was mixed for 1 hr at 4
C.degree., subsequently fractionated by two different speeds of
centrifugation at 4.degree. C. for 30 min, i.e., 15000.times.g and
48000.times.g, and the resultant supernatant was designated as L
and H, respectively. Each supernatant was divided into aliquots and
stored at -80.degree. C. This method was also used to prepare
extracts from AM jelly, which was easily scraped from the adherent
material on the AM stroma.
Example 11: Total Soluble Human Amniotic Membrane and Amniotic
Membrane Jelly Extracts by Different Buffers and Fractionation
Methods
[0286] In examining preparations in different extraction buffers,
the powder as prepared from above was weighed and mixed with Buffer
A (Isotonic Low salt): 100 mM Tris-HCl, pH 7.6, 150 mM NaCl, 4 mM
EDTA, 1% Triton X-100 at the wet weight (g) of AM to the buffer
(ml) at 1:1 ratio by stirring at 4.degree. C. for 1 hr. After
centrifugation at 48000.times.g, the resultant pellet was
subsequently extracted by Buffer B (high salt): 100 mM Tris-HCl, pH
7.6, 1 M NaCl, 4 mM EDTA, 1% Triton X-100 by stirring at 4 C for 1
hr. Again after centrifugation at 48000.times.g, the pellet was
finally extracted by Buffer C (4 M guanidine hydrochloride): 100 mM
sodium acetate, pH 5.8, 4 M guanidine hydrochloride, 4 mM EDTA, 1%
Triton X-100 by stirring at 4.degree. C. for 24 hr. All the above
three buffers were supplemented with the following protease and
phosphatase inhibitors: 1 .mu.g/ml aprotinin, 1 .mu.g/ml
leupeptins, 1 .mu.g/ml pepstatin A, 0.5 mM PMSF, 50 .mu.M sodium
fluoride and 0.2 .mu.M sodium vanadate. The resultant supernatants,
designated as Extract A, B, and C, respectively, were dialyzed
against the dialysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl)
supplemented with 0.5 mM PMSF at 4.degree. C. for 6 hr and dialysis
buffer was changed twice, each with 500.times.(the volume ratio of
dialysis buffer:samples). After dialysis, the volume of each sample
was measured and adjusted to the same volume with the dialysis
buffer. The same method was also used to prepare extracts from AM
jelly, which was the adherent material on the AM stroma that could
be easily scraped off.
Example 12: Preparation of Total Soluble Human Amniotic Membrane
Extracts in PBS
[0287] The entire procedure for preparation of total soluble human
AM extracts (T) was carried out aseptically so as to be used for
subsequent cell culture-based experiments. Frozen human placenta
was obtained from Bio-Tissue, Inc. (Miami, Fla.), from which AM was
retrieved. AM was sliced into small pieces to fit into the barrel
of a BioPulverizer (Biospec Products, Inc., Bartlesville, Okla.),
frozen in the liquid nitrogen, and then pulverized into a fine
powder. The powder was weighed and mixed with cold PBS buffer
(prepared by adding distilled H.sub.2O to 1.times.PBS, pH7.4, from
10.times.PBS, cat #70011-044, Invitrogen, Carlsbad, Calif.) with
protease inhibitors (protease inhibitor cocktail, P8340, Sigma, and
supplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM
sodium fluoride and 0.2 mM sodium vanadate) at 1:1 (ml/g). The
mixture was kept on ice and homogenized with a Tissue Tearor
(Biospec Products, Inc., Dremel, Wis.) for 5 times, 1 min each with
a 2 min interval cooling. This water-soluble extract was designated
as "Total" (T). The total water-soluble extract was mixed for 1 hr
at 4.degree. C., centrifuged at 4.degree. C. for 30 min at
48000.times.g. The supernatant was divided into aliquots and stored
at -80.degree. C.
Example 13: Preparation of Water-Soluble AM Stromal Extracts
[0288] Using aseptic techniques, frozen human AM obtained from
Bio-Tissue, Inc. (Miami, Fla.) was briefly washed 2-3 times with
HBSS to remove the original storage medium. The AM stroma was
scraped by spatula, frozen in the air phase of liquid nitrogen and
grounded to fine particles by BioPulverizer (Biospec Products,
Inc., Bartlesville, Okla.) followed by homogenization on ice with
Tissue Tearor (Biospec Products, Inc., Dremel, Wis.) in PBS, pH
7.4, for 1 min. The homogenate was mixed by rotation for 1 h and
centrifuged at 14,000.times.g for 30 min at 4.degree. C. The
supernatant in PBS was then collected, and stored in aliquots at
-80.degree. C. The protein concentration was determined by BCA
assay. This water-soluble protein extract, designated as amniotic
stromal extract (ASE), was used for experiments described
herein.
Example 14: AM Stromal Extract Preparation
[0289] The complete procedure for preparation of protein extracts
was carried out aseptically. Frozen human AM obtained from
Bio-Tissue (Miami, Fla.) was briefly washed 2-3 times with HBSS
(Invitrogen, Carlsbad, Calif.) to remove the storage medium. AM
stroma was scraped from the stromal side of the AM by spatula for
AM stroma extract preparation. Human placenta as well as chorion
obtained from Baptist Hospital (Miami, Fla.) was rinsed 3 times
with HBSS to remove blood. To prepare the water-soluble protein
extract, total AM, scraped AM stroma, stroma-removed AM, placenta,
and chorion were each frozen in the air phase of liquid nitrogen
and each ground to fine particles using a BioPulverizer (Biospec
Products, Inc., Bartlesville, Okla.) followed by homogenization on
ice with Tissue Tearor (Biospec Products, Inc., Dremel, Wis.) in
PBS (pH 7.4) for 1 min. Each homogenate was mixed for 1 h and
centrifuged at 14,000 g for 30 min at 4.degree. C. Each supernatant
(in PBS) was then collected and stored in aliquots (0.5 ml) at
-80.degree. C. The BCA assay (Pierce, Rockford, Ill.) was used to
quantitate the total protein in different extracts.
Example 15: Preparing Water-Soluble and Lyophilized Powder Forms of
Human AM Extracts
[0290] To prepare human AM extracts, the entire procedure was
carried out aseptically. Unless otherwise noted, the AM extracts
were handled at room temperature during the steps of the procedure.
First, fresh or frozen human AM was obtained, preferably from
Bio-Tissue, Inc. (Miami, Fla.). Frozen AM was briefly washed 2-3
times with HBSS (Invitrogen, Carlsbad, Calif.) to remove the
storage medium. Fresh human placenta or chorion was rinsed 3 times
with HBSS to remove blood.
[0291] To prepare the water-soluble form of AM extracts, the AM
(e.g., AM stroma, stroma-removed AM, placenta, chorion) was
transferred to a sterile 50 ml centrifuge tube and centrifuged at
4.degree. C. for 5 min at 5000.times.g to remove the excess fluid.
The AM was weighed, transferred to a 100 mm or 150 mm sterile Petri
dish, and frozen in the air phase of a liquid nitrogen container
for 20 min to facilitate the subsequent homogenization. The frozen
AM was then sliced into small pieces with a disposable scalpel or
ground to fine particles using a BioPulverizer (Biospec Products,
Inc., Bartlesville, Okla.) or other suitable device, and
homogenized with Tissue Tearor (Biospec Products, Inc., Dremel,
Wis.), or other suitable device, in phosphate buffered saline (PBS)
or DMEM without phenol red (Invitrogen, Carlsbad, Calif.) at
neutral pH. For biochemical characterization and purification, the
above solutions were supplemented with the following proteinase
inhibitors: 1 .mu.g/ml aprotinin, 1 .mu.g/ml leupeptin, 1 .mu.g/ml
pepstatin A, and 1 mM PMSF. However, if the extract was to be
directly added to cell culture, no protease inhibitors were added.
The homogenate was mixed at 4.degree. C. for 30 min and centrifuged
at 15,000.times..g for 30 min. The supernatant (i.e., AM extract)
was collected and stored in aliquots (0.5 ml) at -80.degree. C. The
BCA assay (Pierce, Rockford, Ill.) was used to quantitate the total
protein in each AM extract.
[0292] To prepare the lyophilized powder form of AM extracts,
frozen AM was ground to fine particles using a BioPulverizer
(Biospec Products, Inc., Bartlesville, Okla.), or other suitable
device, and further homogenized as described herein. Aliquots of
approximately 0.5 ml were lyophilized by SpeedVac (Savant
Instruments Inc., Farmingdale, N.Y.) at 4.degree. C. for 4 h to
decrease the weight from 280 mg to 32 mg (about 89% reduction). The
lyophilized powder was weighed and stored at -80.degree. C. Before
use, the lyophilized powder was reconstituted in a suitable
buffer.
[0293] To prepare AM stromal extracts, the AM stroma was scraped
from the stromal surface of intact total AM leaving the basement
membrane and the amniotic epithelium intact, and the frozen AM
stroma was ground using a BioPulverizer as described herein. The
stroma was extracted with PBS at a neutral pH at 4.degree. C. for
30 min and centrifuged at 15,000.times.g for 30 min. The
supernatant was collected and stored in aliquots (0.5 ml) at
-80.degree. C. The BCA assay (Pierce, Rockford, Ill.) was used to
quantitate the total protein in the AM stromal extract.
Example 16: Suppression of TGF-.beta.1 Promoter Activity
[0294] The fetal support preparations and compositions described
herein suppress of TGF-.beta.1 promoter activity as shown herein;
thus the fetal support preparations and compositions described
herein can be used for anti-scarring, anti-inflammatory, and
anti-angiogenic therapies. The fetal portion of the frozen amniotic
membrane has a significantly higher anti-scarring effect than that
of fresh amniotic membrane; the placental portion of the frozen
amniotic membrane also has a significantly higher anti-scarring
effect than the fresh amniotic membrane. Therefore, the frozen
fetal support tissue, either the placental or fetal portion, showed
more potent suppressive effects in TGF-.beta. than the fresh fetal
support tissue. This suppressive effect mediated by total fetal
support tissue extract obtained from frozen fetal support tissue
was dose-dependent over a range of 0.4 to 125 .mu.g/ml (FIG. 8).
Furthermore, such a suppressive effect could not be substituted by
high MW HA alone (exceeding 100.times. of equivalent AM extract),
and was lost after digestion with hyaluronidase (FIG. 9),
suggesting that it was mediated by a complex between HA-I.alpha.I.
Centrifugation at low or high speed did not affect the suppressive
effect significantly. However, subsequent lyophilization and
reconstitution produced a more potent suppressive effect.
Additionally, the overall suppressive effect of AM was more potent
than that of AM jelly.
Example 17: Fetal Support Tissue Preparations and Purified
Compositions Used to Culture Cells
[0295] To examine the effect of fetal support tissue on the cell
differentiation process, myofibroblasts differentiated from AMSCs
at passage 2 were subcultured onto the stromal matrix of AM, and
compared to those subcultured on collagen I-coated dish as a
control. After 7 days of cultivation in DMEM with 10% FBS, AMSCs on
collagen I still maintained a myofibroblastic shape. In contrast,
cells seeded on fetal support tissue stromal matrix exhibited a
mixture of round, spindle, elongated, and dendritic shapes. Thus,
in some embodiments, fetal support tissue preparations have
dedifferentiation abilities, and are used to slow cell
differentiation.
Example 18: Effect of HC-HA/PTX3 on Cell Migration and Collagen Gel
Contraction
[0296] Cell Culture and Treatment
[0297] ARPE-19, a human diploid RPE cell line, was cultured in
HEPES-buffered DMEM and Ham's F-12 (1:1) supplemented with 10% FBS,
50 units/ml penicillin, and 50 .mu.g/ml streptomycin at 37.degree.
C. in humidified air with 5% CO.sub.2. For post-confluence
experiments, cells were continuously cultured for 7 days upon 100%
confluence before being tested. For low cell density assays, cells
were seeded at 1.times.10.sup.4/cm.sup.2 or other densities
overnight (20-24 h) followed by treatment with growth factors and
cytokines for 24-120 h or 48 h (after optimization). In the case of
serum starvation, cells were incubated in serum-free (SF) medium
for 24 h followed by treatment with growth factors and cytokines
for 24-120 h. BrdU (10 .mu.M) labeling was performed for 4 h prior
to the termination of the growth factors/cytokines treatment.
[0298] Purification of HC-HA/PTX3 from Human AM
[0299] HC-HA/PTX3 was prepared from cryopreserved human placentas
provided by Bio-Tissue, Inc. (Miami, Fla.). AM from the same donor
was extracted by PBS (pH 7.4) to generate the PBS extract as
reported. The extract was then fractionated by ultracentrifugation
in a CsCl gradient at an initial density of 1.35 g/ml in 4 M GnHCl
at 35,000 rpm for 48 h at 15.degree. C. (LM8 model, SW41 rotor,
Beckman Coulter, Indianapolis, Ind.). A total of 12 fractions (1
ml/fraction) was collected from each ultracentrifuge tube. The
weight of each fraction was measured to calculate the density.
After the biochemical analysis (HA ELISA, BCA protein assay, and
Western blot, see below), fractions containing HA but little or no
proteins were pooled and subjected to the second run of
ultracentrifugation in a CsCl gradient at an initial density of
1.40 g/ml. Selective fractions (containing HA but undetectable
proteins measured by BCA assay and designated as HC-HA/PTX3) were
pooled and dialyzed against distilled water, lyophilized, and
stored at -80.degree. C. Therefore, the amount of HC-HA/PTX3 was
expressed based on the HA amount present in the complex.
[0300] Cell Migration
[0301] The migration assay was performed in 24-well transwell plate
(8 .mu.m pore size, Costar, Kennebunk, Me.) when 0.5 ml DMEM/F12
(1:1) without or with EGF (10 ng/ml), FGF-2 (20 ng/ml), and
TGF-.beta.1 (10 ng/ml) was added in the lower compartment while 0.1
ml of ARPE-19 cell re-suspended in DMEM/F12 (2.times.10.sup.6/ml)
treated with PBS (vehicle control), HA (25 .mu.g/ml), or HC-HA/PTX3
(25 .mu.g/ml) was added to the upper compartment. After incubation
at 37.degree. C. for 4 h, cells not migrating through the pores
were removed by a cotton swap, while cells on the filter facing the
lower compartment were fixed with 5% glutaraldehyde, stained with
1% crystal violet, and counted from six random microscopic fields
for each control or treatment group.
[0302] Collagen Gel Contraction
[0303] 0.25 ml of collagen type I solution (Corning, Bedford,
Mass.) in cold DMEM/F12 (2.5 mg/ml) was added to each well of
24-well plates, followed by incubation at 37.degree. C. for 1 h
before adding 0.5 ml of ARPE-19 cells or primary human RPE cells
(each at 5.times.10.sup.5/ml) without or with TGF-.beta.1 (10
ng/ml) and treatment of PBS (vehicle control), HA (25 .mu.g/ml), or
HC-HA/PTX3 (25 .mu.g/ml) on the top of collagen gel. After 24 h,
the gels were freed from the walls of the culture wells with a
small spatula. The photographic images of collagen gels were
digitalized and the area was measured with NIH ImageJ 1.45
software. The percentage of gel contraction was determined by
measuring the gel size at 72 h when compared to the initial size
(at 0 h) and compared among groups.
[0304] Results
[0305] HC-HA/PTX3 (25 .mu.g/ml) as well as HA (25 .mu.g/ml)
completely suppressed migration of ARPE-19 cells under the
stimulation of EGF (10 ng/ml), FGF-2 (20 ng/ml), and TGF-.beta.1
(10 ng/ml) (FIG. 21). In contrast, HC-HA/PTX3, but not HA,
significantly reduced the TGF-.beta.1-induced collagen gel
contraction in both ARPE-19 cells and primary human RPE cells (FIG.
22).
[0306] While preferred embodiments have been shown and described
herein, it will be obvious to those skilled in the art that such
embodiments are provided by way of example only. Numerous
variations, changes, and substitutions may now occur. It should be
understood that various alternatives to the embodiments described
herein can be employed in practicing the described methods. It is
intended that the following claims define the scope of the
embodiments and that methods and structures within the scope of
these claims and their equivalents be covered thereby.
* * * * *