U.S. patent application number 13/285738 was filed with the patent office on 2012-07-26 for placental or umbilical cord tissue compositions.
This patent application is currently assigned to Anthrogenesis Corporation. Invention is credited to Qing Liu, Cynthia Ray.
Application Number | 20120189583 13/285738 |
Document ID | / |
Family ID | 39301475 |
Filed Date | 2012-07-26 |
United States Patent
Application |
20120189583 |
Kind Code |
A1 |
Liu; Qing ; et al. |
July 26, 2012 |
PLACENTAL OR UMBILICAL CORD TISSUE COMPOSITIONS
Abstract
The present invention encompasses compositions, including
solutions, gels, and pastes, manufactured from amniotic membrane,
umbilical cord membrane, or both. The present invention also
encompasses methods of making such compositions, and methods of
using the compositions to treat conditions of the eye.
Inventors: |
Liu; Qing; (Hillsborough,
NJ) ; Ray; Cynthia; (Flemington, NJ) |
Assignee: |
Anthrogenesis Corporation
|
Family ID: |
39301475 |
Appl. No.: |
13/285738 |
Filed: |
October 31, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11973125 |
Oct 4, 2007 |
8071135 |
|
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13285738 |
|
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60849521 |
Oct 4, 2006 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61P 31/00 20180101;
A61K 35/44 20130101; A61P 25/00 20180101; A61P 37/08 20180101; A61P
41/00 20180101; A61K 35/50 20130101; A61P 31/10 20180101; A61P
31/04 20180101; A61P 27/02 20180101; A61P 29/00 20180101; A61L
27/3604 20130101; A61P 31/12 20180101; A61L 27/3683 20130101; A61P
35/00 20180101; A61L 27/3641 20130101; A61L 27/52 20130101; A61P
17/02 20180101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/50 20060101
A61K035/50; A61P 31/12 20060101 A61P031/12; A61P 31/00 20060101
A61P031/00; A61P 31/10 20060101 A61P031/10; A61P 41/00 20060101
A61P041/00; A61P 35/00 20060101 A61P035/00; A61P 37/08 20060101
A61P037/08; A61P 25/00 20060101 A61P025/00; A61P 17/02 20060101
A61P017/02; A61P 27/02 20060101 A61P027/02; A61P 31/04 20060101
A61P031/04; A61P 29/00 20060101 A61P029/00 |
Claims
1.-31. (canceled)
32. A composition comprising (i) placental tissue components
soluble in aqueous solution at acidic and neutral pH, and
substantially lacking placental tissue components insoluble in
aqueous solution at acidic and neutral pH, and (ii) one or more
biologically active compounds, wherein the composition is prepared
according to the following method: (a) contacting placental tissue
with an acidic solution; (b) bringing the solution to neutral pH;
and (c) removing insoluble matter within said solution to yield
said placental tissue components.
33. The composition of claim 32, wherein said placental tissue is
amniotic membrane (AM).
34. The composition of claim 32, wherein said composition is
formulated as a liquid, gel, paste, slurry, cream, or ointment.
35. The composition of claim 32, wherein the composition is an
ophthalmologically-acceptable liquid.
36. The composition of claim 32, wherein the insoluble matter in
said solution is removed following step (a).
37. The composition of claim 32, wherein the insoluble matter in
said solution is removed following step (b).
38. The composition of claim 32, wherein said method further
comprises adding said one or more biologically active compounds to
said placental tissue components.
39. The composition of claim 38, wherein the insoluble matter in
said solution is removed following the step of adding said one or
more biologically active compounds to said placental tissue
components.
40. The composition of claim 32, wherein said biologically active
compound is an organic molecule, an antibiotic, an anti-viral
agent, an anti-microbial agent, an anti-fungal agent, an
anti-inflammatory agent, an antiproliferative agent, a cytokine
receptor modulator, a cross-linking agent, an enzyme or protein
inhibitor, an antihistamine, a hormone, a growth factor, a pain
medication, a wound healing agent, a wound sealant, or a
scaffolding reagent.
41. The composition of claim 40, wherein said biologically active
compound is a wound healing agent.
42. The composition of claim 41, wherein said wound healing agent
is iron, zinc, vitamin C, arginine, thymosin, PDGF, TGF, or
hyaluronic acid.
43. The composition of claim 32, wherein said biologically active
compound is a wound sealant.
44. The composition of claim 43, wherein said wound sealant is
fibrin.
45. The composition of claim 32, wherein said biologically active
compound is a cross-linking agent.
46. The composition of claim 45, wherein said cross-linking agent
is riboflavin.
47. A method for treating an injury, wound, or discontinuity in or
on a tissue of a subject, comprising contacting said injury, wound,
or discontinuity with the composition of claim 41.
48. The method of claim 47, wherein said injury, wound, or
discontinuity is a laceration, scrape, thermal or chemical burn,
incision, or puncture.
49. A method for treating an injury, wound, or discontinuity in or
on a tissue of a subject, comprising contacting said injury, wound,
or discontinuity with the composition of claim 43.
50. The method of claim 49, wherein said injury, wound, or
discontinuity is a laceration, scrape, thermal or chemical burn,
incision, or puncture.
51. A method for treating an ocular condition or symptom thereof in
a subject. comprising contacting the affected area of said
subject's eye(s) with the composition of claim 45.
52. The method of claim 51, wherein said ocular condition is
discontinuity of the sclera or cornea, a congenital defect, or an
injury resulting from ocular surgery.
53. The method of claim 51, wherein said condition or symptom is
irritation, inflammation, dryness, stickiness, or symblepharon.
Description
1. PRIOR RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 60/849,521,
filed Oct. 4, 2006, which is incorporated herein by reference in
its entirety.
2. FIELD OF THE INVENTION
[0002] The present invention generally relates to compositions,
e.g., solutions, gels or pastes, made from placenta, umbilical
cord, or both, or tissues from placenta, umbilical cord, or
both.
3. BACKGROUND OF THE INVENTION
[0003] The repair or treatment of various body tissues, such as
skin, organs, and the like, has been accomplished using collagen
compositions, including tissue membranes comprising collagen, e.g.,
amniotic membrane, pericardium, dura mater, and the like. A need
exists, however, for additional, more versatile compositions that
can be used in medical applications in addition to, or in place of,
membranes.
4. SUMMARY OF THE INVENTION
[0004] The present invention provides compositions comprising
aqueous-soluble components, including acid- or base-soluble
components, of placental tissue and/or umbilical cord tissue, e.g.,
amniotic membrane and umbilical cord membrane, including umbilical
cord membrane in combination with Wharton's jelly, and methods of
making and using the same.
[0005] In one embodiment, the invention provides a composition
comprising placental tissue components or umbilical cord components
that are soluble in aqueous solution at neutral pH, and are
substantially lacking placental tissue components or umbilical cord
components that are insoluble in aqueous solution at neutral pH. In
another embodiment, the invention provides a composition comprising
placental tissue components or umbilical cord components that are
soluble in aqueous solution at neutral pH, and placental tissue
components or umbilical cord components that are insoluble in
aqueous solution at neutral pH. In another embodiment, the
invention provides a composition comprising placental tissue
components or umbilical cord components that are soluble in aqueous
solution at acidic and neutral pH, or basic and neutral pH, and are
substantially lacking placental tissue components or umbilical cord
components that are insoluble in aqueous solution at acidic and
neutral pH or basic and neutral pH. In another embodiment, the
invention provides a composition comprising placental tissue
components or umbilical cord components that are soluble in aqueous
solution at acidic and neutral pH, or basic and neutral pH, and
placental tissue components or umbilical cord components that are
insoluble in aqueous solution at acidic and neutral pH or basic and
neutral pH. In another embodiment, said placental components or
said umbilical cord components substantially lack acid- or
base-insoluble amniotic membrane components.
[0006] In a specific embodiment of the above compositions, said
placental tissue is amniotic membrane (AM). In another embodiment,
said compositions are derived from a mixture of amniotic membrane
and umbilical cord tissue components.
[0007] In other specific embodiments, the composition is a liquid,
gel, paste or slurry. In other specific embodiments, the
composition is a cream or ointment.
[0008] In certain specific embodiments, the composition comprises
at least, about, or at most 1.times.10.sup.-9, 5.times.10.sup.-9,
1.times.10.sup.-8, 5.times.10.sup.-8, 1.times.10.sup.-7,
5.times.10.sup.-7, 1.times.10.sup.-6, 5.times.10.sup.-6,
1.times.10.sup.-5, 5.times.10.sup.-5, 10.sup.-4, 5.times.10.sup.-4,
1.times.10.sup.-3, 5.times.10.sup.-3, 1.times.10.sup.-2,
5.times.10.sup.-2, or 1.times.10.sup.-1 grams of placental tissue
components or umbilical cord tissue components, or a particular
tissue component, or a particular type of tissue component, per
gram of composition or per milliliter of composition. In a specific
embodiment, the tissue component is a cytokine or growth
factor.
[0009] In other specific embodiments, the composition comprises at
least or at most 1%, 5%, 10%, 20%, or 50% placental tissue
components or umbilical cord tissue components by weight. In
another specific embodiment, the composition is a lyophilized
composition. In a specific embodiment, the composition comprises
less than about 20% water by weight.
[0010] In another specific embodiment, the composition comprises an
ophthalmologically-acceptable liquid, e.g., an isotonic buffer
liquid. In a specific embodiment, the composition comprises less
than 1% solids by weight. In a more specific embodiment, said
liquid comprises saline solution, glycerin, hypromellose, or
polyethylene glycol. In another more specific embodiment, said
liquid comprises saline solution, glycerin, hypromellose, and
polyethylene glycol. In another specific embodiment, the
composition comprises an ophthalmologically-acceptable lubricant.
In a more specific embodiment, said lubricant is hydrophobic. In
another more specific embodiment, said lubricant is hydrophilic. In
another more specific embodiment, said composition comprises one or
more of an anti-inflammatory compound, an analgesic, an anesthetic
or an immune-suppressing agent.
[0011] The invention further provides a bandage contact lens,
wherein the contact lens comprises a solution, gel or paste of the
invention.
[0012] The invention further provides a composition of the
invention that comprises riboflavin as a crosslinking agent. The
composition preferably comprises collagen. In a specific
embodiment, the composition of the invention comprises collagen
that is crosslinked by riboflavin. In another specific example, the
invention provides a bandage contact lens, comprising collagen,
wherein the collagen is crosslinked by riboflavin. Such a bandage
contact lens can be made, e.g., by immersing the contact lens in a
solution of the invention, wherein the solution comprises
riboflavin, and exposing the lens to ultraviolet light (UVA) for a
time sufficient for the UVA to crosslink a plurality of the
collagens in the lens.
[0013] The invention further provides methods of preparing the
compositions of the invention. In one embodiment, the invention
provides a method of preparing a composition, comprising: (a) in
either order: (i) contacting a placental tissue, umbilical cord
tissue, or portion of either, with an acidic or basic solution;
(ii) disrupting the placental tissue, umbilical cord tissue or
portion thereof in said solution to form a suspension; (b) bringing
the suspension to neutral pH; and (c) removing particulate matter
within said suspension to form the composition.
[0014] In another embodiment, the invention provides a method of
preparing a composition, comprising: (a) disrupting umbilical cord
tissue in a pH-neutral aqueous solution to form a suspension; and
(b) removing umbilical cord tissue components insoluble in said
suspension to form the composition.
[0015] In a specific embodiment of these methods, said acidic
solution is an acetic acid solution. In a more specific embodiment,
said acidic solution is between about pH 4.5 and about pH 6. In
another specific embodiment of the methods, said basic solution is
a sodium hydroxide solution. In a more specific embodiment, said
basic solution is between about pH 8 and about pH 9.5. In another
specific embodiment, said disrupting is performed at a temperature
of between about the freezing point of said solution to about
10.degree. C. In other specific embodiments, the method comprises
adjusting the water content of the solution to at most about 99%,
95%, 90%, 75%, 50% or 10% by weight. In another specific
embodiment, the method comprises decellularizing said placental
tissue or said umbilical cord tissue prior to said disruption. In a
specific embodiment of the method, said placental tissue is
amniotic membrane. In another specific embodiment of the method,
said umbilical cord tissue is umbilical cord membrane in
combination with Wharton's jelly.
[0016] The invention further comprises methods of using the
compositions of the invention. In one embodiment, for example, the
invention provides a method of treating a condition of an eye
comprising contacting a tissue of said eye having or affected by
said condition with a composition of the invention, preferably a
solution of the invention. In specific embodiments. said ocular
condition is irritation, dry eye, blepharitis, symblepharon, red
eye, inflammation of a tissue of an eye or injury of a tissue of an
eye. In a more specific embodiment, said injury is an injury caused
by or related to refractive surgery. In an even more specific
embodiment, said refractive surgery is photorefractive keratectomy
(PRK), laser-assisted sub-epithelial keratectomy (LASEK),
laser-assisted in situ keratomileusis (LASEK), automated lamellar
keratoplasty (ALK), laser thermal keratoplasty (LTK), or conductive
keratoplasty (CK). In another specific embodiment, the invention
provides a method of treating a injury, wound or discontinuity in a
tissue of an individual, comprising contacting said injury, wound
or discontinuity with a compound of the invention. In a specific
embodiment, said contacting is performed after said wound, injury
or discontinuity is closed. In specific embodiments, said injury,
wound or discontinuity is a laceration, scrape, thermal or chemical
bum, incision, puncture, wound caused by a projectile.
[0017] The invention further provides a method of preparing a
contact lens prior to placement on an eye, comprising contacting
said contact lens with the composition of the invention. In a
specific embodiment, said contact lens is a bandage contact
lens.
[0018] As used herein, the term "components," as in "amniotic
membrane components," means any part of a placenta or umbilical
cord that is not an intact organ or tissue of the organ; the term
can include, e.g., placental or umbilical cord biomolecules, or
placental material in suspension produced by homogenization or
disruption of placental or umbilical cord.
[0019] As used herein, "tissue" means an anatomically-distinct part
or division of an organ. For example, "placental tissue" comprises,
e.g., amniotic membrane or chorion.
[0020] As used herein, "treat" or "treatment" refers to the use of
a composition of the invention on or in, e.g., a burn, injury,
wound, or discontinuity in a tissue of an individual, e.g.,
contacting the injury, wound or discontinuity with the composition,
such that at least one aspect of the injury, wound, or
discontinuity is measurably improved compared to that of an
individual on which the composition is not used. An "aspect of the
injury, wound, or discontinuity" includes aspects such as, for
example, the degree of inflammation, leakage of fluid, perceived
discomfort, irritation, degree of tissue repair, degree of
re-epithelialization, and the like. The term also encompasses the
use of a composition of the invention to, e.g., improve the
appearance of normal skin.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention provides for the use of placental
tissue, e.g., whole placenta, amniotic membrane, chorion, and the
like, and/or umbilical cord tissue, e.g., whole umbilical cord,
umbilical cord membrane, Wharton's jelly, umbilical vessels, and
the like, or a combination of any of the foregoing, to produce
useful compositions such as solutions, gels or pastes. In one
embodiment, the compositions are useful in treating a disease,
disorder or condition of the eye. In another preferred embodiment,
the compositions can be used as wound-healing agents.
5.1. AMNIOTIC MEMBRANE AND/OR UMBILICAL CORD MEMBRANE COMPOSITIONS
AND METHODS OF MAKING THE SAME
5.1.1. Obtaining Umbilical Cord and Amniotic Tissue
[0022] Placental tissue, e.g., amniotic membrane, to be used in
making the compositions of the invention, can be obtained as
follows. In a preferred embodiment, the placenta for use in the
methods of the invention is taken as soon as possible after
delivery of the newborn. The placenta may be used immediately, or
may be stored for 2-5 days from the time of delivery prior to any
further treatment. The placenta is typically exsanguinated, that
is, drained of the cord blood remaining after birth. Preferably,
the expectant mother is screened prior to the time of birth, using
standard techniques known to one skilled in the art, for
communicable diseases including but not limited to, HIV, HBV, HCV,
HTLV, syphilis, CMV, and other pathogens known to contaminate
placental tissue.
[0023] One exemplary method of obtaining placental tissue comprises
the following steps. First, the umbilical cord is separated from
the placental disc; optionally, the amniotic membrane is separated
from the chorionic membrane. Following separation of the amniotic
membrane from the chorionic membrane and placental disc, the
umbilical cord stump is cut, e.g., with scissors, and detached from
the placental disc. The placental tissue, e.g., amniotic membrane
may then be stored in a sterile, preferably buffered, saline
solution, such as 0.9% sterile NaCl solution. The placental tissue,
e.g., amniotic membrane can stored by refrigeration, at a
temperature of at least 2.degree. C. in preparation for making a
solution, gel or paste of the invention.
[0024] The umbilical cord can be collected as part of the
collection of placental tissue, e.g., amniotic membrane. The
umbilical cord is separated from the placenta as soon as possible
after delivery of the newborn, and is typically massaged to remove
umbilical cord blood. Optionally, the umbilical cord is sectioned
into pieces of about 10 cm to about 15 cm in length. The umbilical
cord or umbilical cord sections can then be stored for up to about
72 hours in a sterile, preferably buffered, saline solution, such
as 0.9% sterile NaCl solution. Preferably, the umbilical cord is
stored under refrigeration, at a temperature of about 1.degree. C.
to about 5.degree. C. At this time, the umbilical cord is
preferably slit or cut longitudinally using, e.g., a scalpel and
forceps, grooved director, or the like, allowing removal of the
Wharton's jelly, and/or removal of one or more of the umbilical
cord vessels, e.g., with a forceps. Preferably, the vessels are
removed, and the Wharton's jelly is retained. The umbilical cord
membrane can also be processed further without cutting and opening
the membrane. An umbilical cord vessel, for example. can be removed
from the cord by grasping the vessels with a forceps and gently
pulling and massaging until the vessel is removed, leaving the
umbilical cord membrane as an intact tube. In one embodiment of
deveining, the umbilical vein of a fresh (less than 48 hours after
delivery) umbilical cord is canalized using the blunt probe of a
vein stripper. The blunt probe is replaced with a small bullet
probe, and the vein is tied to the probe with thread. The stripper
is then removed, and the process is repeated with the umbilical
arteries.
[0025] In preferred embodiments, the placental tissue or umbilical
tissue used to make the composition of the invention, e.g.,
suspension, solution, slurry gel or paste, retains the tissue's
native cells. In other embodiments, the placental tissue or
umbilical cord tissue can be substantially decellularized; that is,
substantially all cellular material and cellular debris (e.g., all
visible cellular material and cellular debris) can removed from the
tissue prior to production of a solution, gel or paste of the
invention. Any decellularizing process known to one skilled in the
art may be used, e.g., decellularization methods disclosed in U.S.
Application Publication No. 2002/0160510. Preferably the process
used for decellularizing the umbilical cord or umbilical cord
membrane does not disrupt the native conformation of the proteins
making up the biomaterial. In one embodiment, placental or
umbilical cord tissue to be used in making a composition of the
invention can be decellularized by contacting, e.g., placing the
tissue in, a solution of about 0.5% to about 2.0% deoxycholic acid
for a period of 1 hour to about 20 days, preferably about 1 hour to
about 10 days, optionally in combination with physical scraping to
remove cellular material. "Substantially decellularized," as used
herein, means removal of at least 90% of the cells, more preferably
at least 95% of the cells, and most preferably at least 99% of the
cells associated with the umbilical cord membrane.
Decellularization can leave cellular material on the membrane; for
example, decellularization can leave nuclear material detectable by
4',6-diamidino-2-phenylindole (DAPI).
5.1.2. Solutions. Gels and Pastes
[0026] The present invention provides compositions, such as, e.g.,
solutions, suspensions, gels and pastes, that are made from
placental tissue or umbilical cord tissue, e.g., amniotic membrane,
chorion, amnion chorion, umbilical cord membrane, umbilical cord
membrane combined with Wharton's jelly, etc. The present invention
further provides methods of making these, compositions.
5.1.2.1. Compositions Comprising Placental Tissue Components and/or
Umbilical Cord Components Soluble at Neutral pH
[0027] The invention generally provides compositions made from
umbilical cord tissue or a combination of umbilical cord tissue and
placental tissue. In one embodiment, the invention provides a
suspension of umbilical cord tissue, or of umbilical cord tissue
and placental tissue. Such suspensions comprise umbilical cord
tissue, or placental and umbilical cord tissue, that has been
disrupted, e.g., homogenized in aqueous solution such that the
suspension comprises tissue components that are soluble in aqueous
solution at neutral pH (i.e., about pH 7.0 to about pH 7.4), and
components that are insoluble in aqueous solution at neutral pH.
Such suspensions can be made by any method known to those of skill
in the art. In one embodiment, the placental tissue or umbilical
cord tissue is placed in a volume of a solution and disrupted,
e.g., homogenized, to produce a suspension
[0028] In certain embodiments, the suspension comprises components,
both soluble and insoluble in aqueous solution, of a part of, or
the whole of, only placental tissue, e.g., components of a whole
placenta, or components only of parts of a placenta, such as
amniotic membrane, chorion, or combination of amniotic membrane and
chorion, and the like. In another embodiment, the suspension can
comprise components of a part of, or the whole of, only umbilical
cord tissue, e.g., components of a whole umbilical cord, or
components only of umbilical cord membrane, Wharton's jelly,
umbilical vessels, or a combination thereof. In one embodiment, the
suspension comprises components from an amniotic membrane. In
another embodiment, the suspension comprises components from an
umbilical cord membrane in combination with Wharton's jelly. The
invention also comprises suspensions that are combinations of the
foregoing, as well, for example, a combination of amniotic membrane
and umbilical cord membrane components.
[0029] In one embodiment, the suspension of the invention comprises
umbilical cord tissue components, or a combination of umbilical
cord tissue components and placenta tissue components, wherein said
suspension is made by a process comprising (1) contacting umbilical
cord tissue, or umbilical cord and placental tissue, with a
solution at neutral pH, and (2) disrupting said tissue in said
solution to form a suspension.
[0030] In other embodiments, the invention provides a suspension
that comprises components of umbilical cord tissue, or both
placental tissue and umbilical cord tissue, that are soluble in
aqueous solution at neutral pH, and components thereof that are
insoluble in aqueous solution at neutral pH. For example, the
suspension can comprise components of a whole placenta and a whole
umbilical cord; components of a whole placenta and a portion of an
umbilical cord (e.g., membrane only, membrane and Wharton's jelly
only, Wharton's jelly only, umbilical vessels only, etc.);
components of a whole umbilical cord and a portion of a placenta
(e.g., components of amniotic membrane only, chorion only, amnion
and chorion, etc.). In another embodiment, the suspension can also
comprise components of a portion of a placenta and a portion of an
umbilical cord, e.g., components of an amniotic membrane and
components of an umbilical cord membrane; components of an amniotic
membrane and components of an umbilical cord membrane and Wharton's
jelly, components of an amniotic membrane and components of
Wharton's jelly, and the like, in any combination.
[0031] In embodiments in which the suspension comprises components
of both placental tissue and umbilical cord tissue, the components,
either or both of components soluble or insoluble in aqueous
solution at neutral pH, can be present in the suspension in any
ratio. Thus, the suspension can comprise predominantly (i.e.,
greater than 50%) placental tissue components relative to umbilical
cord tissue, or predominantly umbilical cord tissue components
relative to placental tissue. In other embodiments, the suspension
comprises about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 96%, 97%, 98%, or about 99% placental
components or umbilical cord components out of total placental and
umbilical cord components in the suspension.
[0032] In another embodiment, the composition provided by the
present invention is a solution comprising components of placental
tissue and/or umbilical cord tissue that are soluble in aqueous
solution at neutral pH (i.e., about pH 7.0 to about pH 7.4), but
substantially lacking components of placental tissue and/or
umbilical cord tissue that are insoluble in aqueous solution at
neutral pH and removable by, e.g., centrifugation and/or
filtration. For example, such solutions, according to the
invention, can comprise about 0.5%, 0.1%, 0.05% or 0.01% or less
placental tissue components and/or umbilical cord tissue
components, insoluble in aqueous solution at neutral pH (i.e.,
about pH 7.0 to about pH 7.4), by weight. Thus, in one embodiment,
the invention provides a solution that comprises part or all of the
fraction of placental tissue and/or umbilical cord tissue
components that are soluble in pH-neutral aqueous solution and
lacks components of placental tissue and/or umbilical cord tissue
that are insoluble in aqueous solution at neutral pH.
[0033] In one embodiment, the solution of the invention comprises
umbilical cord tissue components, or a combination of umbilical
cord tissue components and placenta tissue components, wherein said
solution is made by a process comprising (1) contacting umbilical
cord tissue, or umbilical cord and placental tissue, with a
solution at neutral pH, (2) disrupting said tissue in said solution
to form a suspension; and (3) removing tissue components that are
insoluble at neutral pH from said suspension.
[0034] In certain embodiments, the solution comprises components of
a part of, or the whole of, only placental tissue, wherein the
components are soluble in pH-neutral aqueous solution, e.g.,
components only of a whole placenta, or components only of parts of
a placenta, such as amniotic membrane, chorion, or combination of
amniotic membrane and chorion, and the like. Similarly, the
solution can comprise components of a part of, or the whole of,
only umbilical cord tissue, that are soluble in pH-neutral aqueous
solution, e.g., components of only a whole umbilical cord, or
components only of umbilical cord membrane, Wharton's jelly,
umbilical vessels, or a combination thereof. In one embodiment, the
solution comprises components from an amniotic membrane that are
soluble in pH-neutral aqueous solution. In another embodiment, the
solution comprises components from an umbilical cord membrane in
combination with Wharton's jelly that are soluble in pH-neutral
aqueous solution. The invention comprises solutions that are
combinations of the foregoing, as well.
[0035] In certain other embodiments, the solution comprises
components of placental tissue and umbilical cord tissue that are
soluble in pH-neutral aqueous solution. For example, the solution
can comprise components, soluble in pH-neutral aqueous solution, of
a whole placenta and a whole umbilical cord; components of a whole
placenta and a portion of an umbilical cord (e.g., membrane only,
membrane and Wharton's jelly only, Wharton's jelly only, umbilical
vessels only, etc.); components of a whole umbilical cord and a
portion of a placenta (e.g., components of amniotic membrane only,
chorion only, amnion and chorion, etc.). In another embodiment, the
solution can also comprise components, soluble in pH-neutral
aqueous solution, of a portion of a placenta and a portion of an
umbilical cord, e.g., components of an amniotic membrane and
components of an umbilical cord membrane; components of an amniotic
membrane and components of an umbilical cord membrane and Wharton's
jelly, components of an amniotic membrane and components of
Wharton's jelly, and the like, in any combination.
[0036] In embodiments in which the solution comprises components of
both placental tissue and umbilical cord tissue, the components can
be present in the solution in any ratio. Thus, the solution can
comprise predominantly (i.e., greater than 50%) placental tissue
components soluble in aqueous solution relative to umbilical cord
tissue soluble in aqueous solution, or predominantly umbilical cord
tissue components soluble in aqueous solution at neutral pH
relative to placental tissue soluble in aqueous solution at neutral
pH. In other embodiments, the solution can comprise about 1%, 2%,
3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,
97%, 98%, or about 99% placental components or umbilical cord
components, soluble in aqueous solution at neutral pH, out of a
total of placental and umbilical cord components soluble in aqueous
solution at neutral pH.
[0037] In various embodiments, the solutions comprise about, no
more than, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%,
1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%
placental tissue components, umbilical cord components, or a
combination thereof, by weight. In other embodiments, the solutions
comprise at least, about, or at most 1.times.10.sup.-9,
5.times.10.sup.-9, 1.times.10.sup.-8, 5.times.10.sup.-8,
1.times.10.sup.-7, 5.times.10.sup.-7, 1.times.10.sup.-6,
5.times.10.sup.-6, 1.times.10.sup.-5, 5.times.10.sup.-5, 10.sup.-4,
5.times.10.sup.-4, 1.times.10.sup.-3, 5.times.10.sup.-3,
1.times.10.sup.-2, 5.times.10.sup.-2, or 1.times.10.sup.-1 grams of
tissue components, or a particular tissue component, or particular
type of tissue component, per gram of composition or per milliliter
of composition. Such tissue components can be, e.g., soluble
proteins such as, e.g., cytokines or growth factors.
[0038] In another embodiment, the invention provides compositions
comprising components of placental tissue and/or of umbilical cord
tissue, soluble in aqueous solution at neutral pH, wherein the
composition is a slurry, paste or gel. Generally, the components of
placental tissue and/or of umbilical cord tissue, soluble in
aqueous solution at neutral pH in the slurry or paste are the
components, soluble in aqueous solution at neutral pH, of the
solutions and suspensions described above. In various embodiments,
such compositions are about 99%, 98%, 97%, 96%, 96%, 90%, 85%, 80%,
75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,
10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or about 1%, or are at least
about, or at most about, 99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%,
80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,
15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or about 1% water by
weight. Similarly, in other embodiments, such compositions are
about 99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%, 80%, 75%, 70%, 65%,
60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%,
6%, 5%, 4%, 3%, 2% or about 1%, or is at least about, or at most
about, 99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%, 80%, 75%, 70%,
65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2% or about 1% tissue components, insoluble at
neutral pH, by weight.
[0039] The invention further provides combinations of the above
compositions. For example, a slurry, gel or paste can be combined
with a suspension; a solution of the invention can be combined with
a suspension; a solution of the composition can be combined with a
slurry, paste, gel etc. of the invention. In such embodiments, the
respective compositions can each be from the same individual, or
can be from different individuals. For example, in a combination of
a solution of the invention and a paste of the invention, the
solution can be from placental tissue and/or umbilical cord tissue
of a single individual or from a plurality of individuals, and the
paste can be placental tissue and/or umbilical cord tissue of a
single individual or from a plurality of individuals. In a
preferred embodiment, the compositions are from a single
individual. Other combinations of suspensions, solutions, slurries
and/or pastes can be from single individuals or pluralities of
individuals in like fashion.
5.1.2.2. Compositions Comprising Placental Tissue Components And/Or
Umbilical Cord Components Soluble at Acidic or Basic pH
[0040] In another embodiment, the invention provides compositions
comprising components of placental tissue and/or umbilical cord
tissue that are soluble at neutral pH and are soluble in an acidic
or basic aqueous solution, e.g., an aqueous solution having a pH of
about pH 6 or less, or a pH of about pH 8 or more. In various
embodiments, the acidic solution in which the components are
soluble is between about pH 4 and about pH 6; about pH 6.0, about
pH 5.3, about pH 5.0, about pH 4.3, about pH 4.0, about pH 3.3 or
about pH 3.0. In various other embodiment, the basic solution in
which the components are soluble is between about pH 8 and about pH
11; about pH 8.0, about ph 8.3, about pH 9.0, about pH 9.3, about
pH 10, about pH 10.3, or about pH 11.
[0041] In one embodiment, the composition of the invention
comprising such placental tissue and/or umbilical cord tissue
components, soluble in an neutral and acidic or neutral and basic
aqueous solution, is a suspension of placental tissue and/or
umbilical cord tissue. Such a suspension comprises, e.g., placental
tissue and/or umbilical cord tissue that has been disrupted or
homogenized in aqueous solution such that the suspension comprises
placental tissue and/or umbilical cord tissue components that are
soluble in neutral and acidic or neutral basic aqueous solution,
and components that are insoluble in aqueous neutral and acidic or
neutral and basic solution.
[0042] In one embodiment, the invention provides a composition
comprising placental and/or umbilical cord tissue components that
are soluble neutral and acidic or neutral basic aqueous solution,
and components that are insoluble in aqueous neutral and acidic or
neutral and basic solution, wherein said composition is made by a
process comprising the steps of disrupting placental and/or
umbilical cord tissue components in aqueous solution at neutral pH
to form a suspension, acidifying or basifying the suspension, and
then bringing said composition to neutral pH.
[0043] In another embodiment, the composition comprises placental
and/or umbilical cord tissue components that are soluble in acidic
and neutral, or basic and neutral, aqueous solution, and components
that are insoluble in neutral and acidic, or neutral and basic,
aqueous solution, is made by a process comprising the steps of
disrupting placental and/or umbilical cord tissue components in
acidic or basic aqueous solution to form a suspension, and bringing
said suspension to a neutral pH.
[0044] In certain embodiments, the suspension comprises components,
both soluble and insoluble in acidic or basic aqueous solution, of
a part of, or the whole of, only placental tissue, e.g., components
of a whole placenta, or components only of parts of a placenta,
such as amniotic membrane, chorion, or combination of amniotic
membrane and chorion, and the like. Similarly, the suspension can
comprise components of a part of, or the whole of, only umbilical
cord tissue, e.g., components of a whole umbilical cord, or
components only of umbilical cord membrane, Wharton's jelly,
umbilical vessels, or a combination thereof. In one embodiment, the
suspension comprises components from an amniotic membrane. In
another embodiment, the suspension comprises components from an
umbilical cord membrane in combination with Wharton's jelly. The
invention comprises suspension that are combinations of the
foregoing, as well.
[0045] In other embodiments, the invention provides a suspension
that comprises components of both placental tissue and umbilical
cord tissue that are soluble in acidic or basic aqueous solution,
and components that are insoluble in acidic or basic aqueous
solution. For example, the suspension can comprise components of a
whole placenta and a whole umbilical cord; components of a whole
placenta and a portion of an umbilical cord (e.g., membrane only,
membrane and Wharton's jelly only, Wharton's jelly only, umbilical
vessels only, etc.); components of a whole umbilical cord and a
portion of a placenta (e.g., components of amniotic membrane only,
chorion only, amnion and chorion, etc.). In another embodiment, the
suspension can also comprise components of a portion of a placenta
and a portion of an umbilical cord, e.g., components of an amniotic
membrane and components of an umbilical cord membrane; components
of an amniotic membrane and components of an umbilical cord
membrane and Wharton's jelly, components of an amniotic membrane
and components of Wharton's jelly, and the like, in any
combination.
[0046] In embodiments in which the suspension comprises components
of both placental tissue and umbilical cord tissue, the components,
either or both of components soluble or insoluble in acidic or
basic aqueous solution, can be present in the suspension in any
ratio. Thus, the suspension can comprise predominantly (i.e.,
greater than 50%) placental tissue components relative to umbilical
cord tissue, or predominantly umbilical cord tissue components
relative to placental tissue. In other embodiments, the suspension
comprises about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 96%, 97%, 98%, or about 99% placental
components and/or umbilical cord components out of total placental
and/or umbilical cord components in the suspension.
[0047] In another embodiment, the composition provided by the
present invention is a solution comprising components of placental
tissue and/or umbilical cord tissue that are soluble in basic or
acidic aqueous solution. Generally, the solution lacks components
of placental tissue and/or umbilical cord tissue that are insoluble
in basic or acidic aqueous solution and removable by, e.g.,
centrifugation and/or filtration. For example, such solutions,
according to the invention, comprise 0.5%, 0.1%, 0.05% or 0.01% or
less placental tissue components and/or umbilical cord tissue
components, insoluble in acidic or basic aqueous solution, by
weight. Thus, in one embodiment, the invention provides a solution
that comprises part or all of the fraction of placental tissue
and/or umbilical cord tissue components that are soluble in aqueous
solution and lacks components of placental tissue and/or umbilical
cord tissue that are insoluble in acidic or basic aqueous
solution.
[0048] In one embodiment, the invention provides a composition
comprising placental and/or umbilical cord tissue components that
are soluble neutral and acidic or neutral basic aqueous solution,
and components that are insoluble in aqueous neutral and acidic or
neutral and basic solution, wherein said composition is made by a
process comprising the steps of (1) disrupting placental and/or
umbilical cord tissue components in aqueous solution at neutral pH
to form a suspension; (2) acidifying or basifying the suspension,
(3) bringing said composition to neutral pH; and (4) removing
tissue components insoluble in said aqueous solution at acidic and
neutral pH, or at basic and neutral pH.
[0049] In certain embodiments, the solution comprises components of
a part of, or the whole of, placental tissue, wherein the
components are soluble in acidic or basic aqueous solution, e.g.,
components of a whole placenta, or components only of parts of a
placenta, such as amniotic membrane, chorion, or combination of
amniotic membrane and chorion, and the like. Similarly, the
solution can comprise components of a part of or the whole of,
umbilical cord tissue, that are soluble in acidic or basic aqueous
solution, e.g., components of a whole umbilical cord, or components
only of umbilical cord membrane, Wharton's jelly, umbilical
vessels, or a combination thereof. In one embodiment, the solution
comprises components from an amniotic membrane that are soluble in
acidic or basic aqueous solution. In another embodiment, the
solution comprises components from an umbilical cord membrane in
combination with Wharton's jelly that are soluble in aqueous
solution. The invention comprises solutions that are combinations
of the foregoing, as well.
[0050] In certain other embodiments, the invention provides a
solution that comprises components of placental tissue and
umbilical cord tissue that are soluble in acidic or basic aqueous
solution. For example, the solution can comprise components,
soluble in acidic or basic aqueous solution, of a whole placenta
and a whole umbilical cord; components of a whole placenta and a
portion of an umbilical cord (e.g., membrane only, membrane and
Wharton's jelly only, Wharton's jelly only, umbilical vessels only,
etc.); components of a whole umbilical cord and a portion of a
placenta (e.g., components of amniotic membrane only, chorion only,
amnion and chorion, etc.). In another embodiment, the solution can
also comprise components, soluble in acidic or basic aqueous
solution, of a portion of a placenta and a portion of an umbilical
cord, e.g., components of an amniotic membrane and components of an
umbilical cord membrane; components of an amniotic membrane and
components of an umbilical cord membrane and Wharton's jelly,
components of an amniotic membrane and components of Wharton's
jelly, and the like, in any combination.
[0051] In embodiments in which the solution comprises components of
both placental tissue and umbilical cord tissue, the components can
be present in the solution in any ratio. Thus, the solution can
comprise predominantly (i.e., greater than 50%) placental tissue
components soluble in acidic and neutral, or basic and neutral,
aqueous solution relative to umbilical cord tissue soluble in said
solution, or predominantly umbilical cord tissue components soluble
in acidic and neutral, or basic and neutral, aqueous solution
relative to placental tissue soluble in said aqueous solution. In
other embodiments, the solution comprises about 1%, 2%, 3%, 4%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or
about 99% placental components and/or umbilical cord components,
soluble in acidic and neutral, or basic and neutral, aqueous
solution, out of a total of placental components and umbilical cord
components soluble in said aqueous solution.
[0052] In various embodiments, the solutions comprise no more than
about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%,
1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%
placental tissue components, umbilical cord components, or a
combination thereof, by weight. In other embodiments, the solutions
comprise at least, about, or at most 1.times.10.sup.-9,
5.times.10.sup.-9, 1.times.10.sup.-8, 5.times.10.sup.-8,
1.times.10.sup.-7, 5.times.10.sup.-7, 1.times.10.sup.-6,
5.times.10.sup.-6, 1.times.10.sup.-5, 5.times.10.sup.-5, 10.sup.-4,
5.times.10.sup.-4, 1.times.10.sup.-3, 5.times.10.sup.-3,
1.times.10.sup.-2, 5.times.19.sup.-2, or 1.times.10.sup.-1 grams of
tissue components, or a particular tissue component, or particular
type of tissue component, per gram of composition or per milliliter
of composition. Such tissue components can be, e.g., soluble
proteins such as, e.g., cytokines or growth factors.
[0053] In another embodiment, the invention provides compositions
comprising components of placental tissue and/or of umbilical cord
tissue, soluble in acidic or basic aqueous solution, wherein the
composition is a slurry, paste or gel. Generally, the components of
placental tissue and/or of umbilical cord tissue, soluble in acidic
or basic aqueous solution in the slurry or paste are the
components, soluble in acidic or basic aqueous solution, of the
solutions and suspensions described above. In various embodiments,
such compositions are about 99%, 98%, 97%, 96%, 96%, 90%, 85%, 80%,
75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,
10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or about 1%, or are at least
about, or at most about, 99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%,
80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,
15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or about 1% water by
weight. Similarly, in other embodiments, such compositions are
about 99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%, 80%, 75%, 70%, 65%,
60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%,
6%, 5%, 4%, 3%, 2% or about 1%, or is at least about, or at most
about, 99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%, 80%, 75%, 70%,
65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2% or about 1% tissue components, insoluble at
acidic and neutral pH, or insoluble at basic and neutral pH, by
weight.
[0054] The invention further provides combinations of the above
compositions. For example, a slurry, gel or paste can be combined
with a homogenate; a solution can be combined with a homogenate; a
solution can be combined with a slurry, paste, gel, etc. In such
embodiments, the respective compositions can each be from the same
individual, or can be from different individuals. For example, in a
combination of a solution of the invention and a paste of the
invention, the solution can be from placental tissue and/or
umbilical cord tissue of a single individual or from a plurality of
individuals, and the paste can be placental tissue and/or umbilical
cord tissue of a single individual or from a plurality of
individuals. In a preferred embodiment, the compositions are from a
single individual. Other combinations of suspensions, solutions,
slurries and/or pastes can be from single individuals or
pluralities of individuals in like fashion.
5.1.2.3. Other Compositions of the Invention
[0055] The invention further provides compositions comprising
placental tissue and/or umbilical cord tissue components soluble at
neutral pH, e.g., obtained by disruption of tissue at neutral pH,
in combination with placental tissue and/or umbilical cord
components soluble at acidic and neutral pH or basic and neutral
pH, e.g., obtained by acidification or basification and subsequent
neutralization of tissue. These compositions comprise a suspension,
solution, slurry, gel or paste, comprising such components soluble
in aqueous solution at neutral pH in combination with a suspension,
solution, slurry, gel or paste comprising such components soluble
in acidic or basic solution. For example, a suspension of the
invention comprising such components soluble at neutral pH can be
combined, in any ratio, with a suspension of the invention
comprising such components soluble at acidic or basic pH.
Similarly, a solution of the invention comprising such components
soluble at neutral pH can be combined, in any ratio, with a
solution of the invention comprising such components soluble at
acidic or basic pH.
5.1.3. Methods of Making Compositions of The Invention
[0056] The invention further provides methods of making the
compositions of the present invention.
5.1.3.1. Suspensions and Solutions
[0057] In one embodiment, the invention provides a method of making
a suspension from placental tissue, umbilical cord tissue, or a
combination thereof, comprising disrupting, e.g., homogenizing,
such tissue in aqueous solution at neutral pH. Disruption of
placental tissue, umbilical cord tissue, or combination thereof,
can be accomplished by any art-recognized method of disrupting
tissue, such as maceration, homogenization, use of a blender,
sonication, and the like. Preferably, during disruption, the
temperature of the suspension is kept below room temperature, e.g.,
below 25.degree. C., more preferably below about 10.degree. C., and
more preferably between about 10.degree. C. and the freezing point
of the solution in which the placental tissue or umbilical cord
tissue is disrupted.
[0058] The aqueous solution in which the tissue is disrupted can
comprise one or more compositions that act to reduce protein
degradation. For example, the solution can comprise one or more
protease inhibitors. The solution can also comprise one or more
antioxidants, e.g., thiourea, sodium bisulfite, sodium
metabisulfite, and the like. The solution can comprise one or more
chelators, e.g., EDTA and/or EGTA.
[0059] The time for which the placenta, umbilical cord, placental
tissue, umbilical cord tissue, or combination thereof, is contacted
with a pH-neutral aqueous solution affects the concentration of the
components soluble in aqueous solution, with a longer contact time
generally leading to a more concentrated solution. Such contact can
take place for, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, 50 or 55 minutes, or about 1, 1.5, 2, 2.5, 3,
3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or about 1, 2,
3, 4, 5, 6 days. The degree of disruption can also affect the
concentration of such components.
[0060] In another embodiment, the invention provides a method of
preparing a solution comprising placental or umbilical membrane
components soluble at neutral pH, comprising contacting placental
tissue, umbilical cord tissue, or combination thereof, with a
pH-neutral aqueous solution; disrupting the placental tissue,
umbilical cord tissue, or combination thereof in said solution to
form a suspension, and removing substantially all of the solid
material in said suspension to form a solution. Generally, the
solution is made after making the suspension, as described above.
As used herein, "substantially all solid material" means at least
99%, preferably at least 99.5%, more preferably 99.9% of the weight
of the starting placental or umbilical cord material used to make
the suspension. In one embodiment, the method comprises disrupting
umbilical cord membrane comprising Wharton's jelly in the solution.
In other embodiments, a combination of umbilical cord membrane and
placental tissue, e.g., amniotic membrane, is used to make the
solution. In other embodiments the solution is made with placental
tissue only. In embodiments in which both umbilical cord tissue and
placental tissue are used, the umbilical cord tissue and placental
tissue are from the same individual. In other embodiments in which
both umbilical cord tissue and placental tissue are used, the
placental tissue and umbilical tissue are from different
individuals.
[0061] The solution used for disruption of placental tissue,
umbilical cord tissue, or combination thereof can be any
pharmaceutically-acceptable solution pH-neutral, e.g., a solution
suitable for ocular, topical or internal use. Such solutions
include, e.g., sterile, deionized water, a buffer solution (e.g.,
phosphate-buffered saline, carbonate buffer, or the like), a saline
solution (e.g., a 0.9% saline solution), or the like.
[0062] In the method of making a solution of the invention, various
aspects can be altered to change the concentration of the
components, soluble in aqueous solution, present in the final
solution. For example, the ratio of the weight of placenta and/or
umbilical cord material to volume of solution, in which the tissue
is disrupted, can be varied; a low ratio will produce a solution
with a relatively low concentration of components that are soluble
in aqueous solution, and a high ratio will produce a solution with
a relatively high concentration of components, soluble in aqueous
solution, in the final solution. In one embodiment, for example,
the ratio of aqueous solution to wet solid (that is, wet placental
tissue and/or umbilical cord tissue) is between about 1:100 and
about 1000:1, preferably between about 1:20 and about 600:1. In
various embodiments, for example, the ratio of solution to wet
solid weight (w/w) is about 1:100, 1:90, 1:80, 1:70, 1:60, 1:50,
1:40, 1:30, 1:20, 1:15, 1:10, 1:5, 1:1, 5:1, 10:1, 15:1, 20:1,
25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1,
125:1, 150:1, 175:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1,
500:1, 550:1, 600:1, 700:1, 800:1, 900:1 or about 1000:1. In
another embodiment, for example, a placental tissue or umbilical
cord tissue is disrupted in about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
liters of solution. In specific embodiments, the placental tissue
is amniotic membrane, chorion, or amnion and chorion. In another
specific embodiment, the amniotic membrane, chorion, or amnion and
chorion are from a single placenta. In another specific embodiment,
the amniotic membrane, chorion, or amnion and chorion are from a
plurality of placentas. In another specific embodiment, the
umbilical cord tissue is umbilical cord membrane, Wharton's jelly,
or umbilical vessels, or a combination thereof. As for the
placental tissue, the umbilical cord tissue can be derived from a
single umbilical cord or a plurality of umbilical cords. In
particular embodiments, the ratio of weight of placental tissue,
umbilical cord tissue or combination thereof to the volume of
solution is about 1:1 to about 1:10.
[0063] Removal of the solid components of the suspension can be
accomplished by any means known to those of skill in the art,
including, e.g., centrifugation or filtration. Centrifugation can
be accomplished using any commercially-available centrifugation
apparatus; preferably, the centrifuge accepts bottles or other
containers able to contain 100 ml or more of solution to facilitate
batch processing or clarification of larger volumes of suspension.
Similarly, filtration can be accomplished using any art-recognized
method, such as forced filtration or vacuum filtration. Preferably
a filtration apparatus used to accomplish such filtration has the
capacity to process at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 liters
or more of solution per hour, and comprises a filter that does not
adsorb biomolecules. In embodiments, filtration or centrifugation
removes suspended matter in the suspension wherein the suspended
matter is larger than about 500 microns, 250 microns, 200 microns,
100 microns, 75 microns, 50 microns or 25 microns.
[0064] The invention further encompasses a pH shifting method of
preparing a suspension of the invention comprising contacting
placental tissue and/or umbilical cord tissue with an acidic or
basic aqueous solution to solubilize tissue components that are
soluble in acidic or basic aqueous solution. The method generally
comprises, in either order, disruption, e.g., homogenization of
placental and/or umbilical cord tissue and contacting the tissue
with an acidic or basic solution; allowing the solution to disrupt
the tissue; and shifting the pH of the solution to neutral pH.
[0065] In one embodiment of the pH shifting method, placental
tissue, umbilical cord tissue, or combination thereof, is contacted
with an acidic or basic solution and disrupted, e.g., homogenized,
as described above, to produce a suspension. The tissue can either
be contacted with the acidic or basic solution and disrupted, or
disrupted at neutral pH and then contacted with an acidic or basic
solution, e.g., the solution in which the tissue is disrupted can
be acidified or basified. Preferably, the solution with which the
placental tissue or umbilical tissue is contacted is buffered so
that acidic or basic pH is easily maintained. The resulting
suspension is then, after a period of time, brought to neutral pH
(that is, about pH 7.0 to about pH 7.4).
[0066] The acid used in this method can be any acid, and can be,
e.g., hydrochloric acid, phosphoric acid, acetic acid, sulfuric
acid, picric acid, iodic acid, periodic acid, hydroiodic acid,
bromic acid, hydrobromic acid, perbromic acid, hydrofluoric acid,
chloric acid, perchloric acid, nitric acid, and the like. Acetic
acid and HCl are preferred. Extremely strong acids, such as
fluoroantimonic acid, magic acid or fluorosulfuric acid can also be
used, but are not preferred. Weak acids can also be used, such as
nicotinic acid, salicylic acid, pyruvic acid, or ethanoic acid. The
base can be any base, e.g., sodium hydroxide, sodium acetate,
ammonium hydroxide, potassium hydroxide, barium hydroxide, cesium
hydroxide, strontium hydroxide, calcium hydroxide or lithium
hydroxide. Extremely strong bases can also be used, e.g., sodium
hydride, lithium diispropylamide or lithium amide. Sodium hydroxide
and sodium acetate are preferred bases.
[0067] In certain embodiments, an amount of acid or base is used to
acidify the solution to between about pH 4 and about pH 6, or to
basify the solution to between about pH 8 and about pH 10. In other
embodiments, the solution is acidified to about pH 6.0, about pH
5.3, about pH 5.0, about pH 4.3, about pH 4.0, about pH 3.3 or
about pH 3.0. In other embodiments, the solution is basified to
about pH 8.0, about ph 8.3, about pH 9.0, about pH 9.3, about pH
10, about pH 10.3, or about pH 11.
[0068] The strength (e.g., normality or molarity) of the base or
acid used is not critical; however, a dilute acid or base is
preferable to a more concentrated base or acid so as to avoid local
concentrations of acid or base. during acidification or
basification, that are potentially damaging, e.g., to aqueous
solution-soluble proteins. In various embodiments, an acid or base
used to acidify or basify the solution is between about 0.1N and
about 6N acid or base, or between about 0.1M and about 6M acid or
base. In a specific embodiment, the acid is about IN or about
1M.
[0069] The placental or umbilical cord tissue, or combination
thereof, is contacted with the acidic or basic solution for a time
sufficient for the solution to cause a disruption of the placental
or umbilical cord tissue over and above that caused by physical
disruption of the tissue. For example, such additional disruption
can occur when the placental or umbilical cord tissue is contacted
with the acidic or basic solution for at least about 1, 2, 3, 4, 5,
10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes, or for at least
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22 or 23 hours, or for at least about 1, 2, 3 or
more days. Preferably, the placental tissue and/or umbilical cord
tissue is disrupted at a temperature of between about the freezing
point of the solution and about 10.degree. C.
[0070] Neutralization of the acidic or basic homogenate can be
accomplished by any means known in the art. For example, an acidic
solution can be brought to neutral pH by the addition of an
appropriate amount of, e.g., NaOH. A basic solution can be
neutralized by the addition of an appropriate amount of, e.g.,
hydrochloric acid. In certain embodiments, the base chosen to
neutralize an acidic solution, or the acid chosen to neutralize a
basic solution, results in the formation of a water-soluble salt.
Preferred acid/base pairs include HC1 and NaOH; HAc and NaOH; and
HAc and NaAc.
[0071] The resulting suspension can be used without further
modification. In an embodiment in which the suspension is to be
used for a medical purpose, the suspension is treated to remove
concentrations of salt above physiological, e.g., by dialysis or by
passage over a desalting column.
[0072] The invention further provides a method of making a solution
of the invention. In one embodiment, a suspension made as described
above by the pH shifting method can be clarified, for example, by
centrifugation and/or filtration to substantially remove the solid
components of the suspension, thereby producing a solution of the
invention. In certain embodiments, filtration or centrifugation
removes particulate matter in the suspension wherein the
particulate matter is larger on average than about 500 microns, 250
microns, 200 microns, 100 microns, 75 microns, 50 microns or 25
microns in diameter. The resulting solution comprises components
(that is, proteins, lipids, glycolipids, glycoproteins, and the
like) of placental tissue, umbilical cord tissue or a combination
thereof, wherein the components are soluble in an acidic solution
or a basic solution, and substantially lacks placental tissue or
umbilical tissue components insoluble in an acidic or basic
solution.
[0073] Thus, in one embodiment, the invention provides a method of
preparing a composition, comprising: (a) in either order: (i)
contacting placental tissue, umbilical cord tissue or a combination
thereof, with an acidic or basic solution; (ii) disrupting the
placental tissue, umbilical cord tissue or a combination thereof in
said solution to form a suspension; (b) bringing the suspension to
neutral pH; and (c) removing particulate matter within said
suspension. In another embodiment, the invention provides a method
of preparing a composition, comprising (a) contacting placental
tissue, umbilical cord tissue, or a combination thereof, with an
acidic or basic solution; (b) disrupting the tissue in said
solution to form a suspension; and (c) bringing the suspension to a
neutral pH. In a specific embodiment of the latter method, the
method additionally comprises removing particulate matter within
said suspension after step (c). In a specific embodiment of either
method, the acidic solution is an acetic acid solution. In another
specific embodiment, the acidic solution is between about pH 3.0
and about pH 6.0. In another specific embodiment, the acidic
solution is between about pH 4.5 and about pH 6.0. In another
specific embodiment, said basic solution is a sodium hydroxide
solution. In another specific embodiment, said basic solution is
between about pH 8.0 and pH 11.0. In another specific embodiment,
said basic solution is between about pH 80. and pH 9.5. In another
specific embodiment, said disrupting is performed at a temperature
of between about 10.degree. C. and the freezing point of the
solution in which said tissue is disrupted.
5.1.3.2. Slurries, Pastes and Gels
[0074] The invention further provides methods of making slurries,
gels and pastes from the placental tissue components and/or
umbilical tissue components described above.
[0075] The water content of any of the compositions described in
Section 4.1.2, above, in certain embodiments, is adjusted, e.g.,
the water content increased or reduced. In embodiments in which the
water content is reduced, water can be removed from the composition
by any method known to the art. For example, water can be removed
by evaporation or lyophilization. Water can be removed from the
solution such that the water content of the composition is about
99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,
55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2% or about 1%, or is at least about, or at most about,
99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,
55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2% or about 1% water by weight. Similarly, water can be
removed from the composition such that the solid content of the
composition is about 99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%, 80%,
75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,
10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or about 1%, or is at least
about, or at most about, 99.5%, 99%, 98%, 97%, 96%, 96%, 90%, 85%,
80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,
15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or about 1% solid by
weight.
[0076] The invention, in one embodiment, provides pastes comprising
placental components, umbilical cord components, or a combination
thereof, that are soluble in aqueous solution (either at neutral
pH, or at acidic and neutral pH or basic and neutral pH). In a
specific embodiment, water is removed from a solution made from the
solution made from placental tissue, umbilical tissue or a
combination thereof, described above, until the remaining placental
or umbilical cord material becomes paste-like. As water is removed
from the composition, solutes, such as proteins, start to
precipitate to form a slurry. As the water content of the
composition is reduced below about 10%, the composition becomes
paste-like. In one embodiment, water is removed until the water
content of the composition is between about 1% and about 20%,
preferably between about 3% and about 10%. In another specific
embodiment, the solution comprising placental tissue components,
umbilical cord components or a combination thereof can be
substantially dehydrated, that is, dehydrated until further water
cannot be removed or until the remaining material in solution
appears dry, e.g., until the water content of the material is about
1% or less.
[0077] Dehydration of the solution can be accomplished by any
method known in the art, such as vacuum drying or lyophilization.
Preferably, vacuum drying is carried out at a temperature of
between about 10.degree. C. and the freeing point of the solution,
e.g., about 4.degree. C. Vacuum drying is preferred for larger
volumes of solution, e.g., volumes of 100 ml and larger.
Lyophilization can be accomplished by any art-accepted method; see,
e.g., U.S. Pat. No. 4,001,944. For example, the clarified solution
can be quickly frozen in 100% ethanol and dry ice, then lyophilized
at -20.degree. C. in a sterile lyophilizer until dry.
[0078] Once substantially dehydrated, the material can, for
example, be reconstituted into a paste or solution by adding the
appropriate amount of liquid, e.g., water, buffer, saline solution,
and the like. In one embodiment, the dried water-soluble placental
material or umbilical cord material can be supplemented with
another composition, preferably a composition present in, or
derived from, a collagen-containing tissue. For example, the dried
placental material or umbilical cord material, soluble in aqueous
solution, can be supplemented with, e.g., a collagen composition
such as purified collagen, dried and pulverized collagen-containing
tissue, e.g., pericardium, dura mater, skin, amniotic membrane,
umbilical cord membrane, intestine, and the like. In one
embodiment, placental and/or umbilical cord tissue components
supplemented by an amount of purified collagen not to exceed, e.g.,
100 times the weight of the amniotic membrane material obtained
from the amniotic membrane solution, and water is added so as to
achieve between 3% and 10% water by weight. The water, tissue
material and purified collagen are thoroughly mixed to form a
paste.
[0079] In another embodiment, the placental material or umbilical
cord material that is soluble in aqueous solution is supplemented
with placental or umbilical cord material, insoluble in soluble in
aqueous solution (neutral, acidic or basic) removed, e.g., by
centrifugation and/or filtration during production of the solution,
as described above. In certain embodiments, supplementation using
such material results in the formation of a composition having
relatively less or more insoluble material than is present in the
placental or umbilical cord suspension from which the material,
soluble in pH neutral, or acidic or basic, aqueous solution, was
originally obtained.
[0080] In various embodiments, the ratio of the weight of the
soluble placental tissue or umbilical tissue components to the
supplemental materials is, e.g., about 1:1, 1:2, 1:3, 1:4, 1:5,
1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70,
1:80, 1:90 or about 1:100. The dried soluble placental material or
umbilical cord material can also be supplemented with other
biomolecules, e.g., fibronectin, laminin, or other extracellular
matrix proteins; other proteins such as cytokines, growth factors,
and the like; lipids, glycoproteins, glycolipids, hyaluronic acid,
glycosaminoglycans, and the like.
[0081] The dried water-soluble placental material or umbilical cord
material, alone or supplemented as described above, can, for
example, be combined with a liquid, e.g., water, buffer, saline
solution, or the like, to achieve a water content of between about
3% and about 10% to form a paste.
[0082] In another embodiment, the invention provides gels
comprising, e.g., any of the suspensions, solutions, or soluble
components of placental tissue or umbilical cord tissue of the
invention. In one embodiment, the gel comprises placental material
and/or umbilical cord material that is soluble in aqueous solution,
supplemented with placental or umbilical cord material, insoluble
in soluble in aqueous solution (neutral, acidic or basic) removed,
e.g., by centrifugation and/or filtration during production of the
solution, as described above. In certain embodiments,
supplementation using such material results in the formation of a
composition having relatively less or more insoluble material than
is present in the placental or umbilical cord suspension from which
the material, soluble in pH neutral, or acidic or basic, aqueous
solution, was originally obtained.
[0083] In one embodiment, a gelling compound is added in a
weight/weight ratio to a composition of the invention at a
weight/weight or weight/volume ratio of about 1:10 to about 10:1.
In another embodiment, a gelling compound is added in an amount
such that the water content of the resulting gel to about 60% to
about 99% by weight In embodiments in which the composition is
dehydrated, water is then added to the composition and gelling
compound in an amount that brings the water content of the
resulting gel to about 60% to about 99% by weight. In various
embodiments, the ratio in the gel of the weight of the soluble
placental tissue or umbilical tissue components to the supplemental
materials is, e.g., about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,
1:9, 1:10, 1:15, 1:20 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or
about 1:100. The dried soluble placental material or umbilical cord
material can also be supplemented with other biomolecules, e.g.,
fibronectin, laminin, or other extracellular matrix proteins; other
proteins such as cytokines, growth factors, and the like; lipids,
glycoproteins, glycolipids, hyaluronic acid, glycosaminoglycans,
and the like.
[0084] Gelling agents, and methods of making gels from solutions,
are well-known in the art. Examples of gelling agents that can be
used to make the gels of the invention include, but are not limited
to, hydroxymethylcellulose, hydroxyethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, polyvinyl alcohol,
polyvinylpyrrolidone, pectin, agar, alginic acid, alginate,
amylose, high amylose starch, gum arabic, carrageenan, processed
euchema seaweed, casein, carboxymethyl cellulose, carboxyvinyl
copolymer, hydroxypropylcellulose, microcrystalline cellulose,
sodium carboxymethyl cellulose, natural celluloses, chitin,
chitosan, collagen, dextran, polydextran, elsinan, gelatin, gellan
gum, guar gum, gelatin, ghatti gum, karaya gum, gluten, konjac,
levan, locust bean gum, maltodextrin, methylmethacrulate copolymer,
oat gum, pectin, low methoxy pectin, polyethylene glycol,
polylysine, polybrene, polyacrylic acid, propylene glycol, protein,
pullulan, starch, modified starches, soy protein, tara gum,
tamarind gum, tragacanth, whey protein, xanthan gum, zein, and the
like. Typically, the gelling agents are employed in amount of about
0.3 to about 30 weight percent.
[0085] In certain embodiments, the gel of the invention comprises a
crosslinking agent. In a specific embodiment, the crosslinking
agent is riboflavin. In another specific embodiment, the matrix of
the gel comprising the riboflavin comprises collagen. The
riboflavin can be used, e.g., to crosslink collagen proteins in a
collagen-containing gel, e.g., to improve or increase the
mechanical strength of the gel. Such gels would be useful in, e.g.,
wound healing, wherein the wound to be healed experiences
mechanical forces such as, e.g., stretching, rotation, and the
like.
[0086] In certain embodiments, the invention provides a method of
making a gel, comprising contacting a gel of the invention with
riboflavin, wherein said gel comprises a matrix comprising
collagen, and exposing the gel to ultraviolet light (UVA) for a
time and in an amount sufficient for a plurality of the collagen
molecules in the gel to become crosslinked. The gel comprising the
collagen matrix to be crosslinked can be produced so as to include
riboflavin during manufacture. The riboflavin can also be added to
the gel at any time prior to crosslinking, e.g., immediately before
crosslinking, e.g., by immersion of the gel in a solution of
riboflavin. Such a riboflavin solution can be about 0.01% to about
1% riboflavin, e.g., about 0.1% riboflavin (weight/volume). As
riboflavin is sensitive to light, the gel comprising riboflavin is
preferably handled or processed in the dark prior to crosslinking.
In certain embodiments, the gel comprising riboflavin are
crosslinked by exposure to UVA light for about 10 to about 60
minutes, about 20 to about 40 minutes, or for about 30 minutes. In
certain other embodiments, the maximum irradiance for crosslinking
is about 2.0 mW/cm.sup.2, about 2.5 mW/cm.sup.2, or about 3.0
mw/cm.sup.2.
[0087] Other compositions of the invention, to the extent they
comprise collagens, can be treated with riboflavin in the same
manner to crosslink the collagen proteins.
5.1.4. Formulations
[0088] The compositions described above can be formulated for use
in any medical context.
[0089] Solutions made from placental tissue or umbilical cord
tissue as described herein can, for example, be formulated as eye
drops. In one embodiment, the solution used to make eye drops is a
solution obtained from disruption, e.g., homogenization, of
placental tissue and/or umbilical cord tissue, wherein the solution
comprises placental tissue components, umbilical cord components,
or both, that are soluble in aqueous solution (either at neutral
pH, or at basic and neutral pH or acidic and neutral pH), and
substantially lacks placental tissue components and/or umbilical
cord components that are insoluble in said aqueous solution. In a
specific embodiment, the components substantially lack acid- or
base-insoluble placental tissue components or umbilical cord tissue
components. In yet another embodiment, the placental tissue is
amniotic membrane. In another more specific example, the umbilical
cord tissue is umbilical cord membrane and Wharton's jelly.
[0090] The solutions described herein, used as eye drops, can
comprise other compounds suitable for ocular use. For example, in
one embodiment, the solution can comprises an
ophthalmologically-acceptable liquid or solution. In another
embodiment, the solution comprises saline solution, glycerin,
hypromellose, or polyethylene glycol, or a combination of any of
the foregoing. In a more specific embodiment, the solution
comprises saline solution, glycerin, hypromellose, and polyethylene
glycol. In another specific example, the solution comprises a
lubricant. In a more specific example, the lubricant is
hydrophobic. In another specific example, the lubricant is
hydrophilic. In another specific example, the solution comprises an
antibiotic, an analgesic, and anti-inflammatory compound.
[0091] In other embodiments, the compositions of the invention,
e.g., the solutions provided herein, or the soluble placental
tissue components, umbilical tissue components or combination
thereof in the solutions, can be formulated into creams, ointments
or other medically-useful forms, including transdermal, topical,
and mucosal forms including, but not limited to, sprays, aerosols,
creams, lotions, ointments, emulsions, or other forms. Such forms,
and methods of preparing such forms, are known to those of skill in
the art. See, e.g., Remington: The Science and Practice of
Pharmacy, 20th ed., Lippincott Williams & Wilkins, Baltimore,
Md. (2000); and Introduction to Pharmaceutical Dosage Forms, 4th
ed., Lea & Febiger, Philadelphia (1985). Forms suitable for
contacting mucosal tissues within the oral cavity can be formulated
as mouthwashes or as oral gels.
[0092] Suitable excipients (e.g., carriers and diluents) and other
materials that can be used to provide transdermal, topical, and
mucosal forms encompassed by this invention are well known to those
skilled in the pharmaceutical arts, and depend on the particular
tissue to which a given form will be applied. Typical excipients
include, but are not limited to, water, acetone, ethanol, ethylene
glycol, propylene glycol, butane-1,3-diol, isopropyl myristate,
isopropyl palmitate, mineral oil, and mixtures thereof to form
lotions, tinctures, creams, emulsions, gels or ointments, which are
non-toxic and pharmaceutically acceptable. Moisturizers or
humectants can also be added to pharmaceutical compositions and
dosage forms if desired. Examples of such additional ingredients
are well known in the art. See, e.g., Remington: The Science and
Practice of Pharmacy, 20th ed., Lippincott Williams & Wilkins,
Baltimore, Md. (2000).
[0093] Sterilization of the compositions of the invention can be
accomplished by any means known in the art for sterilizing similar
compositions; however, sterilization methods that generally do not
deactivate proteins or polypeptides are preferred. For example, the
solutions of the invention can be sterilized by, e.g., filtration.
Such a filter preferably excludes at least bacteria, and preferably
excludes bacteria and viruses, e.g., a filter having a pore size of
less than about 0.45 microns or a filter having a pore size of less
than about 20 nanometers. To the extent that the gels, slurries
and/or pastes of the present invention are made from the solutions
of the invention and additional components, the solutions can be
filtered, and the additional components can be sterilized, e.g., by
filtration, heat, radiation, sterilizing chemical, etc., as
appropriate for the component.
[0094] Sterility of the compositions of the invention is greatly
enhanced by topical sterilization of placental and/or umbilical
cord tissues used to prepare the compositions of the invention. For
example, placental and/or umbilical cord tissues can be thoroughly
swabbed with a disinfectant such as 70% alcohol during collection
and processing steps prior to making the compositions of the
invention.
5.1.5. Bioactive Compounds
[0095] The compositions of the invention can comprise one or more
bioactive or medicinal compounds, such as small organic molecules
(e.g., drugs), antibiotics, antiviral agents, antimicrobial agents,
anti-inflammatory agents, antiproliferative agents, cytokines,
enzyme or protein inhibitors, antihistamines, and the like. In
various embodiments, the compositions may comprise antibiotics
(such as Clindamycin, Minocycline, Doxycycline, Gentamycin),
hormones, growth factors, anti-tumor agents, anti-fungal agents,
anti-viral agents, pain medications (including XYLOCAINE.RTM.,
Lidocaine, Procaine, Novocaine, etc.), antihistamines (e.g.,
antazoline, azelastine, diphenhydramine, BENADRYL.RTM., emedastine,
levocabastine, phenylephrine HCL, naphazoline HCL, toxymetazoline
HCL, tetrahydrozoline HCL, etc.), anti-inflammatory agents,
anti-infectives including but not limited to silver (such as silver
salts, including but not limited to silver nitrate and silver
sulfadiazine), elemental silver, antibiotics, bactericidal enzymes
(such as lysozome), wound healing agents (such as cytokines
including but not limited to PDGF (e.g., REGRANEX.RTM.), TGF;
thymosin), hyaluronic acid as a wound healing agent, wound sealants
(such as fibrin with or without thrombin), cellular attractant and
scaffolding reagents (such as fibronectin), and the like, or
combinations of any of the foregoing, or of the foregoing and other
compounds not listed. A person of skill in the art will know which
compounds are useful for specific applications, e.g., which
compounds are useful for ocular applications of the compositions of
the invention.
[0096] The compositions of the invention can comprise any of the
compounds listed herein, without limitation, individually or in any
combination. Any of the bioactive compounds listed herein may be
formulated by known methods for immediate release or extended
release. Additionally, the placental biomaterial may comprise two
or more biologically active compounds in different manners; e.g.,
the biomaterial or membrane may be impregnated with one
biologically active compound and coated with another. In another
embodiment, the placental biomaterial comprises one biologically
active compound formulated for extended release, and a second
biologically active compound formulated for immediate release.
[0097] Wound healing requires adequate nutrition, particularly the
presence of iron, zinc, vitamin C, arginine, and the like. Thus,
the compositions of the invention can comprise a
physiologically-available form of one or more nutrients required
for wound healing. Preferably, the nutrient is formulated for
extended release.
[0098] The compositions of the invention can comprise an
antibiotic. In certain embodiments, the antibiotic is a macrolide
(e.g., tobramycin (TOBI.RTM.)), a cephalosporin (e.g., cephalexin
(KEFLEX.RTM.)), cephradine (VELOSEF.RTM.)), cefuroxime
(CEFTIN.RTM., cefprozil (CEFZIL.RTM.), cefaclor (CECLOR.RTM.),
cefixime (SUPRAX.RTM. or cefadroxil (DURICEF.RTM.), a
clarithromycin (e.g., clarithromycin (Biaxin)), an erythromycin
(e.g., erythromycin (EMYCIN.RTM.)), a penicillin (e.g., penicillin
V (V-CILLINK.RTM. or PEN VEEK.RTM.)) or a quinolone (e.g.,
ofloxacin (FLOXIN.RTM.), ciprofloxacin (CIPRO.RTM.) ornorfloxacin
(NOROXIN.RTM.)), aminoglycoside antibiotics (e.g., apramycin,
arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin,
undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and
spectinomycin), amphenicol antibiotics (e.g., azidamfenicol,
chloramphenicol, florfenicol, and thiamphenicol), ansamycin
antibiotics (e.g., rifamide and rifampin), carbacephems (e.g.,
loracarbef), carbapenems (e.g., biapenem and imipenem),
cephalosporins (e.g., cefaclor, cefadroxil, cefamandole,
cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, and
cefpirome), cephamycins (e.g., cefbuperazone, cefinetazole, and
cefminox), monobactams (e.g., aztreonam, carumonam, and tigemonam),
oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g.,
amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,
benzylpenicillinic acid, benzylpenicillin sodium, epicillin,
fenbenicillin, floxacillin, penamccillin, penethamate hydriodide,
penicillin o-benethamine, penicillin 0, penicillin V, penicillin V
benzathine, penicillin V hydrabamine, penimepicycline, and
phencihicillin potassium), lincosamides (e.g., clindamycin, and
lincomycin), macrolides (e.g., azithromycin, carbomycin,
clarithomycin, dirithromycin, erythromycin, and erythromycin
acistrate), amphomycin, bacitracin, capreomycin, colistin,
enduracidin, enviomycin, tetracyclines (e.g., apicycline,
chlortetracycline, clomocycline, and demeclocycline),
2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans (e.g.,
furaltadone, and furazolium chloride), quinolones and analogs
thereof (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine,
and grepagloxacin), sulfonamides (e.g., acetyl
sulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,
phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones
(e.g., diathymosulfone, glucosulfone sodium, and solasulfone),
cycloserine, mupirocin and tuberin.
[0099] The compositions of the invention can comprise an antifungal
agent. Suitable antifungal agents include but are not limited to
amphotericin B, itraconazole, ketoconazole, fluconazole,
intrathecal, flucytosine, miconazole, butoconazole, clotrimazole,
nystatin, terconazole, tioconazole, ciclopirox, econazole,
haloprogrin, naftifine, terbinafine, undecylenate, and
griseofuldin.
[0100] The compositions of the invention can comprise an
anti-inflammatory agent. Useful anti-inflammatory agents include,
but are not limited to, non-steroidal anti-inflammatory drugs such
as salicylic acid, acetylsalicylic acid, methyl salicylate,
diflunisal, salsalate, olsalazine, sulfasalazine, acetaminophen,
indomethacin, sulindac, etodolac, mefenamic acid, meclofenamate
sodium, tolmetin, ketorolac, dichlofenac, ibuprofen, naproxen,
naproxen sodium, fenoprofen, ketoprofen, flurbinprofen, oxaprozin,
piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, tenoxicam,
nabumetome, phenylbutazone, oxyphenbutazone, antipyrine,
aminopyrine, apazone and nimesulide; leukotriene antagonists
including, but not limited to, zileuton, aurothioglucose, gold
sodium thiomalate and auranofin; and other anti-inflammatory agents
including, but not limited to, methotrexate, colchicine,
allopurinol, probenecid, sulfinpyrazone and benzbromarone.
[0101] The compositions of the invention can comprise an antiviral
agent. Useful antiviral agents include, but are not limited to,
nucleoside analogs, such as zidovudine, acyclovir, gangcyclovir,
vidarabine, idoxuridine, trifluridine, and ribavirin, as well as
foscarnet, amantadine, rimantadine, saquinavir, indinavir,
ritonavir, and the alpha-interferons.
[0102] The compositions of the invention can comprise a cytokine
receptor modulator. Examples of cytokine receptor modulators
include, but are not limited to, soluble cytokine receptors (e.g.,
the extracellular domain of a TNF-.alpha. receptor or a fragment
thereof, the extracellular domain of an IL-10 receptor or a
fragment thereof, and the extracellular domain of an IL-6 receptor
or a fragment thereof), cytokines or fragments thereof (e.g.,
interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-15, TNF-.alpha., TNF-.beta., interferon
(IFN)-.alpha., IFN-.beta., IFN-.gamma., and GM-CSF), anti-cytokine
receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2
receptor antibodies (e.g., Zenapax (Protein Design Labs)),
anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies,
anti-IL-10 receptor antibodies, and anti-IL-12 receptor
antibodies), anti-cytokine antibodies (e. g., anti-IFN antibodies,
anti-TNF-.alpha. antibodies, anti-IL-10 antibodies, anti-IL-6
antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8 (Abgenix)), and
anti-IL-12 antibodies). In a specific embodiment, a cytokine
receptor modulator is IL-4, IL-10, or a fragment thereof. In
another embodiment, a cytokine receptor modulator is an anti-IL-1
antibody, anti-IL-6 antibody, anti-IL-12 receptor antibody, or
anti-TNF-.alpha. antibody. In another embodiment, a cytokine
receptor modulator is the extracellular domain of a TNF-.alpha.
receptor or a fragment thereof. In certain embodiments, a cytokine
receptor modulator is not a TNF-.alpha. antagonist.
[0103] In one embodiment, proteins, polypeptides or peptides
(including antibodies) that are utilized as immunomodulatory agents
are derived from the same species as the recipient of the proteins,
polypeptides or peptides so as to reduce the likelihood of an
immune response to those proteins, polypeptides or peptides. In
another embodiment, when the subject is a human, the proteins,
polypeptides, or peptides that are utilized as immunomodulatory
agents are human or humanized.
[0104] The compositions of the invention can comprise a cytokine.
Examples of cytokines include, but are not limited to, colony
stimulating factor 1 (CSF-1), interleukin-2 (IL-2), interleukin-3
(IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6
(IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-10
(IL-10), interleukin-12 (IL-12), interleukin 15 (IL-15),
interleukin 18 (IL-18), insulin-like growth factor 1 (IGF-1),
platelet derived growth factor (PDGF), erythropoietin (Epo),
epidermal growth factor (EGF), fibroblast growth factor (FGF)
(basic or acidic), granulocyte macrophage stimulating factor
(GM-CSF), granulocyte colony stimulating factor (G-CSF), heparin
binding epidermal growth factor (HEGF), macrophage colony
stimulating factor (M-CSF), prolactin, and interferon (IFN), e.g.,
IFN-alpha, and IFN-gamma), transforming growth factor alpha
(TGF-.alpha.), TGF.beta.1, TGF.beta.2, tumor necrosis factor alpha
(TNF-.alpha.), vascular endothelial growth factor (VEGF),
hepatocyte growth factor (HGF), etc.
[0105] The compositions of the invention can comprise a hormone.
Examples of hormones include, but are not limited to, luteinizing
hormone releasing hormone (LHRH), growth hormone (GH), growth
hormone releasing hormone, ACTH, somatostatin, somatotropin,
somatomedin, parathyroid hormone, hypothalamic releasing factors,
insulin, glucagon, enkephalins, vasopressin, calcitonin, heparin,
low molecular weight heparins, heparinoids, synthetic and natural
opioids, insulin thyroid stimulating hormones, and endorphins.
Examples of .beta.-interferons include, but are not limited to,
interferon .beta.1-a and interferon .beta.1-b.
[0106] The compositions of the invention can comprise an alkylating
agent. Examples of alkylating agents include, but are not limited
to nitrogen mustards, ethylenimines, methylmelamines, alkyl
sulfonates, nitrosoureas, triazenes, mechlorethamine,
cyclophosphamide, ifosfamide, melphalan, chlorambucil,
hexamethylmelaine, thiotepa, busulfan, carmustine, streptozocin,
dacarbazine and temozolomide.
[0107] The compositions of the invention can comprise an
immunomodulatory agent, including but not limited to methothrexate,
leflunomide, cyclophosphamide, cyclosporine A, macrolide
antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP),
corticosteroids, steroids, mycophenolate mofetil, rapamycin
(sirolimus), mizoribine, deoxyspergualin, brequinar,
malononitriloamindes (e.g., leflunamide), T cell receptor
modulators, and cytokine receptor modulators. peptide mimetics, and
antibodies (e.g., human, humanized, chimeric, monoclonal,
polyclonal, Fvs, ScFvs, Fab or F(ab).sub.2 fragments or epitope
binding fragments), nucleic acid molecules (e.g., antisense nucleic
acid molecules and triple helices), small molecules, organic
compounds, and inorganic compounds. In particular, immunomodulatory
agents include, but are not limited to, methothrexate, leflunomide,
cyclophosphamide, cytoxan, Immuran, cyclosporine A, minocycline,
azathioprine, antibiotics(e.g., FK506 (tacrolimus)),
methylprednisolone (MP), corticosteroids, steroids, mycophenolate
mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin,
brequinar, malononitriloamindes (e.g., leflunamide), T cell
receptor modulators, and cytokine receptor modulators. Examples of
T cell receptor modulators include, but are not limited to, anti-T
cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412
(Boehringer), IDEC-CE9.Is (IDEC and SKB), mAb 4162W94, Orthoclone
and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion
(Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan
(IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked
immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)),
anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g.,
IDEC-131(IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)),
anti-CD2 antibodies, anti-CD11a antibodies (e.g., Xanelim
(Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC))) and
CTLA4-immunoglobulin. In a specific embodiment, a T cell receptor
modulator is a CD2 antagonist. In other embodiments, a T cell
receptor modulator is not a CD2 antagonist. In another specific
embodiment, a T cell receptor modulator is a CD2 binding molecule,
preferably MEDI-507. In other embodiments, a T cell receptor
modulator is not a CD2 binding molecule.
[0108] In another embodiment, a composition of the invention can
comprise a crosslinking agent, e.g., riboflavin.
[0109] In various embodiments, the biomaterial, e.g., umbilical
cord membrane or umbilical cord biomaterial may comprise at least
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
300, 400, 500, 600, 700, 800, 900, 100, 1250, 1500, 2000, 2500,
300, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,
8500, 9000, 9500, 10000, 20000, 30000, 40000, 50000, 60000, 70000,
80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000,
700000, 800000, 900000 or at least 1000000 nanograms of a bioactive
compound. In another embodiment, the placental biomaterial, e.g.,
umbilical cord membrane or umbilical cord biomaterial may be coated
with, or impregnated with, no more than 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,
35, 40. 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700,
800, 900, 100, 1250, 1500, 2000, 2500, 300, 3500, 4000, 4500, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000,
30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000,
300000, 400000, 500000, 600000, 700000, 800000, 900000 or at least
1000000 nanograms of a bioactive compound.
5.2. Method of Use
[0110] The invention further provides methods of using the
compositions described herein.
[0111] In one embodiment, the invention provides a method of
treatment of an ocular condition comprising contacting said
condition with a solution of the invention, e.g., a placental
tissue solution, an umbilical cord tissue solution, or combination
thereof. For example, the solutions described herein can be used in
any ocular condition in which the introduction of placental tissue
or umbilical cord tissue, e.g., amniotic membrane, umbilical cord
membrane, or amniotic membrane or umbilical membrane components,
would be useful to encourage ocular tissue repair, regrowth, or to
reduce a symptom, e.g., inflammation of a disorder or condition of
an eye or a tissue of an eye. In a specific embodiment, the
placental tissue solution is an amniotic membrane solution
comprising amniotic membrane components soluble at neutral pH, or
acidic and neutral pH or basic and neutral pH. In another specific
embodiment, the umbilical cord tissue solution is an umbilical cord
membrane solution comprising soluble umbilical cord membrane and
Wharton's jelly components.
[0112] In specific embodiments, the ocular condition or symptom
thereof is irritation, inflammation, dryness or stickiness or
symblepharon. In another specific embodiment, the ocular condition
is a discontinuity of the sclera or cornea. Such a discontinuity
can be caused accidentally, e.g., an injury, or can be cause
deliberately, e.g., during ocular surgery. In another embodiment,
the ocular condition is, or is the result of, a congenital defect.
In a specific embodiment, the ocular surgery is refractive surgery.
In a specific embodiment, refractive surgery is photorefractive
keratectomy (PRK), laser-assisted sub-epithelial keratectomy
(LASEK) or laser-assisted in situ keratomileusis (LASIK). In
another specific embodiment, said ocular surgical procedure is
automated lamellar keratoplasty (ALK), laser thermal keratoplasty
(LTK), or conductive keratoplasty (CK). In another embodiment, said
ocular surgery is a surgery involving the creation of an incision
or hole in the eye, e.g., a cataract surgery, a glaucoma surgery, a
vitreoretinal surgery.
[0113] Administration of the solution of the invention to the
ocular condition can be done a single time or a plurality of times.
Administration can be performed as needed, e.g., hourly; once,
twice, three times or more daily; weekly, and the like.
Administration of the solution can be a sole method of treatment,
or can be part of a plurality of treatments administered at the
same time or separately.
[0114] In one embodiment, the solution of the invention comprises a
crosslinking agent. In a specific embodiment, the crosslinking
agent is riboflavin. In a more specific embodiment, the solution of
the invention comprises riboflavin at a concentration suitable for
enabling or facilitating crosslinking of collagens in the eye in,
e.g., ultraviolet light-mediated corneal collagen crosslinking. The
solution, comprising riboflavin, can be used in the eye to treat
any condition in which the cornea of the eye would benefit from
increased mechanical strength. Such a condition can be, for
instance, keratoconus or corneal ectasia. The solution comprising
riboflavin can also be used as an adjunct to a corneal surgery or
other ocular procedure such as, e.g., intracorneal ring
implantation, conductive keratoplasty, LTK, orthokeratoplasty,
prevention of central island, extended PRK, EpiLASIK or LASEK. In a
preferred embodiment, the cornea has a pachymetry (thickness) of at
least 400 micrometers.
[0115] In one embodiment, such crosslinking is accomplished as
follows. An eye is treated with an anesthetic, e.g., proparacaine
0.5%. A solution of the invention, comprising riboflavin, is then
applied to the eye. Within about 20 minutes, the eye is exposed to
UVA light at, e.g., about 370 nm fluence at about 3 mW/cm.sup.2.
The solution comprising riboflavin is applied to the eye dropwise
about every 3 minutes during UVA exposure. Variations of this
method will be apparent to those of skill in the art.
[0116] In one embodiment, the invention provides a bandage contact
lens, wherein the contact lens comprises a composition of the
invention, e.g., has been soaked or rehydrated in a solution of the
invention, or coated with a composition of the invention (e.g., a
gel or paste). In a specific embodiment, the bandage contact lens
comprises a low water (i.e., <50% water) nonionic polymer lens,
a high water (>50% water) nonionic polymer lens, a low-water
ionic polymer lens or a high-water ionic polymer lens. In a more
specific embodiment, the low-water nonionic polymer lens comprises
a teflicon, tetrafilcon A, crofilcon, helfilcon A&B, mafilcon,
polymacon, hioxifilcon B, or lotrafilcon A lens. In another more
specific embodiment, the high-water nonionic polymer lens comprises
a lidofilcon A, lidofilcon B, surfilcon A, netrafilcon A, hefilcon
C, alfafilcon A, omafilcon A, vasurfilcon A, hioxifilcon A,
nelfilcon A, hilafilcon A, or hilafilcon B lens. In another
specific embodiment, the low-water ionic polymer lens comprises a
bufilcon A, deltafilcon A, or phemfilcon lens. In another specific
embodiment, the high-water ionic polymer lens comprises a bufilcon
A, perfilcon A, stafilcon A, focofilcon A, ocufilcon B, ocufilcon
C, ocufilcon D, ocufilcon E, ocufilcon F, phemfilcon A, methafilcon
A, methafilcon B, or vilfilcon A lens. In another specific
embodiment, the contact lens is made from biomaterial derived from
placental tissue, or umbilical cord tissue, e.g., amniotic
membrane, chorion, amnion chorion, umbilical cord membrane,
umbilical membrane combined with Wharton's jelly, or the like. In a
more specific embodiment, the contact lens is made from biomaterial
which is decellularized and dehydrated according to the methods
described herein. In another specific embodiment, the contact lens
has a hole in the center sufficient to allow a wearer to see
clearly for at least a portion of the user's visual field.
[0117] In another embodiment, the bandage contact lens comprises
collagen, wherein the collagen is crosslinked by riboflavin. Such a
bandage contact lens can be made, e.g., by immersing the contact
lens in a solution of the invention, wherein the solution comprises
riboflavin, and exposing the lens to ultraviolet light (UVA) for a
time sufficient for the UVA to crosslink a plurality of the
collagens in the lens.
[0118] In another embodiment, the invention provides a bandage or
wound dressing comprising a composition, e.g., a solution, of the
invention. Such a bandage can comprise, for example, a composition
of the invention in a gel that can be contacted with the site of a
wound, injury, or other discontinuity in, e.g., the skin.
[0119] The invention further provides use of the compositions of
the invention, e.g., suspensions, solutions, slurries, pastes or
gels, in wound healing. For example, the invention provides a
method of treating an injury, wound or discontinuity in a tissue of
an individual comprising contacting said injury, wound or
discontinuity with a composition, e.g., gel or paste, of the
invention. In various embodiments, the wound, injury or
discontinuity is a laceration, scrape, thermal or chemical burn,
incision, puncture, wound caused by a projectile, and the like.
Such wounds can be accidental or deliberate, e.g., wounds caused
during or as an adjunct to a surgical procedure. Preferably, the
wound, injury or discontinuity is closed prior to contacting with
the gel or paste of the invention.
[0120] In other embodiments, a composition of the invention, e.g.,
gel or paste, is used as a filler, e.g., for tissue removed during
surgery or lost to injury.
[0121] In other embodiments, the composition of the invention,
e.g., gel or paste, ointment, cream, and the like, comprises a
moisturizer or emollient, and is used as a skin softener or
soothing agent, e.g., a soothing agent for sunburn.
4.3 KITS
[0122] The present invention further provides kits comprising the
placental or umbilical cord tissue compositions described
herein.
[0123] In one embodiment, the invention provides a kit comprising a
composition of the invention, e.g., a composition comprising
placental tissue components or umbilical cord components soluble in
aqueous solution at neutral pH and substantially lacking placental
tissue components or umbilical cord components insoluble in aqueous
solution at neutral pH, in an appropriately labeled container. In
certain embodiments, such kits comprise one or more individually
labeled containers containing unit-dosage or multi-dosage aliquots
of the composition, useful for administering a defined amount of
the composition to an individual. The kit can additionally comprise
instructions for administering the compositions of the invention to
an individual, including, e.g., instructions on the frequency and
dose of administration.
[0124] In a more specific embodiment, the invention provides a kit
for the treatment of an ocular condition. The kit may comprise a
placental or umbilical cord tissue composition of the invention,
and any other medical device that would facilitate treatment of an
ocular condition treatable with the composition. In a specific
embodiment, the kit additionally comprises a bandage contact lens,
and includes instructions for use of the bandage contact lens in
combination with the placental or umbilical cord tissue composition
for the treatment of an ocular condition. In one embodiment, the
kit comprises instructions for contacting a tissue of an eye of an
individual having or affected by an ocular condition with the
composition. In a more specific embodiment, the kit comprises a
bandage contact lens, wherein the bandage contact lens comprises a
composition of the invention, e.g., has been soaked or rehydrated
in a solution of the invention, or coated with a composition of the
invention (e.g. a gel or paste). In another specific embodiment,
the kit comprises instructions for contacting a tissue of an eye of
an individual having or affected by an ocular condition with the
bandage contact lens. In another specific embodiment, the kit
additionally comprises a cross-linking agent, e.g. riboflavin.
[0125] In another specific embodiment, the invention provides a kit
for dressing or healing a wound. For example, the kit may comprise
one or more bandages comprising a composition, e.g. a solution of
the invention. Preferably, the bandage is individually wrapped in,
e.g., a peel wrapper or other easily manipulable and openable
packaging.
[0126] The container in which kit components are handled and sold
is preferably labeled per applicable Food and Drug Administration
standards.
6. EXAMPLES
6.1. Example 1
PH Shifting Method of Making an Umbilical Cord or Amniotic Membrane
Solution
[0127] This Example demonstrates a pH shifting method of preparing
a suspension or solution of the invention.
6.1.1. Example Method 1
[0128] A cleaned, substantially bloodless (exsanguinated) placenta
from a normal birth is obtained. The amniotic membrane is removed,
roughly chopped, and placed in a volume of a 0.9%
phosphate-buffered saline solution containing 10 mM EDTA and 10 mM
EGTA, at a ratio of about 100 ml solution for every gram of tissue,
at 4.degree. C. The solution is acidified to about pH 4.5 using
glacial acetic acid. The pH of the solution is determined using a
standard pH meter. The solution and amniotic membrane is placed
into a blender, and the amniotic membrane is homogenized for one
minute at the highest setting to produce an amniotic membrane
suspension. The acidified suspension is allowed to stand at
4.degree. C. for 24 hours. After 24 hours, the suspension is
brought to about pH 7.4 with lON NaOH, and divided into culture
bottles suitable for use in a swinging bucket centrifuge rotor. The
suspension is centrifuged at 4.degree. C. for 10 minutes at 8000 g
to produce a supernatant. The supernatant is dialyzed over a
semipermeable membrane against about 20 volumes of deionized water,
and stored for further use.
6.1.2. Example Method 2
[0129] An umbilical cord is obtained from a cleaned, substantially
bloodless (exsanguinated) placenta from a normal birth. Umbilical
cord veins are removed with a vein stripper, or are removed by
longitudinally cutting the umbilical cord to expose the veins for
removal. The umbilical cord membrane and Wharton's jelly is cut
into sections of approximately 10 cm to 15 cm in length. The
umbilical cord is then rinsed in cold 0.9% saline, and placed in a
0.9% phosphate-buffered saline solution containing 10 mM EDTA and
10 mM EGTA, at a ratio of about 100 ml solution for every gram of
tissue, at 4.degree. C. The solution is acidified to about pH 4.5
using glacial acetic acid. The pH of the solution is determined
using a standard pH meter. The solution and umbilical cord is
placed into a blender, and the umbilical cord is homogenized for
one minute at the highest setting to produce an umbilical cord
suspension. The acidified suspension is allowed to stand at
4.degree. C. for 24 hours. After 24 hours, the suspension is
brought to about pH 7.4 with 10N NaOH, and divided into four
culture bottles suitable for use in a swinging bucket centrifuge
rotor. The suspension is centrifuged at 4.degree. C. for 10 minutes
at 5000 g to produce a supernatant. The supernatant is optionally
centrifuged again at 4.degree. C. and 10000 g. The supernatant is
dialyzed over a semipermeable membrane against 20 volumes of
deionized water, and stored for further use.
6.2. Example 2
Solutions, Gels and Pastes
[0130] 6.2.1. Amniotic Membrane Solution
[0131] This Example demonstrates a method of obtaining an amniotic
membrane and the production of a solution of the invention made
from amniotic membrane.
[0132] A single placenta from a full-term placenta is obtained with
donor consent. The donor is pre-screened for viral pathogens
including hepatitis A virus, hepatitis B virus, Hepatitis C virus,
human immunodeficiency virus types 1 and 2, and
cytomegalovirus.
[0133] Following normal birth, the placenta, umbilical cord and
umbilical cord blood are spontaneously expelled from the
contracting uterus. The placenta, umbilical cord, and umbilical
cord blood are collected following birth. The materials are
transported to a laboratory and processed under aseptic conditions
in a clean room having a HEPA filtration system that is turned on
at least one hour prior to processing. Gloves (sterile or
non-sterile, as appropriate) are worn at all times while handling
the placenta. All unused (waste) segments of the amnion/chorion and
contaminated liquids generated during tissue processing are
disposed of as soon as feasible.
[0134] A sterile field is set up with sterile Steri-Wrap sheets and
the following instruments and accessories for processing were
placed on it: sterile tray pack; sterile cell scraper; sterile
scalpel; and a disinfected processing tray.
[0135] The placenta is removed from the transport container and
placed onto the disinfected stainless steel tray. Using surgical
clamps and scissors, the umbilical cord is cut off approximately 2
inches from the placental disc. The umbilical cord is placed into a
separate sterile container for further processing.
[0136] Starting from the edge of the placental membrane, the amnion
is separated from the chorion using blunt dissection with fingers.
This is done prior to cutting the membrane. After the amnion is
separated from the entire surface of the chorion and placental
disc, the amniotic membrane is cut around the umbilical cord stump
with scissors and detached from the placental disc. If separation
of the amnion and chorion is not possible without tearing the
tissue, the amnion and chorion are cut from the placental disc as
one piece and then peeled apart. The chorion is placed into a
separate specimen container to be utilized for other projects.
[0137] The amniotic membrane is optionally decellularized prior to
production of an amniotic membrane solution. In decellularization,
the amniotic membrane is removed from the rinsing tray, and excess
fluid is gently squeezed out with fingers. The membrane is
optionally scraped with a cell scraper and rinsed with sufficient
sterile water to effect removal of substantially all visible cell
material. The amniotic membrane is transferred to a new container,
which is then filled up to the 150 ml mark with decellularizing
solution (1% deoxycholic acid) ensuring that all of the amniotic
membrane is covered. The container is placed in the bin on the
rocking platform. The rocking platform is turned on and the
membrane was agitated in decellularizing solution at 4.degree. C.
for seven days. After decellularization, a new sterile field is set
up with new sterile instruments and disinfected tray in a same
manner as above. The rocking platform is turned off and the
membrane is removed from the container. The membrane is placed into
a new sterile stainless steel processing tray, and sterile 0.9%
NaCl solution is added to cover the bottom of the tray. Using a new
sterile cell scraper, residual decellularization solution and
cellular material is removed from both sides of the tissue. This
step is repeated as many times as needed to remove as much as
possible of visible residual cellular material from the entire
surface on both sides. The membrane is rinsed with sterile 0.9%
NaCl solution in a separate rinsing tray in between cleaning
rounds.
[0138] The amniotic membrane, either decellularized or comprising
cells, is then weighed, and processed according to the pH shifting
protocol described in Example 1, using a weight/volume ratio of
amniotic membrane to solution of 1 g: 10 ml.
6.2.2. Umbilical Cord Suspension and Solution
[0139] The following example demonstrates a method of obtaining an
umbilical cord suspension or solution useful for medical, e.g.,
ocular purposes.
[0140] Human umbilical cord (HUG) harvesting. The umbilical cord is
separated from the placental disc, and rinsed in a sterile saline
solution. The umbilical cord is sectioned into pieces of about 10
cm to about 15 cm in length and is squeezed to remove umbilical
cord blood. The umbilical cord is then slit or cut longitudinally
using a pair of surgical scissors so that the two arteries and one
vein are exposed. The vessels are then removed with a forceps. The
umbilical cord membrane, including Wharton's jelly, is then rinsed
in cold saline or other balanced salt solution at neutral pH. The
solution optionally comprises one or more antibiotics. The
umbilical cord membrane can be further processed immediately, or
can be stored in a refrigerator for up to about 72 hours in a
sterile buffered saline solution, such as 0.9% sterile NaCl
solution.
[0141] Homogenization of HUC at neutral pH. The following
procedures are all carried out at 4.degree. C. and neutral pH
(approximately 7.4) under sterile conditions. Umbilical cord
membrane is cut into small pieces and weighed. Sterile buffer, and
optionally antibiotics, are then added to the chamber of a
homogenizer to reach the desired weight to volume ratio (w/v) of
1:5. The membrane pieces are then homogenized to produce an
umbilical cord membrane suspension. Homogenization is repeated as
necessary in order to obtain a visibly homogenous suspension
without raising the temperature above about 4.degree. C. The
resulting suspension can be used without further treatment.
[0142] The homogenate is centrifuged for 10 minutes at 4.degree. C.
and 3500 rpm and then the supernatant is further centrifuged for
another 5 minutes at 10000 rpm to get rid of any undesired
particulate matter present in the extract to produce an umbilical
cord solution.
[0143] The obtained umbilical cord membrane solution is analyzed by
a Lowry assay to quantify the total protein amount present in the
homogenate. The solution is then filtered through 0.45 micron
filters under a sterile hood, packaged into small vials and stored
at 4.degree. C. until used.
6.2.3. Amniotic Membrane Paste
[0144] This example demonstrates two methods of obtaining an
amniotic membrane paste.
[0145] An amniotic membrane, obtained in the manner described in
Section 5.2.1, above, is homogenized and solubilized in the manner
described in Example 1. The resulting amniotic membrane solution is
dehydrated using a vacuum dehydration apparatus at about 4.degree.
C. Dehydration is continued until the water in the solution is
substantially removed and the remaining amniotic membrane material
appears dry by visual inspection. The mass of the amniotic membrane
material is determined, and water or buffer is then added to the
amniotic membrane matter to achieve a water content of about 3% to
about 10% by weight. The water and amniotic membrane material are
thoroughly mixed to form a paste.
[0146] In a variation of the above method, the amniotic membrane
material is supplemented by an amount of purified collagen not to
exceed 100 times the weight of the amniotic membrane material
obtained from the amniotic membrane solution, and water is added to
as to achieve between 3% and 10% water by weight. The water,
amniotic membrane material and purified collagen are thoroughly
mixed to form a paste.
6.2.4. Umbilical Membrane Paste
[0147] This example demonstrates two methods of obtaining an
umbilical cord paste.
[0148] An umbilical cord membrane comprising Wharton's jelly,
obtained in the manner described in Section 5.2.2, above, is
homogenized and solubilized in the manner described in Example 1.
The resulting umbilical cord solution is dehydrated using a vacuum
dehydration apparatus at about 4.degree. C. Dehydration is
continued until the water in the solution is substantially removed
and the remaining umbilical cord material appears dry by visual
inspection. The mass of the umbilical cord material is determined,
and water or buffer is then added to the umbilical cord material to
achieve a water content of about 3% to about 10% by weight. The
water and umbilical cord material are thoroughly mixed to form a
paste.
[0149] In a variation of the above method, the umbilical cord
material is supplemented by an amount of purified collagen not to
exceed 100 times the weight of the umbilical cord material obtained
from the umbilical cord solution, and water is added to as to
achieve between 3% and 10% water by weight. The water, umbilical
cord material and purified collagen are thoroughly mixed to form a
paste.
6.2.5. Umbilical Cord Gel
[0150] This example demonstrates a method of producing a gel from
umbilical cord.
[0151] An umbilical cord membrane comprising Wharton's jelly,
obtained in the manner described in Section 5.2.2, above, is
homogenized and solubilized in the manner described in Example 1.
The resulting umbilical cord solution is dehydrated using a vacuum
dehydration apparatus at about 4.degree. C. Dehydration is
continued until the water in the solution is substantially removed
and the remaining umbilical cord material appears dry by visual
inspection. The mass of the umbilical cord material is determined,
and a gelling compound is added in a weight/weight ratio to the
umbilical cord material of about 10:1. Water is then added to the
umbilical cord material and gelling compound in an amount that
brings the water content of the resulting gel to about 60% to about
99% by weight.
[0152] Ina variation of the above method, the umbilical cord
material is supplemented with purified collagen in a weight not to
exceed 100 times the weight of the umbilical cord material obtained
from the umbilical cord solution. Water is added to the umbilical
cord material, purified collagen, and gelling agent to bring the
water content of the resulting gel to 60% to about 99% by
weight.
6.2.6. Combination Compositions
[0153] This Example demonstrates methods of producing combinations
of compositions made from placental tissue and umbilical cord
tissue.
[0154] A solution of the invention is produced as follows. A
solution comprising amniotic membrane components is made according
to the method described in Section 5.2.1, above, and a solution
comprising umbilical cord membrane components is made according to
the method described in Section 5.2.2, above. The two solutions are
combined in a ratio of from about 1:10 to about 10:1 by volume, and
mixed thoroughly. Alternatively, the protein content of each
solution is determined, and the two solutions re combined in a
ratio of from about 1:10 to about 10:1 by protein content. The
resulting solution is centrifuged to remove visible solid matter
and stored at 4.degree. C. for later use.
[0155] A solution of the invention is produced as follows. A
solution comprising amniotic membrane components is made according
to the method described in Section 5.2.1, and is mixed thoroughly
with an umbilical cord membrane paste containing purified collagen
as described in Section 5.2.4, above. The resulting solution is
centrifuged to remove visible solid matter and stored at 4.degree.
C. for later use.
[0156] Equivalents:
[0157] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described will
become apparent to those skilled in the art from the foregoing
description and accompanying figures. Such modifications are
intended to fall within the scope of the appended claims.
[0158] Various publications, patents and patent applications are
cited herein, the disclosures of which are incorporated by
reference in their entireties.
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