U.S. patent application number 16/853569 was filed with the patent office on 2020-08-20 for therapeutics using adipose cells and cell secretions.
The applicant listed for this patent is Cell Ideas PTY Ltd.. Invention is credited to Richard Lilischkis, Graham Vesey, Rebecca Anne Webster.
Application Number | 20200261510 16/853569 |
Document ID | 20200261510 / US20200261510 |
Family ID | 1000004808677 |
Filed Date | 2020-08-20 |
Patent Application | download [pdf] |
United States Patent
Application |
20200261510 |
Kind Code |
A1 |
Vesey; Graham ; et
al. |
August 20, 2020 |
Therapeutics Using Adipose Cells and Cell Secretions
Abstract
The invention relates to compositions comprising (i) adipose
tissue-derived cell secretions or (ii) an adipose tissue-derived
cell suspension, optionally comprising adipocytes, or (iii) a
combination of adipose tissue-derived cell secretions and an
adipose tissue-derived cell suspension, optionally comprising
adipocytes, and to their use in pharmaceutical compositions and
methods for treatment of various conditions. The invention also
relates to improved methods, agents and compositions for
cryopreservation of cells.
Inventors: |
Vesey; Graham; (Hornsby,
AU) ; Webster; Rebecca Anne; (Avalon, AU) ;
Lilischkis; Richard; (Westleigh, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cell Ideas PTY Ltd. |
Gordon |
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AU |
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|
Family ID: |
1000004808677 |
Appl. No.: |
16/853569 |
Filed: |
April 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16135700 |
Sep 19, 2018 |
10660921 |
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16853569 |
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14342479 |
Mar 3, 2014 |
10111909 |
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PCT/AU2012/001140 |
Sep 21, 2012 |
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16135700 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0667 20130101;
C12N 5/0653 20130101; A61K 35/35 20130101; A01N 1/0221 20130101;
A61K 35/28 20130101 |
International
Class: |
A61K 35/35 20060101
A61K035/35; A61K 35/28 20060101 A61K035/28; C12N 5/077 20060101
C12N005/077; C12N 5/0775 20060101 C12N005/0775; A01N 1/02 20060101
A01N001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 23, 2011 |
AU |
2011903938 |
Apr 4, 2012 |
AU |
2012901350 |
Aug 23, 2012 |
AU |
2012903646 |
Claims
1.-60. (canceled)
61. A method of alleviating pain in a mammalian subject, the method
comprising, administering to the subject a pharmaceutical
composition which comprises adipose tissue-derived cell secretions,
wherein said adipose tissue-derived cell secretions are prepared by
culturing multiply-passaged adherent progeny cells from an adipose
tissue-derived cell suspension and harvesting supernatant from the
cell culture after about 3 or more days, said adherent progeny
cells having a fibroblast-like appearance.
62. The method according to claim 61, wherein said administration
to said subject is by intra-articular injection or by intramuscular
injection or by subcutaneous injection or by topical
administration.
63. The method according to claim 61, wherein the pain is back
pain, or is pain associated with a burn injury, an inflammatory
disorder, a ligament injury, a tendon injury, or the sciatic nerve,
or is neuropathic pain.
64. The method according to claim 63, wherein the neuropathic pain
is facial pain, pain of the teeth, jaw, gum or of a limb.
65. The method according to claim 61, wherein the adipose
tissue-derived cell suspension (i) comprises adipocytes or (ii) is
substantially free of adipocytes.
66. The method according to claim 61, wherein the subject is
selected from the group consisting of (i) a human, (ii) poultry,
(iii) an equine animal, (iv) a feline animal, (v) a canine animal,
(vi) a bovine animal, and (vii) a porcine animal.
67. The method according to claim 61, wherein the adipose
tissue-derived cell secretions or the adipose tissue-derived cell
suspension is (i) derived from adipose tissue allogeneic to the
recipient subject or animal, or (ii) derived from the intended
recipient subject or animal, or (iii) derived from adipose tissue
xenogeneic to the recipient subject or animal.
68. The method according to claim 61, wherein the adipose
tissue-derived cell secretions is a preparation concentrated by
between 2-fold and 20-fold.
69. The method according to claim 61, wherein said adipose
tissue-derived cell secretions comprises concentrated media from
culture of an adipose tissue-derived cell suspension, optionally
wherein said adipose tissue-derived cell suspension (i) comprises
adipocytes and adipose tissue-derived mesenchymal stem cells or
(ii) is substantially free of adipocytes, optionally wherein said
media from culture of an adipose tissue-derived cell suspension is
concentrated between 2-fold and 20-fold.
70. A kit comprising (a) a pharmaceutical composition comprising
adipose tissue-derived cell secretions from an adipose
tissue-derived cell suspension, wherein said adipose tissue-derived
cell secretions are prepared by culturing multiply-passaged
adherent progeny cells from an adipose tissue-derived cell
suspension and harvesting supernatant from the cell culture after
about 3 or more days, said adherent progeny cells comprising cells
having a fibroblast-like appearance; and (b) instructions for use
of said kit in alleviating pain in a mammalian subject.
71. The kit according to claim 70, wherein the pain is back pain,
or is pain associated with a burn injury, an inflammatory disorder,
a ligament injury, a tendon injury, or the sciatic nerve, or is
neuropathic pain, optionally wherein the neuropathic pain is facial
pain, pain of the teeth, jaw, gum or of a limb.
72. The kit according to any claim 70, wherein the adipose
tissue-derived cell suspension (i) comprises adipocytes, or (ii) is
substantially free of adipocytes.
73. The kit according to claim 70, wherein the adipose
tissue-derived cell secretions is (i) derived from adipose tissue
allogeneic to the recipient subject or animal, or (ii) derived from
the intended recipient subject or animal, or (iii) derived from
adipose tissue xenogeneic to the recipient subject or animal.
74. The kit according to claim 70, wherein the adipose
tissue-derived cell secretions is a preparation concentrated by
between 2-fold and 20-fold.
75. The kit according to claim 70, wherein said adipose
tissue-derived cell secretions comprises concentrated media from
culture of an adipose tissue-derived cell suspension, optionally
wherein said adipose tissue-derived cell suspension (i) comprises
adipocytes and adipose tissue-derived mesenchymal stem cells or
(ii) is substantially free of adipocytes, optionally wherein said
media from culture of an adipose tissue-derived cell suspension is
concentrated between 2-fold and 20-fold.
Description
RELATED APPLICATIONS
[0001] The present application claims benefit from Australian
provisional patent application No. 2011903938 entitled "Therapeutic
methods and compositions" filed on 23 Sep. 2011 and from Australian
provisional patent application No. 2012901350 entitled "Therapeutic
methods and compositions" filed on 4 Apr. 2012 and from Australian
provisional patent application No. 2012903646 entitled "Therapeutic
methods and compositions" filed on 23 Aug. 2012, the entire
contents of each of which is incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to compositions comprising (i)
adipose tissue-derived cell secretions or (ii) an adipose
tissue-derived cell suspension, optionally comprising adipocytes or
(iii) a combination of adipose tissue-derived cell secretions and
an adipose. tissue-derived cell suspension, optionally comprising
adipocytes, and to their use in pharmaceutical compositions and
methods for treatment of various conditions such as inflammatory
disorders, ligament injuries, tendon injuries, or for alleviating
pain associated with such conditions, an inflammatory disorder, a
ligament injury or a tendon injury, in a mammalian subject. In the
methods of the invention the compositions are administered to a
site on a patient remote to the site affected by the inflammatory
disorder or condition. The invention also relates to the use of
such compositions in pharmaceutical compositions and methods for
the treatment or prevention of disease in an intensively farmed
animal, wherein said administration is by subcutaneous or
intramuscular injection. The invention also relates to the
treatment of pain in a subject, the treatment being by subcutaneous
injection or intramuscular injection. The invention also relates to
treatment of neuropathic pain in a subject. The invention also
relates to improved methods, agents and compositions for
cryopreservation of cells.
BACKGROUND OF THE INVENTION
[0003] Adipose tissue contains a cell population of large lipid
filled adipocytes, and a non-adipocyte cell population, which
comprises cells associated with various connective fibres and cells
associated with capillaries and larger blood vessels. The
non-adipocyte cell population is also thought to comprise a
population of adipose-derived adult stem cells and consequently
there has been interest in using adipose tissue as a source of
isolated stem cells for various therapeutic applications.
[0004] In general, methods for obtaining adipose tissue derived
presumptive stem cells involves depleting adipocytes from
adipose-derived non-adipocyte cells, which requires digesting
adipose tissue with enzymes such as collagenase, and then
separating the liberated cells by centrifuging the digested sample.
During centrifugation, the adipose-derived non-adipocyte cells
separate from the adipocytes to form a pellet, whereas the lipid
containing adipocytes float. The non-adipocyte cell fraction is
then used as a source of tissue stem cells.
[0005] The present inventors have earlier described the use of an
adipose tissue-derived cell suspension which comprises adipocytes
for the preparation of a pharmaceutical composition for use in the
treatment of an inflammatory disorder or the alleviation of pain
associated with an inflammatory disorder in a subject, and for the
treatment and alleviation of pain of conditions such as a cartilage
or bone disorders. This is described in Australian Patent
Application No. 2009201915 and in International Publication No.
WO2010/020005, the contents of which are incorporated herein by
cross-reference. The present inventors have also earlier described
the use of adipose tissue-derived cells secretions for the
preparation of compositions for use in treatment of various
conditions and diseases, including in the alleviation of pain
associated with such conditions.
[0006] Generations of selective breeding of animals for certain
desirable traits, such as rapid growth, efficient feed conversion
and muscle mass accumulation in animals raised for meat production,
or milk quality and volume in dairy animals, has also resulted in
modern breeds of animals often being prone to a higher incidence of
detrimental health conditions than less intensively bred or
selected animals, such as wild populations. The clinical incidence
or effect of such detrimental traits can be exacerbated by the
manner in which the animals are farmed, such as in intensive
farming operations. Modern pig breeds, farmed under intensive
conditions for example, are prone to leg weakness, such as
osteochondrosis (OCD), arthritis, a high risk of clinical and
sub-clinical bacterial infection, all of which have the potential
to detrimentally affect the general well-being of the animal and
hence detrimentally affect the farming operation.
[0007] There remains a need for improved methods for the treatment
of inflammatory conditions, ligament and tendon injuries and
compositions for use therein. There remains a need for improved
methods for the treatment and prevention of detrimental conditions
associated with intensive animal farming. There remains a need for
the treatment of pain in a subject and compositions for use
therein.
SUMMARY OF THE INVENTION
[0008] Methods previously described for the treatment of
inflammatory disorders using adipose tissue-derived cell
suspensions and cell free compositions teach the administration of
the composition or suspension to the afflicted area, such as by
intra-articular injection in the case of an arthritic joint.
Surprisingly, the inventor has now identified that direct
administration of the therapeutic composition to the afflicted area
is not required. The inventor has surprisingly identified that the
remote delivery of a composition comprising secretions from an
adipose tissue-derived cell suspension or of a composition
comprising an adipose tissue-derived cell suspension, optionally
comprising adipocytes, or of a combination thereof, can also be
effective in the treatment of such conditions.
[0009] The inventors have surprisingly identified that frozen stem
cells, such as mesenchymal stem cells, such as adipose
tissue-derived stem cells, may be used as therapeutic agents in
treatment of various conditions. Surprisingly such frozen cells may
be used without the need for culturing the cells after retrieval
from frozen storage. The inventors have also identified that
storage of cells in the presence of cell-derived secretions
improves the viability and proliferation potential of cryopreserved
stem cells, including cells derived from adipose tissue.
[0010] Accordingly, in a first aspect of the invention there is
provided a method of treating a condition selected from the group
consisting of an inflammatory disorder, a ligament injury and a
tendon injury, or alleviating pain associated with an inflammatory
disorder, a ligament injury or a tendon injury, in a subject,
comprising administering to the subject a pharmaceutical
composition which comprises (i) adipose tissue-derived cell
secretions, or (ii) an adipose tissue-derived cell suspension,
optionally comprising adipocytes or (iii) a combination of (i) and
(ii), wherein said administration to said subject is at a site
remote from the site of said condition. In an embodiment the
subject is a mammalian subject. In an embodiment the adipose
tissue-derived cell suspension, optionally comprising adipocytes
comprises aggregates of cells and or comprises pieces of adipose
tissue. In an embodiment the cell suspension comprises adipocytes.
In an embodiment the cell suspension is substantially free of
adipocytes. In an embodiment the adipose tissue-derived cell
secretions comprises a carrier liquid selected from cell culture
media and distilled water. In an embodiment an adipose
tissue-derived cell suspension, optionally comprising adipocytes,
or a combination of an adipose tissue-derived cell secretions and
an adipose tissue-derived cell suspension, optionally comprising
adipocytes comprises a carrier liquid being a cell culture medium,
such as DMEM.
[0011] In an embodiment the inflammatory disorder or condition is
selected from the group consisting of osteoarthritis, stifle
disease, wobblers, a tendon injury and a ligament injury. In an
embodiment the inflammatory disorder is atopic dermatitis. In an
embodiment the inflammatory disorder or condition is selected from
the group consisting of rheumatoid arthritis, back pain, and
multiple sclerosis. In an embodiment the inflammatory disorder or
condition is an immune driven disease. In an embodiment the method
comprises administration of adipose tissue-derived cell secretions.
In an embodiment the method comprises administration of an adipose
tissue-derived cell suspension. In an embodiment the administration
is subcutaneous administration. In an embodiment the administration
is intra-muscular administration. In an embodiment the
administration is in the rump, arm, or buttocks. In an embodiment
the administration is into the neck of the subject, such as the
nape of the subject, such as the scruff of the neck when the
subject is a dog or cat.
[0012] In a second aspect there is provided a method of treating a
joint disease or condition in a subject, the method comprising
administering to the subject a pharmaceutical composition which
comprises (i) adipose tissue-derived cell secretions, or (ii) an
adipose tissue-derived cell suspension, optionally comprising
adipocytes, or (iii) a combination of (i) and (ii), wherein said
administration to said subject is at a site remote from the site of
said condition. In an embodiment the subject is a mammalian
subject. In an embodiment the adipose tissue-derived cell
suspension, optionally comprising adipocytes, comprises aggregates
of cells and or comprises pieces of adipose tissue. In an
embodiment the adipose tissue-derived cell secretions comprises a
carrier liquid selected from cell culture media and distilled
water. In an embodiment an adipose tissue-derived cell suspension,
optionally comprising adipocytes, or a combination of an adipose
tissue-derived cell secretions and an adipose tissue-derived cell
suspension, optionally comprising adipocytes, comprises a carrier
liquid being a cell culture medium, such as DMEM. In an embodiment
the treatment comprises administering a pharmaceutical composition
comprising an adipose tissue-derived cell suspension to said mammal
by subcutaneous injection. In an embodiment the subcutaneously
administered cell suspension is substantially free of adipocytes.
In an embodiment the subcutaneously administered cell suspension
comprises adipocytes.
[0013] In an embodiment the mammalian subject is an equine, feline,
canine, bovine or porcine animal. In an embodiment the subject is a
human. In an embodiment the subject is poultry.
[0014] In an embodiment the administration is subcutaneous
administration. In an embodiment the administration is
intra-muscular administration. In an embodiment the administration
is in the rump, arm, or buttocks. In an embodiment the
administration is into the neck of the subject, such as the nape of
the subject, such as the scruff of the neck when the subject is a
dog or cat.
[0015] In a third aspect of the invention there is provided a
method for the treatment or prevention of a disease in an
intensively farmed animal, the method comprising administering to
the animal a pharmaceutical composition which comprises (i) adipose
tissue-derived cell secretions, or (ii) an adipose tissue-derived
cell suspension, optionally comprising adipocytes, or (iii) a
combination of adipose tissue-derived cell secretions and an
adipose tissue-derived cell suspension, optionally comprising
adipocytes, wherein said administration is by subcutaneous
injection or intramuscular injection.
[0016] In an embodiment the disease of an intensively farmed animal
is an orthopeadic developmental disease. In an embodiment the
disease of an intensively farmed animal is selected from the group
consisting of leg weakness, lameness, arthritis, developmental
diseases and bacterial infection. In an embodiment the
developmental disease is osteochondrosis (OCD).
[0017] In an embodiment the pharmaceutical composition is
administered to an animal prior to the onset of clinical symptoms
of the disease. In an embodiment the intensively farmed animal is a
pig and the pharmaceutical composition is administered prior to the
onset of clinical symptoms of the orthopeadic developmental
disease, such as osteochondrosis.
[0018] In an embodiment the intensively farmed animal is selected
from the group consisting of pigs, cattle, sheep, and poultry,
[0019] In an embodiment the intensively farmed animal is a breeder
female. In an embodiment the intensively farmed animal is a
pregnant female. In an embodiment the animal is a pregnant sow. In
an embodiment the pregnant sow has clinical symptoms of
osteochondrosis or arthritis.
[0020] In an embodiment the administration is subcutaneous
administration. In an embodiment the administration is
intra-muscular administration. In an embodiment the administration
is into the neck of the subject, such as the nape of the
subject.
[0021] The following embodiments apply to all aspects of the
invention herein, unless the context clearly indicates
otherwise.
[0022] In an embodiment the adipose tissue-derived cell secretions
are prepared from an adipose tissue-derived cell suspension. In an
embodiment the adipose tissue-derived cell suspension is
substantially free of adipocytes. In an embodiment the adipose
tissue-derived cell suspension further comprises adipocytes. In an
embodiment the adipose tissue-derived cell suspension comprises
mature adipocytes. In an embodiment the adipose tissue-derived cell
secretions are prepared by culture of adipose tissue-derived cell
suspension. In an embodiment the adipose tissue-derived cell
suspension is substantially free of adipocytes. In an embodiment
the adipose tissue-derived cell suspension further comprises
adipocytes. In an embodiment the adipose tissue-derived cell
suspension, optionally comprising adipocytes comprises aggregates
of cells and or comprises pieces of adipose tissue.
[0023] In an embodiment the adipose tissue-derived cell suspension
is prepared by a method that comprises removal of (i) part of the
adipocyte content or (ii) substantially all of the adipocyte
content during preparation of the adipose tissue-derived cell
suspension.
[0024] In an embodiment the adipose tissue-derived cell secretions
is a concentrated preparation. In an embodiment the concentrated
preparation is concentrated in comparison to the cell secretions as
initially harvested from the adipose tissue-derived cell
suspensions or culture thereof. In an embodiment the adipose
tissue-derived cell secretions is a preparation concentrated by
between about 2-fold and about 20-fold. In an embodiment the
adipose tissue-derived cell secretions is a preparation
concentrated by about 10-fold.
[0025] In an embodiment the adipose tissue-derived cell secretions
is of bovine, canine, porcine or equine origin. In an embodiment
the adipose tissue-derived cell secretions is of human origin.
[0026] In an embodiment, the adipose tissue-derived cell secretions
is derived from adipose tissue autologous to the recipient subject
or animal. In an embodiment the adipose tissue-derived cell
secretions is derived from adipose tissue allogeneic to the
recipient subject or animal. In an embodiment the adipose
tissue-derived cell secretions is derived from adipose tissue
xenogeneic to the recipient subject or animal.
[0027] In an embodiment the adipose tissue-derived cell suspension,
optionally comprising adipocytes, comprises mature adipocytes.
[0028] In an embodiment the adipose tissue-derived cell suspension,
optionally comprising adipocytes is of bovine, canine, porcine or
equine origin. In an embodiment the adipose tissue-derived cell
suspension, optionally comprising adipocytes is derived from
adipose tissue autologous to the recipient subject or animal. In an
embodiment the adipose tissue-derived cell suspension, optionally
comprising adipocytes is derived from adipose tissue allogeneic to
the recipient subject or animal. In an embodiment the adipose
tissue-derived cell suspension, optionally comprising adipocytes is
derived from adipose tissue xenogeneic to the recipient subject or
animal.
[0029] In an embodiment the adipose tissue-derived cell suspension,
optionally comprising adipocytes is a cell suspension obtained by
cell expansion in culture.
[0030] In an embodiment the adipose tissue-derived cell secretions,
or a pharmaceutical composition thereof are stored frozen prior to
administration.
[0031] In an embodiment the adipose tissue-derived cell suspension,
optionally comprising adipocytes, or a pharmaceutical composition
thereof are stored frozen prior to administration.
[0032] In an embodiment the adipose tissue-derived cell secretions
in combination with adipose tissue-derived cell suspension,
optionally comprising adipocytes, or a pharmaceutical composition
thereof is stored frozen prior to administration. In an embodiment
the cell secretions in said combination are a concentrated
preparation. In an embodiment the preparation is concentrated by
between 2-fold and 20-fold in comparison to the secretions prior to
concentration.
[0033] In an embodiment the method further comprises (i) thawing
frozen adipose tissue-derived cell secretions, or (ii) thawing
frozen adipose tissue-derived cell suspension, optionally
comprising adipocytes, or (iii) thawing a frozen combination of
adipose tissue-derived cell secretions and an adipose
tissue-derived cell suspension, optionally comprising adipocytes,
or (iv) thawing a frozen pharmaceutical composition of any of (i),
(ii) or (iii), prior to administration to the recipient subject or
animal.
[0034] In an embodiment the frozen secretions, cell suspension,
combination thereof, or pharmaceutical composition thereof, is
administered to the recipient subject or animal soon after thawing,
such as within about 10 minutes after thawing, or within about 20
minutes after thawing, or within about 30 minutes after thawing or
within about one hour of thawing or within about two hours of
thawing.
[0035] In an embodiment the method further comprises combining (i)
a composition comprising adipose tissue-derived cell secretions and
(ii) an adipose tissue-derived cell suspension, optionally
comprising adipocytes, prior to administering said combination to
the recipient subject or animal. In an embodiment said combining
occurs within 2 hours before said administration. In an embodiment
one or both of said composition comprising adipose tissue-derived
cell secretions and said adipose tissue-derived cell suspension,
optionally comprising adipocytes, is stored frozen prior to said
combining. In a further embodiment the composition comprising
adipose tissue-derived cell secretions and said adipose
tissue-derived cell suspension, optionally comprising adipocytes,
are mixed together before the composition is frozen.
[0036] In an embodiment the pharmaceutical composition is a
veterinary composition and the subject is a non-human animal.
[0037] In a fourth aspect of the invention there is provided use of
(i) adipose tissue-derived cell secretions, or (ii) an adipose
tissue-derived cell suspension, optionally comprising adipocytes,
or (iii) a combination of (i) and (ii), for the preparation of a
pharmaceutical composition for use in the treatment of a condition
selected from the group consisting of an inflammatory disorder, a
ligament injury and a tendon injury, or alleviating pain associated
with an inflammatory disorder, a ligament injury or a tendon
injury, in a mammalian subject, wherein the composition is suitable
for administration to a site of said subject remote from the site
of said condition.
[0038] In a fifth aspect there is provided use of (i) adipose
tissue-derived cell secretions, or (ii) an adipose tissue-derived
cell suspension, optionally comprising adipocytes, or (iii) a
combination of (i) and (ii), for the preparation of a
pharmaceutical composition for use in the treatment of a joint
disease or condition in a mammalian subject, wherein the
composition is suitable for administration to a site of said
subject remote from the site of said condition. In an embodiment
the adipose tissue-derived cell suspension, optionally comprising
adipocytes, comprises aggregates of cells and or comprises pieces
of adipose tissue. In an embodiment the adipose tissue-derived cell
suspension is substantially free of adipocytes. In an embodiment
the adipose tissue-derived cell suspension comprises
adipocytes.
[0039] In a sixth aspect of the invention there is provided Use of
(i) adipose tissue-derived cell secretions, or (ii) an adipose
tissue-derived cell suspension, optionally comprising adipocytes,
or (iii) a combination of (i) and (ii), for the preparation of a
pharmaceutical composition for use in the treatment or prevention
of disease in an intensively fanned animal, wherein the composition
is suitable for subcutaneous injection or intramuscular
injection.
[0040] In a seventh aspect of the invention there is provided a
composition comprising (i) adipose tissue-derived cell secretions,
or (ii) an adipose tissue-derived cell suspension, optionally
comprising adipocytes, or (iii) a combination of (i) and (ii), for
treatment of a condition selected from the group consisting of an
inflammatory disorder, a ligament injury and a tendon injury, or
alleviating pain associated with an inflammatory disorder, a
ligament injury or a tendon injury, wherein the composition is
administered to a site on a subject remote from the site afflicted
by the condition. In an embodiment the composition is an injectable
composition. In an embodiment the administration is by subcutaneous
injection or intramuscular injection.
[0041] In an eighth aspect of the invention there is provided a
composition comprising (i) adipose tissue-derived cell secretions,
or (ii) an adipose tissue-derived cell suspension, optionally
comprising adipocytes, or (iii) a combination of (i) and (ii), for
treatment of a joint disease or condition in a mammalian subject,
wherein said composition is administered a site of said subject
remote from the site of said condition. In an embodiment the
adipose tissue-derived cell suspension, optionally comprising
adipocytes, comprises aggregates of cells and or comprises pieces
of adipose tissue. In an embodiment the adipose tissue-derived cell
suspension is substantially free of adipocytes. In an embodiment
the adipose tissue-derived cell suspension comprises
adipocytes.
[0042] In a ninth aspect of the invention there is provided a
composition comprising (i) adipose tissue-derived cell secretions,
or (ii) an adipose tissue-derived cell suspension, optionally
comprising adipocytes, or (iii) a combination of (i) and (ii); for
treatment or prevention of a disease in an intensively farmed
animal, wherein in said treatment or prevention the composition is
administered by subcutaneous injection or intramuscular
injection.
[0043] In a tenth aspect of the invention there is provided a
pharmaceutical composition comprising (i) adipose tissue-derived
cell secretions, or (ii) an adipose tissue-derived cell suspension,
optionally comprising adipocytes, or (iii) a combination of (i) and
(ii), together with a pharmaceutically acceptable carrier, diluent,
excipient or adjuvant. In an embodiment the composition comprising
adipose tissue-derived cell secretions further comprises
adipocytes. In an embodiment the composition is a frozen
composition.
[0044] In an eleventh aspect of the invention there is provided a
kit comprising (a) a pharmaceutical composition selected from the
group consisting of (i) a composition comprising adipose
tissue-derived secretions, (ii) a composition comprising an adipose
tissue-derived cell suspension, optionally comprising adipocytes,
and (iii) a combination of (i) and (ii); and (b) instructions for
use of said kit in treatment of a condition selected from the group
consisting of an inflammatory disorder, a ligament injury and a
tendon injury, or alleviating pain associated with an inflammatory
disorder, a ligament injury or a tendon injury; wherein said
treatment comprises administration of said pharmaceutical
composition to a site on a subject remote from the site afflicted
by the condition.
[0045] In a twelfth aspect of the invention there is provided a kit
comprising (a) a pharmaceutical composition selected from the group
consisting of (i) a composition comprising adipose tissue-derived
secretions, (ii) a composition comprising an adipose tissue-derived
cell suspension, optionally comprising adipocytes, and (iii) a
combination of (i) and (ii); and (b) instructions for use of said
kit in treatment of a joint disease or condition in a mammalian
subject, wherein said treatment comprises administration of said
pharmaceutical composition to a site on a subject remote from the
joint afflicted by the joint disease or condition. In an embodiment
the adipose tissue-derived cell suspension, optionally comprising
adipocytes, comprises aggregates of cells and or comprises pieces
of adipose tissue. In an embodiment the adipose tissue-derived cell
suspension is substantially free of adipocytes. In an embodiment
the adipose tissue-derived cell suspension comprises
adipocytes.
[0046] In a thirteenth aspect of the invention there is provided a
kit comprising (a) a pharmaceutical composition selected from the
group consisting of (i) a composition comprising adipose
tissue-derived secretions, (ii) a composition comprising an adipose
tissue-derived cell suspension, optionally comprising adipocytes,
and (iii) a combination of (i) and (ii); and (b) instructions for
use of said kit in treatment or prevention of disease in an
intensively farmed animal, wherein in said treatment or prevention
the composition is administered by subcutaneous injection or
intramuscular injection.
[0047] In an embodiment the kit comprises one or more frozen
compositions. In an embodiment the kit comprises instructions for
combining a composition comprising adipose tissue-derived
secretions and a composition comprising an adipose tissue-derived
cell suspension, optionally comprising adipocytes, prior to
administration of a combined composition. In an embodiment the kit
further comprises one or more injection devices, such as one or
more syringes. In an embodiment the injection device contains a
composition of the kit.
[0048] In a fourteenth aspect of the invention there is provided a
method of alleviating pain in a mammalian subject, the method
comprising administering to the subject a pharmaceutical
composition which comprises (i) adipose tissue-derived cell
secretions, or (ii) an adipose tissue-derived cell suspension,
optionally comprising adipocytes, or (iii) a combination of (i) and
(ii), wherein said administration to said subject is by
intramuscular injection or by subcutaneous injection or by an
appropriate form of administration at or near a site of the pain.
In an embodiment the pain is associated with a condition selected
from the group consisting of an inflammatory disorder, a ligament
injury and a tendon injury. In an embodiment the pain is associated
with osteoarthritis, stifle disease, wobblers, a tendon injury or a
ligament injury. In an embodiment the pain is associated with
atopic dermatitis. In an embodiment the pain is associated with
rheumatoid arthritis, back pain, or multiple sclerosis. In an
embodiment the pain is associated with a condition selected from
the group consisting of leg weakness, lameness, arthritis,
developmental diseases and bacterial infection. In an embodiment
the pain is associated with osteochondrosis (OCD). In an embodiment
the pain is associated with a burn injury. In an embodiment the
pain is neck and or shoulder pain, whiplash associated disorder, or
complex regional pain syndrome. In an embodiment the pain is back
pain. In an embodiment the pain is lower back pain. In an
embodiment the pain is pain associated with a sciatic disorder. In
an embodiment the treatment is of pain for which there is no
discernable causative clinical condition. In an embodiment the
treatment is of pain for which there is no discernable causative
clinical condition in the part or region of the body in which the
subject experiences the pain. In an embodiment the pain is
neuropathic pain. In an embodiment the neuropathic pain is pain for
which there is no discernable causative clinical condition. In an
embodiment the appropriate form of administration is injection. In
an embodiment the appropriate form of administration is topical
application. The neuropathic pain may be localised to one area of
the body or it may be experienced in multiple sites of the
subject's body. When experienced in multiple sites, the intensity
of the pain may be similar at multiple sites or it may be different
at multiple sites. In an embodiment the neuropathic pain is
neuropathic facial pain. In an embodiment the pain is neuropathic
facial pain and administration to said subject is by injection into
the jaw or the gum. In an embodiment the injection into the jaw or
the gum is at the original site of the pain. In an embodiment the
pain is associated with a joint disease or joint disorder and the
composition is administered to a site on said subject which is
remote to said joint. In an embodiment the adipose tissue-derived
cell suspension, optionally comprising adipocytes, comprises
aggregates of cells and or comprises pieces of adipose tissue.
[0049] In a fifteenth aspect of the invention there is provided use
of (i) adipose tissue-derived cell secretions, or (ii) an adipose
tissue-derived cell suspension, optionally comprising adipocytes,
or (iii) a combination of (i) and (ii), for the preparation of a
pharmaceutical composition for use in alleviating pain in a
mammalian subject, wherein the pharmaceutical composition is
suitable for administration to said subject by intramuscular
injection or by subcutaneous injection or by an appropriate form of
administration, such as topical administration, at or near a site
of the pain.
[0050] In a sixteenth aspect of the invention there is provided a
composition comprising (i) adipose tissue-derived cell secretions,
or (ii) an adipose tissue-derived cell suspension, optionally
comprising adipocytes, or (iii) a combination of (i) and (ii), for
alleviating pain in a mammalian subject, wherein the pharmaceutical
composition is administered to said subject by intramuscular
injection or by subcutaneous injection or by an appropriate form of
administration, such as topical administration, at or near a site
of the pain.
[0051] In a seventeenth aspect of the invention there is provided a
kit comprising (a) a pharmaceutical composition selected from the
group consisting of (i) a composition comprising adipose
tissue-derived secretions, (ii) a composition comprising an adipose
tissue-derived cell suspension, optionally comprising adipocytes,
and (iii) a combination of (i) and (ii); and (b) instructions for
use of said kit in alleviating pain in a mammalian subject, wherein
the pharmaceutical composition is administered to said subject by
intramuscular injection or by subcutaneous injection or by an
appropriate form of administration, such as topical administration,
at or near a site of the pain.
[0052] In a further aspect the invention provides a composition
comprising adipose tissue-derived cells and adipose tissue-derived
cell secretions. In an embodiment the cells are adherent cells. In
an embodiment the cells are mesenchymal stem cells. In an
embodiment the composition is a frozen composition. In an
embodiment the adipose tissue-derived cell secretions comprise
clarified media from culture of adipose tissue-derived cells. In an
embodiment the adipose tissue-derived cell secretions is a
concentrated preparation of media from culture of adipose
tissue-derived cells. In an embodiment the adipose tissue-derived
cell secretions is a preparation concentrated between 2-fold and
20-fold. In an embodiment the composition further comprises
adipocytes. In an embodiment the cells are progeny cells from
culture of an adipose tissue-derived cell suspension. In an
embodiment the cells comprise a cell line obtained by culture of an
adipose tissue-derived cell suspension. In an embodiment the
progeny cells are from multiple passaging of cells derived from an
adipose tissue-derived cell suspension. In an embodiment the
multiple passaging comprises five or more passages. In an
embodiment the multiple passaging comprises ten or more passages.
In an embodiment the composition comprises cells of an adipose
tissue-derived cell line which has been frozen multiple times.
[0053] In a further aspect the invention provides a method for the
cryopreservation of a stored cell, the method comprising combining
said cell with a composition comprising cell secretions and storing
said combination in a frozen state. In an embodiment the method is
for the cryopreservation of a cell line. In an embodiment, prior to
said storing, the combination is held at room temperature for up to
one hour. In an embodiment the said stored cell is an adherent
cell. In an embodiment the stored cell is a mesenchymal stem cell.
In an embodiment the stored cell is an adipose tissue-derived cell.
In an embodiment the cell line is an adipose tissue-derived cell
line. In an embodiment the composition comprising cell secretions
comprises clarified media from culture of an adipose tissue-derived
cell suspension. In an embodiment the culture of an adipose
tissue-derived cell suspension is culture of progeny cells of an
adipose tissue-derived cell suspension. In an embodiment the
composition comprising cell secretions comprises concentrated media
from culture of an adipose tissue-derived cell suspension. In an
embodiment the composition comprising cell secretions comprises
media from culture of an adipose tissue-derived cell suspension
concentrated between 2-fold and 20-fold. In an embodiment the cell
line has been passaged multiple times. In an embodiment the cell
line has been passaged more than five times. In an embodiment the
cell line has been passaged more than ten times. In an embodiment
the cell line has been passaged more than fifteen times. In an
embodiment the cell line has been frozen multiple times.
[0054] It will be understood that embodiments described herein
apply equally to any and all aspects of the invention described
herein, they are simply not repeated under each aspect of the
invention for the sake of brevity.
[0055] The summary of the invention described above is not limiting
and other features and advantages of the invention will be apparent
from the following detailed description of the preferred
embodiments, as well as from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0056] FIG. 1: Proliferation of cells after freezing. Cells that
had not been passaged and were frozen without secretions (left
graph) and with secretions (right graph) were stained with the
Click-iT ERD assay that identifies proliferating cells. The
proliferating cells appear in the upper right quadrant.
[0057] FIG. 2: Recovery of cells after freezing. Cells that had
been cultured until a cumulative cell doubling of approximately 13
times had been reached were frozen without secretions (top image)
and with secretions (bottom image) and then thawed and cultured for
72 hours.
[0058] FIG. 3: Paw volume measurements from a collagen antibody
induced arthritis (CAIA) mouse model treated intramuscularly with
SVF+adipocyte secretions (.box-solid.) or vehicle control
(.circle-solid.).
[0059] FIG. 4: Ankle size measurements from a collagen antibody
induced arthritis (CAIA) mouse model treated intramuscularly with
SVF+adipocyte secretions (.box-solid.) or vehicle control
(.circle-solid.).
[0060] FIG. 5: Clinical arthritis scores from a collagen antibody
induced arthritis (CAIA) mouse model treated intramuscularly with
SVF+adipocyte secretions (.box-solid.) or vehicle control
(.circle-solid.).
[0061] FIG. 6: Paw volume measurements from CAIA mice treated IV
with cells or cells with concentrated secretions. Cells
(.box-solid.); cells plus secretions (.circle-solid.).
[0062] FIG. 7: Paw volume Area Under the Curve results from CAIA
mice treated IV with cells or cells with concentrated
secretions.
[0063] FIG. 8: Ankle size measurements from CAIA mice treated IV
with cells or cells with concentrated secretions. Cells
(.box-solid.); cells plus secretions (.circle-solid.).
[0064] FIG. 9: Ankle size Area Under the Curve results from CAIA
mice treated IV with cells or cells with concentrated
secretions.
[0065] FIG. 10: Clinical arthritis scores from CAIA mice treated IV
with cells or cells with concentrated secretions. Cells
(.box-solid.); cells plus secretions (.circle-solid.).
[0066] FIG. 11: Clinical arthritis score Area Under the Curve
results from CAIA mice treated IV with cells or cells with
concentrated secretions.
ABBREVIATIONS
[0067] DMEM Dulbecco's Modified Eagles Medium.
[0068] SVCs stromal vascular cells.
[0069] SVF stromal vascular fraction.
[0070] OCD osteochondrosis.
[0071] MSC mesenchymal stem cell(s).
Definitions
[0072] In the context of the present invention reference to a
composition comprising "adipose tissue-derived secretions" will be
understood to mean a composition which includes one or more factors
released from cells of the adipose tissue. The material used in the
preparation of the composition comprising the secretions may or may
not include adipocytes.
[0073] The term "pharmaceutically acceptable" as used herein in the
context of various components relevant to the invention, such as
carriers, diluents, cryopreservatives, is intended to encompass not
only such components which are suitable for administration to a
human subject, but also those suitable for administration to a
non-human mammalian subject. In particular embodiments, the
pharmaceutically acceptable component is suitable for
administration to a non-human mammalian subject. In particular
embodiments the pharmaceutically acceptable component is suitable
for administration to a human subject. In particular embodiments,
the pharmaceutically acceptable component is suitable for
administration to a non-human mammalian subject and to a human
subject.
[0074] The terms "treating", "treatment", "therapy" and the like in
the context of the present specification refer to the alleviation
of the symptoms and/or the underlying cause of the condition or
disease, such as inflammatory disorder, ligament injury, or tendon
injury or disease of an intensively farmed animal. In certain
embodiments a treatment will slow, delay or halt the progression of
a disorder or the symptoms of the disorder or injury, or reverse
the progression of the disorder or injury, at least temporarily.
Hence, in the context of this invention the word "treatment" or
derivations thereof such as "treating" when used in relation to a
therapeutic application includes all aspects of a therapy, such as
the alleviation of pain associated with the condition being
treated, alleviation of the severity of the condition being
treated, improvement in one or more symptoms of the condition being
treated, etc. Use of the word "treatment" or derivatives thereof
will be understood to mean that the subject being "treated" may
experience any one or more of the aforementioned benefits.
[0075] The term "preventing" and the like, in the context of the
"prevention" of disease, refers to hindrance of the progression of
the symptoms or the underlying cause of the disease. It will be
understood that complete prevention of a disease may occur, such
that the disease does not occur in a treated animal or subject.
Equally, it will be understood that the term includes partial
prevention, such as the failure of a disease to progress to the
typical state observed in an animal or subject left untreated.
[0076] Throughout this specification, reference to "a" or "one"
element does not exclude the plural, unless context determines
otherwise. Similarly, reference to "an embodiment" does not exclude
the characteristic of that described embodiment applying in
combination with one or more other embodiments described, unless
the context determines otherwise.
[0077] The term "therapeutically effective amount" as used herein
includes within its meaning a non-toxic but sufficient amount of a
compound or composition for use in the invention to provide the
desired therapeutic effect. The exact amount required will vary
from subject to subject depending on factors such as the species
being treated, the age and general condition of the subject,
co-morbidities, the seventy of the condition being treated, the
particular agent being administered and the mode of administration
and so forth. Thus, for any given case, an appropriate "effective
amount" may be determined by one of ordinary skill in the art using
only routine methods.
[0078] In the context of this specification, the term "comprising"
means including, but not necessarily solely including. Furthermore,
variations of the word "comprising", such as "comprise" and
"comprises", have correspondingly varied meanings. Hence, the term
"comprising" and variations thereof is used in an inclusive rather
than exclusive meaning such that additional integers or features
may optionally be present in a composition, method, etc. that is
described as comprising integer A, or comprising integer A and B,
etc.
[0079] In the context of this specification the term "about" will
be understood as indicating the usual tolerances that a skilled
addressee would associate with the given value.
[0080] In the context of this specification, where a range is
stated for a parameter it will be understood that the parameter
includes all values within the stated range, inclusive of the
stated endpoints of the range. For example, a range of "5 to 10"
will be understood to include the values 5, 6, 7, 8, 9, and 10 as
well as any sub-range within the stated range, such as to include
the sub-range of 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc, and
inclusive of any value and range between the integers which is
reasonable in the context of the range stated, such as 5.5, 6.5,
7.5, 5.5 to 8.5 and 6.5 to 9, etc.
[0081] In the context of this specification, the term "plurality"
means any number greater than one.
[0082] It is to be noted that reference herein to use of the
inventive methods and compositions in treatment or therapy will be
understood to be applicable to human and non-human, such as
veterinary, applications. Hence it will be understood that, except
where otherwise indicated, reference to a patient, subject or
individual means a human or a non-human, such as an individual of
any species of social, economic, agricultural or research
importance including but not limited to members of the
classifications of ovine, bovine, equine, porcine, feline, canine,
primates, rodents, especially domesticated or farmed members of
those classifications, such as sheep, cattle, horses, pigs and
dogs.
[0083] Where examples of various embodiments or aspects of the
invention are described to herein they will generally be prefaced
by appropriate terms including "such as" or "for example", or
"including". It will be understood that the examples are being
described as inclusive possibilities, such as for the purpose of
illustration or understanding and are not, unless the context
indicates otherwise, being provided as limiting.
[0084] The pharmaceutical composition referred to herein may also
be referred to as a medicament when intended for therapeutic use.
Hence, it will be understood that where the invention is described
as including the use of a composition of described components for
the preparation of a pharmaceutical composition for an intended
therapeutic purpose, that description equally means use for the
preparation of a medicament for that intended therapeutic purpose,
unless the context indicates otherwise.
[0085] To the extent that it is permitted, all references cited
herein are incorporated by reference in their entirety.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0086] The present inventors have identified that surprisingly the
remote delivery of a composition comprising secretions from an
adipose tissue-derived cell suspension can be effective in the
treatment of various conditions including inflammatory diseases and
bone and joint disorders, including ligament injuries and tendon
injuries. The inventors have also surprisingly identified that
remote delivery of an adipose tissue-derived cell suspension,
optionally comprising adipocytes, or of a combination of adipose
tissue-derived secretions and an adipose tissue-derived cell
suspension, optionally comprising adipocytes, can be effective in
the treatment of such conditions. Previously described methods for
the treatment of such conditions have described the administration
of the therapeutic to a site of disease or pain by direct
application, such as intra-articular injection in the case of a
joint disease. The present invention thus pertains to methods of
treating such conditions by remote administration of (i) a
composition comprising adipose tissue-derived cell secretions, or
(ii) a composition comprising an adipose tissue-derived cell
suspension, optionally comprising adipocytes, or (iii) a
combination of (i) and (ii), to a subject in need thereof. The
invention also provides for the use of (i) adipose tissue-derived
cell secretions, or (ii) an adipose tissue-derived cell suspension,
optionally comprising adipocytes, or (iii) a combination of (i) and
(ii), for the preparation of a medicament for the treatment of a
condition selected from the group consisting of an inflammatory
disorder, a ligament injury and a tendon injury, or alleviating
pain associated with an inflammatory disorder, a ligament injury, a
tendon injury, neuropathic pain, or a burn injury, the medicament
suitable for remote administration to a subject.
[0087] As described herein, the inventor has surprisingly
identified that administration of such a therapeutic agent does not
necessarily need to be direct administration of the agent to the
diseased or affected site, such as a joint. By administration of
the therapeutic agent, such as by subcutaneous injection or
intramuscular injection, the inventor has identified that various
diseases may be treated or prevented. In the case of a disease
affecting a joint, the description herein of the administration as
remote simply means that it is not administered directly into the
joint, but rather is typically administered by subcutaneous
injection or intramuscular injection. Hence, the site of
administration by subcutaneous injection or intramuscular injection
may or may not be particularly distant from the affected joint. The
invention pertains also to methods for the treatment or prevention
of disease in an intensively farmed animal, the method comprising
administration by subcutaneous injection or intramuscular injection
of (i) a composition comprising adipose tissue-derived cell
secretions, or (ii) an adipose tissue-derived cell suspension,
optionally comprising adipocytes, or (iii) a combination of (i) and
(ii).
[0088] The inventor has identified that allogeneic and xenogeneic
compositions can be used in the treatment and further that the
therapeutic compositions can be stored frozen prior to use. It will
be understood that in an aspect the invention herein relates to
such compositions per se, regardless of the manner in which they
may be used or may be intended to be used. In other aspects, the
invention relates to the use of compositions of the invention in
the methods disclosed herein. In this manner so-called "off the
shelf" or "ready to use" therapeutic products offering advantages,
such as of supply, ease of use, less patient discomfort, and a
lower requirement for technical skills, compared to an autologous
patient-derived therapeutic agent, can be made available. The
present invention thus permits the preparation of the therapeutic
agent in advance of patient contact, such that a product comprising
adipose tissue-derived cell secretions or an adipose tissue-derived
cell suspension, optionally comprising adipocytes, may be made
available without the need to anesthetize a subject or animal for
extraction of adipose tissue. Similarly, where the therapeutic
agent is a combination of adipose tissue-derived cell secretions
and an adipose tissue-derived cell suspension, optionally
comprising adipocytes, that combination may be made available to
the user, such as a clinician, veterinarian, or farmer in advance
or the separate compositions of cell secretions and of cell
suspension may be supplied, with the user then preparing the
combination shortly before administration. As described herein the
cell secretions, the cell suspension, or the combination can be
stored, for example at -20.degree. C. until required for use.
Alternatively, the cell secretions, the cell suspension, or the
combination may be stored at a lower temperature, such as in a
freezer at -70.degree. C. to -90.degree. C., or in liquid nitrogen
storage, either in the vapour phase or in the liquid phase, until
required for use. Compositions comprising cells will typically be
stored in liquid nitrogen. In a preferred embodiment the
composition comprising adipose tissue-derived cell secretions, or
the adipose tissue-derived cell suspension, optionally comprising
adipocytes; or the combination of adipose tissue-derived cell
secretions and adipose tissue-derived cell suspension, optionally
comprising adipocytes, is stored in the liquid phase of liquid
nitrogen storage. In an embodiment the adipose tissue-derived cell
secretions is a concentrated preparation. In an embodiment the
concentrated preparation is concentrated in comparison to the cell
secretions as initially harvested from the adipose tissue-derived
cell suspensions or culture thereof. In an embodiment the adipose
tissue-derived cell secretions is a preparation concentrated by
between about 2-fold and about 20-fold. In an embodiment the
adipose tissue-derived cell secretions is a preparation
concentrated by about 10-fold.
[0089] Without wishing to be bound by any proposed mechanism of
action, it is proposed that the adipose tissue-derived cell
secretions comprise cytokines, such as anti-inflammatory cytokines,
that are able to migrate to a source of injury or disease and there
effect improvement in the underlying condition, or alleviation of
pain associated with the condition. Similarly, the adipose
tissue-derived cell suspension, optionally comprising adipocytes,
when injected subcutaneously or intramuscularly then operate to
secrete various cell factors, such as cytokines, which are able to
migrate to the site of injury or disease, be it clinical or
subclinical, thereby effecting improvement of the underlying
condition or prevention of clinical occurrence, for example in an
intensively farmed animal. As described in the Examples herein, the
method is also effective in improving lameness of a treated
individual and may provide benefits in performance enhancement, as
demonstrated by the improved agility and mobility of treated
individuals.
[0090] Adipose Tissue
[0091] The cell secretions of the invention are adipose
tissue-derived cell secretions. The cell suspensions of the
invention are adipose tissue-derived cell suspensions. Adipose
tissue may be human adipose tissue or mammalian animal adipose
tissue. The human or animal may be alive or dead, provided that
there are still viable cells within the adipose tissue. The adipose
tissue may comprise "white" adipose tissue, or "brown" adipose
tissue.
[0092] The adipose tissue may originate from any source in the body
which is accessible. Subcutaneous fat, for example, is readily
accessible with only superficial wounding, or by using "keyhole
surgery" techniques. For example adipose tissue may be tissue
collected using liposuction techniques, or adipose tissue which is
removed with reproductive tissue when de-sexing a male or female
animal. The adipose tissue may be rinsed with a tissue culture
medium or buffered isotonic solution to remove adherent blood
cells, and may be trimmed or coarsely processed to remove large
blood vessels or connective tissue elements prior to generating an
adipose tissue-derived cell suspension.
[0093] The adipose tissue may be derived from a mature or from a
juvenile animal.
[0094] In particular embodiments the mammal is a companion animal,
such as a canine or a feline domestic animal, or a working animal.
In other particular embodiments the mammal is a, farm animal or
racing animal selected from a horse, donkey, ass, cow, buffalo,
sheep, goat, camel or pig.
[0095] Adipose Tissue-Derived Cell Suspension
[0096] The adipose tissue-derived cell secretions and hence the
compositions comprising such secretions, are preferably prepared by
first obtaining or preparing an adipose tissue-derived cell
suspension. As described herein the methods, kits, uses, and
compositions of the invention may comprise adipose tissue-derived
cell secretions, an adipose tissue-derived cell suspension or a
combination of the cell secretions and the cell suspension. The
adipose tissue-derived cell suspension may or may not comprise
adipocytes.
[0097] The term "adipose tissue-derived cell suspension" as used
herein encompasses isolated cells from adipose tissue or small
aggregates or pieces of adipose tissue, or a mixture of two or more
of: isolated cells, small aggregates and pieces of adipose tissue.
The cell suspension may be obtained by mechanically dissociating
adipose tissue using techniques which are readily available in the
art. Any suitable method for the mechanical dissociation of adipose
tissue may be used, for example by mincing adipose tissue with
blades, or with scissors, or by forcing adipose tissue through
screens or meshes with a pore size sufficient to break the tissue
into isolated cells or small pieces of adipose tissue, or a
combination of these techniques. Small aggregates of adipose tissue
may form when dissociated adipose-derived cells reassociate into
larger assemblies, for example on standing in a medium. Small
pieces or aggregates of adipose tissue may be less than ten
millimetres in diameter, less than five millimetres in diameter,
less than one millimetre in maximum diameter, less than 500 .mu.m
in maximum diameter or less than 250 .mu.m in maximum diameter.
[0098] The adipose tissue-derived cell suspension may be filtered
through a mesh or screen to remove cell aggregates or tissue pieces
which are greater than the mesh or screen pore size.
[0099] Proteolytic enzymes may be used to promote the dissociation
of adipose tissue into an adipose tissue-derived cell suspension.
Enzymes which are suitable for such a use are well known in the
art, and include but are not limited to trypsin, and collagenase.
It is usual to remove and/or otherwise inactivate the proteolytic
enzymes before using the adipose-tissue-derived cell extract, as
these enzymes may not be compatible with a desired in vivo use of
the cells. The proteolytic enzymes may be used in combination with
techniques for the mechanical dissociation of adipose tissue to
generate an adipose tissue-derived cell suspension.
[0100] A mechanical dissociation technique may be used without
using one or more proteolytic enzymes. The technique used in this
manner may be used to rapidly generate an adipose tissue-derived
cell suspension.
[0101] The cell suspension may be suspended in a liquid. The liquid
may be added to the adipose tissue before, during or after the
dissociation of the adipose tissue. The liquid may comprise a
medium which is capable of maintaining adipose tissue cell survival
for at least 24 hours under appropriate culture conditions. The
liquid may comprise an isotonic buffered solution, such as a
phosphate or a HEPES buffered saline, which is capable of
maintaining adipose tissue cell survival for at least one hour. The
liquid may comprise a tissue culture medium. The liquid may
comprise serum or serum components which support or extend adipose
tissue cell survival in the cell suspension. The serum or serum
components may be autologous serum or serum components.
[0102] In some embodiments the cell suspension may not have added
liquid, but instead the cells are suspended in liquid which is
formed during the dissociation of the tissue.
[0103] The preparation of an adipose tissue-derived cell suspension
may comprise a centrifugation step. The centrifugation of isolated
cells or small aggregates or pieces of adipose tissue suspended in
a liquid, such as a medium, is at approximately 500 g for 10
minutes, or for sufficient time and at a sufficient g-force to
generate a cell pellet which comprises adipose-derived
non-adipocyte cells, above which is a layer of medium, floating
above which in turn is a layer which comprises the viable
adipocytes, and floating at the top is a layer of lipid which is
derived from ruptured adipocytes. Following centrifugation, in
certain embodiments the lipid layer and the medium layer will be
discarded and the retained cells are mixed, leaving an adipose
tissue-derived cell suspension which comprises viable adipocytes
and adipose-derived non-adipocyte cells. In other embodiments, only
the layer comprising the viable adipocytes will be retained. In
other embodiments, the layer comprising adipocytes may be removed
and hence not included in the adipose tissue-derived cell
suspension. This will typically occur when preparing an adipose
tissue-derived cell suspension which is substantially free of
adipocytes. A cell suspension referred to herein as being
substantially free of adipocytes means that the cell suspension has
been significantly depleted of adipocytes compared to the starting
material, such as by removal of the adipocyte fraction after
centrifugation. It will be understood that substantially free of
adipocytes when used in relation to a cell suspension includes
complete absence of adipocytes and also includes the situation
where minimal retention of adipocytes in the material has occurred.
In other embodiments, only part of the adipocyte content of the
adipose tissue may be removed in the preparation of the adipose
tissue-derived cell suspension. In this case, the resultant cell
suspension will comprise adipocytes, but at a reduced proportion
relative to other retained components, such as the stem cells,
compared to the proportion in the starting material. In an
embodiment the adipose tissue-derived cell suspension comprises at
least 10% adipocytes by volume. In an embodiment the adipose
tissue-derived cell suspension comprises between 10% and 30%
adipocytes by volume
[0104] One centrifugation step or multiple centrifugation steps may
be used, for example to provide additional cell separation steps.
In other embodiments, the preparation of an adipose tissue-derived
cell suspension does not include a centrifugation step.
[0105] The adipose tissue-derived cell suspension may or may not
comprise viable adipocytes. When present, the adipocytes may retain
detectable quantities of lipid in their cytoplasm, and may be
separated from adipose-derived non-adipocyte cells on the basis of
the different density provided by the lipid. Lipid may be
detectable using light microscopy techniques, including phase
contrast microscopy, or by staining a sample of cells with a
lipophilic dye such as Oil Red O. Adipocytes which retain lipid in
their cytoplasm are considerably more fragile than other
adipose-derived cells, and accordingly where viable adipocytes are
desired techniques for dissociating tissue which damage or kill a
large proportion of the adipocytes should be avoided. The
ultrasonic dissociation of adipose tissue or techniques in which
adipose tissue is vigorously shaken, for example, are unlikely to
provide a cell suspension which contains large numbers of viable
adipocytes. The viability of adipocytes may readily be determined
using readily available techniques, such as the LIVE/DEAD cell
viability assays (Molecular Probes).
[0106] The adipose tissue-derived cell suspension may comprise both
adipocytes and adipose-derived non-adipocyte cells. The
adipose-derived non-adipocyte sells typically include cells of the
stromal vascular fraction, including mesenchymal stem cells. Cells
of the stromal vascular fraction typically pellet upon
centrifugation conditions described herein of an adipose
tissue-derived cell suspension.
[0107] In embodiments which comprise both adipocytes and
adipose-derived non-adipocyte cells, the adipose tissue-derived
cell suspension may be conveniently prepared by methods which
comprise a centrifugation step, as described herein, in which both
the adipocyte cell layer and the pelleted adipose-derived
non-adipocyte cells are collected. Alternatively, in these
embodiments the adipose tissue-derived cell suspension may be
prepared by dissociating adipose tissue as described herein without
a centrifugation step.
[0108] The adipose tissue-derived cell suspension, optionally
comprising adipocytes, may be stored under appropriate conditions.
The storage conditions typically permit the retention of cell
viability of some or all cells in the cell suspension, such as
greater than 50%, greater than 60%, greater than 70%, greater than
80%, greater than 90%, or greater than 95%.
[0109] Where the adipose tissue-derived cell suspension is to be
stored frozen it may be in any carrier liquid appropriate for
freezing of cells. As an illustrative but not limiting example, the
cells may be suspended in culture medium, which may be
serum-containing or serum-free, such as DMEM, RPMI, minimal
essential media, or in serum prior to freezing.
[0110] Where the adipose tissue-derived cell suspension is to be
stored frozen it typically also comprises a cryopreservative, for
example, dimethylsulfoxide (DMSO) or glycerol, at an appropriate
concentration, such as 5% to 10%. As described herein cell
secretions such as those obtained or derived from cell culture of
an adipose tissue-derived cell suspension can also be used as a
cryopreservative for cell storage. Such secretions, optionally
clarified and or optionally concentrated, can be combined with a
cell population intended for frozen storage, such as mesenchymal
cells, such as adipose tissue-derived cells, including a cell line
resulting from culturing of an adipose tissue-derived cell
suspension. The combination may be held at an appropriate
temperature, such as room temperature, for example approximately 18
C to 25 C, prior to freezing for any suitable time, such as up to
about one or two hours, to permit interaction between the
secretions and the cells, for example approximately 10 minutes, 20
minutes, 30 minutes, 60 minutes, 90 minutes or two hours. In
preferred embodiments the combination is held at room temperature
for about 30 minutes.
[0111] The constituents of the cell suspension, such as the liquid
medium and the cryopreservative, are typically pharmaceutically
acceptable at the concentrations used. This has the advantage that
the adipose tissue-derived cell suspension can be administered to a
recipient subject or animal after thawing with minimal post-thaw
processing.
[0112] The cell suspension is typically frozen under controlled
conditions to minimize cell damage, for example by slow freezing,
typically at a rate of about PC/min, such as by placing in a
programmable freezing device, or in an insulated container in a
-70.degree. C. to -90.degree. C. freezer. For storage, frozen cells
are typically then transferred to liquid nitrogen storage.
[0113] A cell processing method and device which may be used for
the preparation of adipose tissue-derived cell suspensions is
described in co-pending application PCT/AU2012/000272, the contents
of which are incorporated herein by reference.
[0114] Bovine Adipose Tissue Derived Cell Suspensions
[0115] The adipose tissue-derived cell suspensions, and hence the
adipose tissue-derived cell secretions, may be derived from any
appropriate source. Bovine adipose tissue is one such source. The
inventor has previously described inventive methods for the
preparation of adipose tissue derived cell suspensions from bovine
sources, particularly from bovine tail base tissue, as that
material was found to be refractory to standard methods appropriate
to multiple other sources of adipose tissue, such as human, canine,
equine, mouse and rat. This is described in co-pending application
PCT/AU2012/000274, the contents of which is incorporated herein by
reference.
[0116] For example, where the present invention utilises bovine
adipose-derived material, a bovine adipose tissue-derived cell
suspension for use as a therapeutic composition or for use in the
preparation of adipose tissue-derived cell secretions may be
prepared according to a method comprising: [0117] exposing a sample
of bovine adipose tissue to a proteolytic enzyme solution to
generate a cell suspension; [0118] centrifuging the suspension of
cells to form a cell pellet, a free lipid layer above a floating
cell layer which comprises adipocytes and an intermediate layer
between the cell pellet and the floating cell layer, said
intermediate layer being to depleted of cells relative to the cell
pellet and the floating cell layer; and [0119] removing the free
lipid layer and the intermediate layer and mixing the cell pellet
and floating cell layer to form an adipose tissue derived cell
suspension which comprises adipocytes.
[0120] In the methods, uses and compositions of the invention where
a cell suspension which does not comprise adipocytes is desired,
the floating cell layer which comprises adipocytes may also be
removed and discarded in the performance of the above method.
[0121] The method may comprise additional steps in the preparation
of adipose tissue-derived cell suspensions as set out elsewhere in
this specification, in particular the previous section headed
"Adipose Tissue-Derived Cell Suspension". These additional steps
include, for example, mechanically dissociating the tissue, and
suspension via a medium or buffer etc.
[0122] The removed intermediate layer may be retained as it
typically includes adipose tissue-derived secretions. As the
concentration of secretions in the removed intermediate layer is
typically low compared to the concentration of secretions produced
from subsequent cell culturing of adipose tissue-derived cell
suspensions, as described below, cell secretions harvested from
cultured cells are typically used in the methods, uses and
compositions of the invention.
[0123] In certain embodiments the proteolytic enzyme solution
comprises collagenase. The collagenase typically is used at a final
concentration of about 0.2% w/v or about 0.25% w/v or greater. In
certain embodiments the exposure of the bovine adipose tissue to
proteolytic enzyme is conducted under conditions which result in
incomplete digestion of the adipose tissue, such as which result in
significant amounts of intact adipose tissue being present.
Typically, for example, there may be pieces of adipose tissue
present that are the same size as they were prior to starting the
digestion. In embodiments of the method anywhere between about 20%
to about 80% of the adipose tissue may not be digested.
[0124] In certain embodiments the cells may be subjected to
multiple centrifugation steps or wash steps, for example in order
to remove excessive free lipid.
[0125] As described further in the following section an adipose
tissue-derived cell suspension which may be of any species origin,
such as mentioned herein, for example bovine, porcine, canine,
feline, equine, human, etc, or an aliquot thereof, may be used in
the preparation of a composition comprising secretions of the
adipose tissue-derived cells.
[0126] Compositions Comprising Adipose Tissue-Derived
Secretions
[0127] A composition comprising secretions from adipose
tissue-derived cells may be prepared from an adipose tissue-derived
cell suspension by any appropriate manner. As noted herein the
liquid components formed during the preparation of an adipose
tissue-derived cell suspension typically includes adipose
tissue-derived secretions, thereby representing one embodiment of a
composition comprising such secretions. In this form the
composition comprising adipose tissue-derived secretions may be
collected at any appropriate stage in the preparation of a cell
suspension, such as by collection of the intermediate liquid layer
between the cell pellet and the floating cell layer after
centrifugation of the adipose tissue-derived material. In this
embodiment the collected material comprising secretions may or may
not include adipocytes.
[0128] Typically, the composition is generated by exposure of a
medium to the adipose tissue-derived cell suspension comprising
adipocytes. Exposure of the medium to the adipocyte tissue-derived
cell suspension may be for any appropriate time and conditions, as
set by the operator. Exposure of the medium to the adipocyte
tissue-derived cell suspension does not require conditions which
enable cell attachment to a substratum. In these embodiments, the
composition comprising adipose tissue-derived secretions may be
generated by exposing a medium to the adipose tissue-derived cell
suspension for any appropriate period of time, such as at least 6
hours, at least 8 hours, at least 10 hours, or at least 12 hours,
followed by removal of the cell suspension from the medium, or vice
versa, for example by centrifugation or by filtration. In an
embodiment, the adipose tissue-derived cell suspension may be
exposed to low oxygen conditions, such as less than 10% oxygen,
less than 5% oxygen or less than 1% oxygen. The removal of the cell
suspension and the medium from each other may result in complete or
incomplete removal of cells. Hence the medium, which comprises the
adipose tissue-derived secretions, may or may not include
adipocytes after removal from the cell suspension. In certain
embodiments the composition is generated by exposing a medium to
the adipose tissue-derived cell suspension for no more than 12
hours, no more than 18 hours or no more than 24 hours. In certain
embodiments the composition may be generated by exposing a medium
to the adipose tissue-derived cell suspension for a longer period
of time, such as any period of time between about 1 and 15 days,
for example for about 1 day, 2 days, 3 days, 4, days, 5 days, 6
days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days,
14 days or 15 days. Typically, the suspension is kept in an
incubator for 5 to 10 days and then the secretions are collected.
Exposure of the medium to the cells may or may not be under
conditions which enable cell attachment to a substratum. Typically,
where exposure of the medium to the cells is for more than about 1
day the conditions will permit cell attachment to the
substratum.
[0129] The composition may comprise cell-derived molecules which
are released from cells following cell death or the breakup of
adipose tissue cells. The composition may comprise secretions of
cells of the adipose tissue-derived cell suspension, optionally
comprising adipocytes. The exposing of a medium to an adipose
tissue-derived cell suspension may be at a temperature of from
4.degree. C. to 50.degree. C., more typically at a temperature of
from 10.degree. C. to 40.degree. C. and most typically at a
temperature of from 20.degree. C. to 37.degree. C.
[0130] For a typical adipose tissue-derived cell suspension, 5 g of
adipose tissue is dissociated and suspended in 50 mls of DMEM
containing 10% autologous serum. The adipose tissue derived cell
suspension typically comprises from 100,000 to 1,000,000
non-adipocyte cells for every gram of adipose tissue source
material. The number of adipocytes per gram of adipose tissue
source material is typically between 100,000 and 5,000,000.
[0131] The term "medium" as used herein is intended to encompass
compositions which support the survival of at least some cells in
an adipose tissue-derived cell suspension for at least one hour.
The medium may be a tissue culture medium, such as DMEM, RPMI, or
minimal essential medium, optionally supplemented with serum. The
medium may be a buffered isotonic solution, such as a phosphate
buffered saline or Hank's buffered saline solution, provided the
medium is suitable for administration to a subject. The medium may
be liquid which is formed during the dissociation of adipose
tissue. The medium may optionally be supplemented with factors
which promote cell survival or attachment and cell division, such
as insulin, progesterone and selenium, or serum or serum
components. In certain embodiments the medium must be suitable for
a pharmaceutical composition, which is acceptable for in vivo use.
Such media will be substantially free of pyrogens or other
impurities which may be harmful to humans or animals.
Pharmaceutically-acceptable media are commercially available. The
phrase "pharmaceutically acceptable" refers to molecular entities
and compositions that do not produce unacceptable adverse,
allergic, or other untoward reactions when administered to an
animal or a human.
[0132] Preparation of a composition comprising adipose
tissue-derived secretions may include a step of lysis of the
adipose tissue-derived cell suspension comprising adipocytes. A
lysate comprising cell secretions may be prepared by any suitable
method. In an example embodiment, an adipose tissue-derived cell
suspension may be exposed to a medium, such as described above.
Cells of the suspension may then be lysed by any suitable means,
such as by mechanical disruption (eg, vigorous shaking or
agitation), ultrasonic disruption, freeze thawing, freeze drying or
the addition of one or more agents capable of inducing cell lysis,
typically adipocyte lysis. Such lysing agents are known in the art
and include urea, sodium dodecyl sulphate and Triton .times.100.
After a lysis step the preparation may be centrifuged or filtered
to assist in the removal of cell debris, or it may be used without
such a clarifying step, in which case the composition comprising
adipose tissue-derived secretions may also include cell debris. In
some cases the cell lysate may be removed from the lysing agent by
precipitation of the cell lysate. Where the lysis step results in
incomplete cell lysis, the composition comprising adipose
tissue-derived secretions may also comprise adipose-derived cells,
such as adipocytes.
[0133] In certain embodiments preparation of the adipose
tissue-derived secretions comprises culturing the cell suspension
comprising adipocytes under appropriate conditions to form an
adherent cell culture, such as a confluent adherent cell culture;
harvesting supernatant of the adherent cell culture and removing
cells from said supernatant to form a composition comprising
adipose tissue-derived secretions. The removal of the cells from
the supernatant to leave a composition comprising adipose
tissue-derived secretions may be complete removal or may be partial
removal. In the latter case, the composition comprising adipose
tissue-derived secretions may therefore also include
adipocytes.
[0134] Prior to commencing the culturing of the cells, the adipose
tissue derived cell suspension may be re-suspended in a desired
volume of an appropriate buffer, such as DMEM, RPMI or minimal
essential media. The cell suspension, or an aliquot thereof, may be
added to a sterile tissue culture flask and incubated under
appropriate conditions, typically until the adherent cells have
reached confluence. The cell culture is preferably in the presence
of sterile serum. The concentration of the serum in the culture may
be any suitable concentration which assists culturing of adipose
tissue-derived cells, such as for example in the range of about 5%
v/v to about 30% v/v, such as about 10% v/v, or about 15% v/v or
about 20% v/v. The serum may be any appropriate serum for the
culturing of adipose tissue-derived cells, such as a commercial
fetal calf serum, or a serum prepared in house, such as by methods
known in the art. The serum may be autologous, having been prepared
from the same individual from which the adipose tissue was
obtained, or it may be allogeneic. In other embodiments the serum
may be xenogeneic, such as the use of calf serum in the culturing
of adipose tissue-derived cells obtained from a canine source.
Typically, the cells are cultured at 37.degree. C. with 5%
CO.sub.2. In a further embodiment the cells are cultured in hypoxic
conditions.
[0135] During culturing the adipose tissue-derived cells secrete
cytokines including anti-inflammatory molecules, pro-inflammatory
molecules, chemokines, growth factors and other cell signalling
molecules into the medium. The supernatant in the culture thereby
comprises adipose tissue-derived secretions.
[0136] In certain embodiments the culture may be frozen and freeze
dried, resulting in a freeze dried preparation that includes cells
and the secretions. Rehydration of the freeze dried preparation
will lyse the majority of the cells resulting in the release of
additional cytokines. Rehydration will typically be performed using
a volume of fluid that is less than the original volume of the
adipose tissue-derived cells, such as a volume of fluid that is
between about 5 and about 20 times less than the original volume,
more typically about 10 times less than the original volume of the
adipose tissue-derived cells which results in a composition that is
10 times concentrated. The composition may then be filtered to
remove cell debris resulting in a composition that contains
concentrated cytokines. This provides a preferred method for
producing large volumes of concentrated secretions.
[0137] In other certain embodiments the supernatant may be
harvested from the culture at any appropriate time, although
typically for an adherent cell culture it will be harvested when
the cells have reached confluence, such as after about 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13 or 14 days. Cells, cell debris and any
remaining adipose tissue may be removed from the supernatant, such
as by filtration. In an embodiment the filtration may be through a
20 micron mesh. If desired, multiple steps of filtration may be
undertaken such as through two or more filters of decreasing mesh
size. The resultant preparation of adipose tissue-derived
secretions is typically filter sterilised, such as through a 0.22
micron filter. The sterilised composition may be used immediately,
or may be aliquoted for use, or for storage. Typically, if stored,
the composition is stored frozen at -20.degree. C. The composition
contains secretions from the adipose tissue-derived cells.
[0138] A composition comprising adipose tissue-derived secretions
may also comprise adipocytes. Where present, the adipocytes may
remain from the original adipose tissue used in the preparation of
the secretions or they may be added to the composition comprising
the secretions.
[0139] As described above the preparation of adipose tissue-derived
cell secretions may be made by culturing an adipose tissue-derived
cell suspension and subsequent harvest and filtration of
supernatant, with or without additional steps described above, such
as filter-sterilizing, freeze-drying and concentrating.
[0140] A composition comprising the secretions, which may be a
harvested supernatant comprising secretions, may, for example, be
freeze-dried and subsequently re-hydrated in a desired volume of an
appropriate liquid. Typically the appropriate liquid will be
pharmaceutically acceptable. The appropriate liquid may be
distilled water.
[0141] Optionally, confluent adipose tissue-derived cell cultures,
after harvest of supernatant, may be passaged into new tissue
culture flasks and cultured again to confluence under appropriate
conditions. Continued passaging of the cells may be undertaken for
the preparation of adipose tissue-derived secretions. For example,
supernatant may be harvested from flasks in any desired passage
number, for example from flasks in passage number three through to
passage five. Harvested supernatants may be pooled as desired, for
example to obtain a greater amount of secretions. With additional
steps, such as concentration steps, in this manner an adipose
tissue-derived cell secretion preparation of higher concentration
may be prepared.
Pharmaceutical Compositions and Other Compositions of the
Invention
[0142] In aspects of the invention the adipose tissue-derived
composition, containing secretions of adipose tissue cells, or the
adipose tissue-derived cell suspension, optionally comprising
adipocytes, is used for the preparation of a pharmaceutical
composition. According to one aspect the invention provides (i) a
composition comprising adipose tissue-derived secretions or (ii) an
adipose tissue-derived cell suspension, optionally comprising
adipocytes or (iii) a combination of (i) and (ii) for the
preparation of a pharmaceutical composition for use in the
treatment of a condition selected from the group consisting of an
inflammatory disorder, a ligament injury and a tendon injury in a
subject, or for use in alleviating pain associated with an
inflammatory disorder, a ligament injury; a tendon injury,
neuropathic pain, or a burn injury, wherein the treatment comprises
remote administration of the pharmaceutical composition to the
subject.
[0143] The pharmaceutical composition may also be referred to as a
medicament. Typically, the remote administration is subcutaneous
administration or intra-muscular administration. Typically the
pharmaceutical composition also comprises one or more of a
pharmaceutically acceptable carrier diluent, excipient or
adjuvant.
[0144] In another aspect the invention provides use of (i) a
composition comprising adipose tissue-derived secretions or (ii) an
adipose tissue-derived cell suspension, optionally comprising
adipocytes, or (iii) a combination of (i) and (ii), for the
preparation of a pharmaceutical composition for use in the
treatment or prevention of disease in an intensively farmed animal,
wherein the pharmaceutical composition is suitable for subcutaneous
injection or intramuscular injection.
[0145] In another aspect the invention provides use of use of (i) a
composition comprising adipose tissue-derived cell secretions, or
(ii) an adipose tissue-derived cell suspension, optionally
comprising adipocytes, or (iii) a combination of (i) and (ii), for
the preparation of a pharmaceutical composition for use in the
treatment of a joint disease or condition in a mammalian subject,
wherein the composition is suitable for administration to a site of
said subject remote from the site of said condition.
[0146] In another aspect the invention provides use of (i) adipose
tissue-derived cell secretions, or (ii) an adipose tissue-derived
cell suspension, optionally comprising adipocytes, or (iii) a
combination of (i) and (ii), for the preparation of a
pharmaceutical composition for use in alleviating pain in a
mammalian subject, wherein the pharmaceutical composition is
suitable for administration to said subject by intramuscular
injection or by subcutaneous injection or by an appropriate form of
administration, such as by topical administration, at or near a
site of the pain.
[0147] The invention thus provides pharmaceutical compositions or
medicaments comprising (i) adipose tissue-derived cell secretions,
or (ii) an adipose tissue-derived cell suspension, optionally
comprising adipocytes, or (iii) a combination of (i) and (ii). The
pharmaceutical composition may be provided as part of a kit, for
example including additional components useful in the intended
treatment, such as for example written instructions, or it may be
provided as a single item, such as a single vial or aliquot of the
composition.
[0148] According to a further aspect the invention provides a
pharmaceutical composition comprising adipose tissue-derived
secretions, together with a pharmaceutically acceptable carrier,
diluent, excipient or adjuvant. In certain embodiments the
composition comprising adipose tissue-derived secretions further
comprises adipocytes. In certain embodiments the composition is
cell-free. In certain embodiments the composition is prepared by
exposing a medium or other liquid to an adipose tissue-derived cell
suspension, which may or may not comprise mature adipocytes. The
pharmaceutical composition has therapeutic properties, such as for
the treatment of a condition selected from the group consisting of
an inflammatory disorder, a ligament injury and a tendon injury in
a subject, or for alleviating pain associated with a condition
selected from the group consisting of an inflammatory disorder, a
ligament injury, a tendon injury, neuropathic pain and a burn
injury. The pharmaceutical composition has therapeutic properties
suitable for treatment or prevention of disease in an intensively
farmed animal. Such diseases include leg weakness, lameness,
arthritis, developmental diseases and bacterial infection. In an
embodiment the developmental disease is osteochondrosis (OCD). In
an embodiment the developmental disease is a bone cyst. In an
embodiment the developmental disease is an orthopaedic
developmental disease.
[0149] In embodiments, the adipose tissue is taken from an
individual subject, and the pharmaceutical composition is
administered to the same individual, and thus the adipose
tissue-derived secretions or adipose tissue-derived cell
suspension, optionally comprising adipocytes, or combination
thereof, is a purely autologous preparation.
[0150] In embodiments, the adipose tissue is taken from one or more
individual subjects and the pharmaceutical composition is
administered to a different subject of the same species, and thus
the adipose tissue-derived secretions or adipose tissue-derived
cell suspension, optionally comprising adipocytes, or combination
thereof is an allogeneic preparation. In embodiments the adipose
tissue is taken from an individual of a different species to that
which is intended to be a recipient of the therapeutic composition.
For example, a composition comprising secretions or cells or a
combination of both prepared from bovine tissue may be for
administration to an individual of a different species, such as a
human, feline, porcine, or canine. In embodiments where the
composition comprising adipose tissue-derived secretions or adipose
tissue-derived cell suspension, optionally comprising adipocytes,
is for use in a different individual of the same species as the
source material or for use in an individual of a different species
as the source material, the composition may typically be devoid of
cells of the immune system in order to minimise the possibility of
host (recipient) immune response to the composition or graft versus
host disease.
[0151] In embodiments the pharmaceutical composition is prepared
from more than one source of adipose tissue, such as from different
preparations taken from the same individual or from different
preparations taken from different individuals. The pooling may
comprise combining multiple adipose tissue-derived cell
suspensions, such as in a pooled culturing step or the pooling may
comprise combining multiple compositions of adipose tissue-derived
secretions, such as may be obtained from separate culturing or
exposure steps.
[0152] The pharmaceutical composition may be administered to the
subject patient at a site remote from the afflicted area. In this
context, "remote" means that the administration is not direct
application of the cell secretions or cell suspension or
combination thereof to the site of inflammation or other injury or
disease being treated where such a site is identifiable. As an
illustration, in the case of treatment of an arthritic joint,
administration as previously described in the art involved
injection of adipose tissue-derived cell suspensions or adipose
tissue-derived cell secretions directly into the afflicted joint.
Such administration requires a high degree of skill on the part of
the treating physician or clinician to ensure appropriate
precision. The handling of the affected limb or joint required in
such administration also increases the distress experienced by the
patient, be they human or non-human. By providing for the remote
administration of adipose tissue-derived cell secretions, or
adipose tissue-derived cell suspension, or combination thereof, the
present invention offers improved methods, uses and compositions
for the treatment of such diseases. For example, the remote
administration may be by subcutaneous injection, such as in the
scruff of the neck of an animal (for example a cat or dog) being
treated, or by intramuscular injection. As a further example,
administration to a dog by intramuscular injection may be in to
thigh of the dog. As a further example, administration to a bovine
by intramuscular injection may be in the caudal fold.
[0153] Where the therapeutic compositions of the invention are
administered to treat or prevent a disease of an intensively farmed
animal, the administration is by subcutaneous injection or
intramuscular injection. Due to the nature of the condition being
treated in such an animal description of the administration being
"remote" may not be appropriate. For example, administration of the
therapeutic composition of the invention to an intensively farmed
animal may be for treatment or prevention of generalized or
localized leg weakness, osteochondrosis. As described herein
administration of the therapeutic composition to such animals is by
subcutaneous injection or intramuscular injection.
[0154] Where the therapeutic compositions of the invention are
administered for treatment of pain for which there is no
discernable clinical cause, or for neuropathic pain which may or
may not have a discernable clinical cause, the composition may be
administered to the subject by any appropriate means, such as by
injection or by topical application, which means may be at or near
a site of the pain or may be remote from a site of the pain.
Neuropathic pain may be experienced by a subject at multiple sites
in the body. Treatment of such pain may be by administration at one
site, such as a site of original pain, which site may also be
remote from another site of pain experienced by the subject. In an
example, the pain is neuropathic pain. The neuropathic pain may be
localised to one area of the body or it may be experienced in
multiple sites of the subject's body. When experienced in multiple
sites, the intensity of the pain may be similar at multiple sites
or it may be different at multiple sites. An example of neuropathic
pain is neuropathic facial pain. In methods of the invention,
neuropathic facial pain in a subject may be treated by
administration of a composition of the invention to the subject by
injection into the jaw or the gum. The injection into the jaw or
the gum of such a patient may comprise administration at the
original site of the pain or may be administration at a secondary
site of the pain.
[0155] As shown in the Examples compositions and methods of the
invention are also effective in alleviating pain and blistering
associated with burns. Where the compositions of the invention are
administered for treatment of a burn injury, or for alleviating
pain associated with a burn of the skin, the composition is
typically administered by topical application, such as in the form
of a cream, gel, or lotion.
[0156] A pharmaceutical composition of the invention may be
supplied to the user as a frozen solution. As described herein, for
example, the cell secretions, the cell suspension, or the
combination can be stored, at approximately -20.degree. C. until
required for use. Alternatively, the cell secretions, the cell
suspension, or the combination may be stored at a lower
temperature, for example in a freezer at -70.degree. C. to
-90.degree. C., or in liquid nitrogen storage, either in the vapour
phase or in the liquid phase, until required for use. Compositions
comprising cells will typically be stored in liquid nitrogen. In a
preferred embodiment the composition comprising adipose
tissue-derived cell secretions, or the adipose tissue-derived cell
suspension, optionally comprising adipocytes, or the combination of
adipose tissue-derived cell secretions and adipose tissue-derived
cell suspension, optionally comprising adipocytes, is stored in the
liquid phase of liquid nitrogen storage. In a preferred embodiment
a composition comprising cells is stored in combination with cell
secretions obtained from a culture of adipose tissue-derived cells.
In another preferred embodiment a composition comprising cells is
stored in a composition which comprises clarified and or
concentrated media from a culture of adipose tissue-derived cells.
The culture of adipose tissue-derived cells will be understood to
include culture of cells freshly obtained from adipose tissue, as
well as culture of cells which have arisen as progeny cells or a
cell line from prior culturing. In use, such as by the treating
physician, clinician, veterinarian, technician, assistant, or
farmer, the composition is typically administered to the subject or
animal as soon as possible after thawing. The pharmaceutical
composition may alternatively be stored, for example on ice or in a
refrigerator or in a cool pack, at approximately 2.degree. C. to
5.degree. C. for a short time between thawing and administration.
In this context a short time would typically be no more than
several hours, such as no more than about half an hour, or no more
than about one hour, or no more than about two hours. As the
cryoprotectants are typically toxic to the cells and can cause loss
of viability if kept thawed, the composition, particularly where it
comprises viable cells, is typically injected to the recipient
animal as soon as possible after thawing.
[0157] It had previously been considered that a stored (eg., a
frozen) suspension comprising mesenchymal stem cells requires a
period of time under appropriate cell culturing conditions before
use in a therapeutic setting, for example to assist in cell
recovery. The inventor has surprisingly identified that culturing
of a previously frozen cell suspension, such as an adipose
tissue-derived cells suspension optionally comprising adipocytes,
is not required prior to use of the cell suspension in a
therapeutic setting. As described herein benefits from the
administration of such a cell suspension are apparent when the cell
suspension is administered after thawing and without intervening
cell culturing. As demonstrated in the Examples herein, storage of
cells in the presence of adipose tissue-derived cell secretions
provides further benefits. For example, frozen cells stored in the
presence of secretions, optionally a clarified and or concentrated
preparation of secretions, demonstrate superior therapeutic
efficacy when utilised after retrieval from storage compared to
frozen cells stored in the absence of secretions.
[0158] A pharmaceutical composition of the invention may be
supplied in a "ready-to-use" form. In such embodiments the user
typically requires only thawing to an acceptable temperature for
administration before the composition is administered. In such
embodiments the composition may be supplied in pre-measured doses,
such as a pre-measured or pre-determined dose suitable for a given
recipient subject or animal, for example pre-determined on the
basis of the recipient species, or on the basis of the recipient
individual, such as a small breed of dog, compared to a large breed
of dog, or a juvenile animal compared to an adult animal. The
pre-measured dose may alternatively or additionally be on the basis
of the disease or condition being treated or intended to be
prevented. A ready-to-use form of the composition may comprise the
composition supplied with or in an injectable device, such as a
syringe. The injectable device may be capable of delivering a
single application to an individual recipient or may be capable of
delivering single or multiple applications to multiple recipients.
The injectable device may be adjustable, for example to permit
delivery of a range of different doses.
[0159] In embodiments where the pharmaceutical composition
comprises a combination of adipose tissue-derived cell secretions
and an adipose tissue-derived cell suspension, optionally
comprising adipocytes, the composition may be supplied to the user
as a combination or as separate compositions for combination by the
user. It will be understood that reference to a "user" in this
context means the individual who actually administers the
therapeutic composition to the recipient subject or animal and also
means a member of the team or group who is undertaking that
administration. For example the user may be any individual who is
assisting in the application of the methods of the invention such
as a clinician, a doctor, a veterinarian, a farmer, a clinical
nurse, a veterinary nurse, a technical assistant, or a
farmhand.
[0160] Inflammatory Disorders
[0161] The pharmaceutical composition may be administered for the
treatment of an inflammatory disorder and/or for alleviating pain
associated with an inflammatory disorder in a subject.
[0162] Inflammation may arise as a response to an injury or
abnormal stimulation caused by a physical, chemical, or biologic
agent. An inflammation reaction may include the local reactions and
resulting morphologic changes, destruction or removal of the
injurious material, and responses that lead to repair and healing.
The term "inflammatory" when used in reference to a disorder refers
to a pathological process which is caused by, resulting from, or
resulting in inflammation that is inappropriate or which does not
resolve in the normal manner. Inflammatory disorders may be
systemic or localized to particular tissues or organs.
[0163] Inflammation is known to occur in many disorders which
include, but are not limited to: Systemic Inflammatory Response
(SIRS); Alzheimer's Disease (and associated conditions and symptoms
including: chronic neuroinflammation, glial activation; increased
microglia; neuritic plaque formation; and response to therapy);
Amyotropic Lateral Sclerosis (ALS), arthritis (and associated
conditions and symptoms including, but not limited to: acute joint
inflammation, antigen-induced arthritis, arthritis associated with
chronic lymphocytic thyroiditis, collagen-induced arthritis,
juvenile arthritis; rheumatoid arthritis, osteoarthritis, prognosis
and streptococcus-induced arthritis, spondyloarthopathies, gouty
arthritis), asthma (and associated conditions and symptoms,
including: bronchial asthma; chronic obstructive airway disease;
chronic obstructive pulmonary disease, juvenile asthma and
occupational asthma); cardiovascular diseases (and associated
conditions and symptoms, including atherosclerosis; autoimmune
myocarditis, chronic cardiac hypoxia, congestive heart failure,
coronary artery disease, cardiomyopathy and cardiac cell
dysfunction, including: aortic smooth muscle cell activation;
cardiac cell apoptosis; and immunomodulation of cardiac cell
function; diabetes and associated conditions, including autoimmune
diabetes, insulin-dependent (Type 1) diabetes, diabetic
periodontitis, diabetic retinopathy, and diabetic nephropathy);
gastrointestinal inflammations (and related conditions and
symptoms, including celiac disease, associated osteopenia, chronic
colitis, Crohn's disease, inflammatory bowel disease and ulcerative
colitis); gastric ulcers; hepatic inflammations such as viral and
other types of hepatitis, cholesterol gallstones and hepatic
fibrosis, HIV infection and associated conditions, including
degenerative responses, neurodegenerative responses, and HIV
associated Hodgkin's Disease, Kawasaki's Syndrome and associated
diseases and conditions, including mucocutaneous lymph node
syndrome, cervical lymphadenopathy, coronary artery lesions, edema,
fever, increased leukocytes, mild anemia, skin peeling, rash,
conjunctiva redness, thrombocytosis; inflammatory disorders of the
skin, including dermatitis, such as atopic dermatitis and
associated conditions; multiple sclerosis, nephropathies and
associated diseases and conditions, including diabetic nephropathy,
endstage renal disease, acute and chronic glomerulonephritis, acute
and chronic interstitial nephritis, lupus nephritis, Goodpasture's
syndrome, hemodialysis survival and renal ischemic reperfusion
injury, neurodegenerative diseases and associated diseases and
conditions, including acute neurodegeneration, induction of IL-I in
aging and neurodegenerative disease, IL-I induced plasticity of
hypothalamic neurons and chronic stress hyperresponsiveness,
ophthalmopathies and associated diseases and conditions, including
diabetic retinopathy, Graves' ophthalmopathy, and uveitis,
osteoporosis and associated diseases and conditions, including
alveolar, femoral, radial, vertebral or wrist bone loss or fracture
incidence, postmenopausal bone loss, mass, fracture incidence or
rate of bone loss, otitis media (adult or paediatric), pancreatitis
or pancreatic acinitis, periodontal disease and associated diseases
and conditions, including adult, early onset and diabetic;
pulmonary diseases, including chronic lung disease, chronic
sinusitis, hyaline membrane disease, hypoxia and pulmonary disease
in SIDS; restenosis of coronary or other vascular grafts;
rheumatism including rheumatoid arthritis, rheumatic Aschoff
bodies, rheumatic diseases and rheumatic myocarditis; thyroiditis
including to chronic lymphocytic thyroiditis; urinary tract
infections including chronic prostatitis, chronic pelvic pain
syndrome and urolithiasis, immunological disorders, including
autoimmune diseases, such as alopecia aerata, autoimmune
myocarditis, Graves' disease, Graves ophthalmopathy, lichen
sclerosis, multiple sclerosis, psoriasis, systemic lupus
erythematosus, systemic sclerosis, thyroid diseases (e.g. goitre
and struma lymphomatosa (Hashimoto's thyroiditis, lymphadenoid
goitre), sleep disorders and chronic fatigue syndrome and obesity
(non-diabetic or associated with diabetes), resistance to
infectious diseases, such as Leishmaniasis, Leprosy, Lyme Disease,
Lyme Carditis, malaria, cerebral malaria, meningitis,
tubulointerstitial nephritis associated with malaria), which are
caused by bacteria, viruses (e.g. cytomegalovirus, encephalitis,
Epstein-Barr Virus, Human Immunodeficiency Virus, Influenza Virus)
or protozoans (e.g., Plasmodium falciparum, trypanosomes), response
to trauma, including cerebral trauma (including strokes and
ischemias, encephalitis, encephalopathies, epilepsy, perinatal
brain injury, prolonged febrile seizures, SIDS and subarachnoid
hemorrhage), low birth weight (e.g. cerebral palsy), lung injury
(acute hemorrhagic lung injury, Goodpasture's syndrome, acute
ischemic reperfusion), myocardial dysfunction, caused by
occupational and environmental pollutants (e.g. susceptibility to
toxic oil syndrome silicosis), radiation trauma, and efficiency of
wound healing responses (e.g. burn or thermal wounds, chronic
wounds, surgical wounds and spinal cord injuries), septicemia,
hypothyroidism, oxygen dependence, cranial abnormality, early onset
menopause, a subject's response to transplant (rejection or
acceptance), acute phase response (e.g. febrile response), general
inflammatory response, acute respiratory distress response, acute
systemic inflammatory response, wound healing, adhesion,
immunoinflammatory response, neuroendocrine response, fever
development and resistance, acute-phase response, stress response,
disease susceptibility, repetitive motion stress, tennis elbow,
ligament and tendon problems, and pain management and response.
[0164] In particular embodiments the inflammatory disorder is a
joint-related inflammatory disorder, such as arthritis.
[0165] The methods and compositions of the invention may be used
for the treatment of ligament injuries and tendon injuries or for
the alleviation of pain associated with such injuries. Ligament
injuries and tendon injuries, in some forms, can be classified as
inflammatory disorders. Some ligament injuries and tendon injuries
may not be considered inflammatory disorders. For the avoidance of
doubt, ligament injuries and tendon injuries contemplated in this
invention may be those which are inflammatory disorders or are
associated therewith and those which may not be considered
inflammatory disorders.
[0166] The methods and compositions of the present invention may be
used in conjunction with other treatments, such as those for
inflammatory diseases. As demonstrated in Australian Patent
Application No. 2009201915 and in International Publication No.
WO2010/020005, the contents of which are incorporated herein by
cross-reference, administration of a composition comprising adipose
tissue-derived cells, which composition comprises adipocytes,
directly into an joint affected by inflammatory diseases, such as
osteoarthritis, provides therapeutic benefits for the patient and
is associated with improvement in the joint. The present invention
additionally offers an adjunct treatment, whereby, for example, a
treatment regime may be commenced by administration of the
composition comprising adipose tissue-derived cells, which
composition comprises adipocytes, into the affected joint, and that
is followed by the less invasive course of treatment utilising the
remote delivery of the present invention.
[0167] The present invention may also offer advantages as an
adjunct treatment for arthritis and other inflammatory diseases in
combination with established treatments. For example, a side-effect
of long term treatment of inflammatory conditions utilising NSAIDS
is renal failure. The present invention offers an alternative to
continued use of NSAIDS in patients with signs of renal failure.
Alternatively, the use of the present invention in combination with
NSAIDS may permit the NSAIDS to be used at a reduced dosage,
thereby delaying the onset of renal failure.
[0168] The present invention may be used in combination with any
therapy known for inflammatory diseases, including use with any of
cartrophen, metacam, previcox, tramadol. The methods and
compositions of the present invention may be used in conjunction
with herbal medications, such as valerian, rosemary oil and yucca
leaves.
[0169] The present invention permits the preparation of cell
secretions in advance, such that a product comprising the adipose
tissue-derived cell secretions may be made available without the
need to anesthetize a subject for extraction of adipose tissue. The
present invention permits the preparation of compositions
comprising (i) adipose tissue-derived cell secretions, or (ii) an
adipose tissue-derived cell suspension, optionally comprising
adipocytes, or (iii) a combination of (i) and (ii), in advance of a
treatment need. As described herein the cell secretions or other
compositions of the invention can be stored, for example at
-20.degree. C. or below, until required for use.
[0170] Intensively Farmed Animals
[0171] As a result of intensive selection of farm animals over many
generations for desirable traits, such as rapid growth, large
muscle mass and efficient feed conversion in animals raised for
meat production (eg., pigs and cattle) and milk quality and volume
in dairy animals, modern breeds of farm animals often have a higher
prevalence of susceptibility to disease and acute and chronic
conditions than do wild or less intensively farmed populations. The
incidence of and severity of manifestation of such diseases and
conditions can be exacerbated by the manner in which an animal is
farmed. Animals raised under intensive farming conditions, such as
in densely populated piggeries or in feed lots are typically
susceptible to a higher incidence of disease and potentially
detrimental growth conditions than animals raised under less
intensive conditions, such as a free range or open grazing
situation. Due to intensive selection, as described above however,
even animals raised in free range or open grazing operations may
still have a higher susceptibility to or prevalence of detrimental
health conditions. In the context of the invention therefore it
will be understood that reference to intensively farmed animals or
intensive farming conditions includes intensively bred animals
which may be raised in densely populated operations, for example
with limited availability for individual movement, and also
includes intensively bred animals in less densely populated
operations, such as where an individual has access to free range or
open grazing.
[0172] The identification by the inventor that the therapeutic
compositions of the invention are effective when administered
subcutaneously or intramuscularly is conducive to the methods and
compositions of the invention having beneficial effects in farming
operations and particularly intensive animal farming, such as
intensive farming of pigs, cattle, sheep and poultry. Whilst a
therapeutic agent for a localized disease, such as an arthritic
joint, can be administered by intra-articular injection into the
afflicted joint, such administration requires a high degree of
skill on the part of the operator, as well as potentially causing
pain to the animal by handling of the afflicted joint, and hence
requiring restraint of the animal being treated. In contrast,
subcutaneous injection or intramuscular injection has a lower skill
requirement on the part of the operator, will generally not require
handling of the afflicted limb, and typically requires only minimal
restraint of the animal being treated. With the absence of
requirement for high degree of operator skills, as compared to
intra-articular injection for example, administration of the
compositions of the invention can be undertaken as part of a
routine on-farm treatment or preventive program. In such an
approach animals may be administered the compositions of the
invention at the same time as the animals are being treated for
other conditions, or at the same time as the animals are undergoing
routine procedures associated with the animal breeding or growing
enterprise, such as vaccination, ear tagging, tattooing, tail
docking, or castration.
[0173] Generations of selectively breeding animals of a particular
species for a desirable trait has contributed to the increased
prevalence of certain undesirable characteristics in the
population. As an example, pigs have for many years been selected
for rapid growth, large muscle mass and efficient feed conversion.
This has, however, led to much greater prevalence of skeletal,
joint and cartilage problems than is seen in a wild population.
These problems can be exacerbated in a farming operation where the
animal has limited room to move. Changes associated with
cartilaginous structures may be referred to as leg weakness or
osteochondrosis (OCD). The term "leg weakness" is also sometimes
used to describe poor leg conformation or to describe a clinical
condition associated with lameness or stiffness. It may arise due
to abnormal changes in the articular cartilage and the growth
(epiphyseal) plates, which are responsible for the growth of bones
both in length and diameter. The exact mechanisms that cause these
changes are not fully understood. They are thought to arise due to
the pressure and sheer stresses that are placed upon these rapidly
growing tissues, which reduces oxygen supply causing abnormal
growth and consistency of the cartilage. Reduced blood supply
through a deficiency of blood vessel is although thought to
contribute to such problems. Damage to the cartilage tends to be
progressive and irreversible, the damaged cartilage being replaced
by fibrous tissue.
[0174] Shortening and bending of the bones near the joints and at
extremities of long bones may follow the cartilage damage. Weak
epiphyseal plates also have a tendency, to fracture and cartilage
covering the joint surfaces may split and form fissures. In modern
pigs, such changes in the cartilage take place from as early as two
months of age. These potentially detrimental changes are not
generally clinically apparent at an early stage. In breeding
enterprises it is not uncommon for 20% to 30% of boars and gilts to
be culled due to leg weakness and leg deformities. Such conditions,
including OCD, thus have the potential for serious economic and
ethical consequences for the farming enterprise. There is currently
no specific treatment for OCD. In advanced stages OCD may also lead
to arthritis and permanent lameness.
[0175] The compositions of the present invention are beneficial in
the growth and repair and maturation of bone and cartilage.
Accordingly, the present invention provides methods for treatment
or prevention of bone and cartilage disorders in an animal,
including leg weakness or OCD. As OCD can lead to arthritis in an
affected animal, the methods and compositions of the invention can
also be effective in preventing the occurrence of or reducing the
severity or incidence of arthritis in an animal at risk of
arthritis. In preferred embodiments the animal is a pig. As
described above, such conditions commence at an early age and, in
early stages of development are typically not associated with any
clinical symptoms. In the methods of the invention the therapeutic
composition may be administered to an animal which exhibits
clinical symptoms of a joint disease or to an animal which does not
exhibit clinical symptoms of a joint disease.
[0176] Clinical symptoms may include separation or fracture of the
bones at the epiphyseal plate (epiphyseolysis) associated with
sudden movement, lameness, sudden fractures (such as of the knee
and elbow joints) which may be more common in young animals,
abnormal leg conformation, abnormal gait, stiffness, pain. Age of
visible onset of OCD can be variable, for example within three
months of gilts being introduced on to a farm, during the first
pregnancy, in lactation or in the first two to three weeks post
weaning.
[0177] In an embodiment the invention provides a method for
reducing the severity of OCD in an animal, the method comprising
administering to said animal a pharmaceutical composition
comprising (i) adipose tissue-derived cell secretions, or (ii) an
adipose tissue-derived cell suspension, optionally comprising
adipocytes, or (iii) a combination of (i) and (ii), wherein the
administering is by subcutaneous injection or intramuscular
injection. In preferred embodiments the animal is a pig.
[0178] Administration of the pharmaceutical composition to the
recipient animal may occur when the animal is at any suitable age.
For prevention of the onset of clinical symptoms of OCD or for
reducing the severity of OCD in a pig, the recipient is preferably
treated at a young age. For example a pig may be treated at, or
soon after weaning, which typically occurs in a pig farming
operation when the piglets are between about two and five weeks
old. Young pigs may be treated at between about one month and six
months of age: or between about three months and six months of age.
In piggeries, the pigs undergo a rapid growth phase at about 12 to
16 weeks of age. It is envisaged that in one embodiment the
administration of the compositions of the invention would be at the
beginning of this growth period, for example at around 8 weeks of
age, or 9 weeks of age, or 10 weeks of age, or 11 weeks of age, or
12 weeks of age, or 13 weeks of age.
[0179] The animal may receive a single administration of the
composition of the invention or may receive multiple
administrations, such as two, three, four or more administrations.
Where multiple administrations of the composition are performed,
they will typically be separated by about one, two, three or four
months in the case of administration to young pigs.
[0180] The method is also beneficial in the treatment of
symptomatic OCD. In such cases the recipient animal is typically an
older animal, such as more than six months old. In an older animals
multiple administration of the compositions of the invention may be
separated by about two to six weeks, or by about one, two, three or
four months or more. The choice of timing of multiple
administration may be determined by the skilled addressee, for
example it may be on the basis of re-appearance of or increased
severity of symptoms, pregnancy, weaning, etc.
[0181] As described above the compositions and methods of the
invention find application in preventing onset of OCD in pigs and
in reducing the severity of OCD and other developmental orthopaedic
conditions in pigs. Other mammals including humans, dogs, cattle,
and horses, may also suffer developmental orthopaedic conditions,
such as OCD, for example through predisposition due to environment
and or genetics. The invention also finds application in preventing
onset of, or the severity of, or in treating, developmental
orthopaedic conditions, such as OCD, in such animals.
[0182] As described herein the compositions of the invention are
also capable of treating joint disease, arthritis and inflammatory
conditions in afflicted mammals. The compositions of the invention
are also useful in alleviating pain in mammalian subjects. The
mammal may be any mammal such as a human, a domestic animal, a farm
animal, such as a stud, breeder or grower animal, such as for meat
or dairy production. In the treatment of clinically relevant
arthritis, for example, the farm animal may be a pig. The pig may
be a young pig or may be a breeder sow. For example a clinically
affected pregnant sow may be treated to assist them to get through
to farrowing.
[0183] Treatment of Pain
[0184] The inventor has identified that compositions of the
invention are useful in the treatment of subjects having pain.
Various specific types of pain are described herein, including the
following.
[0185] Painful Musculoskeletal Conditions Other than Arthritis.
[0186] Painful musculoskeletal conditions are common, with a
prevalence of approximately 30%. For musculoskeletal conditions
other than arthritis, the prognosis is generally good; most people
recover within a few weeks following the onset of symptoms. However
a significant minority do not recover and develop long lasting or
chronic pain, defined as pain lasting longer than 3 months. For
these patients prognosis is poor and recovery is slow. A major
focus of contemporary research is the early identification of
patients who are at a high risk of a poor outcome. A common finding
from this research is that early high pain intensity is a risk
factor for delayed recovery and the development of chronic
pain.
[0187] The most frequent cause of chronic musculoskeletal pain is
low back pain. Over 1 million Australians have a disability
associated with their back problems and it is the leading reason
for Australians leaving the workforce. Other common but less
prevalent chronic pain conditions include: neck and shoulder pain,
whiplash associated disorder (WAD) and complex regional pain
syndrome (CRPS).
[0188] Back pain is an extremely common, difficult-to-manage and
expensive health condition. In Australia back pain is associated
with costs of around $9 billion/year. Over 85% of low back pain is
`non-specific` low back pain (NSLBP) in that a structural source of
the pain cannot be reliably identified. Plausible therapeutic
targets are innervated tissues and include the disc, the facet
joint and the sacroiliac joint, however other tissues such as
muscle and ligament may also be involved. Up to 15% of low back
pain is radiculopathy where impingement of the nerve root causes
symptoms including back and leg pain. Pain associated with
radiculopathy is believed to be associated with a local
inflammatory process. Treatments for radicular pain include advice
to stay active, analgesia including NSAIDS, epidural corticosteroid
injection and transfroaminal peri-radicular injections of
corticosteroid.
[0189] Neck and Shoulder Pain
[0190] Whiplash associated disorder (WAD) are injuries to the neck
caused by acceleration-deceleration energy transfer resulting most
commonly from a motor vehicle accident. As with most painful
musculoskeletal conditions prognosis for new injuries is typically
good in the first few weeks or months, but after 3 months recovery
rates slow markedly and a significant proportion of patients
develops chronic Whiplash Associated Disorder (WAD). High pain
intensity is known to be a predictor or poor outcome. Whiplash is
largely resistant to conservative treatments.
[0191] Complex Regional Pain Syndrome
[0192] Complex regional pain syndrome (CRPS) is a complication of
minor trauma, usually to one limb, characterised by incapacitating
pain, swelling, colour and temperature changes, and bone
demineralisation in the limb. In Australia on average 5000 people
are diagnosed with CRPS every year. The most common inciting minor
trauma is wrist fracture. The incidence of CRPS following wrist
fracture is 5-7%. Prognosis is poor and treatment options are often
highly invasive, have significant side effect profiles and are only
moderately effective. The major cause of CRPS is thought to be an
aberrant inflammatory response to tissue injury and a more
pro-inflammatory balance of inflammatory mediators is expressed by
patients with CRPS compared to those without the condition.
[0193] Neuropathic Pain
[0194] The inventor has identified that compositions of the
invention are useful in the treatment of subjects having pain for
which there is no discernable clinical cause, such as some forms of
neuropathic pain. Neuropathic pain refers to a group of painful
disorders characterized by pain due to dysfunction or disease of
the nervous system at a peripheral level, a central level, or both.
It is a complex entity with many symptoms and signs that fluctuate
in number and intensity over time. The three common components of
neuropathic pain are steady and neuralgic pain; paroxysmal
spontaneous attacks; and hypersensitivity.
[0195] Neuropathic pain can be very disabling, severe and
intractable, causing distress and suffering for individuals,
including dysaesthesia and paraesthesia. Sensory deficits, such as
partial or complex loss of sensation, are also commonly seen. In
addition, there are significant psychological and social
consequences linked to chronic neuropathic pain, which contribute
to a reduction in quality of life.
[0196] Neuropathic pain is quite common in general medical
practice. In some forms, the neuropathic pain is not associated
with any discernable clinical causative condition. As an example it
is demonstrated herein that the compositions of the invention are
effective in alleviating neuropathic facial pain. In some forms,
the neuropathic pain is associated with a discernable clinical
condition. The prevalence of trigeminal neuralgia is 2.1 to 4.7
persons per 100,000 of the population, and of painful diabetic
neuropathy occurs in 11% to 16% of Type 1 diabetics as well as Type
II diabetics and postherpetic neuralgia is found in approximately
34 persons per 100,000 of the population. Treatment of neuropathic
pain is not easy. Patients with neuropathic pain do not always
respond to standard analgesics such as non-steroidal
anti-inflammatory drugs (NSAIDs) and to some extent neuropathic
pain is resistant to opiates. The pharmacologic agents best studied
and longest used for the treatment of neuropathic pain are
antidepressants and anticonvulsants both of which may have serious
side effects.
[0197] A composition of the invention may be administered to a
subject for treatment of such pain at any appropriate site.
Administration may typically be using an appropriate type of
injection or it may be by topical application. For example, an
injection may be subcutaneous, intramuscular, or directly into an
accessible site at or near a site of the pain. As this type of pain
may manifest in multiple areas of the subject's body, for example
jaw pain and limb or shoulder pain, the administration may be at or
near to one site of the pain and remote from another site afflicted
by pain. Typically, where multiple sites of the pain occur in a
patient, the administration is at or near a site identified as an
original or primary site of the pain. As an illustration of this
treatment, the examples herein show treatment of neuropathic facial
pain by injection into the subject's gum. A subject being treated
may be administered a single application of a composition of the
invention, such as a single injection or may be administered
multiple applications, such as multiple injections.
[0198] The invention will now be described in more detail, by way
of illustration only, with respect to the following examples. The
examples are intended to serve to illustrate this invention and
should not be construed as limiting the generality of the
disclosure of the description throughout this specification.
EXAMPLES
Example 1. Preparation of a Cell Free Extract
Preparation of Adipose Tissue
[0199] A 10 g sample of adipose tissue was collected by excision
from the inguinal fat pad of a dog. The adipose tissue was rinsed
with saline and then minced finely using scissors and mixed with 20
ml of Dulbecco's Modified Eagle's Medium (DMEM, Sigma). Collagenase
(Sigma) was added to produce a final concentration of 0.05% w/v and
the sample was incubated at 37.degree. C. for 30 minutes. At the
end of 30 minutes the adipose tissue was partially digested and
consisted of a mixture of partially digested fat particles,
liberated stromal vascular cells (SVCs) and liberated adipocytes.
The sample was then centrifuged at 500 g for 15 minutes. Four
distinct layers were visible within the centrifuged sample: a small
(2 mm thick) layer of free lipid on the surface, below which is was
a white 20 mm thick layer of adipose tissue and adipocytes and then
a large clear layer of DMEM and then a pellet of adipose
tissue-derived non-adipocyte cells. The small layer of lipid was
carefully removed with a pasteur pipette. A fresh pasteur pipette
was then carefully inserted through the adipocytes and the clear
DMEM was removed without disturbing the floating adipose tissue,
adipocytes or the pelleted cells. This resulted in a sample that
contained only the floating pieces of adipose tissue and adipocytes
suspended in a small volume of DMEM and the pelleted cells. The
pieces of adipose tissue and adipocytes and the pelleted cells were
gently mixed with a pasteur pipette and transferred to a 15 ml
centrifuge tube. The pieces of adipose tissue and cells were then
washed in DMEM to remove collagenase as follows. DMEM was added to
a final volume of 14 ml and the sample centrifuged at 500 g for 10
minutes. This resulted in three distinct layers: floating pieces of
adipose tissue and adipocytes, DMEM and pelleted adipose
tissue-derived non-adipocyte cells. The DMEM was carefully removed
by inserting a pasteur pipette through the adipocytes taking care
not to disturb the pieces of adipose tissue, adipocytes or the
pelleted cells.
Tissue Culture
[0200] The floating pieces of adipose tissue and adipocytes and the
pelleted cells were gently resuspended in 10 ml of DMEM and
transferred to a 300 ml tissue culture flask. A 30 ml volume of
DMEM and 10 mls of autologous sterile serum were added and the
flask was then incubated at 37.degree. C. with 5% CO.sub.2. The
flask was examined daily by microscopy. Cells became attached and
fibroblast-like in appearance between days 3 and 6. The attached
cells became confluent between days 5 and 10.
Harvesting Cell Free Cell Secretions
[0201] Once cells were confluent on the base of the flask the
supernatant was harvested and the suspended adipose tissue and
cells were removed by filtration through a 20 micron mesh. The
solution was filter sterilised through a 0.22 micron filter and
then aseptically dispensed into 10 ml vials and stored frozen at
-20.degree.. In the Examples that follow, this material may be
referred to as "CellFree".
Example 2. Production of Concentrated Canine Cell Free Extracts
[0202] Volumes (lop ml) of the frozen cell free extracts in Example
1 were freeze dried in a Telstar Lyobeta freeze dryer for 2 days.
The resulting freeze-dried cake was rehydrated with 10 ml of
distilled water. The concentrated sample was then sonicated in a
sonicating water bath for 20 min. The 10 ml volumes contained a
concentrated mix of cytokines.
Example 3. Subcutaneous Administration of Cell Free Extracts from
Canine Adipose Tissue to Arthritic Dogs
[0203] In this example the safety and efficacy of subcutaneous
injections of the canine cellular secretions prepared according to
Example 1 above, in the treatment of osteoarthritis in dogs was
investigated. Five dogs each received subcutaneous injections in
the scruff of the neck once per week for four weeks at the rate of
0.3 ml/10 kg of body weight. The dogs in this trial were
predominantly advanced in age (>10 years) and had a range of
joint and or bone disorders, including osteoarthritis of the elbow,
chronic hip dysplasia, stifle disease and wobblers. Individually
the dogs in this trial were each on a range of medications for the
treatment of their conditions (Table 1). The existing medication
was stopped whilst the trial was conducted. The dogs were assessed
by their regular treating vets, as well as being observed by their
owners for anecdotal changes, such as changes in behaviour,
mobility, and obvious signs of pain. The trial is summarised in
Table 1.
TABLE-US-00001 TABLE 1 Subcutaneous administration of cell free
extracts from canine adipose tissue to arthritic dogs Continued
Existing or stopped Adverse Dog/Age Condition medication medication
effects Comments Smudge Chronic OA Monthly Stopped None OA did not
12 y of elbow cartrophen observed deteriorate Oz Chronic hip
Monthly Stopped None OA did not 14 y dysplasia cartrophen observed
deteriorate Daisy Stifle disease Metacam Stopped None Marked 13 y
observed improvement; less pain; more active; owners report better
than Metacam Lassie Mild elbow None n/a None Moderately 10 y OA
observed less lame, more mobile; less pained Versace Wobblers
Prednisone Stopped None Large 10 y observed improvement in
stability and activity levels
[0204] No adverse reactions were noted with repeated injections.
All five dogs did not deteriorate even though their current
medications were stopped. Three of the five dogs showed significant
improvement above what was seen from their previous
medications.
Example 4. Subcutaneous Administration of Concentrated Secretions
from Canine Adipose Tissue to Arthritic Dogs
[0205] The results of the trials presented in Example 3
demonstrated that there are no adverse effects associated with the
method of the invention and also demonstrated that in some treated
subjects the remote administration of the adipose tissue-derived
cell secretions was associated with improved activity of the
subject, improved mobility or an apparently lower degree or pain.
The inventor reasoned that administration of a higher dose of
adipose tissue-derived cell secretions may provide further
advantages.
[0206] A concentrated cell free Extract produced as described in
Example 1 and 2 was used to treat 9 arthritic dogs. A 2 ml volume
of the concentrated cell free extract was administered by
subcutaneous injection into the scruff of the neck. The dogs were
re-examined after 10 days by a veterinarian. Results are presented
in Table 2.
TABLE-US-00002 TABLE 2 Subcutaneous administration of concentrated
secretions from canine adipose tissue to arthritic dogs Breed Age
(yrs) Condition Date treated Outcome German 10 OA hips 5, Aug. 2011
Improved mobility and less Shepherd pain. Samoyed 11 Lameness 19,
Jul. 2011 Improved mobility and less pain. Doberman 10 Wobblers 7,
Jul. 2011 Great improvement. Dog more stable, active and has
greater control over bowel movements. Border 12 OA 5, Aug. 2011
More active, improved mobility Collie and less pain. German 6 OA
30, Jul. 2011 Much better. Shepherd More active, improved mobility
and less pain. Dogue de 3 OA 11, Jun. 2011 More active, improved
mobility bordeaux and less pain. Labrador 12 OA, 8, Aug. 2011 More
active, improved mobility neurological and less pain. issues. Bull
13 Arthritic 1, Apr.2011 More active and mobile, pain mastiff hips
free. Effect has not waned cross when assessed after 5 months.
(Chief) Golden 12 Arthritic 28, Apr.2011 More active and mobile,
pain Retriever elbows and free. Effect lasted 4 months. (Ruby) hips
Effect was again apparent after re-treatment.
Example 5. Subcutaneous Administration of Cell Free Extracts from
Canine Adipose Tissue to Dogs with Atopic Dermatitis
[0207] A cell free extract prepared as described in Example 1 was
administered by subcutaneous injection to three dogs with atopic
dermatitis. A 3 ml volume was injected into the scruff of the neck,
once a week for 3 weeks. All three dogs showed an improvement when
assessed by veterinary examination between 2 and 4 weeks after
treatment, their condition was improved, with reduced inflammation
being the primary outcome (Table 3).
TABLE-US-00003 TABLE 3 Subcutaneous administration of cell free
extracts from canine adipose tissue to dogs with atopic dermatitis.
Condition Name Age Breed treated Outcome Comments Gemma 10 Bull
mastiff Atopic Moderate Generally much less years x dermatitis
improvement in inflamed, some skin condition papules still present;
Topaz 6 German Atopic Minor Lesions on ventrum years shepherd
dermatitis improvement in less inflamed, scabby skin tail lesion
dryer. BJ 2 Kelpie Atopic Significant Feet look great, left years
cross dermatitis improvement in arm lesion much skin condition
improved, coat generally looks shinier. Original inflammation along
ventrum, arm, chest and paws improved.
Example 6. Subcutaneous Administration of Concentrated Cell Free
Extracts from Equine Adipose Tissue in a Lame Horse
[0208] An equine cell free extract was prepared as described in
Example 1 using adipose tissue collected from the tail base of a
horse. The adipose tissue was processed exactly as described in
Example 1 except that autologous equine serum was added to the
tissue culture flask instead of canine serum. The cell free extract
was concentrated by freeze drying as described in Example 2.
[0209] A 2 ml volume of the concentrated cell free extract was
administered by subcutaneous injection into the neck of a racehorse
that was mildly lame. The horse had previously been treated with
intra-articular injections of steroids and was no longer responding
to this treatment.
[0210] The horse showed a marked reduction in lameness two days
after administration of the cell free extract.
Example 7: Subcutaneous Administration of Concentrated Cell Free
Extracts from Equine Adipose Tissue in Horses
[0211] Five horses with either suspensory ligament damage or early
stage osteoarthritis of the knees were treated with concentrated
equine secretions (Example 6) by subcutaneous injection into the
neck two days before racing. All horses demonstrated improved
mobility (Table 4).
TABLE-US-00004 TABLE 4 Subcutaneous administration of concentrated
cell free extracts from equine adipose tissue in horses Date
Problem Effect 16, Aug.2011 Early stage Improved mobility and less
lame, OA in knees performed better than expected in race. 19, Aug.
2011 Suspensory Improved mobility and less lame, ligament injury
performed better than expected in race (won). 19, Aug. 2011
Suspensory Improved mobility and less lame. ligament injury 10,
Sep. 2011 Suspensory Improved mobility and less lame, ligament
injury performed better than expected in race. 10, Sep. 2011 Early
stage Improved mobility and less lame, OA in knees performed better
than expected in race.
Example 8. Treatment of Osteoarthritis in Dogs by Subcutaneous
Injection of Cryopreserved Allogeneic Adipose Derived Cells
Including Adipocytes
Processing of Adipose Tissue
[0212] A 10 g sample of falciform adipose tissue was collected from
a female dog during a routine desex procedure. The adipose tissue
was rinsed with saline and then minced finely using scissors and
mixed with 20 mls of Dulbecco's Modified Eagle's Medium (DMEM,
Sigma). Collagenase (Sigma) was added to a final concentration of
0.05% (w/v)' and the sample was incubated at 37.degree. C. for 90
minutes. During the incubation the sample was gently inverted by
hand every 15 minutes.
[0213] Following collagenase treatment the sample was aseptically
filtered through a stainless steel mesh (700 micron pore size),
transferred to a 50 ml centrifuge tube and centrifuged at 500 g for
15 minutes.
[0214] Four distinct layers were visible within the centrifuged
sample: a small (2 mm thick) layer of free lipid on the surface,
below which was a white 10 mm thick layer of adipocytes and then a
large clear layer of DMEM and then a pellet of SVF cells. The small
layer of oil was carefully removed with a pasteur pipette. A fresh
pasteur pipette was then carefully inserted through the adipocytes
and the clear DMEM was removed without disturbing the floating
adipocytes or the pelleted SVF cells. This resulted in a sample
that contained only the floating adipocytes and the pelleted SVF
cells. The floating and the pelleted cells were gently mixed with a
pasteur pipette and transferred to a 15 ml centrifuge tube.
[0215] The cells were then washed in DMEM to remove collagenase.
DMEM was added to a final volume of 14 mls and the sample
centrifuged at 500 g for 10 minutes. This resulted in three
distinct layers: floating adipocytes, DMEM and pelleted SVF cells.
The DMEM was carefully removed by inserting a pasteur pipette
through the adipocytes taking care not to disturb the adipocytes or
the pelleted cells.
[0216] The floating and the pelleted cells were gently resuspended
in 4 mls of DMEM and mixed with a pasteur pipette.
Expansion and Cryopreservation of Cells
[0217] Aliquots (0.5 mls) of the cell suspension were transferred
to a T175 tissue culture flask containing 50 mls of DMEM plus 10%
canine serum and incubated in a CO.sub.2 incubator at 37.degree. C.
until a confluent cell monolayer was present (6 days). The floating
cells (adipocytes) still had a healthy morphology at this time.
[0218] Cells were stripped with 3 mls of TrypLE Express
(Invitrogen), decanted into 50 ml centrifuge tubes and centrifuged
at 500.times.g for 10 minutes. The floating and the pelleted cells
were resuspended in 2 mls of canine serum plus 10% DMSO and
transferred to a cryovial. The cryovials were frozen in a Mr Frosty
slow freezing device (Invitrogen) in a -80.degree. C. freezer for
24 hours and then transferred to a liquid nitrogen dewar.
Administration of Cells to Dogs
[0219] Seven dogs with osteoarthritis were given a single
subcutaneous injection of cells into the scruff of the neck. The
cryovial per dog was thawed at room temperature and the cell
suspension drawn up with a syringe and hypodermic needle. The
entire 2 mls of cell suspension was injected.
Monitoring of Dogs
[0220] Dogs were monitored over an 8 week period by their owners
and were examined by a veterinarian. Six of the seven dogs showed a
marked improvement in their mobility and an apparent reduction in
pain. The seventh dog did not improve but did not show a negative
response.
Example 9. Treatment of Osteoarthritis in Dogs by Subcutaneous
Injection of Cells Mixed with Cell Secretions
[0221] A vial of the cryopreserved cells as described in Example 8
was removed from liquid nitrogen and mixed with 5 mls of warm
(37.degree. C.) canine secretions prepared as in Example 1. The
cells and the secretions were then drawn up into a syringe and
administered subcutaneously into the scruff of a dog with
arthritis. The dog responded well to the treatment and showed a
rapid improvement in mobility and a reduction in pain.
Example 10. Preparation of Adipose Derived Cells that had been
Premixed with Cell Secretions and then Frozen
Processing of Adipose Tissue
[0222] A 10 g sample of falciform adipose tissue was collected from
a female dog during a routine desex procedure. The adipose tissue
was rinsed with saline and then minced finely using scissors and
mixed with 20 mls of Dulbecco's Modified Eagle's Medium (DMEM,
Sigma). Collagenase (Sigma) was added to a final concentration of
0.05% w/v and the sample was incubated at 37.degree. C. for 90
minutes. During the incubation the sample was gently inverted by
hand every 15 minutes.
[0223] Following collagenase treatment the sample was aseptically
filtered through a stainless steel mesh (700 micron pore size),
transferred to a 50 ml centrifuge tube and centrifuged at 500 g for
15 minutes.
[0224] The floating cells and the supernatant were discarded and
the pelleted cells were gently mixed with a pasteur pipette and
transferred to a 15 ml centrifuge tube.
[0225] The cells were then washed in DMEM to remove collagenase.
DMEM was added to a final volume of 14 mls and the sample
centrifuged at 500 g for 10 minutes. The supernatant was discarded
and the pelleted SVF cells were gently resuspended in 4 mls of DMEM
and mixed with a pasteur pipette.
Expansion of Cells
[0226] Aliquots (0.5 mls) of the cell suspension were transferred
to tissue culture flasks containing DMEM plus 10% canine serum and
incubated in a CO2 incubator at 37.degree. C. until a confluent
cell monolayer was present (7 to 10 days). Cells were stripped with
3 mls of TrypLE Express (Invitrogen), decanted into 50 ml
centrifuge tubes and centrifuged at 500.times.g for 10 minutes.
Cells were either cryopreserved at this point, in a Mr Frosty slow
freezing device as described in Example 8, or they were placed into
new tissue culture flasks and passaged further until they had
doubled approximately 8 or 13 times. The passaged cells were then
stripped and centrifuged.
Cryopreservation of Cells
[0227] The pelleted cell samples (no passage, approx. 8 doublings
and approx. 13 doublings) were each divided into two samples, one
was mixed with concentrated secretions whereas the other was not.
The cells were resuspended in either canine serum or a mixture of
canine serum plus concentrated canine secretions that were produced
according to Example 1 but that had been concentrated ten-fold by
centrifuging in a 3 kDa Amicon centrifugal filter tube (Millipore).
The concentrated secretions were mixed with canine serum at a ratio
of either 1 to 1 or at a ratio of 1 part concentrated secretions to
10 parts serum. The cell suspension were mixed with the serum and
secretions and then held at room temperature for 30 minutes to
allow the secretions to interact with the cells. The cell
suspensions were then transferred to cryovials in 2 ml
aliquots.
[0228] DMSO was added to each cryovial to produce a final
concentration of 10% and the cryovials were frozen in a Mr Frosty
slow freezing device (Invitrogen) in a -80.degree. C. freezer for
24 hours and then transferred to a liquid nitrogen dewar for long
term storage.
Thawing Cryopreserved Cells and Analysis of Cell Viability
[0229] Vials were removed from liquid nitrogen and allowed to thaw
at room temperature. The vials were mixed by gently inverting and
then a 0.1 ml volume was removed for viability measurement. The 0.1
ml volume was placed in a flow cytometry tube and mixed with 0.9
mls of Isoflow (Beckman Coulter) that contained propidium iodide
(Sigma Chemical Company, Louisville, USA) at a concentration of 10
.mu.g/mL and Syto11 (Molecular Probes, Eugene, USA) at a
concentration of 1 .mu.g/mL. Samples were analysed on a FACSCan
flow cytometer and the percentage of Syto11 positive (live cells)
and propidium iodide positive (dead cells) recorded.
Culture of Cryopreserved Cells
[0230] A 1 ml volume of the thawed samples were placed into a T75
tissue culture flask with DMEM and 10% fetal calf serum and
incubated at 37.degree. C. with 5% in a CO.sub.2 incubator for 5
days. Flasks were then examined each day using an inverted
microscope and the percentage confluency recorded.
[0231] Samples of tissue culture media from the flasks were removed
and analysed using the Bio-Plex Pro Human 27-plex cytokine,
chemokine and growth factor assay (Biorad, Hercules, USA) for
ILR1a, G-CSF, VEGF and IL-10. Control samples of fresh media with
and without secretions were included as controls.
Assessment of Cell Viability after Cryopreservation
[0232] The viability of cells frozen with the secretions was higher
than the cells frozen without secretions (Table 5).
TABLE-US-00005 TABLE 5 Viability of passaged (13 cell doublings)
and non- passaged cells frozen with and without secretions Average
viability Cell type post-thawing Non-passaged cells with no
secretions 66.83% Non-passaged cells with secretions 75.70%
Passaged cells (13 cell doublings) with no 35.95% secretions
Passaged cells (13 cell doublings) with 71.6% secretions
Assessment of Proliferation after Freezing Using the Click-iT EDU
Assay
[0233] The proliferation rates of thawed cells were assessed using
the Click-iT EDU assay (Life Technologies). A volume of the thawed
cell suspension that contained approximately 500,000 cells was
transferred to a 6 well plate. A 2 ml volume of DMEM plus 10%
canine serum was added to the well. The EDU reagent was added to
the well to create a final concentration of 10 .mu.M. The plate was
incubated at 37.degree. C. with 5% CO.sub.2 in a CO2 incubator at
37.degree. C. for 2 hours. The supernatant was collected and the
adherent cells were stripped and both were combined, washed (in PBS
plus 1% bovine serum albumin; Sigma) and resuspended in 100 .mu.L
of Click-iT fixative and incubated for 15 min at room temperature
to fix the cells. Cells were then washed and resuspended in 100
.mu.L of Click-iT permeabilisation agent to permeabilise cells. The
cells were labelled for detection, by incubating (30 min) in the
Click-iT reaction cocktail (500 .mu.L) containing 2.5 .mu.L of
AlexaFluor 488 Fluorescent dye. Following incubation the cells were
washed in Click-iT permeabilisation and wash reagent and was
analysed by flow cytometry. Cells were then analysed using a
FACScan flow cytometer. Proliferating cells showed an increase in
green fluorescence compared to non-proliferating cells.
[0234] The results of analysing the cells are displayed in FIG. 1.
The sample that had been stored frozen as cells plus secretions
showed a population of proliferating cells equal to 16.8% of the
total cells. The samples that had been stored frozen as cells with
no secretions showed a population of proliferating cells equal to
only 1.8% of the total cells.
Recovery of Cells after Freezing
[0235] The addition of secretions to cells prior to freezing the
cells improved the ability of the cells to survive the freezing
process and to proliferate after thawing. Cells that had been
cultured until they reached cumulative cell doublings of
approximately 8 and that had been frozen with secretions were
observed to attach to the tissue culture flask more rapidly and
grow more rapidly than the same cells frozen without secretions. At
24 hours the cells frozen with secretions had reached 90%
confluency whereas the cells frozen without secretions had reached
only 60% confluency (Table 6).
TABLE-US-00006 TABLE 6 Assessment of cultured cells (approximately
8 cumulative cell doublings) frozen with and without secretions
Confluency after 24 hours Cell type in culture post-thaw Cells
frozen with no secretions 60% Cells frozen with secretions 90%
Comparison of Cytokines Produced from Cells Frozen with and without
Secretions
[0236] Cells that had been cultured through approximately 8
cumulative cell doublings and that were frozen with secretions
produced a statistically significant (two-tailed t-test) greater
amount of the cytokines ILr1a, IL-6, IL-8, IL-9, IL-12, IL-13, FGF
basic, TNF-a and VEGF than the same cells frozen without secretions
(Table 7), upon post-thaw culturing for 5 days.
TABLE-US-00007 TABLE 7 Comparison of cytokines produced by cells
that had been frozen with or without secretions. The numbers are
the means of three replicates. FGF Sample IL-1ra IL-6 IL-8 IL-9
IL-10 IL-12 IL-13 basic TNF-a VEGF Cells with 80 8 17 10 43 91 8 14
20 5166 secretions Cells 20 0 0 0 18 38 3 0 1 988 p-value 0.005
0.000 0.006 0.000 0.115 0.016 0.039 0.000 0.001 0.059
[0237] These sets of data demonstrate that there is a beneficial
effect of combining secretions with cells prior to freezing the
cells. Cells that are frozen without secretions lose their ability
to proliferate and to produce cytokines. Including secretions with
the cells prior to freezing preserves the ability of the cells to
proliferate and to produce cytokines.
Example 11. Production of Secretions from Passaged Cells and Use of
the Secretions to Freeze Cells
[0238] Canine adipose derived cells were isolated and cultured as
described in Example 10. The cells were passaged until the cells
had reached a cumulative cell doubling of approximately 13 times.
The tissue culture supernatant from the cells was concentrated
using a 3 kDa Amicon centrifugal filter tube (Millipore). The
concentrated secretions were mixed with serum at a ratio of 1 to 1.
The cells were stripped, washed and the cell pellet was resuspended
in the mixture of serum and secretions and then held at room
temperature for 30 minutes to allow the secretions to interact with
the cells. The cell suspensions were then transferred to cryovials,
mixed with 10% DMSO and frozen as described in Example 10.
[0239] The cryopreserved cell suspensions were thawed and cultured
in tissue culture flasks as described in Example 10. At 72 hours
the cells frozen with secretions had reached 90% confluency whereas
the cells frozen without secretions had reached only 20% confluency
(FIG. 2). Importantly, the cells without secretions became
senescent and did not reach greater than 20% confluency even after
10 days of incubation.
[0240] This is an important finding. There is a need to be able to
expand mesenchymal stem cells and other cell types in tissue
culture to produce large numbers of cells. However, mesenchymal
stem cells and other cell types cannot be cultured indefinitely.
After a number of cell doublings the cells become senescent and
will not multiply any further. This limits the number of doses that
can be produced from a single culture.
[0241] To produce large numbers of cells from a single starting
culture requires a cell bank of cryopreserved vials to be set up.
Normally a double cell bank is created. A first set of
cryopreserved vials is created and one of these vials is then
thawed and used to produce a second set of cryopreserved vials.
Each time a batch of product is produced one of these second set of
vials is thawed and placed into culture. This cell banking process
typically results in cells that are not as therapeutically
effective as freshly isolated cells. By combining secretions with
the cells during the freezing steps overcomes this problem and
allows the production of a therapeutically effective frozen
product.
Example 12. Treatment of Neuropathic Facial Pain with Autologous
Adipose Derived Cells
[0242] Four human patients suffering from neuropathic facial pain
were treated with an autologous adipose derived cell suspension
comprised of stromal vascular fraction cells and adipocytes.
[0243] Liposuction was used to collect approximately 200 grams of
adipose tissue from the abdomen and or thighs of each patient. The
lipoaspirate was processed immediately after collection by washing
with sterile saline and then digesting by adding sterile
collagenase to a final concentration of 0.05% w/v. The sample was
incubated at 37.degree. C. for 20 minutes, filtered through a 800
micron mesh and transferred to centrifuge tubes.
[0244] The centrifuge tubes were centrifuged at 400 g for 10
minutes, and the layer between the floating adipocytes and the
pelleted cells was removed. The cell pellet and floating adipocytes
were combined and filter sterilized saline was added until the
tubes were full. The samples were centrifuged again at 400 g for 10
minutes and the layer between the pelleted cells and the floating
adipocytes was removed. The resulting cell preparation was diluted
to a volume of 10 ml with sterile saline and dispensed into 2 nil
volumes in sterile syringes.
[0245] The patients were given four or five injections of 2 mL
volume into their gums at the original site of the pain.
[0246] Patients were followed up at between 4 and 8 weeks post
treatment and their level of pain was assessed using a visual
analogue scale (0=no pain, 10=worst pain imaginable). The results
from these assessments are described in the Table 8 below.
TABLE-US-00008 TABLE 8 Administration of autologous adipose derived
cells for treatment of neuropathic pain Pretreatment condition Post
treatment condition Patient 1 Constant sharp neuralgic At one month
post treatment: pain all day, Two or three pain episodes each pain
in tooth and cheek, day (2-3 hours each), pain intensity 8. pain in
tooth only, pain intensity 6-7. Patient 2 Pain in arm, shoulder,
jaw, At two months post treatment: teeth, pain intensity 9, mild
tenderness in tooth only, pain intensity 0-1. Patient 3 Pain in
teeth, jaw and At 3 weeks post treatment: gum, pain intensity 8,
Neurontin has been reduced to needed Neurontin 1200 100 mg and pain
in teeth and mg and Endep 50 m. gum only, pain intensity 4. Patient
4 Facial pain intensity 8-9, At one month post treatment: only 2
hours sleep at night facial pain intensity 2 but and woken by sharp
pain. episode of higher pain intensity from cold weather, sleeps 6
hours.
Example 13. Treatment of Pain with Topical Application of
Secretions from Bovine Adipose Derived Cells
[0247] A 10 g sample of adipose tissue was collected by excision
from the tail-base of a 1 year old steer. The adipose tissue was
rinsed with saline and then minced roughly using scissors into
pieces of approximately 5 mm diameter and mixed with 20 ml of
Dulbecco's Modified Eagle's Medium (DMEM, Sigma). Collagenase
(Sigma) was added to produce a final concentration of 0.2% [w/v]
and the sample was incubated at 37.degree. C. for 30 minutes. At
the end of 30 minutes the adipose tissue was partially digested and
consisted of a mixture of partially digested fat particles,
liberated stromal vascular cells (SVCs) and liberated
adipocytes.
[0248] The sample was then washed to remove collagenase by
centrifuging at 500 g for 15 minutes. Four distinct layers were
visible within the centrifuged sample: a small (2 mm thick) layer
of free lipid on the surface, below which was a white 20 mm thick
layer of adipose tissue and adipocytes and then a large clear layer
of DMEM/collagenase and then a pellet of adipose tissue-derived
non-adipocyte cells. The small layer of lipid was carefully removed
with a pasteur pipette. A fresh pasteur pipette was then carefully
inserted through the adipocytes and the clear DMEM was removed
without disturbing the floating adipose tissue, adipocytes or the
pelleted cells. This resulted in a sample that contained only the
floating pieces of adipose tissue and adipocytes suspended in a
small volume of DMEM and the pelleted cells. The pieces of adipose
tissue and adipocytes and the pelleted cells were gently mixed with
a pasteur pipette and transferred to a 15 ml centrifuge tube.
[0249] The pieces of adipose tissue and cells were then washed in
DMEM to remove collagenase as follows. DMEM was added to a final
volume of 14 ml and the sample centrifuged at 500 g for 10 minutes.
This resulted in three distinct layers: floating pieces of adipose
tissue and adipocytes, DMEM and pelleted adipose tissue-derived
non-adipocyte cells. The DMEM was carefully removed by inserting a
pasteur pipette through the adipocytes taking care not to disturb
the pieces of adipose tissue, adipocytes or the pelleted cells.
Tissue Culture
[0250] The floating and the pelleted cells were gently resuspended
in 10 ml of DMEM and transferred to a 300 nil tissue culture flask.
A 30 ml volume of DMEM and 10 mls of sterile fetal calf serum were
added and the flask was then incubated at 37.degree. C. with 5%
CO.sub.2. The flask was examined daily by microscopy. Cells became
attached and fibroblast-like in appearance between days 3 and
6.
Harvesting Cell Free Cell Secretions
[0251] After 6 days the supernatant was harvested and the suspended
adipose tissue and cells were removed by filtration through a 20
micron mesh. The solution was filter sterilised through a 0.22
micron filter and then aseptically dispensed into 10 ml vials and
stored frozen at -20.degree. C.
Preparation of Cream Containing Bovine Secretions and a Placebo
Cream
[0252] A vial of the bovine secretions was thawed and mixed with an
equal amount of Aqueous Base Cream BP and dispensed into plastic
tubes and stored at 4.degree. C. until used.
[0253] A second lot of cream that did not contain bovine secretions
was prepared as a placebo control. DMEM was mixed with an equal
amount of Aqueous Base Cream BP and dispensed into plastic
tubes.
Treatment of Pain from Burns
[0254] A 46 year old male who had accidentally mildly burnt his
thumb and middle finger was given a tube of each cream. The tubes
were labelled as Cream 1 and Cream 2 and the man did not know which
cream contained the secretions or which was the placebo. The man
applied Cream 1 to the middle finger and Cream 2 to the thumb
approximately 30 minutes after the burns occurred. The man reported
that after 10 minutes there was no longer any pain in his thumb.
The pain in the thumb did not return. The pain in the middle finger
continued for approximately 6 hours. A blister formed on the middle
finger but no blister occurred on the thumb.
[0255] Cream 1 was the placebo and was applied to the middle
finger. Cream 2 contained the bovine secretions and was applied to
the thumb.
Example 14. Intramuscular Administration of Secretions from Human
Adipose-Derived Cells to Mice with Collagen Antibody-Induced
Arthritis
Preparation of Adipose Tissue
[0256] Liposuction was used to collect approximately 200 grams of
adipose tissue from a patient. The lipoaspirate was digested by
adding sterile collagenase to a final concentration of 0.05% w/v.
The sample was incubated at 37.degree. C. for 30 minutes, filtered
through a 800 micron mesh and transferred to centrifuge tubes. The
tubes were centrifuged at 1500 g for 5 minutes, to obtain the
pelleted cells (stromal vascular fraction; SVF) and floating
adipocytes. Four distinct layers were visible within the
centrifuged sample: a small layer of free lipid on the surface,
below which was a thick layer of adipose tissue and adipocytes and
then a large clear layer of saline and a pellet of adipose
tissue-derived non-adipocyte cells (SVF). The lipid layer was
aspirated and discarded. The adipocyte and SVF fractions were
separately collected after the centrifugation. The fractions were
washed separately with saline and centrifuged at 1500.times.g for 5
mins. The SVF pellet was gently resuspended in 10 mL of cell
culture media that consisted of Dulbeccos Modified Eagle Medium
(DMEM) supplemented with 10% foetal bovine serum and 1%
penicillin-streptomycin solution.
[0257] A 300 .mu.L portion of the SVF pellet was filtered through a
35 .mu.m nylon mesh topped tube. A 200 .mu.l, portion of the
filtered sample was enumerated and the viability determined in
TruCount tubes containing isoflow, propidium iodide (10 .mu.g/mL)
and Syto11 (1 .mu.M) using a FacsScan flow cytometer. The total
number of viable nucleated cells in the SVF pellet was
determined.
Tissue Culture and Harvesting of Secretions
[0258] A T175 cm.sup.2 culture flask was seeded with approximately
29 million viable SVF cells and 30 mL of adipocytes. A volume of 50
mL of cell culture media, as described above, was also added to the
flask. The flask was incubated at 37.degree. C. with 5% CO.sub.2
for 72 hours.
[0259] Following the 72-hour incubation, the conditioned medium was
collected from the flask. This conditioned media sample was
centrifuged at 4980 g for 10 mins and stored at -80.degree. C. This
conditioned media was thawed, filter sterilized using 0.22 .mu.m
syringe filter, aliquoted and frozen at -80.degree. C. A vehicle
control, containing DMEM supplemented with 1%
penicillin-streptomycin solution, was also filter sterilized using
a 0.22 micron syringe filter, aliquoted and frozen at -80.degree.
C. These aliquoted samples were given coded names and were shipped
on dry ice to TetraQ where they were administered to mice suffering
from CAIA.
Collagen Antibody-Induced Arthritis Mouse Model
[0260] The collagen antibody-induced arthritis (CAIA) model is a
widely accepted animal model of arthritis which has been reported
in the literature to investigate the pathogenic mechanisms involved
in arthritis and to screen potential therapeutic candidates. The
model targets type I collagen, one of the major constituents of
articular cartilage. In mice, CAIA is induced by the administration
of a cocktail of 5 anti-type II collagen antibodies followed by a
lipopolysaccharide (LPS) injection three days later. The subsequent
administration of LPS following the antibody cocktail not only
increases the severity of the arthritis through the induction of
pro-inflammatory cytokines and complement component activation, but
also reduces the amount of monoclonal antibody required to induce
the arthritis in this model. The arthritis that develops in these
mice closely resembles rheumatoid arthritis in people, including
synovitis with infiltration of polymorphonuclear and mononuclear
cells, pannus formation, fibrosis cartilage degradation and bone
erosion. Significant swelling and redness is observed in the paws
of mice suffering with CAIA. The clinical manifestations of paw
redness and swelling can be assessed to assign a clinical arthritis
score to mice in the CAIA model. Outcome measures of paw volume,
ankle size and clinical arthritis score can be used to determine
the effectiveness of a treatment on reducing the severity of
arthritis in this CAIA model.
[0261] At day zero, each mouse (total of 12) received an
intravenous injection of 1.5 mg (150 .mu.L) of an anti-type II
collagen 5 clone antibody cocktail. This cocktail contains 5
monoclonal antibodies: Clone A2-10 (IgG2a), F10-21 (IgG2a), D8-6
(IgG2a), D1-2G (IgG2b), and D2-112 (IgG2b) recognizing the
conserved epitopes on various species of type II collagen. On day
3, mice received an intraperitoneal injection of 80 .mu.L (40
.mu.g/mouse) of LPS. The trial design consisted of 2 groups of 6
mice. The mice received 50 ILL doses administered via the
intramuscular (1M) route on days 6, 8, 10 and 12 of the following:
group 1 received human secretions from the SVF+adipocytes and group
2 received the vehicle control.
[0262] The mice were monitored throughout the trial period (2
weeks) and the primary outcome measurements of paw volume, ankle
size and clinical arthritis score were taken on days 0 and 2-13.
The measurements were taken prior to administration of the collagen
antibody cocktail (days 0) and LPS (day 3) and prior to
administration of vehicle and the human SVF+adipocyte secretions.
Paw volume was measured using a plethysmometer. The paw size was
measured using microcalipers across the hillock (ankle joint) of
each hindpaw. The mice were assessed and scored for the severity of
arthritis using a standard scale (0--normal; 1--mild redness,
slight swelling of ankle or wrist, redness and swelling limited to
individual joints; 2--moderate swelling of ankle or wrist, redness
in more than one joint, 3--severe swelling including some digits,
ankle or foot; 4--maximal swelling and inflamed, involving multiple
joints).
Data Analysis
[0263] The average and standard deviation (SD) of each of the
primary outcome measures, paw volume (cm.sup.3), ankle size (mm),
and clinical arthritis score were calculated for each time-point
post-treatment for both groups of mice. A two-tailed t-test was
performed on the primary outcome measures at each time-point
post-treatment to compare the effect of the SVF+adipocyte
secretions versus the vehicle control. The statistical significance
criterion was P<0.05.
Results
[0264] The paw volume results for both groups are presented in FIG.
3. An analysis of the data at day 11 revealed a significant
reduction (p-value=0.002) in the paw volume of mice treated with
SVF+adipocyte secretions when compared to the vehicle control
mice.
[0265] The ankle size measurements for both groups are presented in
FIG. 4. An analysis of the data at day 11 revealed a significant
reduction (p-value=0.018) in the ankle size of mice treated with
SVF+adipocyte secretions when compared to the vehicle control
mice.
[0266] The clinical arthritis scores for both treatment groups are
presented in FIG. 5. An analysis of the data at day 11 revealed a
significant reduction (p-value=0.017) in the clinical arthritis
score of mice treated IM with SVF+adipocyte secretions when
compared to the vehicle control mice.
[0267] The data clearly shows that administration of secretions by
intra-muscular injection has a therapeutic effect in the CAIA mouse
model. The injection site is remote from the site of the
disease.
Example 15. Intravenous Administration of Adipose-Derived Cells
Cryopreserved in Standard Cryoprotectants Versus Cells
Cryopreserved with Concentrated Secretions to Mice with Collagen
Antibody-Induced Arthritis
Preparation of Adipose Tissue
[0268] Canine adipose tissue was processed as described in Example
1 to produce canine adherent cells. Furthermore, canine secretions
which were concentrated 10.times., using a 3 kDa Amicon centrifugal
filter tube, from adipose-derived cells, were produced as described
in Example 1. From these cells and secretions, the following test
products were prepared in cryovials and cryopreserved in a Mr
Frosty slow freezing device (Invitrogen) in a -80.degree. C.
freezer for 24 hours and then transferred to a liquid nitrogen
dewar: [0269] 1. 70,000 cells cryopreserved in 90% canine serum and
10% DMSO [0270] 2. 70,000 cells cryopreserved in 45% canine
secretions (10.times. concentrate), 45% canine serum and 10%
DMSO.
Collagen Antibody-Induced Arthritis Mouse Model
[0271] The collagen antibody-induced arthritis mouse (CAIA) model
described in Example 14 was also used to investigate the effects of
administering the above described-test products. A total of 12 mice
were induced with CAIA as described in Example 14. At day 6
post-CAIA induction, the above described cryopreserved test
products were thawed at room temperature immediately before
injection. 6 mice were each injected intravenously with 140 .mu.L
of cells (Which equated to 70,000 cells) which had been
cryopreserved in standard cryoprotectants, and the remaining 6 mice
were each injected intravenously with 140 .mu.L of cells (which
equated to 70, 000 cells) which had been cryopreserved in a
cryoprotectant mixture which included concentrated secretions.
[0272] The mice were monitored daily throughout the trial and the
primary outcome measures of paw volume, ankle size and clinical
arthritis score were taken on days 0 and 2-12 as described in
Example 14.
Data Analysis
[0273] The average and standard deviation (SD) of each of the
primary outcome measures, paw volume (cm.sup.3), ankle size (mm),
and clinical arthritis score were calculated for each time-point
post-treatment for both groups of mice and graphed. The delta (A)
paw volume, ankle size and clinical arthritis score was calculated
by subtracting the pre-CAIA induction score from the post-CAIA
induction scores and expressing this as a percentage change. The
area under the A paw volume, ankle size and clinical arthritis
curves were determined for each group. A two-tailed t-test was used
to compare the A paw volume, ankle size and clinical arthritis area
under the curve (AUC) values from both groups.
Results
[0274] The paw volume results for both treatment groups are
presented in FIG. 6. An area under the curve analysis of this data
revealed a significant reduction in the paw volume of mice treated
with cells and concentrated secretions when compared to cells alone
(FIG. 7).
[0275] The ankle size measurements for both treatment groups are
presented in FIG. 8. A significant reduction in the ankle size of
mice treated with cells and concentrated secretions was observed
when compared to the mice that received cells only (FIG. 9)
[0276] The clinical arthritis scores for both treatment groups are
presented in FIG. 10. A significant reduction in the clinical
arthritis score was observed in the mice treated with cells and
secretions when compared to mice that received cells alone (FIG.
11).
[0277] The data shows an increased therapeutic effect by combining
cells with secretions prior to freezing the cells. The data clearly
shows that a frozen product that combines cells and secretions has
a therapeutic effect in the CAIA mouse model. When cells alone were
administered there was no therapeutic effect. This is due to the
cells being damaged during the freezing process and the damaged
cells are not secreting the cytokines that are required to cause
the therapeutic effect. By freezing the cells with secretions the
cells are able to survive the freezing process and be fully
functional and capable of secreting the cytokines required to cause
the therapeutic effect.
Example 16. Treatment of Pain in Dogs by Subcutaneous
Administration of Adipose Derived Cells
[0278] Frozen cells were prepared according to Example 10. The cell
suspension was thawed and administered subcutaneously in the scruff
of the neck of a dog that was suffering from sciatic nerve pain.
One week after treatment the dog showed no signs of pain.
[0279] A second dog that was suffering from intervertebral disc
disease and was administered a subcutaneous injection of cells in
to the scuff of the neck. One week after treatment the dog showed
no signs of pain.
* * * * *