U.S. patent application number 16/277274 was filed with the patent office on 2019-06-13 for process for preparing metabolized conditioned growth media.
The applicant listed for this patent is Allergan, Inc.. Invention is credited to James Vincent Gruber, Rahul Mehta, Smitha Rao, Sujatha D. Sonti.
Application Number | 20190175672 16/277274 |
Document ID | / |
Family ID | 46047949 |
Filed Date | 2019-06-13 |
![](/patent/app/20190175672/US20190175672A1-20190613-D00001.png)
![](/patent/app/20190175672/US20190175672A1-20190613-D00002.png)
![](/patent/app/20190175672/US20190175672A1-20190613-D00003.png)
![](/patent/app/20190175672/US20190175672A1-20190613-D00004.png)
![](/patent/app/20190175672/US20190175672A1-20190613-D00005.png)
![](/patent/app/20190175672/US20190175672A1-20190613-D00006.png)
United States Patent
Application |
20190175672 |
Kind Code |
A1 |
Gruber; James Vincent ; et
al. |
June 13, 2019 |
PROCESS FOR PREPARING METABOLIZED CONDITIONED GROWTH MEDIA
Abstract
Compositions comprising metabolized conditioned growth medium
and/or metabolized cell extract and methods of use are described.
The metabolized conditioned growth medium and metabolized cell
extract compositions may be formulated with an acceptable carrier
into injectable or topical formulations, for example, as a cream,
lotion or gel, and may be used in cosmeceutical or pharmaceutical
applications. The metabolized conditioned growth medium and
metabolized cell extract may also be further processed to
concentrate or reduce one or more factors or components contained
within the metabolized conditioned growth medium or metabolized
cell extract. The growth medium may be conditioned by any
eukaryotic cell. The metabolized conditioned growth medium and
metabolized cell extract may be used to prevent or treat a
condition, for example, a skin condition.
Inventors: |
Gruber; James Vincent;
(Washington, NJ) ; Rao; Smitha; (Hillsborough,
NJ) ; Mehta; Rahul; (San Marcos, CA) ; Sonti;
Sujatha D.; (San Marcos, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allergan, Inc. |
Irvine |
CA |
US |
|
|
Family ID: |
46047949 |
Appl. No.: |
16/277274 |
Filed: |
February 15, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14047929 |
Oct 7, 2013 |
10206961 |
|
|
16277274 |
|
|
|
|
13294599 |
Nov 11, 2011 |
9408881 |
|
|
14047929 |
|
|
|
|
61413166 |
Nov 12, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 8/9789 20170801;
A61Q 19/02 20130101; A61K 36/06 20130101; A61P 17/14 20180101; A61P
31/00 20180101; A61P 31/10 20180101; A61P 17/10 20180101; A61K
2800/522 20130101; C12P 1/02 20130101; A61K 8/9706 20170801; A61K
36/064 20130101; A61Q 19/00 20130101; A61K 8/99 20130101; A61K
35/33 20130101; A61Q 19/001 20130101; A61P 17/18 20180101; A61K
8/9728 20170801; A61P 17/16 20180101; A61Q 19/08 20130101; A61P
17/00 20180101; A61Q 7/00 20130101; A61K 35/33 20130101; A61K
2300/00 20130101 |
International
Class: |
A61K 36/06 20060101
A61K036/06; A61Q 7/00 20060101 A61Q007/00; A61Q 19/08 20060101
A61Q019/08; A61Q 19/02 20060101 A61Q019/02; A61Q 19/00 20060101
A61Q019/00; A61K 8/9706 20060101 A61K008/9706; A61K 36/064 20060101
A61K036/064; C12P 1/02 20060101 C12P001/02; A61K 35/33 20060101
A61K035/33; A61K 8/99 20060101 A61K008/99 |
Claims
1. A process for preparing a metabolized conditioned growth medium,
the process comprising: (a) culturing cells in a growth medium
sufficient to meet the nutritional needs required to grow the cells
in vitro to form a conditioned growth medium and removing the
conditioned growth medium from the cultured cells; (b) culturing
yeast cells; (c) exposing the yeast cells to the conditioned growth
medium; (d) culturing the yeast cells to metabolize at least a
portion of the conditioned growth medium, and; (e) collecting the
metabolized conditioned growth medium.
2. The process of claim 1, wherein the collected metabolized
conditioned growth medium of step (e) is concentrated, filtered
and/or purified.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/047,929, filed Oct. 7, 2013, which is a
continuation of U.S. patent application Ser. No. 13/294,599 filed
Nov. 11, 2011, now issued as U.S. Pat. No. 9,408,881 which claims
the benefit of U.S. Provisional Application No. 61/413,166, filed
Nov. 12, 2010, the entire disclosure of which is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The disclosure relates to compositions comprising
metabolized conditioned growth medium and/or metabolized cell
extract, and methods for preventing or treating a condition, for
example a skin condition, by administering the compositions to a
subject.
BACKGROUND OF THE INVENTION
[0003] Culture medium compositions typically include essential
amino acids, salts, vitamins, minerals, trace metals, sugars,
lipids and nucleosides. Cell culture medium attempts to supply the
components necessary to meet the nutritional needs required to grow
cells in a controlled, artificial and in vitro environment.
Nutrient formulations, pH, and osmolarity vary in accordance with
parameters such as cell type, cell density, and the culture system
employed. Many cell culture medium formulations are documented in
the literature and a number of media are commercially available.
Once the culture medium is incubated with cells, it is known to
those skilled in the art as "spent" or "conditioned medium".
Conditioned medium contains many of the original components of the
medium, as well as a variety of cellular metabolites and secreted
proteins, including, for example, growth factors, inflammatory
mediators and other extracellular proteins.
SUMMARY OF THE INVENTION
[0004] In one embodiment, the disclosure provides a composition
comprising metabolized conditioned growth medium and an acceptable
carrier. In some embodiments, the metabolized conditioned growth
medium is conditioned growth medium metabolized by yeast cells. In
some embodiments, the conditioned growth medium is prepared by
culturing cells in a growth medium sufficient to meet the
nutritional needs required to grow the cells in vitro to form a
conditioned growth medium. In other embodiments, the composition is
a topical or injectable composition. In some embodiments, the
composition is used to treat or prevent a skin condition. In other
embodiments, the skin condition is a cosmetic defect, a congenital
defect, hair loss or an acquired defect. In further embodiments,
the skin condition is fine lines and wrinkles; age spots and
dyspigmentation; decreased skin texture, tone and elasticity;
roughness and photo damage; decreased ability of skin to regenerate
itself; environmental damage; decreased smoothness and tightness of
skin; age spots; fine and coarse lines and wrinkles; fine and
coarse periocular wrinkles; nasolabial folds; facial fine and
coarse lines; decreased skin radiance and evenness; decreased skin
firmness; hyperpigmentation; dark spots and/or patches; decreased
skin brightness and youthful appearance; photoaged skin;
intrinsically and extrinsically aged skin; abnormal skin cellular
turnover; decreased skin barrier; decrease of skin's ability to
retain moisture; brown and red blotchiness; redness; abnormal skin
epidermal thickness; reduction of dermal epidermal junction;
increased pore size and number of pores; or a combination thereof.
In one embodiment, the composition rejuvenates sun damaged and
aging skin; improves the appearance of fine lines and wrinkles;
promotes cell renewal; diminishes the appearance of age spots and
dyspigmentation; improves skin tone, texture and elasticity;
reduces roughness and photo damage; prevents or reduces
environmental damage; plumps the skin; brightens the skin; lightens
the skin; strengthens the ability of skin to regenerate itself;
improves the appearance of age spots; brightens and lightens age
spots; improves skin firmness, elasticity, resiliency; smoothes,
tightens, or fills in fine lines on the skin; reduces the
appearance of dark circles under the eye; improves lip texture or
condition; enhances natural lip color; increases lip volume;
promotes epithelialization of post-procedure skin; restores the
skin's barrier or moisture balance; improves the appearance of age
spots; improves the appearance of skin pigmentation, or a
combination thereof. In one embodiment, the compositions reduce the
appearance of fine lines and wrinkles; diminish the appearance of
age spots and dyspigmentation; improve skin texture, tone and
elasticity; reduce roughness and photo damage; strengthen the
ability of skin to regenerate itself; prevent or reduce
environmental damage; smooth and tightens skin; brighten and
lighten age spots; reduce fine and coarse lines and wrinkles;
improve appearance of fine and coarse periocular wrinkles; improve
appearance of nasolabial folds; improve perioral wrinkles; improve
facial fine and coarse lines; improve skin tone, radiance and
evenness; improve skin firmness; reduce tactile roughness; improve
skin texture, overall photodamage, overall hyperpigmentation;
global improvement; reduction in appearance of dark spots and/or
patches; improve appearance of skin brightness and youthful
appearance; improve overall condition of skin; improve the
appearance of photoaged skin; improve appearance of instrincally
and extrinsically aged skin; improve skin cellular turnover;
improve skin barrier; improve skin's ability to retain moisture;
reduce the appearance of brown and red blotchiness, redness;
increase skin epidermal thickness; strengthen dermal epidermal
junction; reduce the appearance of pore size and pores; improve
smoothness, or a combination thereof.
[0005] In certain embodiments, administration of a composition
described herein may result in at least a 2-fold improvement of one
or more symptoms or conditions. Folds improvement of one or more
symptoms or conditions include, but are not limited to, 3-fold,
5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 75-fold, 100-fold or
more, or any number therebetween. In certain embodiments,
administration of a composition described herein may result in
improvement of about 1% to about 100%, about 10% to about 90%,
about 20% to about 80%, about 30% to about 70%, about 40% to about
60%, or about 50%. In other embodiments, administration of a
composition described herein may result in improvement of one or
more symptoms or conditions of about 5%, about 10%, about 15%,
about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 100%, about 125%, about 150%
or more of one or more symptoms or conditions.
[0006] In other embodiments, the disclosure also provides a
composition comprising metabolized cell extract and an acceptable
carrier. In some embodiments, the metabolized cell extract is cell
extract metabolized by yeast cells. In some embodiments, the cell
extract is derived from animal cells, skin cells or fibroblasts. In
other embodiments, the composition is a topical or injectable
composition. In some embodiments, the composition is used to treat
or prevent a skin condition. In other embodiments, the skin
condition is a cosmetic defect, a congenital defect, hair loss or
an acquired defect. In further embodiments, the skin condition is
fine lines and wrinkles; age spots and dyspigmentation; decreased
skin texture, tone and elasticity; roughness and photo damage;
decreased ability of skin to regenerate itself; environmental
damage; decreased smoothness and tightness of skin; age spots; fine
and coarse lines and wrinkles; fine and coarse periocular wrinkles;
nasolabial folds; facial fine and coarse lines; decreased skin
radiance and evenness; decreased skin firmness; hyperpigmentation;
dark spots and/or patches; decreased skin brightness and youthful
appearance; photoaged skin; intrinsically and extrinsically aged
skin; abnormal skin cellular turnover; decreased skin barrier;
decrease of skin's ability to retain moisture; brown and red
blotchiness; redness; abnormal skin epidermal thickness; reduction
of dermal epidermal junction; increased pore size and number of
pores; or a combination thereof.
[0007] In one embodiment, the disclosure provides metabolized
conditioned growth medium prepared by a process comprising: (a)
culturing cells in a growth medium sufficient to meet the
nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells; (b) culturing yeast cells; (c)
exposing the yeast cells to the conditioned growth medium; (d)
culturing the yeast cells to metabolize at least a portion of the
conditioned growth medium; and (e) collecting the metabolized
conditioned growth medium.
[0008] In another embodiment, the disclosure provides processes for
preparing metabolized conditioned growth medium comprising: (a)
culturing cells in a growth medium sufficient to meet the
nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells; (b) culturing yeast cells; (c)
exposing the yeast cells to the conditioned growth medium; (d)
culturing the yeast cells to metabolize at least a portion of the
conditioned growth medium; and (e) collecting the metabolized
conditioned growth medium.
[0009] In another embodiment, the disclosure provides compositions
comprising metabolized conditioned growth medium, and an acceptable
carrier. In another embodiment, the disclosure provides
compositions comprising metabolized conditioned growth medium, and
an acceptable carrier, wherein the topical composition is used to
treat or prevent a skin condition. In some embodiments, the
composition is used to treat or prevent a skin condition. In some
embodiments, the skin condition is a cosmetic defect, a congenital
defect, hair loss or an acquired defect. In further embodiments,
the skin condition is fine lines and wrinkles; age spots and
dyspigmentation; decreased skin texture, tone and elasticity;
roughness and photo damage; decreased ability of skin to regenerate
itself; environmental damage; decreased smoothness and tightness of
skin; age spots; fine and coarse lines and wrinkles; fine and
coarse periocular wrinkles; nasolabial folds; facial fine and
coarse lines; decreased skin radiance and evenness; decreased skin
firmness; hyperpigmentation; dark spots and/or patches; decreased
skin brightness and youthful appearance; photoaged skin;
intrinsically and extrinsically aged skin; abnormal skin cellular
turnover; decreased skin barrier; decrease of skin's ability to
retain moisture; brown and red blotchiness; redness; abnormal skin
epidermal thickness; reduction of dermal epidermal junction;
increased pore size and number of pores; or a combination
thereof.
[0010] In another embodiment, the disclosure provides compositions
comprising metabolized conditioned growth medium, and an acceptable
carrier, wherein the topical composition is used to treat or
prevent a skin condition, the metabolized conditioned growth medium
is present in an amount of about 0.01% to about 50% by weight of
the composition, and the metabolized conditioned growth medium is
prepared by culturing yeast cells in a growth medium sufficient to
meet the nutritional needs required to grow the cells in vitro to
form a conditioned growth medium
[0011] In another embodiment, the disclosure provides methods for
preventing or treating a skin condition in a subject comprising
administering to the subject a therapeutically effective amount of
metabolized conditioned growth medium. In some embodiments, the
composition is used to treat or prevent a skin condition. In other
embodiments, the skin condition is a cosmetic defect, a congenital
defect, hair loss or an acquired defect. In further embodiments,
the skin condition is fine lines and wrinkles; age spots and
dyspigmentation; decreased skin texture, tone and elasticity;
roughness and photo damage; decreased ability of skin to regenerate
itself; environmental damage; decreased smoothness and tightness of
skin; age spots; fine and coarse lines and wrinkles; fine and
coarse periocular wrinkles; nasolabial folds; facial fine and
coarse lines; decreased skin radiance and evenness; decreased skin
firmness; hyperpigmentation; dark spots and/or patches; decreased
skin brightness and youthful appearance; photoaged skin;
intrinsically and extrinsically aged skin; abnormal skin cellular
turnover; decreased skin barrier; decrease of skin's ability to
retain moisture; brown and red blotchiness; redness; abnormal skin
epidermal thickness; reduction of dermal epidermal junction;
increased pore size and number of pores; or a combination
thereof.
[0012] In one embodiment, the disclosure provides methods for
treating hair loss comprising administering to a subject a
therapeutically effective amount of metabolized conditioned growth
medium. In another embodiment, the disclosure provides methods for
stimulating hair growth comprising administering to a subject a
therapeutically effective amount of metabolized conditioned growth
medium. In another embodiment, the disclosure provides methods for
preventing or treating a congenital defect in a subject comprising
administering to the subject a therapeutically effective amount of
metabolized conditioned growth medium. In another embodiment, the
disclosure provides methods for preventing or treating a cosmetic
defect in a subject comprising administering to the subject a
therapeutically effective amount of metabolized conditioned growth
medium. In one embodiment, treatment of the cosmetic defect
rejuvenates sun damaged and aging skin; improves the appearance of
fine lines and wrinkles; promotes cell renewal; diminishes the
appearance of age spots and dyspigmentation; improves skin tone,
texture and elasticity; reduces roughness and photo damage;
prevents or reduces environmental damage; plumps the skin;
brightens the skin; lightens the skin; strengthens the ability of
skin to regenerate itself; improves the appearance of age spots;
brightens and lightens age spots; improves skin firmness,
elasticity, resiliency; smoothes, tightens, or fills in fine lines
on the skin; reduces the appearance of dark circles under the eye;
improves lip texture or condition; enhances natural lip color;
increases lip volume; promotes epithelialization of post-procedure
skin; restores the skin's barrier or moisture balance; improves the
appearance of age spots; improves the appearance of skin
pigmentation, or a combination thereof. In another embodiment, the
disclosure provides methods for preventing or treating an acquired
defect in a subject comprising administering to the subject a
therapeutically effective amount of metabolized conditioned growth
medium. Said methods may rejuvenate sun damaged and aging skin;
improve the appearance of fine lines and wrinkles; promote cell
renewal; improve skin tone, texture and/or firmness; plump the
skin; brightens the skin; lighten the skin; strengthen the skin's
ability to regenerate itself; improve the appearance of age spots;
improve skin firmness, elasticity, resiliency; smooth, tighten, or
fill in fine lines on the skin; reduce the appearance of dark
circles under the eye; improve lip texture or condition; enhance
natural lip color; increases lip volume; promote epithelialization
of post-procedure skin; restore the skin's barrier or moisture
balance; improve the appearance of age spots; improve the
appearance of skin pigmentation, reduce the appearance of fine
lines and wrinkles; diminish the appearance of age spots and
dyspigmentation; improve skin texture, tone and elasticity; reduce
roughness and photo damage; strengthen the ability of skin to
regenerate itself; prevent or reduce environmental damage; smooth
and tightens skin; brighten and lighten age spots, reduce in fine
and coarse lines and wrinkles, improve appearance of fine and
coarse periocular wrinkles, improve appearance of nasolabial folds,
improve perioral wrinkles, improve facial fine and coarse lines,
improve skin tone, radiance and evenness, improve skin firmness,
reduce tactile roughness, improve skin texture, overall
photodamage, overall hyperpigmentation, global improvement, reduce
in appearance of dark spots and/or patches, improve appearance of
skin brightness and youthful appearance, improve overall condition
of skin, improve the appearance of photoaged skin, improve
appearance of instrincally and extrinsically aged skin, improve
skin cellular turnover, improve skin barrier, improve skin's
ability to retain moisture, reduce the appearance of brown and red
blotchiness, redness, increase skin epidermal thickness, strengthen
dermal epidermal junction, reduce the appearance of pore size and
pores, improve smoothness, or a combination thereof.
[0013] In another embodiment, the disclosure provides methods for
reducing the appearance of fine lines and wrinkles comprising
administering to a subject a therapeutically effective amount of
metabolized conditioned growth medium. In another embodiment, the
disclosure provides methods for diminishing the appearance of age
spots and dyspigmentation comprising administering to a subject a
therapeutically effective amount of metabolized conditioned growth
medium. In another embodiment, the disclosure provides methods for
improving skin texture, tone and elasticity comprising
administering to a subject a therapeutically effective amount of
metabolized conditioned growth medium. In another embodiment, the
disclosure provides methods for reducing roughness and photo damage
comprising administering to a subject a therapeutically effective
amount of metabolized conditioned growth medium. In another
embodiment, the disclosure provides methods for strengthening the
ability of skin to regenerate itself comprising administering to a
subject a therapeutically effective amount of metabolized
conditioned growth medium. In another embodiment, the disclosure
provides methods for preventing or reducing environmental damage
comprising administering to a subject a therapeutically effective
amount of metabolized conditioned growth medium. In another
embodiment, the disclosure provides methods for smoothing and
tightening skin comprising administering to a subject a
therapeutically effective amount of metabolized conditioned growth
medium. In another embodiment, the disclosure provides methods for
brightening and lightening age spots comprising administering to a
subject a therapeutically effective amount of metabolized
conditioned growth medium.
[0014] In another embodiment, the disclosure provides metabolized
cell extract prepared by a process comprising: (a) providing a cell
extract; (b) culturing yeast cells; (c) exposing the yeast cells to
the cell extract; (d) culturing the yeast cells to metabolize at
least a portion of the cell extract; and (e) collecting the
metabolized conditioned cell extract.
[0015] In another embodiment, the disclosure provides processes for
preparing the metabolized cell extract comprising: (a) providing a
cell extract; (b) culturing yeast cells; (c) exposing the yeast
cells to the cell extract; (d) culturing the yeast cells to
metabolize at least a portion of the cell extract; and (e)
collecting the metabolized cell extract.
[0016] In another embodiment, the disclosure provides compositions
comprising metabolized cell extract, and an acceptable carrier. In
another embodiment, the disclosure provides compositions comprising
metabolized cell extract, and an acceptable carrier, wherein the
topical composition is used to treat or prevent a skin condition.
In some embodiments, the composition is used to treat or prevent a
skin condition. In other embodiments, the skin condition is a
cosmetic defect, a congenital defect, hair loss or an acquired
defect. In further embodiments, the skin condition is fine lines
and wrinkles; age spots and dyspigmentation; decreased skin
texture, tone and elasticity; roughness and photo damage; decreased
ability of skin to regenerate itself; environmental damage;
decreased smoothness and tightness of skin; age spots; fine and
coarse lines and wrinkles; fine and coarse periocular wrinkles;
nasolabial folds; facial fine and coarse lines; decreased skin
radiance and evenness; decreased skin firmness; hyperpigmentation;
dark spots and/or patches; decreased skin brightness and youthful
appearance; photoaged skin; intrinsically and extrinsically aged
skin; abnormal skin cellular turnover; decreased skin barrier;
decrease of skin's ability to retain moisture; brown and red
blotchiness; redness; abnormal skin epidermal thickness; reduction
of dermal epidermal junction; increased pore size and number of
pores; or a combination thereof.
[0017] In another embodiment, the disclosure provides compositions
comprising metabolized cell extract, and an acceptable carrier,
wherein the topical composition is used to treat or prevent skin
condition, the metabolized cell extract is present in an amount of
about 0.01% to about 50% by weight of the composition, and the
metabolized cell extract is cell extract metabolized by yeast
cells. In other embodiments, the skin condition is a cosmetic
defect, a congenital defect, hair loss or an acquired defect. In
further embodiments, the skin condition is fine lines and wrinkles;
age spots and dyspigmentation; decreased skin texture, tone and
elasticity; roughness and photo damage; decreased ability of skin
to regenerate itself; environmental damage; decreased smoothness
and tightness of skin; age spots; fine and coarse lines and
wrinkles; fine and coarse periocular wrinkles; nasolabial folds;
facial fine and coarse lines; decreased skin radiance and evenness;
decreased skin firmness; hyperpigmentation; dark spots and/or
patches; decreased skin brightness and youthful appearance;
photoaged skin; intrinsically and extrinsically aged skin; abnormal
skin cellular turnover; decreased skin barrier; decrease of skin's
ability to retain moisture; brown and red blotchiness; redness;
abnormal skin epidermal thickness; reduction of dermal epidermal
junction; increased pore size and number of pores; or a combination
thereof. In one embodiment, treatment of the cosmetic defect
rejuvenates sun damaged and aging skin; improves the appearance of
fine lines and wrinkles; promotes cell renewal; diminishes the
appearance of age spots and dyspigmentation; improves skin tone,
texture and elasticity; reduces roughness and photo damage;
prevents or reduces environmental damage; plumps the skin;
brightens the skin; lightens the skin; strengthens the ability of
skin to regenerate itself; improves the appearance of age spots;
brightens and lightens age spots; improves skin firmness,
elasticity, resiliency; smoothes, tightens, or fills in fine lines
on the skin; reduces the appearance of dark circles under the eye;
improves lip texture or condition; enhances natural lip color;
increases lip volume; promotes epithelialization of post-procedure
skin; restores the skin's barrier or moisture balance; improves the
appearance of age spots; improves the appearance of skin
pigmentation, or a combination thereof.
[0018] In another embodiment, the disclosure provides methods for
preventing or treating a skin condition in a subject comprising
administering to the subject a therapeutically effective amount of
metabolized cell extract. In another embodiment, the disclosure
provides methods for treating hair loss comprising administering to
a subject a therapeutically effective amount of metabolized cell
extract. In another embodiment, the disclosure provides methods for
stimulating hair growth comprising administering to a subject a
therapeutically effective amount of metabolized cell extract. In
another embodiment, the disclosure provides methods for preventing
or treating a congenital defect in a subject comprising
administering to the subject a therapeutically effective amount of
metabolized cell extract. In another embodiment, the disclosure
provides methods for preventing or treating a cosmetic defect in a
subject comprising administering to the subject a therapeutically
effective amount of metabolized cell extract. In one embodiment,
treatment of the cosmetic defect rejuvenates sun damaged and aging
skin; improves the appearance of fine lines and wrinkles; promotes
cell renewal; diminishes the appearance of age spots and
dyspigmentation; improves skin tone, texture and elasticity;
reduces roughness and photo damage; prevents or reduces
environmental damage; plumps the skin; brightens the skin; lightens
the skin; strengthens the ability of skin to regenerate itself;
improves the appearance of age spots; brightens and lightens age
spots; improves skin firmness, elasticity, resiliency; smoothes,
tightens, or fills in fine lines on the skin; reduces the
appearance of dark circles under the eye; improves lip texture or
condition; enhances natural lip color; increases lip volume;
promotes epithelialization of post-procedure skin; restores the
skin's barrier or moisture balance; improves the appearance of age
spots; improves the appearance of skin pigmentation, or a
combination thereof. In another embodiment, the disclosure provides
methods for preventing or treating a acquired defect in a subject
comprising administering to the subject a therapeutically effective
amount of metabolized cell extract. Said methods may rejuvenate sun
damaged and aging skin; improve the appearance of fine lines and
wrinkles; promote cell renewal; improve skin tone, texture and/or
firmness; plump the skin; brightens the skin; lighten the skin;
strengthen the skin's ability to regenerate itself; improve the
appearance of age spots; improve skin firmness, elasticity,
resiliency; smooth, tighten, or fill in fine lines on the skin;
reduce the appearance of dark circles under the eye; improve lip
texture or condition; enhance natural lip color; increases lip
volume; promote epithelialization of post-procedure skin; restore
the skin's barrier or moisture balance; improve the appearance of
age spots; improve the appearance of skin pigmentation, reduce the
appearance of fine lines and wrinkles; diminish the appearance of
age spots and dyspigmentation; improve skin texture, tone and
elasticity; reduce roughness and photo damage; strengthen the
ability of skin to regenerate itself; prevent or reduce
environmental damage; smooth and tightens skin; brighten and
lighten age spots, reduce in fine and coarse lines and wrinkles,
improve appearance of fine and coarse periocular wrinkles, improve
appearance of nasolabial folds, improve perioral wrinkles, improve
facial fine and coarse lines, improve skin tone, radiance and
evenness, improve skin firmness, reduce tactile roughness, improve
skin texture, overall photodamage, overall hyperpigmentation,
global improvement, reduce in appearance of dark spots and/or
patches, improve appearance of skin brightness and youthful
appearance, improve overall condition of skin, improve the
appearance of photoaged skin, improve appearance of instrincally
and extrinsically aged skin, improve skin cellular turnover,
improve skin barrier, improve skin's ability to retain moisture,
reduce the appearance of brown and red blotchiness, redness,
increase skin epidermal thickness, strengthen dermal epidermal
junction, reduce the appearance of pore size and pores, improve
smoothness, or a combination thereof.
[0019] In another embodiment, the disclosure provides methods for
reducing the appearance of fine lines and wrinkles comprising
administering to a subject a therapeutically effective amount of
metabolized cell extract. In another embodiment, the disclosure
provides methods for diminishing the appearance of age spots and
dyspigmentation comprising administering to a subject a
therapeutically effective amount of metabolized cell extract. In
another embodiment, the disclosure provides methods for improving
skin texture, tone and elasticity comprising administering to a
subject a therapeutically effective amount of metabolized cell
extract. In another embodiment, the disclosure provides methods for
reducing roughness and photo damage comprising administering to a
subject a therapeutically effective amount of metabolized cell
extract. In another embodiment, the disclosure provides methods for
strengthening the ability of skin to regenerate itself comprising
administering to a subject a therapeutically effective amount of
metabolized cell extract. In another embodiment, the disclosure
provides methods for preventing or reducing environmental damage
comprising administering to a subject a therapeutically effective
amount of metabolized cell extract. In another embodiment, the
disclosure provides methods for smoothing and tightening skin
comprising administering to a subject a therapeutically effective
amount of metabolized cell extract. In another embodiment, the
disclosure provides methods for brightening and lightening age
spots comprising administering to a subject a therapeutically
effective amount of metabolized cell extract.
[0020] In one embodiment, also provided are compositions comprising
a metabolized conditioned growth medium and an acceptable carrier,
wherein the metabolized conditioned growth medium is conditioned
growth medium metabolized by yeast cells. In one embodiment, the
conditioned growth medium is prepared by culturing cells in a
growth medium sufficient to meet the nutritional needs required to
grow the cells in vitro to form a conditioned growth medium. In
another embodiment, the composition is an injectable composition or
a topical composition. In yet another embodiment, the topical
composition is an ointment, a cream, a hydrogel, or a lotion. In
still another embodiment, the metabolized condition growth medium
is encapsulated within an encapsulant. In one embodiment, the
encapsulant is at least one of liposomes, niosomes, sub-micron
emulsions, polymeric encapsulates, gels, creams and lotions. In
some embodiments, the composition is used to treat or prevent a
skin condition. In other embodiments, the composition is used to
treat or prevent fine lines and wrinkles; age spots and
dyspigmentation; decreased skin texture, tone and elasticity;
roughness and photo damage; decreased ability of skin to regenerate
itself; environmental damage; decreased smoothness and tightness of
skin; age spots; fine and coarse lines and wrinkles; fine and
coarse periocular wrinkles; nasolabial folds; facial fine and
coarse lines; decreased skin radiance and evenness; decreased skin
firmness; hyperpigmentation; dark spots and/or patches; decreased
skin brightness and youthful appearance; photoaged skin;
intrinsically and extrinsically aged skin; abnormal skin cellular
turnover; decreased skin barrier; decrease of skin's ability to
retain moisture; brown and red blotchiness; redness; abnormal skin
epidermal thickness; reduction of dermal epidermal junction;
increased pore size and number of pores; or a combination thereof.
In still other embodiments, the composition is used to treat or
prevent a cosmetic defect, a congenital defect, hair loss or an
acquired defect. In some embodiments, the cosmetic defect is a
glabellar frown line, deep nasolabial crease, circum-oral
geographical wrinkle, sunken cheeks or mammary hypoplasia. In other
embodiments, treatment of the cosmetic defect rejuvenates sun
damaged and aging skin; improves the appearance of fine lines and
wrinkles; promotes cell renewal; diminishes the appearance of age
spots and dyspigmentation; improves skin tone, texture and
elasticity; reduces roughness and photo damage; prevents or reduces
environmental damage; plumps the skin; brightens the skin; lightens
the skin; strengthens the ability of skin to regenerate itself;
improves the appearance of age spots; brightens and lightens age
spots; improves skin firmness, elasticity, resiliency; smoothes,
tightens, or fills in fine lines on the skin; reduces the
appearance of dark circles under the eye; improves lip texture or
condition; enhances natural lip color; increases lip volume;
promotes epithelialization of post-procedure skin; restores the
skin's barrier or moisture balance; improves the appearance of age
spots; improves the appearance of skin pigmentation, or a
combination thereof. In some embodiments, the acquired defect is a
medical condition that occurs post-trauma, post-surgery or
post-infection. In other embodiments, the acquired defect is a
post-medical procedure defect. In yet other embodiments, the
acquired defect is a depressed scar, subcutaneous atropy, a
keratotic lesion, enophthalmos in an unucleated eye, acne pitting
of the face, linear scleroderma with subcutaneous atrophy,
saddle-nose deformity, Romberg's disease or unilateral vocal cord
paralysis.
[0021] Also provided are compositions comprising a metabolized cell
extract and an acceptable carrier, wherein the metabolized cell
extract is cell extract metabolized by yeast cells. In some
embodiments, the cell extract is derived from animal cells. In
other embodiments, the cell extract is derived from skin cells. In
another embodiment, the cell extract is derived from fibroblasts.
In some embodiments, the composition is an injectable composition
or a topical composition. In some embodiments, the topical
composition is an ointment, a cream, a hydrogel, or a lotion. In
other embodiments, the metabolized condition growth medium is
encapsulated within an encapsulant. In one embodiment, the
encapsulant is at least one of liposomes, niosomes, sub-micron
emulsions, polymeric encapsulates, gels, creams and lotions. In
some embodiments, the composition is used to treat or prevent a
skin condition. In other embodiments, the skin condition is a
cosmetic defect, a congenital defect, hair loss or an acquired
defect. In further embodiments, the skin condition is fine lines
and wrinkles; age spots and dyspigmentation; decreased skin
texture, tone and elasticity; roughness and photo damage; decreased
ability of skin to regenerate itself; environmental damage;
decreased smoothness and tightness of skin; age spots; fine and
coarse lines and wrinkles; fine and coarse periocular wrinkles;
nasolabial folds; facial fine and coarse lines; decreased skin
radiance and evenness; decreased skin firmness; hyperpigmentation;
dark spots and/or patches; decreased skin brightness and youthful
appearance; photoaged skin; intrinsically and extrinsically aged
skin; abnormal skin cellular turnover; decreased skin barrier;
decrease of skin's ability to retain moisture; brown and red
blotchiness; redness; abnormal skin epidermal thickness; reduction
of dermal epidermal junction; increased pore size and number of
pores; or a combination thereof.
[0022] In yet other embodiments, the composition is used to treat
or prevent a cosmetic defect, a congenital defect, hair loss or an
acquired defect. In some embodiments, the cosmetic defect is a
glabellar frown line, deep nasolabial crease, circum-oral
geographical wrinkle, sunken cheeks or mammary hypoplasia. In some
embodiments, treatment of the cosmetic defect rejuvenates sun
damaged and aging skin; improves the appearance of fine lines and
wrinkles; promotes cell renewal; diminishes the appearance of age
spots and dyspigmentation; improves skin tone, texture and
elasticity; reduces roughness and photo damage; prevents or reduces
environmental damage; plumps the skin; brightens the skin; lightens
the skin; strengthens the ability of skin to regenerate itself;
improves the appearance of age spots; brightens and lightens age
spots; improves skin firmness, elasticity, resiliency; smoothes,
tightens, or fills in fine lines on the skin; reduces the
appearance of dark circles under the eye; improves lip texture or
condition; enhances natural lip color; increases lip volume;
promotes epithelialization of post-procedure skin; restores the
skin's barrier or moisture balance; improves the appearance of age
spots; improves the appearance of skin pigmentation, or a
combination thereof. In some embodiments, the acquired defect is a
medical condition that occurs post-trauma, post-surgery or
post-infection. In other embodiments, the acquired defect is a
post-medical procedure defect. In yet other embodiments, the
acquired defect is a depressed scar, subcutaneous atropy, a
keratotic lesion, enophthalmos in an unucleated eye, acne pitting
of the face, linear scleroderma with subcutaneous atrophy,
saddle-nose deformity, Romberg's disease or unilateral vocal cord
paralysis. In still other embodiments, the composition is used to
treat or prevent fine lines and wrinkles; age spots and
dyspigmentation; decreased skin texture, tone and elasticity;
roughness and photo damage; decreased ability of skin to regenerate
itself; environmental damage; decreased smoothness and tightness of
skin; age spots; fine and coarse lines and wrinkles; fine and
coarse periocular wrinkles; nasolabial folds; facial fine and
coarse lines; decreased skin radiance and evenness; decreased skin
firmness; hyperpigmentation; dark spots and/or patches; decreased
skin brightness and youthful appearance; photoaged skin;
intrinsically and extrinsically aged skin; abnormal skin cellular
turnover; decreased skin barrier; decrease of skin's ability to
retain moisture; brown and red blotchiness; redness; abnormal skin
epidermal thickness; reduction of dermal epidermal junction;
increased pore size and number of pores; or a combination
thereof.
[0023] Also provided herein is a metabolized conditioned growth
medium prepared by a process comprising:
[0024] (a) culturing cells in a growth medium sufficient to meet
the nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells;
[0025] (b) culturing yeast cells;
[0026] (c) exposing the yeast cells to the conditioned growth
medium;
[0027] (d) culturing the yeast cells to metabolize at least a
portion of the conditioned growth medium; and
[0028] (e) collecting the metabolized conditioned growth medium. In
some embodiments, the yeast used in the process above is of the
Pichia genus. In other embodiments, the yeast is Pichia pastoris.
In other embodiments, the cells in step (a) are skin cells. In yet
other embodiments, the cells in step (a) are fibroblasts. In still
other embodiments, the cells in step (a) are animal cells. In still
other embodiments, the process to prepare the metabolized
conditioned growth medium further comprises (e) processing the
metabolized conditioned growth medium, wherein processing is
concentrating, filtering, purifying, or a combination thereof. In
some embodiments, the metabolized conditioned growth medium
includes a continuous flow system or a bioreactor system in the
culturing step of (a) or (b). In yet other embodiments, the cells
are suspended or floated in the growth medium.
[0029] Provided herein is a process for preparing the metabolized
conditioned growth medium above, comprising:
[0030] (a) culturing cells in a growth medium sufficient to meet
the nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells;
[0031] (b) culturing yeast cells;
[0032] (c) exposing the yeast cells to the conditioned growth
medium;
[0033] (d) culturing the yeast cells to metabolize at least a
portion of the conditioned growth medium; and
[0034] (e) collecting the metabolized conditioned growth medium. In
one embodiment, a composition is provided comprising the
metabolized conditioned growth medium and an acceptable carrier. In
some embodiments, the metabolized conditioned growth medium is
present in an amount of about 0.0001% to about 95% by weight of the
composition. In other embodiments, the metabolized conditioned
growth medium is present in an amount of about 0.01% to about 50%
by weight of the composition. In some embodiments, the metabolized
conditioned growth medium is present in an amount of about 0.01% to
about 10% by weight of the composition. In some embodiments, the
metabolized conditioned growth medium is present in an amount of
about 10% to about 20% by weight of the composition. In some
embodiments, the metabolized conditioned growth medium is present
in an amount of about 0.01%, 0.5%, 1.0%, 5.0%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or
95% by weight of the composition. In one non-limiting example, the
metabolized conditioned growth medium is present in the composition
in an amount of about 10%, about 15%, or about 20% by weight of the
composition. In other embodiments, the composition further
comprises at least one of water, surfactants, emulsifiers,
conditioners, emollients, waxes, oils, polymers, thickeners,
fixatives, colorants, nutraceuticals, cosmeceuticals, therapeutics,
pharmaceuticals, antifungals, antimicrobials, steroidal hormones,
antidandruff agents, anti-acne components, sunscreens, and
preservatives. In some embodiments, the composition is an
injectable composition or a topical composition. In one embodiment,
the topical composition is an ointment, a cream, a hydrogel, or a
lotion. In other embodiments, the metabolized conditioned growth
medium is encapsulated within an encapsulant. In some embodiments,
the encapsulant is at least one of liposomes, niosomes, sub-micron
emulsions, polymeric encapsulates, gels, creams, and lotions.
[0035] Also provided herein is a metabolized cell extract prepared
by a process comprising:
[0036] (a) providing a cell extract;
[0037] (b) culturing yeast cells;
[0038] (c) exposing the yeast cells to the cell extract;
[0039] (d) culturing the yeast cells to metabolize at least a
portion of the cell extract; and
[0040] (e) collecting the metabolized cell extract. In some
embodiments, the yeast is of the Pichia genus. In other
embodiments, the yeast is Pichia pastoris. In yet other
embodiments, the cell extract is derived from skin cells. In still
other embodiments, the cell extract is derived from fibroblasts. In
still other embodiments, the cell extract is derived from animal
cells. In yet other embodiments, the process further comprises (e)
processing the metabolized cell extract, wherein processing is
concentrating, filtering, purifying, or a combination thereof. In
one embodiment, the culturing comprises a continuous flow system or
a bioreactor system.
[0041] In still other embodiments, a process for preparing the
metabolized cell extract above comprises:
[0042] (a) providing a cell extract;
[0043] (b) culturing yeast cells;
[0044] (c) exposing the yeast cells to the cell extract;
[0045] (d) culturing the yeast cells to metabolize at least a
portion of the cell extract; and
[0046] (e) collecting the metabolized cell extract.
[0047] Also provided herein are compositions comprising: an
acceptable carrier and a metabolized conditioned growth medium
prepared by a process comprising:
[0048] (a) culturing cells in a growth medium sufficient to meet
the nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells;
[0049] (b) culturing yeast cells;
[0050] (c) exposing the yeast cells to the conditioned growth
medium;
[0051] (d) culturing the yeast cells to metabolize at least a
portion of the conditioned growth medium; and
[0052] (e) collecting the metabolized conditioned growth medium. In
some embodiments, the yeast is of the Pichia genus. In other
embodiments, the yeast is Pichia pastoris. In some embodiments, the
cells in step (a) are skin cells. In other embodiments, the cells
in step (a) are fibroblasts. In yet another embodiment, the cells
in step (a) are animal cells. In some embodiments, the process
further comprises (e) processing the metabolized conditioned growth
medium, wherein processing is concentrating, filtering, purifying,
or a combination thereof. In some embodiments, the culturing of
step (a) or (b) comprises a continuous flow system or a bioreactor
system. In yet other embodiments, the cells are suspended or
floated in the growth medium. In still other embodiments, the
metabolized conditioned growth medium is present in an amount of
about 0.0001% to about 95% by weight of the composition. In still
other embodiments, the metabolized conditioned growth medium is
present in an amount of about 0.01% to about 50% by weight of the
composition. In yet other embodiments, the metabolized conditioned
growth medium is present in an amount of about 0.01% to about 10%
by weight of the composition. In some embodiments, the metabolized
conditioned growth medium is present in an amount of about 0.01%,
0.5%, 1.0%, 5.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% by weight of the
composition. In one non-limiting example, the metabolized
conditioned growth medium is present in the composition in an
amount of about 10%, about 15%, or about 20% by weight of the
composition. In still other embodiments, the composition further
comprises at least one of water, surfactants, emulsifiers,
conditioners, emollients, waxes, oils, polymers, thickeners,
fixatives, colorants, nutraceuticals, cosmeceuticals, therapeutics,
pharmaceuticals, antifungals, antimicrobials, steroidal hormones,
antidandruff agents, anti-acne components, sunscreens, and
preservatives. In still other embodiments, the composition is an
injectable composition or a topical composition. In yet other
embodiments, the topical composition is an ointment, a cream, a
hydrogel, or a lotion. In some embodiments, the metabolized
conditioned growth medium is encapsulated within an encapsulant. In
one embodiment, the encapsulant is at least one of liposomes,
niosomes, sub-micron emulsions, polymeric encapsulates, gels,
creams, and lotions.
[0053] Also provided herein are compositions comprising: an
acceptable carrier and a metabolized cell extract prepared by a
process comprising:
[0054] (a) providing a cell extract;
[0055] (b) culturing yeast cells;
[0056] (c) exposing the yeast cells to the cell extract;
[0057] (d) culturing the yeast cells to metabolize at least a
portion of the cell extract; and
[0058] (e) collecting the metabolized cell extract. In some
embodiments, the yeast is of the Pichia genus. In other
embodiments, the yeast is Pichia pastoris. In other embodiments,
the cell extract is derived from skin cells. In yet other
embodiments, the cell extract is derived from fibroblasts. In still
other embodiments, the cell extract is derived from animal cells.
In one embodiment, the process further comprises (e) processing the
metabolized cell extract, wherein processing is concentrating,
filtering, purifying, or a combination thereof. In another
embodiment, the culturing comprises a continuous flow system or a
bioreactor system. In still other embodiments, the metabolized
conditioned growth medium is present in an amount of about 0.0001%
to about 95% by weight of the composition. In yet another
embodiment, the metabolized cell extract is present in an amount of
about 0.01% to about 50% by weight of the composition. In still
other embodiments, the metabolized cell extract is present in an
amount of about 0.01% to about 10% by weight of the composition. In
some embodiments, the metabolized cell extract is present in an
amount of about 10% to about 20% by weight of the composition. In
some embodiments, the metabolized cell extract is present in an
amount of about 0.01%, 0.5%, 1.0%, 5.0%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%
by weight of the composition. In one non-limiting example, the
metabolized cell extract is present in the composition in an amount
of about 10%, about 15%, or about 20% by weight of the composition.
In one embodiment, the composition further comprises at least one
of water, surfactants, emulsifiers, conditioners, emollients,
waxes, oils, polymers, thickeners, fixatives, colorants,
nutraceuticals, cosmeceuticals, therapeutics, pharmaceuticals,
antifungals, antimicrobials, steroidal hormones, antidandruff
agents, anti-acne components, sunscreens, and preservatives. In
still other embodiments, the composition is an injectable
composition or a topical composition. In yet another embodiment,
the topical composition is an ointment, a cream, a hydrogel, or a
lotion. In one embodiment, the metabolized conditioned growth
medium is encapsulated within an encapsulant. In another
embodiment, the encapsulant is at least one of liposomes, niosomes,
sub-micron emulsions, polymeric encapsulates, gels, creams, and
lotions.
[0059] Also provided herein are methods for preventing or treating
a skin condition in a subject comprising administering to the
subject a therapeutically effective amount of a composition
comprising: an acceptable carrier and a metabolized conditioned
growth medium prepared by a process comprising:
[0060] (a) culturing cells in a growth medium sufficient to meet
the nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells;
[0061] (b) culturing yeast cells;
[0062] (c) exposing the yeast cells to the conditioned growth
medium;
[0063] (d) culturing the yeast cells to metabolize at least a
portion of the conditioned growth medium; and
[0064] (e) collecting the metabolized conditioned growth medium. In
one embodiment, the skin condition is a cosmetic defect. In another
embodiment, the condition is hair loss. In another embodiment, the
condition is a congenital defect or an acquired defect. In one
embodiment, the acquired defect is a medical condition that occurs
post-trauma, post-surgery or post-infection. In another embodiment,
the acquired defect is a post-medical procedure defect. In yet
another embodiment, the acquired defect is a depressed scar,
subcutaneous atrophy, a keratotic lesion, enophthalmos in an
unucleated eye, acne pitting of the face, linear scleroderma with
subcutaneous atrophy, saddle-nose deformity, Romberg's disease or
unilateral vocal cord paralysis. In one embodiment, the cosmetic
defect is a glabellar frown line, deep nasolabial crease,
circum-oral geographical wrinkle, sunken cheeks or mammary
hypoplasia. In certain embodiments, the condition is fine lines and
wrinkles; age spots and dyspigmentation; decreased skin texture,
tone and elasticity; roughness and photo damage; decreased ability
of skin to regenerate itself; environmental damage; decreased
smoothness and tightness of skin; age spots; fine and coarse lines
and wrinkles; fine and coarse periocular wrinkles; nasolabial
folds; facial fine and coarse lines; decreased skin radiance and
evenness; decreased skin firmness; hyperpigmentation; dark spots
and/or patches; decreased skin brightness and youthful appearance;
photoaged skin; intrinsically and extrinsically aged skin; abnormal
skin cellular turnover; decreased skin barrier; decrease of skin's
ability to retain moisture; brown and red blotchiness; redness;
abnormal skin epidermal thickness; reduction of dermal epidermal
junction; increased pore size and number of pores; or a combination
thereof. In certain embodiments, administration of a composition
described herein may result in at least a 2-fold improvement of one
or more symptoms or conditions. Folds improvement of one or more
symptoms or conditions include, but are not limited to, 3-fold,
5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 75-fold, 100-fold or
more, or any number therebetween. In certain embodiments,
administration of a composition described herein may result in
improvement of about 1% to about 100%, about 10% to about 90%,
about 20% to about 80%, about 30% to about 70%, about 40% to about
60%, or about 50%. In other embodiments, administration of a
composition described herein may result in improvement of one or
more symptoms or conditions of about 5%, about 10%, about 15%,
about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 100%, about 125%, about 150%
or more of one or more symptoms or conditions. In some embodiments,
the yeast is of the Pichia genus. In some embodiments, the yeast is
Pichia pastoris. In one embodiment, the cells in step (a) are skin
cells. In another embodiment, the cells in step (a) are
fibroblasts. In some embodiments, the cells in step (a) are animal
cells. In other embodiments, the process further comprises (e)
processing the metabolized conditioned growth medium, wherein
processing is concentrating, filtering, purifying, or a combination
thereof. In one embodiment, the culturing of step (a) or (b)
comprises a continuous flow system or a bioreactor system. In
another embodiment, the cells are suspended or floated in the
growth medium. In still other embodiments, the metabolized
conditioned growth medium is present in the composition in an
amount of about 0.0001% to about 95% by weight of the composition.
In another embodiment, the metabolized conditioned growth medium is
present in the composition in an amount of about 0.01% to about 50%
by weight of the composition. In one embodiment, the metabolized
conditioned growth medium is present in the composition in an
amount of about 0.01% to about 10% by weight of the composition. In
some embodiments, the metabolized conditioned growth medium is
present in an amount of about 10% to about 20% by weight of the
composition. In some embodiments, the metabolized conditioned
growth medium is present in an amount of about 0.01%, 0.5%, 1.0%,
5.0%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90% or 95% by weight of the composition. In one
non-limiting example, the metabolized conditioned growth medium is
present in the composition in an amount of about 10%, about 15%, or
about 20% by weight of the composition. In some embodiments, the
composition further comprises at least one of water, surfactants,
emulsifiers, conditioners, emollients, waxes, oils, polymers,
thickeners, fixatives, colorants, nutraceuticals, cosmeceuticals,
therapeutics, pharmaceuticals, antifungals, antimicrobials,
steroidal hormones, antidandruff agents, anti-acne components,
sunscreens, and preservatives. In one embodiment, the composition
is an injectable composition or a topical composition. In another
embodiment, the topical composition is an ointment, a cream, a
hydrogel, or a lotion. In one embodiment, the metabolized
conditioned growth medium is encapsulated within an encapsulant. In
some embodiments, the encapsulant is at least one of liposomes,
niosomes, sub-micron emulsions, polymeric encapsulates, gels,
creams, and lotions. In some embodiments, at least one
extracellular matrix protein is up-regulated by the administration
of the composition to the subject. In some embodiments, the
extracellular matrix protein is up-regulated by about 5% to about
100%. In one embodiment, the extracellular matrix protein is
up-regulated by about 10% to about 50%. In another embodiment, the
extracellular matrix protein is up-regulated by about 60% to about
100%. In yet another embodiment, the extracellular matrix protein
is up-regulated by about 5%, by about 10%, by about 15%, by about
20%, by about 25%, by about 30%, by about 35%, by about 40%, by
about 45%, by about 50%, by about 55%, by about 60%, by about 65%,
by about 70%, by about 75%, by about 80%, by about 85%, by about
90%, by about 95% or by about 100%. In one embodiment, the
extracellular matrix protein is a collagen protein or a lysyl
hydroxylase protein. In some embodiments, the extracellular matrix
protein is encoded by COL4A1 gene or PLOD1 gene. In other
embodiments, at least one repair protein is up-regulated by the
administration of the composition to the subject. In one
embodiment, the repair protein is up-regulated by about 10% to
about 70%. In another embodiment, the repair protein is
up-regulated by about 25% to about 50%. In some embodiments, the
repair-protein is up-regulated by about 5%, by about 10%, by about
15%, by about 20%, by about 25%, by about 30%, by about 35%, by
about 40%, by about 45%, by about 50%, by about 55%, by about 60%,
by about 65%, by about 70%, by about 75%, by about 80%, by about
85%, by about 90%, by about 95% or by about 100%. In one
embodiment, the repair protein is a fibronectin protein. In yet
another embodiment, the repair protein is encoded by FN1 gene. In
some embodiments, at least one cellular connectivity protein is
up-regulated by the administration of the composition to the
subject. In one embodiment, the cellular connectivity protein is
up-regulated by about 5% to about 200%. In one embodiment, the
cellular connectivity protein is up-regulated by about 10% to about
80%. In some embodiments, the cellular connectivity protein is
up-regulated by about 30% to about 50%. In one embodiment, the
cellular connectivity protein is up-regulated by about 5%, by about
10%, by about 15%, by about 20%, by about 25%, by about 30%, by
about 35%, by about 40%, by about 45%, by about 50%, by about 55%,
by about 60%, by about 65%, by about 70%, by about 75%, by about
80%, by about 85%, by about 90%, by about 95%, by about 100%, by
about 110%, by about 120%, by about 130%, by about 140%, by about
150%, by about 160%, by about 170%, by about 180%, by about 190% or
by about 200%. In other embodiments, the cellular connectivity
protein is involucrin protein. In some embodiments, the cellular
connectivity protein is encoded by IVL gene. In one embodiment, at
least one antioxidant protein is up-regulated by the administration
of the composition to the subject. In yet another embodiment, the
antioxidant protein is up-regulated by about 5% to about 300%. In
some embodiments, the antioxidant protein is up-regulated by about
10% to about 50%. In other embodiments, the antioxidant protein is
up-regulated by about 200% to about 250%. In some embodiments, the
antioxidant protein is up-regulated by about 5%, by about 10%, by
about 15%, by about 20%, by about 25%, by about 30%, by about 35%,
by about 40%, by about 45%, by about 50%, by about 55%, by about
60%, by about 65%, by about 70%, by about 75%, by about 80%, by
about 85%, by about 90%, by about 95%, by about 100%, by about
125%, by about 150%, by about 175%, by about 200%, by about 250% or
by about 300%. In one embodiment, the antioxidant protein is a
superoxide dismutase protein. In another embodiment, the
antioxidant protein is encoded by SOD2 gene.
[0065] Also provided herein are methods for preventing or treating
a skin condition in a subject comprising administering to the
subject a therapeutically effective amount of a composition
comprising: an acceptable carrier and a metabolized cell extract
prepared by a process comprising:
[0066] (a) providing a cell extract;
[0067] (b) culturing yeast cells;
[0068] (c) exposing the yeast cells to the cell extract;
[0069] (d) culturing the yeast cells to metabolize at least a
portion of the cell extract; and
[0070] (e) collecting the metabolized cell extract.
[0071] In one embodiment, the condition is a cosmetic defect. In
another embodiment, the condition is hair loss. In another
embodiment, the condition is a congenital defect or an acquired
defect. In one embodiment, the acquired defect is a medical
condition that occurs post-trauma, post-surgery or post-infection.
In another embodiment, the acquired defect is a post-medical
procedure defect. In yet another embodiment, the acquired defect is
a depressed scar, subcutaneous atrophy, a keratotic lesion,
enophthalmos in an unucleated eye, acne pitting of the face, linear
scleroderma with subcutaneous atrophy, saddle-nose deformity,
Romberg's disease or unilateral vocal cord paralysis. In one
embodiment, the cosmetic defect is a glabellar frown line, deep
nasolabial crease, circum-oral geographical wrinkle, sunken cheeks
or mammary hypoplasia. In certain embodiments, the skin condition
is a cosmetic defect, a congenital defect, hair loss or an acquired
medical defect. In further embodiments, the skin condition is fine
lines and wrinkles; age spots and dyspigmentation; decreased skin
texture, tone and elasticity; roughness and photo damage; decreased
ability of skin to regenerate itself; environmental damage;
decreased smoothness and tightness of skin; age spots; fine and
coarse lines and wrinkles; fine and coarse periocular wrinkles;
nasolabial folds; facial fine and coarse lines; decreased skin
radiance and evenness; decreased skin firmness; hyperpigmentation;
dark spots and/or patches; decreased skin brightness and youthful
appearance; photoaged skin; intrinsically and extrinsically aged
skin; abnormal skin cellular turnover; decreased skin barrier;
decrease of skin's ability to retain moisture; brown and red
blotchiness; redness; abnormal skin epidermal thickness; reduction
of dermal epidermal junction; increased pore size and number of
pores; or a combination thereof. In certain embodiments,
administration of a composition described herein may result in at
least a 2-fold improvement of one or more symptoms or conditions.
Folds improvement of one or more symptoms or conditions include,
but are not limited to, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold,
25-fold, 75-fold, 100-fold or more, or any number therebetween. In
certain embodiments, administration of a composition described
herein may result in improvement of about 1% to about 100%, about
10% to about 90%, about 20% to about 80%, about 30% to about 70%,
about 40% to about 60%, or about 50%. In other embodiments,
administration of a composition described herein may result in
improvement of one or more symptoms or conditions of about 5%,
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 100%,
about 125%, about 150% or more of one or more symptoms or
conditions. In some embodiments, the yeast is of the Pichia genus.
In some embodiments, the yeast is Pichia pastoris. In one
embodiment, the cells in step (a) are derived from skin cells. In
another embodiment, the cells in step (a) are derived from
fibroblasts. In some embodiments, the cells in step (a) are derived
from animal cells. In other embodiments, the process further
comprises (e) processing the metabolized cell extract, wherein
processing is concentrating, filtering, purifying, or a combination
thereof. In one embodiment, the culturing of step (a) or (b)
comprises a continuous flow system or a bioreactor system. In
another embodiment, the cells are suspended or floated in the
growth medium. In still other embodiments, the metabolized cell
extract is present in the composition in an amount of about 0.0001%
to about 95% by weight of the composition. In another embodiment,
the metabolized cell extract is present in the composition in an
amount of about 0.01% to about 50% by weight of the composition. In
one embodiment, the metabolized cell extract is present in the
composition in an amount of about 0.01% to about 10% by weight of
the composition. In some embodiments, the metabolized cell extract
is present in an amount of about 10% to about 20% by weight of the
composition. In some embodiments, the metabolized cell extract is
present in an amount of about 0.01%, 0.5%, 1.0%, 5.0%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90% or 95% by weight of the composition. In one non-limiting
example, the metabolized cell extract is present in the composition
in an amount of about 10%, about 15%, or about 20% by weight of the
composition. In some embodiments, the composition further comprises
at least one of water, surfactants, emulsifiers, conditioners,
emollients, waxes, oils, polymers, thickeners, fixatives,
colorants, nutraceuticals, cosmeceuticals, therapeutics,
pharmaceuticals, antifungals, antimicrobials, steroidal hormones,
antidandruff agents, anti-acne components, sunscreens, and
preservatives. In one embodiment, the composition is an injectable
composition or a topical composition. In another embodiment, the
topical composition is an ointment, a cream, a hydrogel, or a
lotion. In one embodiment, the metabolized cell extract is
encapsulated within an encapsulant. In some embodiments, the
encapsulant is at least one of liposomes, niosomes, sub-micron
emulsions, polymeric encapsulates, gels, creams, and lotions. In
some embodiments, at least one extracellular matrix protein is
up-regulated by the administration of the composition to the
subject. In some embodiments, the extracellular matrix protein is
up-regulated by about 5% to about 100%. In one embodiment, the
extracellular matrix protein is up-regulated by about 10% to about
50%. In another embodiment, the extracellular matrix protein is
up-regulated by about 60% to about 100%. In yet another embodiment,
the extracellular matrix protein is up-regulated by about 5%, by
about 10%, by about 15%, by about 20%, by about 25%, by about 30%,
by about 35%, by about 40%, by about 45%, by about 50%, by about
55%, by about 60%, by about 65%, by about 70%, by about 75%, by
about 80%, by about 85%, by about 90%, by about 95% or by about
100%. In one embodiment, the extracellular matrix protein is a
collagen protein or a lysyl hydroxylase protein. In some
embodiments, the extracellular matrix protein is encoded by COL4A1
gene or PLOD1 gene. In other embodiments, at least one repair
protein is up-regulated by the administration of the composition to
the subject. In one embodiment, the repair protein is up-regulated
by about 10% to about 70%. In another embodiment, the repair
protein is up-regulated by about 25% to about 50%. In some
embodiments, the repair-protein is up-regulated by about 5%, by
about 10%, by about 15%, by about 20%, by about 25%, by about 30%,
by about 35%, by about 40%, by about 45%, by about 50%, by about
55%, by about 60%, by about 65%, by about 70%, by about 75%, by
about 80%, by about 85%, by about 90%, by about 95% or by about
100%. In one embodiment, the repair protein is a fibronectin
protein. In yet another embodiment, the repair protein is encoded
by FN1 gene. In some embodiments, at least one cellular
connectivity protein is up-regulated by the administration of the
composition to the subject. In one embodiment, the cellular
connectivity protein is up-regulated by about 5% to about 200%. In
one embodiment, the cellular connectivity protein is up-regulated
by about 10% to about 80%. In some embodiments, the cellular
connectivity protein is up-regulated by about 30% to about 50%. In
one embodiment, the cellular connectivity protein is up-regulated
by about 5%, by about 10%, by about 15%, by about 20%, by about
25%, by about 30%, by about 35%, by about 40%, by about 45%, by
about 50%, by about 55%, by about 60%, by about 65%, by about 70%,
by about 75%, by about 80%, by about 85%, by about 90%, by about
95%, by about 100%, by about 110%, by about 120%, by about 130%, by
about 140%, by about 150%, by about 160%, by about 170%, by about
180%, by about 190% or by about 200%. In other embodiments, the
cellular connectivity protein is involucrin protein. In some
embodiments, the cellular connectivity protein is encoded by IVL
gene. In one embodiment, at least one antioxidant protein is
up-regulated by the administration of the composition to the
subject. In yet another embodiment, the antioxidant protein is
up-regulated by about 5% to about 300%. In some embodiments, the
antioxidant protein is up-regulated by about 10% to about 50%. In
other embodiments, the antioxidant protein is up-regulated by about
200% to about 250%. In some embodiments, the antioxidant protein is
up-regulated by about 5%, by about 10%, by about 15%, by about 20%,
by about 25%, by about 30%, by about 35%, by about 40%, by about
45%, by about 50%, by about 55%, by about 60%, by about 65%, by
about 70%, by about 75%, by about 80%, by about 85%, by about 90%,
by about 95%, by about 100%, by about 125%, by about 150%, by about
175%, by about 200%, by about 250% or by about 300%. In one
embodiment, the antioxidant protein is a superoxide dismutase
protein. In another embodiment, the antioxidant protein is encoded
by SOD2 gene.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIG. 1 is a graph representing the kinetics of the
deposition of glycosaminoglycans and collagen laid down over time
by the three-dimensional tissue products Transcyte.TM. and
Dermagraft.RTM.. The deposition volumes of the glycosaminoglycans
are dependent on the period of growth while the deposition of
collagen is not dependent on the period of growth.
[0073] FIG. 2 is a graph representing the effect of extracellular
matrix (removed from Transcyte.TM.) and added at dilutions of 1:2,
1:5, 1:10, and 1:100 to monolayer cultures of human fibroblasts and
keratinocytes. The most significant effect illustrated is at a 1:10
dilution of the matrix.
[0074] FIG. 3 is a graph representing relative proliferation of
human fibroblasts and keratinocytes exposed to conditioned medium
(cell culture medium which has previously supported the growth of
cells in Transcyte.TM.). An increase in cell response was revealed
in as little as three days.
[0075] FIG. 4 is a graph demonstrating investigator assessments of
fine and coarse wrinkles. Significant reductions in mean scores
were observed for fine and coarse wrinkles at all visits (all
P<0.0001). In each data set, fine wrinkle assessment is shown on
the left and coarse wrinkle assessment is shown in the right.
[0076] FIG. 5 a graph demonstrating investigator assessments of
skin tone, firmness and radiance. Significant improvements in mean
scores for skin firmness, skin tone and radiance at all visits (all
P<0.02). In each data set, week 4 results are shown on the left,
week 8 results are shown in the middle and week 12 results are
shown on the right.
[0077] FIG. 6 is a graph demonstrating investigator assessments of
tactile roughness. Significant improvements in mean scores for skin
firmness, skin tone and radiance at all visits (all P<0.0001).
Percent change from baseline at week 4 is shown in the left-hand
column, week 8 in the middle column and week 12 in the right-hand
column, respectively.
[0078] FIG. 7 is a graph providing the results at week 12 of a
subject self-assessment questionnaire from the 12-week clinical
usage (Combined/Combination Product) study described in Example
18.
DETAILED DESCRIPTION OF THE INVENTION
[0079] The present disclosure relates to novel compositions
comprising metabolized conditioned growth medium and/or metabolized
cell extract, cultured using any eukaryotic cell type or
three-dimensional tissue construct, and methods for using the
compositions. The cells are cultured in monolayer or in
three-dimensions. The cells are preferably human and include
stromal cells, parenchymal cells, mesenchymal stem cells, liver
reserve cells, neural stem cells, pancreatic stem cells and/or
embryonic stem cells. Medium conditioned by cell and tissue
cultures will contain a variety of naturally secreted proteins,
such as biologically active growth factors. Also disclosed are
novel compositions comprising products derived from the conditioned
cell media and uses for these compositions.
[0080] The "pre-conditioned" cell culture medium may be any cell
culture medium which adequately addresses the nutritional needs of
the cells being cultured. Examples of cell media include, but are
not limited to Dulbecco's Modified Eagle's Medium (DMEM), Ham's
F12, RPMI 1640, Iscove's, McCoy's and other media formulations
readily apparent to those skilled in the art, including those found
in Methods For Preparation of Media, Supplements and Substrate For
Serum-Free Animal Cell Culture Alan R. Liss, New York (1984) and
Cell & Tissue Culture: Laboratory Procedures, John Wiley &
Sons Ltd., Chichester, England 1996, both of which are incorporated
by reference herein in their entirety. The medium may be
supplemented, with any components necessary to support the desired
cell or tissue culture. Additionally serum, such as bovine serum,
which is a complex solution of albumins, globulins, growth
promoters and growth inhibitors may be added if desired. The serum
should be pathogen free and carefully screened for mycoplasma
bacterial, fungal, and viral contamination. Also, the serum should
generally be obtained from the United States and not obtained from
countries where indigenous livestock carry transmittable agents.
Hormone addition into the medium may or may not be desired. The
medium may also be serum-free, i.e., cells are grown in the absence
of a serum supplement.
[0081] The "conditioned growth medium" may be any growth medium
conditioned as disclosed herein, and can be prepared, for example,
as disclosed in U.S. Pat. Nos. 6,372,494; 7,118,746; 7,160,726, all
of which are incorporated herein by reference in its entirety.
[0082] Other ingredients, such as vitamins, growth and attachment
factors, proteins etc., can be selected by those of skill in the
art in accordance with his or her particular need. In some
embodiments, any cell type appropriate to achieve the desired
conditioned medium may be used. Genetically engineered cells may be
used to culture the media. Such cells can be modified, for example,
to express a desired protein or proteins so that the concentration
of the expressed protein or proteins in the medium is optimized for
the particular desired application. In accordance with the present
disclosure, the cells and tissue cultures used to condition the
medium may be engineered to express a target gene product which may
impart a wide variety of functions, including but not limited to,
improved properties in expressing proteins resembling physiological
reactions, increased expression of a particular protein useful for
a specific application, such as wound healing or inhibiting certain
proteins such as proteases, lactic acid, etc.
[0083] The cells may be engineered to express a target gene product
which is biologically active which provides a chosen biological
function, which acts as a reporter of a chosen physiological
condition, which augments deficient or defective expression of a
gene product, or which provides an anti-viral, anti-bacterial,
anti-microbial, or anti-cancer activity. In accordance with the
present disclosure, the target gene product may be a peptide or
protein, such as an enzyme, hormone, cytokine, antigen, or
antibody, a regulatory protein, such as a transcription factor or
DNA binding protein, a structural protein, such as a cell surface
protein, or the target gene product may be a nucleic acid such as a
ribosome or antisense molecule. The target gene products include,
but are not limited to, gene products which enhance cell growth.
For example, the genetic modification may upregulate an endogenous
protein, introduce a new protein or regulate ion concentration by
expressing a heterologous ion channel or altering endogenous ion
channel function. Examples include, but are not limited to
engineered tissues that express gene products which are delivered
systemically (e.g., secreted gene products such as proteins
including growth factors, hormones, Factor VIII, Factor IX,
neurotransmitters, and enkaphalins).
[0084] In some embodiments, cells are grown in a two-dimensional
monolayer according to any method known to the skilled artisan.
See, e.g., cell culture techniques such as those described in
Sambrook J et al. (2000) Molecular Cloning: A Laboratory Manual
(Third Edition); Goeddel, ed. (1990) Methods in Enzymology 185,
Current Protocols In Molecular Biology; F. M. Ausubel et al., eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc. (1994,
supplemented through 1999); Animal Cell Culture: A Practical
Approach (Freshney ed. 1986).
[0085] In some embodiments, cells are grown on a three-dimensional
stromal support and grow in multiple layers, forming a cellular
matrix. This matrix system approaches physiologic conditions found
in vivo to a greater degree than previously described monolayer
tissue culture systems. Three-dimensional cultures, such as
Dermagraft.RTM. (Advanced Tissue Sciences, Inc., La Jolla,
Calif.)"Dermagraft.RTM.", and TransCyte.TM. (Smith & Nephew,
PLC, United Kingdom) "Transcyte.TM.", produce numerous growth
factors and other proteins that are secreted into the medium at
physiological ratios and concentrations. Dermagraft.RTM. is
composed of allogeneic neonatal fibroblasts cultured on
biodegradable polyglactin. TransCyte.TM. is a temporary living skin
replacement comprising a three-dimensional stromal tissue bonded to
a transitional covering as described in U.S. Pat. No. 5,460,939.
Additionally, the three-dimensional tissue cultures which condition
the cell media may contain mesenchymal stem cells, liver reserve
cells, neural stem cells, pancreatic stem cells, and/or embryonic
stem cells and/or parenchymal cells and/or parenchymal stem cells
found in many tissue types, including but not limited to bone
marrow, skin, liver, pancreas, kidney, adrenal and neurologic
tissue, as well as tissues of the gastrointestinal and
genitourinary tracts, and the circulatory system. See U.S. Pat.
Nos. 4,721,096; 4,963,489; 5,032,508; 5,266,480; 5,160,490; and
5,559,022, each of which is incorporated by reference herein in
their entirety.
I. Definitions
[0086] The following terms used herein shall have the meanings
indicated:
[0087] Cells: includes cells from any organism, including animals,
plants, fungi, protists, or monera. In one embodiment, the source
of the cells is an animal or a plant. In another embodiment, the
animal is a mammal. In another embodiment, the mammal is a
human.
[0088] Cell Extract: includes lysed or fragmented cell content that
may or may not include cell particulate matter.
[0089] Adherent Layer: cells attached directly to the
three-dimensional support or connected indirectly by attachment to
cells that are themselves attached directly to the support.
[0090] Conditioned Medium: a formulation containing extracellular
protein(s) and cellular metabolites, which has previously supported
the growth of any desired eukaryotic cell type, said cells having
been cultured in either two or three dimensions. Also called
"Conditioned Cell Medium" or "Conditioned Cell and Tissue Culture
Medium".
[0091] Stromal Cells: fibroblasts with or without other cells
and/or elements found in loose connective tissue, including but not
limited to, endothelial cells, pericytes, macrophages, monocytes,
plasma cells, mast cells, adipocytes, mesenchymal stem cells, liver
reserve cells, neural stem cells, pancreatic stem cells,
chondrocytes, prechondrocytes, etc.
[0092] Tissue-Specific or Parenchymal Cells: the cells which form
the essential and distinctive tissue of an organ as distinguished
from its supportive framework.
[0093] Two-Dimensional Cell Culture: cells cultured in a monolayer,
for example, on a flat surface or the like.
[0094] Three-Dimensional Framework: a three-dimensional scaffold
composed of any material and/or shape that (a) allows cells to
attach to it (or can be modified to allow cells to attach to it);
and (b) allows cells to grow in more than one layer. This support
is inoculated with stromal cells to form the living
three-dimensional stromal tissue. The structure of the framework
can include a mesh, a sponge or can be formed from a hydrogel.
[0095] Three-Dimensional Stromal Tissue or Living Stromal Matrix: a
three-dimensional framework which has been inoculated with stromal
cells that are grown on the support. The extracellular matrix
proteins elaborated by the stromal cells are deposited onto the
framework, thus forming a living stromal tissue. The living stromal
tissue can support the growth of tissue-specific cells later
inoculated to form the three-dimensional cell culture.
[0096] Tissue-Specific Three-Dimensional Cell Culture or
Tissue-Specific Three-Dimensional Construct: a three-dimensional
living stromal tissue which has been inoculated with
tissue-specific cells and cultured. In general, the tissue specific
cells used to inoculate the three-dimensional stromal matrix should
include the "stem" cells (or "reserve" cells) for that tissue;
i.e., those cells which generate new cells that will mature into
the specialized cells that form the parenchyma of the tissue.
[0097] The following abbreviations shall have the meanings
indicated:
[0098] BCS=bovine calf serum
[0099] BFU-E=burst-forming unit-erythroid
[0100] TGF-.beta.=transforming growth factor-.beta.
[0101] CFU-C=colony forming unit-culture
[0102] CFU-GEMM=colony forming unit-granuloid, erythroid, monocyte,
megakaryocyte
[0103] CSF=colony-stimulating factor
[0104] DMEM=Dulbecco's Modified Eagle's Medium
[0105] EDTA=ethylene diamine tetraacetic acid
[0106] FBS=fetal bovine serum
[0107] FGF=fibroblast growth factor
[0108] GAG=glycosaminoglycan
[0109] GM-CSF=granulocyte/macrophage colony-stimulating factor
[0110] HBSS=Hank's balanced salt solution
[0111] HS=horse serum
[0112] IGF=insulin-like growth factor
[0113] LTBMC=long term bone marrow culture
[0114] MEM=minimal essential medium
[0115] PBL=peripheral blood leukocytes
[0116] PBS=phosphate buffered saline
[0117] PDGF=platelet-derived growth factor
[0118] RPMI 1640=Roswell Park Memorial Institute medium number 1640
(GIBCO, Inc., Grand Island, N.Y.)
[0119] SEM=scanning electron microscopy
[0120] VEGF=vascular endothelial growth factor
II. Metabolized Conditioned Growth Medium and Metabolized Cell
Extract
[0121] In one embodiment, the disclosure provides metabolized
conditioned growth medium prepared by a process comprising: (a)
culturing cells in a growth medium sufficient to meet the
nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells; (b) culturing yeast cells; (c)
exposing the yeast cells to the conditioned growth medium; (d)
culturing the yeast cells to metabolize at least a portion of the
conditioned growth medium; and (e) collecting the metabolized
conditioned growth medium. In some embodiments, the cells may
originate from a plant and/or animal source, including human.
[0122] In another embodiment, the disclosure provides metabolized
cell extract prepared by a process comprising: (a) providing a cell
extract; (b) culturing yeast cells; (c) exposing the yeast cells to
the cell extract; (d) culturing the yeast cells to metabolize at
least a portion of the cell extract; and (e) collecting the
metabolized cell extract. In some embodiments, the cell extract is
obtained by: culturing cells in a growth medium sufficient to meet
the nutritional needs required to grow the cells in vitro and
removing the resulting cell extract from the growth medium. In some
embodiments, the cells may originate from a plant and/or animal
source, including human.
[0123] Metabolizing the extracellular proteins in conditioned
growth media or cell extracts, such as growth factors, cytokines,
and stress proteins, opens new possibilities in the preparation of
products for use in a large variety of areas including tissue
repair, e.g., in the treatment of wounds and other tissue defects
such as cosmetic defects as well as human and animal feed
supplements. For example, growth factors are known to play an
important role in the wound healing process. At least one activity
of these growth factors is imparted to the conditioned cell media
or cell extracts through the metabolism processes of the
disclosure.
[0124] Cellular cytokines and growth factors are involved in a
number of critical cellular processes including cell proliferation,
adhesion, morphologic appearance, differentiation, migration,
inflammatory responses, angiogenesis, and cell death. Studies have
demonstrated that hypoxic stress and injury to cells induce
responses including increased levels of mRNA and proteins
corresponding to growth factors such as PDGF (platelet-derived
growth factor), VEGF (vascular endothelial growth factor), FGF
(fibroblast growth factor), and IGF (insulin-like growth factor)
(Gonzalez-Rubio, M. et al., 1996, Kidney Int. 50(1):164-73;
Abramovitch, R. et al., 1997, Int J Exp. Pathol. 78(2):57-70;
Stein, I. et al., 1995, Mol Cell Biol. 15(10):5363-8; Yang, W. et
al., 1997, FEBS Lett. 403(2):139-42; West, N. R. et al., 1995, J
Neurosci. Res. 40(5):647-59).
[0125] Growth factors, such as transforming growth factor-.beta.,
also known as TGF-.beta., are induced by certain stress proteins
during wound healing. Two known stress proteins are GRP78 and
HSP90. These proteins stabilize cellular structures and render the
cells resistant to adverse conditions. The TGF-.beta. family of
dimeric proteins includes TGF-.beta. 1, TGF-.beta.2, and
TGF-.beta.3 and regulates the growth and differentiation of many
cell types. Furthermore, this family of proteins exhibits a range
of biological effects, stimulating the growth of some cell types
(Noda et al., 1989, Endocrinology 124:2991-2995) and inhibiting the
growth of other cell types (Goey et al., 1989, J. Immunol.
143:877-880; Pietenpol et al., 1990, Proc. Natl. Acad. Sci. USA
87:3758-3762). TGF-.beta. has also been shown to increase the
expression of extracellular matrix proteins including collagen and
fibronectin (Ignotz et al., 1986, J. Biol. Chem. 261:4337-4345) and
to accelerate the healing of wounds (Mustoe et al., 1987, Science
237:1333-1335).
[0126] Another such growth factor is PDGF. PDGF was originally
found to be a potent mitogen for mesenchymal-derived cells (Ross R.
et al., 1974, Proc. Natl. Acad. Sci. USA 71(4):1207-1210; Kohler N.
et al., 1974, Exp. Cell Res. 87:297-301). Further studies have
shown that PDGF increases the rate of cellularity and granulation
in tissue formation. Wounds treated with PDGF have the appearance
of an early stage inflammatory response including an increase in
neutrophils and macrophage cell types at the wound site. These
wounds also show enhanced fibroblast function (Pierce, G. F. et
al., 1988, J. Exp. Med. 167:974-987). Both PDGF and TGF-.beta. have
been shown to increase collagen formation, DNA content, and protein
levels in animal studies (Grotendorst, G. R. et al., 1985, J. Clin.
Invest. 76:2323-2329; Sporn, M. B. et al., 1983, Science (Wash DC)
219:1329). PDGF has been shown to be effective in the treatment of
human wounds. In human wounds, PDGF-AA expression is increased
within pressure ulcers undergoing healing. The increase of PDGF-AA
corresponds to an increase in activated fibroblasts, extracellular
matrix deposition, and active vascularization of the wound.
Furthermore, such an increase in PDGF-AA is not seen in chronic
non-healing wounds (Principles of Tissue Engineering, R. Lanza et
al. (eds.), pp. 133-141 (R.G. Landes Co. TX 1997). A number of
other growth factors having the ability to induce angiogenesis and
wound healing include VEGF, KGF and basic FGF.
[0127] A. Growth Media
[0128] Growth media formulations for cell culture are well known in
the literature and many are commercially available.
[0129] Preconditioned media ingredients include, but are not
limited to those described below. Additionally, the concentration
of the ingredients is well known to one of ordinary skill in the
art. See, for example, Methods For Preparation Of Media,
Supplements and Substrate for Serum free Animal Cell Cultures,
supra. The ingredients include amino-acids (both D and/or L-amino
acids) such as glutamine, alanine, arginine, asparagine, cysteine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, and valine and their derivatives; acid soluble subgroups
such as thiamine, ascorbic acid, ferric compounds, ferrous
compounds, purines, glutathione and monobasic sodium
phosphates.
[0130] Additional ingredients include sugars, deoxyribose, ribose,
nucleosides, water soluble vitamins, riboflavin, salts, trace
metals, lipids, acetate salts, phosphate salts, HEPES, phenol red,
pyruvate salts and buffers.
[0131] Other ingredients often used in media formulations include
fat soluble vitamins (including A, D, E and K) steroids and their
derivatives, cholesterol, fatty acids and lipids Tween 80,
2-mercaptoethanol pyramidines as well as a variety of supplements
including serum (fetal, horse, calf, etc.), proteins (insulin,
transferrin, growth factors, hormones, etc.) antibiotics
(gentamicin, penicillin, streptomycin, amphotericin B, etc.) whole
egg ultra filtrate, and attachment factors (fibronectins,
vitronectins, collagens, laminins, tenascins, etc.).
[0132] The media may or may not need to be supplemented with growth
factors and other proteins such as attachment factors since many of
the cell constructs, particularly the three-dimensional cell and
tissue culture constructs described in this application themselves
elaborate such growth and attachment factors and other products
into the media.
[0133] B. Cell Cultures
[0134] 1. The Cells
[0135] Any cells may be used in the cell cultures. For example, the
cells may be stromal cells, parenchymal cells, mesenchymal stem
cells (lineage committed or uncommitted progenitor cells), liver
reserve cells, neural stem cells, pancreatic stem cells, and/or
embryonic stem cells. The cells may include, but are not limited
to, bone marrow, skin, liver, pancreas, kidney, neurological
tissue, adrenal gland, mucosal epithelium, and smooth muscle, to
name but a few. The fibroblasts and fibroblast-like cells and other
cells and/or elements that comprise the stroma may be fetal or
adult in origin, and may be derived from convenient sources such as
skin, liver, pancreas, mucosa, arteries, veins, umbilical cord, and
placental tissues, etc. Such tissues and/or organs can be obtained
by appropriate biopsy or upon autopsy. In fact, cadaver organs may
be used to provide a generous supply of stromal cells and
elements.
[0136] Embryonic stem cells and/or other elements that comprise the
stroma may be isolated using methods known in the art. For
instance, recently human embryonic stem cell populations and
methods for isolating and using these cells have been reported in
Keller et al., Nature Med., 5:151-152 (1999), Smith Curr. Biol.
8:R802-804 (1998); isolated from primordial germ cells, Shamblatt
et al., PNAS 95:13726-1373 (1998), isolated from blastocytes,
Thomason et al., Science 282:1145-1147 (1988). The isolation and
culture of mesenchymal stem cells are known in the art. See Mackay
et al., Tissue Eng. 4:415-428 (1988); William et al., Am Surg.
65:22-26 (1999). Inoculation of these cells is described infra, in
Section 5.3. Likewise, neural stem cells may be isolated in the
manner described in Flax et al., Nature Biotechnol., 16:1033-1039
(1998); and Frisen et al., Cell. Mol. Life Sci., 54:935-945
(1998).
[0137] The cells may be cultured in any manner known in the art
including in monolayer, beads or in three-dimensions and by any
means (i.e., culture dish, roller bottle, a continuous flow system,
etc.). Methods of cell and tissue culturing are well known in the
art, and are described, for example, in Cell & Tissue Culture:
Laboratory Procedures, supra, Freshney (1987), Culture of Animal
Cells: A Manual of Basic Techniques, infra.
[0138] In general, the cell lines utilized are carefully screened
for human and animal pathogens. Depending upon the application,
such screening may be of critical importance where only pathogen
free cells are acceptable (e.g., for wound healing, food additives,
etc.) Methods of screening for pathogens are well known in the art.
The cell type, whether cultured in two-dimensions or
three-dimensions, will affect the properties of the conditioned
medium. In some embodiments, the cells are cultured in two
dimensions. In other embodiments, the cells are cultured in three
dimensions.
[0139] a. Genetically Engineered Cells
[0140] In some embodiments, the cells can act as vehicles for
introducing gene products into the media or extract that promote
the repair and/or regeneration of tissue defects, for example. The
cells can be genetically engineered to express, for example,
inflammatory mediators, such as IL-6, IL-8 and G-CSF. The cells
could also or alternatively be genetically engineering to express
anti-inflammatory factors, e.g., anti-GM-CSF, anti-TNF, anti-IL-1,
anti-IL-2, etc.
[0141] In another embodiment, the cells can be genetically
engineered to express a gene into the media or extract which would
exert a therapeutic effect, e.g., in the production of TGF-.beta.
to stimulate cartilage production, or other factors such as BMP-13
to promote chondrogenesis or stimulatory factors that promote
migration of stromal cells and/or matrix deposition. Since the
constructs comprise eukaryotic cells, the gene product will be
properly expressed and processed to form an active product.
Preferably, the expression control elements used should allow for
the regulated expression of the gene so that the product can be
over-synthesized in culture. The transcriptional promoter chosen,
generally, and promoter elements specifically, depend, in part,
upon the type of tissue and cells cultured. Cells and tissues which
are capable of secreting proteins are preferable (e.g., those
having abundant rough endoplasmic reticulum and Golgi complex
organelles). The over-produced gene product will then be secreted
by the engineered cell into the conditioned media.
[0142] The cells may be genetically engineered to regulate one or
more genes; or the regulation of gene expression may be transient
or long-term; or the gene activity may be non-inducible or
inducible.
[0143] The cells can also be genetically engineered to "knock out"
expression of factors that promote inflammation. Negative
modulatory techniques for the reduction of target gene expression
levels or target gene product activity levels are discussed below.
"Negative modulation", as used herein, refers to a reduction in the
level and/or activity of target gene product relative to the level
and/or activity of the target gene product in the absence of the
modulatory treatment. The expression of a gene native to the cell
can be reduced or knocked out using a number of techniques, for
example, expression may be inhibited by inactivating the gene
completely (commonly termed "knockout") using standard homologous
recombination techniques. Usually, an exon encoding an important
region of the protein (or an exon 5' to that region) is interrupted
by a positive selectable marker (for example neo), preventing the
production of normal mRNA from the target gene and resulting in
inactivation of the gene. A gene may also be inactivated by
creating a deletion or an inactivating insertion in part of a gene,
or by deleting the entire gene. By using a construct with two
regions of homology to the target gene that are far apart in the
genome, the sequences intervening the two regions can be deleted.
Mombaerts et al., 1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084-3087.
Alternatively, a gene may also be inactivated by deletion of
upstream or downstream expression elements.
[0144] Antisense and ribozyme molecules which inhibit expression of
the target gene can also be used to reduce the level of target gene
activity. For example, antisense RNA molecules which inhibit the
expression of major histocompatibility gene complexes (HLA) have
been shown to be most versatile with respect to immune responses.
Furthermore, appropriate ribozyme molecules can be designed as
described, e.g., by Haseloff et al., 1988, Nature 334:585-591; Zaug
et al., 1984, Science 224:574-578; and Zaug and Cech, 1986, Science
231:470-475. Still further, triple helix molecules can be utilized
in reducing the level of target gene activity. These techniques are
described in detail by L. G. Davis et al., eds, Basic Methods in
Molecular Biology, 2nd ed., Appleton & Lange, Norwalk, Conn.
1994.
[0145] Methods that may be useful to genetically engineer the cells
are well-known in the art and are further detailed in co-owned U.S.
Pat. Nos. 4,963,489 and 5,785,964, the disclosures of which are
incorporated herein by reference. For example, a recombinant DNA
construct or vector containing an exogenous nucleic acid, e.g.,
encoding a gene product of interest, may be constructed and used to
transform or transfect the stromal cells. Such transformed or
transfected cells that carry the exogenous nucleic acid, and that
are capable of expressing said nucleic acid, are selected and
clonally expanded in the three-dimensional constructs of this
disclosure.
[0146] Methods for preparing DNA constructs containing the gene of
interest, for transforming or transfecting cells, and for selecting
cells carrying and expressing the gene of interest are well-known
in the art. See, for example, the techniques described in Maniatis
et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al.,
1989, Current Protocols in Molecular Biology, Greene Publishing
Associates & Wiley Interscience, N.Y.; and Sambrook et al.,
1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0147] The cells can be engineered using any of a variety of
vectors including, but not limited to, integrating viral vectors,
e.g., retrovirus vector or adeno-associated viral vectors; or
non-integrating replicating vectors, e.g., papilloma virus vectors,
SV40 vectors, adenoviral vectors; or replication-defective viral
vectors. Where transient expression is desired, non-integrating
vectors and replication defective vectors may be preferred, since
either inducible or constitutive promoters can be used in these
systems to control expression of the gene of interest.
Alternatively, integrating vectors can be used to obtain transient
expression, provided the gene of interest is controlled by an
inducible promoter. Other methods of introducing DNA into cells
include the use of liposomes, lipofection, electroporation, a
particle gun, or by direct DNA injection.
[0148] The cells are preferably transformed or transfected with a
nucleic acid, e.g., DNA, controlled by, i.e., in operative
association with, one or more appropriate expression control
elements such as promoter or enhancer sequences, transcription
terminators, polyadenylation sites, among others, and a selectable
marker. Following the introduction of the foreign DNA, engineered
cells may be allowed to grow in enriched media and then switched to
selective media. The selectable marker in the foreign DNA confers
resistance to the selection and allows cells to stably integrate
the foreign DNA as, for example, on a plasmid, into their
chromosomes and grow to form foci which, in turn, can be cloned and
expanded into cell lines. This method can be advantageously used to
engineer cell lines which express the gene product into the
media.
[0149] Any promoter may be used to drive the expression of the
inserted gene. For example, viral promoters include but are not
limited to the CMV promoter/enhancer, SV40, papillomavirus,
Epstein-Barr virus, elastin gene promoter and .beta.-globin.
Preferably, the control elements used to control expression of the
gene of interest should allow for the regulated expression of the
gene so that the product is synthesized only when needed in vivo.
If transient expression is desired, constitutive promoters are
preferably used in a non-integrating and/or replication-defective
vector. Alternatively, inducible promoters could be used to drive
the expression of the inserted gene when necessary. Inducible
promoters can be built into integrating and/or replicating vectors.
For example, inducible promoters include, but are not limited to,
metallothionein and heat shock protein.
[0150] According to one embodiment, the inducible promoters used
for expressing exogenous genes of interest are those that are the
native promoters of those regulatory proteins as disclosed herein
that are induced as a result of cryopreservation and subsequent
thawing. For example, the promoter of TGF-.beta., VEGF, or various
known heat shock proteins can be used as the expression control
element, i.e., can be operatively linked to an exogenous gene of
interest in order to express a desired gene product in the tissue
constructs conditioning the cell media.
[0151] A variety of methods may be used to obtain the constitutive
or transient expression of gene products engineered into the cells.
For example, the transkaryotic implantation technique described by
Seldon et al., 1987, Science 236:714-718 can be used.
"Transkaryotic", as used herein, suggests that the nuclei of the
implanted cells have been altered by the addition of DNA sequences
by stable or transient transfection. Preferably, the cells are
engineered to express such gene products transiently and/or under
inducible control during the post-operative recovery period, or as
a chimeric fusion protein anchored to the stromal cells, for
example, as a chimeric molecule composed of an intracellular and/or
transmembrane domain of a receptor or receptor-like molecule, fused
to the gene product as the extracellular domain.
[0152] Furthermore, it may be desirable to prepare a construct
having an extracellular matrix containing a foreign gene product,
growth factor, regulatory factor, etc., which is then found in the
conditioned media. This embodiment is based on the discovery that,
during the growth of human stromal cells on a three-dimensional
support framework, the cells synthesize and deposit on the
framework a human extracellular matrix as produced in normal human
tissue. The extracellular matrix is secreted locally by cells and
not only binds cells and tissue together but also influences the
development and behavior of the cells it contacts. The
extracellular matrix contains various connective tissue proteins,
e.g., fiber-forming proteins interwoven in a hydrated gel composed
of a network of glycosaminoglycans chains. The glycosaminoglycans
are a heterogeneous group of long, negatively charged
polysaccharide chains, which (except for hyaluronic acid) are
covalently linked to protein to form proteoglycans molecules.
According to this embodiment, the stromal cells may be genetically
engineered to express a desired gene product, or altered forms of a
gene product, which will be present in the extracellular matrix and
ultimately the cell medium.
[0153] 2. Culturing the Cells
[0154] In some embodiments, the cells are grown in two-dimensional
cell cultures. In other embodiments, the cells are grown in
three-dimensional cell cultures. See, e.g., U.S. Pat. No.
6,372,494, herein incorporated by reference in its entirety. The
stromal cells used in the cell cultures preferably comprise
fibroblasts, mesenchymal stem cells, liver reserve cells, neural
stem cells, pancreatic stem cells, and/or embryonic stem cells with
or without additional cells and/or elements described more fully
herein.
[0155] The cells can be cultured by any means known in the art.
Preferably, the cells are cultured in an environment which enables
aseptic processing and handling. Conventional means of cell and
tissue culture have been limited by the need for human supervision
and control of the media. This limits the amount of cells and
tissue that can be cultured at a single time and consequently the
volume of conditioned cell media that can be obtained at a single
time. For this reason, it is preferred that the media be
conditioned in a manner allowing for large scale growth (yielding
large scale conditioned media) using, for example, an apparatus for
aseptic large scale culturing like that described in co-owned U.S.
Pat. No. 5,763,267 (the '267 patent) which is incorporated by
reference herein in its entirety for all purposes. Using the
aseptic closed system described in the '267 patent, preconditioned
culture media is transported from a fluid reservoir to an inlet
manifold and evenly distributed to the cultures in a continuous
flow system and is useful in culturing three-dimensional cell and
tissue cultures, such as Dermagraft.RTM. for example. In
particular, the apparatus described in the '267 patent includes a
plurality of flexible or semi-flexible treatment chambers
comprising one or more individual culture pockets, a plurality of
rigid spacers, an inlet fluid manifold, an outlet fluid manifold, a
fluid reservoir, and a means for transporting fluid within the
system.
[0156] During treatment, liquid medium is transported from the
fluid reservoir to the inlet manifold, which in turn evenly
distributes the media to each of the connected treatment chambers
and internal culture pockets. An outlet fluid manifold is also
provided to ensure that each treatment chamber is evenly filled and
to ensure that any air bubbles formed during treatment are removed
from the treatment chambers. The treatment chambers are flexible or
semi-flexible so as to provide for easy end-user handling during
rinsing and application of the cultured transplants. Due to the
flexibility of the treatment chambers, rigid spacers are also
provided which ensure even fluid distribution within the chambers
during treatment.
[0157] In another embodiment, the tissue is cultivated in an
apparatus for aseptic growth of tissue cultures as described in
U.S. Pat. No. 5,843,766 (the '766 patent) incorporated herein in
its entirety for all purposes. The '766 patent discloses a tissue
culture chamber in which the chamber is a casing that provides for
growth of tissue that can be grown, preserved in frozen form, and
shipped to the end user in the same aseptic container. The tissue
culture chamber includes a casing comprising a substrate within the
casing designed to facilitate three-dimensional tissue growth on
the surface of the substrate. The casing includes an inlet and an
outlet port which assist the inflow and outflow of medium. The
casing also includes at least one flow distributor. In one
embodiment, the flow distributor is a baffle, which is used to
distribute the flow of the medium within the chamber to create a
continuous, uniform piece of tissue. In a second embodiment, the
flow distributor is a combination of deflector plates, distribution
channels, and a flow channel. In each embodiment, the casing
further includes a seal so as to ensure an aseptic environment
inside the chamber during tissue growth and storage.
[0158] Fibroblasts will support the growth of many different cells
and tissues in the three-dimensional culture system, and,
therefore, can be inoculated onto the matrix to form a "generic"
stromal support matrix for culturing any of a variety of cells and
tissues. However, in certain instances, it may be preferable to use
a "specific" rather than "generic" stromal support matrix, in which
case stromal cells and elements can be obtained from a particular
tissue, organ, or individual. Moreover, fibroblasts and other
stromal cells and/or elements may be derived from the same type of
tissue to be cultured in the three-dimensional system. This might
be advantageous when culturing tissues in which specialized stromal
cells may play particular structural/functional roles; e.g., smooth
muscle cells of arteries, glial cells of neurological tissue,
Kupffer cells of liver, etc.
[0159] Once inoculated onto the three-dimensional support, the
stromal cells will proliferate on the framework and deposit the
connective tissue proteins naturally secreted by the stromal cells
such as growth factors, regulatory factors and extracellular matrix
proteins. The stromal cells and their naturally secreted connective
tissue proteins substantially envelop the framework thus forming
the living stromal tissue which will support the growth of
tissue-specific cells inoculated into the three-dimensional culture
system of the disclosure. In fact, when inoculated with the
tissue-specific cells, the three-dimensional stromal tissue will
sustain active proliferation of the culture for long periods of
time. Importantly, because openings in the mesh permit the exit of
stromal cells in culture, confluent stromal cultures do not exhibit
contact inhibition, and the stromal cells continue to grow, divide,
and remain functionally active.
[0160] Growth and regulatory factors are elaborated by the stromal
tissue into the media. Growth factors (for example, but not limited
to, .alpha.FGF, .beta.FGF, insulin growth factor or TGF-betas), or
natural or modified blood products or other bioactive biological
molecules (for example, but not limited to, hyaluronic acid or
hormones), enhance the colonization of the three-dimensional
framework or scaffolding and condition the culture media.
[0161] The extent to which the stromal cells are grown prior to use
of the cultures in vivo may vary depending on the type of tissue to
be grown in three-dimensional tissue culture. The living stromal
tissues which condition the medium may be used as corrective
structures by implanting them in vivo. Alternatively, the living
stromal tissues may be inoculated with another cell type and
implanted in vivo. The stromal cells may be genetically engineered
to adjust the level of protein products secreted into the culture
medium to improve the concentration of recovered product obtained
from the conditioned medium. For example, stromal cells may be
genetically engineered to improve concentration of one or more or
the following products obtained from the conditioned medium.
anti-inflammatory factors, e.g., anti-GM-CSF, anti-TNF, anti-IL-1,
anti-IL-2, etc. Alternatively, the stromal cells may be genetically
engineered to "knock out" expression of native gene products that
promote inflammation, e.g., GM-CSF, TNF, IL-1, IL-2, or "knock out"
expression of MHC in order to lower the risk of rejection.
[0162] Growth of the stromal cells in three-dimensions will sustain
active proliferation of both the stromal and tissue-specific cells
in culture for much longer time periods than will monolayer
systems. Moreover, the three-dimensional system supports the
maturation, differentiation, and segregation of cells in culture in
vitro to form components of adult tissues analogous to counterparts
found in vivo and secure proteins into the conditional medium more
closely resembling physiological ratios.
[0163] a. Establishment of Three-Dimensional Stromal Tissue
[0164] The three-dimensional support or framework may be of any
material and/or shape that: (a) allows cells to attach to it (or
can be modified to allow cells to attach to it); and (b) allows
cells to grow in more than one layer. A number of different
materials may be used to form the framework, including but not
limited to: non-biodegradable materials, e.g., nylon (polyamides),
dacron (polyesters), polystyrene, polypropylene, polyacrylates,
polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC),
polytetrafluorethylene (PTFE; teflon), thermanox (TPX),
nitrocellulose, cotton; and biodegradable materials, e.g.,
polyglycolic acid (PGA), collagen, collagen sponges, cat gut
sutures, cellulose, gelatin, dextran, polyalkanoates, etc. Any of
these materials may be woven braided, knitted, etc., into a mesh,
for example, to form the three-dimensional framework. The
framework, in turn can be fashioned into any shape desired as the
corrective structure, e.g., tubes, ropes, filaments, etc. Certain
materials, such as nylon, polystyrene, etc., are poor substrates
for cellular attachment. When these materials are used as the
three-dimensional framework, it is advisable to pre-treat the
framework prior to inoculation of stromal cells in order to enhance
the attachment of stromal cells to the support. For example, prior
to inoculation with stromal cells, nylon frameworks could be
treated with 0.1M acetic acid, and incubated in polylysine, FBS,
and/or collagen to coat the nylon. Polystyrene could be similarly
treated using sulfuric acid.
[0165] When the cultures are to be implanted in vivo, it may be
preferable to use biodegradable matrices such as polyglycolic acid,
collagen, collagen sponges, woven collagen, catgut suture material,
gelatin, polylactic acid, or polyglycolic acid and copolymers
thereof, for example. Where the cultures are to be maintained for
long periods of time or cryopreserved, non-degradable materials
such as nylon, dacron, polystyrene, polyacrylates, polyvinyls,
teflons, cotton, etc., may be preferred. A convenient nylon mesh
which could be used in accordance with the disclosure is
NITEX.RTM., a nylon filtration mesh having an average pore size of
210 .mu.m and an average nylon fiber diameter of 90 .mu.m
(#3-210/36, Tetko, Inc., N.Y.).
[0166] Stromal cells comprising fibroblasts, mesenchymal stem
cells, liver reserve cells, neural stem cells, pancreatic stem
cells and/or embryonic stem cells with or without other cells and
elements described below, are inoculated onto the framework. Also,
cells found in loose connective tissue may be inoculated onto the
three-dimensional support along with fibroblasts. Such cells
include but are not limited to smooth muscle cells, endothelial
cells, pericytes, macrophages, monocytes, plasma cells, mast cells,
adipocytes, etc. As previously explained, fetal fibroblasts can be
used to form a "generic" three-dimensional stromal matrix that will
support the growth of a variety of different cells and/or tissues.
However, a "specific" stromal tissue may be prepared by inoculating
the three-dimensional framework with fibroblasts derived from the
same type of tissue to be cultured and/or from a particular
individual who is later to receive the cells and/or tissues grown
in culture in accordance with the three-dimensional system.
[0167] Thus, in one embodiment, stromal cells which are specialized
for the particular tissue may be cultured. For example, stromal
cells of hematopoietic tissue, including but not limited to
fibroblasts, endothelial cells, macrophages/monocytes, adipocytes
and reticular cells, could be used to form the three-dimensional
subconfluent stroma for the long term culture of bone marrow in
vitro. Hematopoietic stromal cells may be readily obtained from the
"buffy coat" formed in bone marrow suspensions by centrifugation at
low forces, e.g., 3000.times.g. In the stromal layer that makes up
the inner wall of arteries, a high proportion of undifferentiated
smooth muscle cells can be added to provide the protein elastic.
Stromal cells of liver may include fibroblasts, Kupffer cells, and
vascular and bile duct endothelial cells. Similarly, glial cells
could be used as the stroma to support the proliferation of
neurological cells and tissues; glial cells for this purpose can be
obtained by trypsinization or collagenase digestion of embryonic or
adult brain (Ponten and Westermark, 1980, in Federof, S. Hertz, L.,
eds, "Advances in Cellular Neurobiology," Vol. 1, New York,
Academic Press, pp. 209-227). The growth of cells in the
three-dimensional stromal cell culture may be further enhanced by
adding to the framework, or coating the support with proteins
(e.g., collagens, elastic fibers, reticular fibers) glycoproteins,
glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,
chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), a
cellular matrix, and/or other materials.
[0168] Further, mesenchymal stem cells (lineage committed or
uncommitted progenitor cells) are advantageous "stromal" cells for
inoculation onto the framework. The cells may differentiate into
osteocytes, fibroblasts of the tendons and ligaments, marrow
stromal cells, adipocytes and other cells of connective tissue,
chondrocytes, depending on endogens or supplemented growth and
regulatory factors and other factors including prostaglandin,
interleukins and naturally-occurring chalones which regulate
proliferation and/or differentiation.
[0169] Fibroblasts may be readily isolated by disaggregating an
appropriate organ or tissue which is to serve as the source of the
fibroblasts. This may be readily accomplished using techniques
known to those skilled in the art. For example, the tissue or organ
can be disaggregated mechanically and/or treated with digestive
enzymes and/or chelating agents that weaken the connections between
neighboring cells making it possible to disperse the tissue into a
suspension of individual cells without appreciable cell breakage.
Enzymatic dissociation can be accomplished by mincing the tissue
and treating the minced tissue with any of a number of digestive
enzymes either alone or in combination. These include but are not
limited to trypsin, chymotrypsin, collagenase, elastase, and/or
hyaluronidase, DNase, pronase, dispase etc. Mechanical disruption
can also be accomplished by a number of methods including, but not
limited to, the use of grinders, blenders, sieves, homogenizers,
pressure cells, or insonators to name but a few. For a review of
tissue disaggregation techniques, see Freshney, Culture of Animal
Cells: A Manual of Basic Technique, 2d Ed., A.R. Liss, Inc., New
York, 1987, Ch. 9, pp. 107-126.
[0170] Once the tissue has been reduced to a suspension of
individual cells, the suspension can be fractionated into
subpopulations from which the fibroblasts and/or other stromal
cells and/or elements can be obtained. This also may be
accomplished using standard techniques for cell separation
including, but not limited to, cloning and selection of specific
cell types, selective destruction of unwanted cells (negative
selection), separation based upon differential cell agglutinability
in the mixed population, freeze-thaw procedures, differential
adherence properties of the cells in the mixed population,
filtration, conventional and zonal centrifugation, centrifugal
elutriation (counterstreaming centrifugation), unit gravity
separation, countercurrent distribution, electrophoresis and
fluorescence-activated cell sorting. For a review of clonal
selection and cell separation techniques, see Freshney, Culture of
Animal Cells: A Manual of Basic Techniques, 2d Ed., A.R. Liss,
Inc., New York, 1987, Ch. 11 and 12, pp. 137-168.
[0171] The isolation of fibroblasts may, for example, be carried
out as follows: fresh tissue samples are thoroughly washed and
minced in Hanks balanced salt solution (HBSS) in order to remove
serum. The minced tissue is incubated from 1-12 hours in a freshly
prepared solution of a dissociating enzyme such as trypsin. After
such incubation, the dissociated cells are suspended, pelleted by
centrifugation and plated onto culture dishes. All fibroblasts will
attach before other cells, therefore, appropriate stromal cells can
be selectively isolated and grown. The isolated fibroblasts can
then be grown to confluency, lifted from the confluent culture and
inoculated onto the three-dimensional matrix (see, Naughton et al.,
1987, J. Med. 18 (3 and 4) 219-250). Inoculation of the
three-dimensional framework with a high concentration of stromal
cells, e.g., approximately 10.sup.6 to 5.times.10.sup.7 cells/ml,
will result in the establishment of the three-dimensional stromal
tissue in shorter periods of time.
[0172] After inoculation of the stromal cells, the
three-dimensional framework should be incubated in an appropriate
nutrient medium. As previously mentioned, many commercially
available media such as RPMI 1640, Fisher's, Iscove's, McCoy's, and
the like may be suitable for use. It is important that the
three-dimensional stromal cell cultures be suspended or floated in
the medium during the incubation period in order to maximize
proliferative activity. The culture is "fed" periodically and the
conditioned media is recovered and processed as described below in
Sections 5.6 and 5.7. Thus, depending upon the tissue to be
cultured and the collagen types desired, the appropriate stromal
cell(s) may be selected to inoculate the three-dimensional
matrix.
[0173] During incubation of the three-dimensional stromal cell
cultures, proliferating cells may be released from the matrix.
These released cells may stick to the walls of the culture vessel
where they may continue to proliferate and form a confluent
monolayer. This should be prevented or minimized, for example, by
removal of the released cells during feeding, or by transferring
the three-dimensional stromal culture to a new culture vessel. The
presence of a confluent monolayer in the vessel will "shut down"
the growth of cells in the three-dimensional matrix and/or culture.
Removal of the confluent monolayer or transfer of the culture to
fresh media in a new vessel will restore proliferative activity of
the three-dimensional culture system. It should be noted that the
conditioned media is processed, if necessary, so that it does not
contain any whole cells (unless whole cells are used for a specific
application). Such removal or transfers should be done in any
culture vessel which has a stromal monolayer exceeding 25%
confluency. Alternatively, the culture system could be agitated to
prevent the released cells from sticking, or instead of
periodically feeding the cultures, the culture system could be set
up so that fresh media continuously flows through the system. The
flow rate could be adjusted to both maximize proliferation within
the three-dimensional culture, and to wash out and remove cells
released from the culture, so that they will not stick to the walls
of the vessel and grow to confluence.
[0174] Other cells, such as parenchymal cells, may be inoculated
and grown on the three-dimensional living stromal tissue.
[0175] b. Innoculation of Additional Cells into the Cell
Cultures
[0176] Once the cell culture has reached a particular degree of
growth, additional cells may be inoculated into the cell culture
and cultured along with the cells of the cell culture. The
additional cells are grown on the living tissue in vitro to form a
cultured counterpart of the native tissue and condition the media
by elaborating extracellular products into the media at ratios
resembling physiological levels. A high concentration of cells in
the inoculum will advantageously result in increased proliferation
in culture much sooner than will low concentrations.
[0177] The cells chosen for inoculation will depend upon the tissue
to be cultured, which may include, but is not limited to, bone
marrow, skin, liver, pancreas, kidney, neurological tissue, adrenal
gland, mucosal epithelium, and smooth muscle, to name but a few.
Such cells with elaborate characteristic extracellular proteins
such as certain growth factors into the media resulting in media
optimized for certain tissue specific applications.
[0178] For example, and not by way of limitation, a variety of
epithelial cells can be cultured on the living tissue. Examples of
such epithelial cells include, but are not limited to,
keratinocytes, oral mucosa and gastrointestinal (G.I.) tract cells.
Such epithelial cells may be isolated by enzymatic treatment of the
tissue according to methods known in the art, followed by expansion
of these cells in culture and application of epithelial cells to,
for example, the three-dimensional stromal support cell matrix. The
presence of the stromal support provides growth factors and other
proteins which promote normal division and differentiation of
epithelial cells.
[0179] In general, this inoculum should include the "stem" cell
(also called the "reserve" cell) for that tissue; i.e., those cells
which generate new cells that will mature into the specialized
cells that form the various components of the tissue.
[0180] The parenchymal or other surface layer cells used in the
inoculum may be obtained from cell suspensions prepared by
disaggregating the desired tissue using standard techniques
described above for obtaining stromal cells. The entire cellular
suspension itself could be used to inoculate the three-dimensional
living stromal tissue. As a result, the regenerative cells
contained within the homogenate will proliferate, mature, and
differentiate properly on the matrix, whereas non-regenerative
cells will not. Alternatively, particular cell types may be
isolated from appropriate fractions of the cellular suspension
using standard techniques described above for fractionating stromal
cells. Where the "stem" cells or "reserve" cells can be readily
isolated, these may be used to preferentially inoculate the
three-dimensional stromal support. For example, when culturing bone
marrow, the stroma may be inoculated with bone marrow cells, either
fresh or derived from a cryopreserved sample. When culturing skin,
the three-dimensional stroma may be inoculated with melanocytes and
keratinocytes. When culturing liver, the three-dimensional stroma
may be inoculated with hepatocytes. When culturing pancreas, the
three-dimensional stroma may be inoculated with pancreatic
endocrine cells. For a review of methods which may be utilized to
obtain parenchymal cells from various tissues, see, Freshney,
Culture of Animal Cells. A Manual of Basic Technique, 2d Ed., A.R.
Liss, Inc., New York, 1987, Ch. 20, pp. 257-288.
[0181] In fact, different proportions of the various types of
collagen deposited on the stromal matrix prior to inoculation can
affect the growth of the later-inoculated tissue-specific cells.
For example, for optimal growth of hematopoietic cells, the matrix
should preferably contain collagen types III, IV and I in an
approximate ratio of 6:3:1 in the initial matrix. For three
dimensional skin culture systems, collagen types I and III are
preferably deposited in the initial matrix. The proportions of
collagen types deposited can be manipulated or enhanced by
selecting fibroblasts which elaborate the appropriate collagen
type. This can be accomplished using monoclonal antibodies of an
appropriate isotype or subclass that is capable of activating
complement, and which define particular collagen types. These
antibodies and complement can be used to negatively select the
fibroblasts which express the desired collagen type. Alternatively,
the stromal cells used to inoculate the matrix can be a mixture of
cells which synthesize the appropriate collagen type desired. The
distribution and origins of various types of collagen is shown in
Table I.
TABLE-US-00001 TABLE 1 DISTRIBUTIONS AND ORIGINS OF VARIOUS TYPES
OF COLLAGEN Collagen Type Principal Tissue Distribution Cells of
Origin I Loose and dense ordinary Fibroblasts and reticular cells;
connective tissue; collagen smooth muscle cells fibers
Fibrocartilage Bone Osteoblast Dentin Odontoblasts II Hyaline and
elastic cartilage Chondrocytes Vitreous body of eye Retinal cells
III Loose connective tissue; Fibroblasts and reticular cells
reticular fibers Papillary layer of dermis Blood vessels Smooth
muscle cells; endothelial cells IV Basement membranes Epithelial
and endothelial cells Lens capsule of eye Lens fibers V Fetal
membranes; placenta Fibroblast Basement membranes Bone Smooth
muscle Smooth muscle cells Fibroblasts VI Connective Tissue VII
Epithelial basement Fibroblasts, keratinocytes membranes, anchoring
fibrils Cornea Corneal fibroblasts VIII Cartilage IX Hypertrophic
cartilage X Cartilage Fibroblasts XI Papillary dermis Fibroblasts
XII Reticular dermis Fibroblasts XIV, P 170 bullous pemphigoid
Keratinocytes undulin antigen
[0182] During incubation, the cell culture system should be
suspended or floated in the nutrient medium. Cultures should be fed
with fresh media periodically. Again, care should be taken to
prevent cells released from the culture from sticking to the walls
of the vessel where they could proliferate and form a confluent
monolayer. The release of cells from the culture appears to occur
more readily when culturing diffuse tissues as opposed to
structured tissues. For example, the skin culture is histologically
and morphologically normal; the distinct dermal and epidermal
layers do not release cells into the surrounding media. By
contrast, the bone marrow cultures release mature non-adherent
cells into the medium much the way such cells are released in
marrow in vivo. As previously explained, should the released cells
stick to the culture vessel and form a confluent monolayer, the
proliferation of the three-dimensional culture will be "shut down".
This can be avoided by removal of released cells during feeding,
transfer of the culture to a new vessel, by agitation of the
culture to prevent sticking of released cells to the vessel wall,
or by the continuous flow of fresh media at a rate sufficient to
replenish nutrients in the culture and remove released cells. As
previously mentioned, the conditioned media is processed, if
necessary, so that it is free of whole cells and cellular
debris.
[0183] The growth and activity of cells in culture can be affected
by a variety of growth factors such as insulin, growth hormone,
somatomedins, colony stimulating factors, erythropoietin, epidermal
growth factor, hepatic erythropoietic factor (hepatopoietin), and
liver-cell growth factor. Other factors which regulate
proliferation and/or differentiation include prostaglandins,
interleukins, and naturally-occurring chalones.
[0184] C. Conditioned Growth Media and Cell Extract
[0185] 1. Recovering the Conditioned Growth Media or Cell Extract
from the Cell Cultures
[0186] The resultant conditioned growth medium may be separated
from the cell cultures by any means known to the skilled artisan.
In one embodiment, the conditioned growth medium is pumped out of
the cell culture system and processed for use. In some embodiments,
the conditioned growth medium is recovered once the cell cultures
have conditioned the growth medium to a sufficient extent (i.e.,
once the medium is conditioned so that extracellular proteins such
as growth factors have reached desirable levels in the medium).
Preferably, the conditioned growth medium is recovered at the later
stages of growth of the tissue when the level of certain growth
factors and connective tissue protein secretion is at its highest
level (See FIG. 1). In a preferred embodiment, the conditioned
growth medium is recovered after exposure of the growth medium to
the cells at days 10 through day 14 of culturing.
[0187] The cell extract produced by the cell cultures can be
isolated from the conditioned growth medium by any means known to
the skilled artisan.
[0188] 2. Processing the Conditioned Growth Media or Cell
Extract
[0189] Following recovery of the conditioned growth medium or cell
extract, it may be necessary to further process the resulting
supernatant. Such processing may include, but are not limited to,
concentration by a water flux filtration device or by defiltration
using the methods described in Cell & Tissue Culture:
Laboratory Procedures, supra, pp 29 D:0.1-29D:0.4.
[0190] Additionally, the medium may be concentrated 10 to 20 fold
using a positive pressure concentration device having a filter with
a 10,000 ml cut-off (Amicon, Beverly, Mass.).
[0191] Also, the conditioned growth medium or cell extract may be
further processed for product isolation and purification to remove
unwanted proteases, for example. The methods used for product
isolation and purification so that optimal biological activity is
maintained will be readily apparent to one of ordinary skill in the
art. For example, it may be desirous to purify a growth factor,
regulatory factor, peptide hormone, antibody, etc. Such methods
include, but are not limited to, gel chromatography (using matrices
such as sephadex) ion exchange, metal chelate affinity
chromatography with an insoluble matrix such as cross-linked
agarose, HPLC purification and hydrophobic interaction
chromatography of the conditioned media. Such techniques are
described in greater detail in Cell & Tissue Culture:
Laboratory Procedures, supra. Depending upon the desired
application of the conditioned growth medium or cell extract,
and/or products derived thereof, appropriate measures must be taken
to maintain sterility. Alternatively, sterilization may be
necessary and can be accomplished by methods known to one of
ordinary skill in the art, such as, for example, heat and/or filter
sterilization taking care to preserve the desired biological
activity.
[0192] 3. Some Products Found in the Conditioned Growth Media or
Cell Extract
[0193] Table 2 below lists the concentration of a number of growth
factors determined by ELISA (enzyme linked immuno assay) to be in
Applicants' conditioned medium which previously supported the
growth of the cells grown in Dermagraft.RTM. tissue culture. It is
believed that a cell extract according to the present invention
would also contain these growth factors. It should be understood
that the following list is not an all inclusive list of factors and
is provided solely to further characterize the conditioned medium
by providing the concentration of some of the biologically active
factors present in the medium of the disclosure.
TABLE-US-00002 TABLE 2 Growth Factor Concentrations in Conditioned
Medium as Measured by ELISA VEGF 3.2 ng/ml G-CSF 2.3 ng/ml IL-8 0.9
ng/ml IL-8 3.2 ng/ml KGF 1.67 ng/ml TGF-.beta. 0.8 ng/ml
[0194] A variety of methods have been utilized to quantify and
characterize the major molecular components secreted by fibroblasts
found in the three-dimensional tissue cultures TransCyte.TM. and
Dermagraft.RTM.). The human matrix proteins and glycosaminoglycans
(GAGs) present in TransCyte.TM. and Dermagraft.RTM. include, but
are not limited to, collagen I, III, fibronectin, tenascin,
decorin, versican betaglycan, syndecan as well as other components
(data not shown). These secreted proteins and GAGs serve major
structural functions as well as stimulate cell division, migration,
adhesion and signal transduction. The deposition of
glycosaminoglycans (deposition volume is dependent on period of
growth) and collagen (deposition volume is not dependent on period
of growth) in the three-dimensional growth systems are illustrated
in FIG. 1. The components have been measured by ELISA, Western blot
analysis, immuno histochemistry and PCR. For example, some of the
components found in TransCyte.TM. include collagen I, III, and VII
(RNA), fibronectin, tenascin, thrombospondin 2, elastin,
proteoglycans, decorin, versican as well as other components (data
not shown). Activity of these components in tissue development,
healing, and normal function have been well described.
Additionally, Applicants describe certain effects of the human
bioengineered matrix on cell function in vitro. For example,
Applicants have noted that cell proliferation is increased by
adding bioengineered matrix. To study its effects on cell
proliferation, matrix was physically removed from TransCyte.TM. and
Dermagraft.RTM. and added in varying dilutions to monolayer
cultures of human fibroblasts and keratinocytes. The results of
increased cell proliferation are shown in FIG. 2.
[0195] Further, Applicants note the effect of three dimensional
conditioned medium on the preparation and composition of
three-dimensional tissues was examined by measuring the amount of
collagen secreted into the extracellular matrix of tissues cultured
in the presence of serum-free medium, medium or three dimensional
conditioned medium. The effect of three dimensional conditioned
medium on the preparation and composition of three-dimensional
tissues was examined by measuring the amount of collagen secreted
into the extracellular matrix of tissues cultured in the presence
of serum-free medium, medium or three dimensional conditioned
medium. The conditioned medium of the disclosure significantly
increases collagen deposition of tissue in vitro.
[0196] D. Metabolizing the Conditioned Growth Media or Cell
Extract
[0197] 1. The Yeast
[0198] The process of metabolizing the conditioned growth media or
cell extract can occur with a variety of microorganisms such as,
for example, yeast, bacillus, molds, plant cells and the like.
Especially preferred for the present disclosure are ferments made
using yeast. As used herein, the term "yeast" is meant to encompass
a single yeast cell, multiple yeast cells and/or a culture of yeast
cells. The yeast can be of various fungal families, known to those
skilled in the art including, but not limited to: Neurospora,
Ceratostomella, Claviceps, Xylaria, Rosellinia, Helotium,
Sclerotinia, Tulostoma, Rhizopogon, Truncocolumella, Mucor,
Rhizopus, Entomophthora, Dictostylium, Blastocladia, Synchytrium,
Saprolegnia, Peronospora, Albugo, Pythium, Phytophthora,
Plasmodiophora, Tuber, Hydnum, Lecanora, Roccella, Pertusaria,
Usnea, Evernia, Ramalina, Alectoria, Cladonia, Parmelia, Cetraria,
Agaricus, Cantharellus, Omphalotus, Coprinus, Lactarius, Marasmius,
Pleurotus, Pholiota, Russula, Lactarius, Stropharia, Entoloma,
Lepiotaceae, Corticium, Pellicularia, Tricholoma, Volvaria,
Clitocybe, Flammulina, Saccharomyces, Schizosaccharomyces,
Saccharomycetaceae, Eurotium, Aspergillus, Thielavia, Peziza,
Plectania, Morchella, Wynnea, Helvella, Gyromitra, Phallales,
Dictyophera, Mutinus, Clathrus, Pseudocolus, Lycoperdon, Calvatia,
Geastrum, Radiigera, Astreus, Nidularia, Gastrocybe, Macowanites,
Gastroboletus, Albatrellus, Neolentinus, Nigroporus, Oligoporus,
Polyporus, Fistulina, Fomes, Boletus, Fuscoboletinus, Leccinum,
Phylloporus, Suillus, Strobilomyces, Boletellus, Tremella,
Auricularia, Dacrymyces, Melampsora, Cronartium, Puccinia,
Gymnosporangium, Tilletia, Urocystis, Septobasidium, Hygrocybe,
Hygrophorus, Hygrotrama, Neohygrophorus, Cortinarius, Gymnopilus,
Trichophyton, Microsporum, Monilia, Candida, Cercosporella,
Penicillium, Blastomyces, Cercospora, Ustilaginoidea, Tuber
cularia, Fusarium, Rhizoctinia, Ozonium, Sclerotium, Geoglossum, or
Armillaria. Of particular interest are the fungi belonging to the
family Saccharomycetaceae. Of greater interest are the fungi
belonging to the genus Pichia. Of most interest are the fungi
belonging to the species pastoris. In a preferred embodiment,
Pichia pastoris is used in the fermentation process. In some
embodiments, the yeast is aerobically grown.
[0199] 2. Fermentation Process
[0200] The yeast fermentation process can be carried out in a
stirred tank bio-reactor. Examples of such bioreactors might
include for example, fermentors available from New Brunswick
Scientific, Edison N.J. or Applikon Biotechnology Foster City
Calif.
[0201] The yeast extracts of the present disclosure include
cytoplasmic and extra-cellular components of the yeast which
include, but are not limited to, the nutrient broth, cellular
protein material, cellular nucleic material, cellular protoplasmic
material and/or cell wall components. In some embodiments, the
extracts are relatively water soluble, for example, equal or more
than 1-gram of yeast extracts dissolve in 1-gram of water. The
extracts may also be soluble in water/organic solvent mixtures such
as, but not limited to, aqueous glycols and aqueous glycerols.
[0202] 3. Recovering/Processing the Metabolized Conditioned Growth
Media or Metabolized Cell Extract
[0203] The metabolized conditioned growth media or metabolized cell
extract may be recovered and/or processed according to the
procedures described above for recovering and processing the
conditioned growth media or cell extract. In some embodiments, the
processing includes several steps and may involve the use of
techniques, including but not limited to continuous flow
centrifugation, lenticular dead-end filtration for clarification of
bio-mass, and tangential flow filtration with sterile and
ultra-filtration capabilities. In one embodiment, the final
processing step includes tangential flow filtration of a specific
molecular weight cut-off in order to selectively target bio-active
components. See, e.g., "Yeast Protocols" W. Xiao ed. (Humana Press
2006), which is incorporated by reference herein.
[0204] E. Activity/Characterization of the Metabolized Conditioned
Growth Media or Metabolized Cell Extract
[0205] During the yeast metabolism described above, therapeutic
products present in the conditioned growth media and cell extract,
including but not limited to enzymes, hormones, cytokines,
antigens, antibodies, clotting factors, and regulatory proteins,
are metabolized by the yeast. The term metabolized is known to
those skilled in the art but essentially means that the yeast begin
the process of digesting the components of the conditioned nutrient
media or cell extract. The process of metabolizing can render
larger molecules as small, more readily available components that
are more useful for the applications of the present invention than
the unmetabolized conditioned growth media or cell extracts.
Therapeutic proteins that may be metabolized include, but are not
limited to, inflammatory mediators, angiogenic factors, Factor
VIII, Factor IX, erythropoietin (EPO), alpha-1 antitrypsin,
calcitonin, glucocerebrosidase, human growth hormone and
derivatives, low density lipoprotein (LDL), and apolipoprotein E,
IL-2 receptor and its antagonists, insulin, globin,
immunoglobulins, catalytic antibodies, the interleukins (ILs),
insulin-like growth factors, superoxide dismutase, immune responder
modifiers, BMPs (bone morphogenic proteins) parathyroid hormone and
interferon, nerve growth factors, tissue plasminogen activators,
and colony stimulating factors (CSFs). These metabolized components
may maintain the activity of their precursors, or may be
transformed to have greater enhanced activity, including the
downregulation or upregulation of various physiological systems
that affect, for example, skin maintenance and condition.
[0206] The medium or extract may be further processed to
concentrate or reduce one or more metabolized factor or component
contained within the medium or extract, for example, enrichment of
a metabolized growth factor using immunoaffinity chromatography or,
conversely, removal of a less desirable component, for any given
application as described herein.
[0207] In other embodiments, the metabolized conditioned growth
media and/or metabolized cell extract of the present disclosure
contains one or more cytoplasmic and extra-cellular components of
the yeast which include, but are not limited to, the nutrient
broth, cellular protein material, cellular nucleic material,
cellular protoplasmic material and/or cell wall components.
III. Compositions
[0208] In one embodiment, the disclosure provides compositions
comprising the metabolized conditioned growth medium or the
metabolized cell extract, and an acceptable carrier. In some
embodiments, the composition is a personal care composition.
[0209] In some embodiments, the metabolized conditioned growth
medium or the metabolized cell extract is present in the
composition in an amount of about 0.0001% to about 95% by weight of
the composition. In other embodiments, the metabolized conditioned
growth medium or the metabolized cell extract is present in the
composition in an amount of about 0.01% to about 50% by weight of
the composition. In other embodiments, the metabolized conditioned
growth medium or the metabolized cell extract is present in the
composition in an amount of about 5% to about 30% by weight of the
composition. In other embodiments, the metabolized conditioned
growth medium or the metabolized cell extract is present in the
composition in an amount of about 10% to about 20% by weight of the
composition. In other embodiments, the metabolized conditioned
growth medium or the metabolized cell extract is present in the
composition in an amount of about 13% to about 17% by weight of the
composition. In other embodiments, metabolized conditioned growth
medium or the metabolized cell extract is present in the
composition in an amount of about 0.01% to about 10% by weight of
the composition. In one embodiment, the metabolized conditioned
growth medium or the metabolized cell extract is present in the
composition in an amount of about 10%, about 15%, or about 20% by
weight of the composition.
[0210] In other embodiments, the composition further comprises at
least one of water, a preservative, a surfactant, an emulsifier, a
conditioner, an emollient, a wax, an oil, a polymer, a thickener, a
fixative, a colorant, a humectant, a moisturizer, a stabilizer, a
diluent, a solvent and a fragrance. Non-limiting examples of
additional ingredients of the compositions include, but are not
limited to, Palmitoyl trippeptide 5, hydrolyzed silk (Sericin),
Dipalmitoyl hydroxyproline, Ergothioneine (EGT), Ubiquinone
(Coenzyme Q10), Camellia Sinensis (Green Tea) Leaf Extract,
Tetrahexyldecyl Ascorbate, Tocopheryl Acetate, Rubus Fruticosus
(Blackberry) Leaf Extract, Saccharomyces Ferment Filtrate Lysate,
Alpha-Arbutin, Gamma Aminobutyric Acid, and/or Hyaluronic Filling
Spheres.
[0211] In one embodiment, the composition further comprises at
least one preservative. Suitable preservatives include, but are not
limited to, acids, alcohols, glycols, parabens, quaternary-nitrogen
containing compounds, isothiazolinones, aldehyde-releasing
compounds and halogenated compounds. Illustrative alcohols include
phenoxyethanol, isopropyl alcohol, and benzyl alcohol; illustrative
glycols include propylene, butylene and pentylene glycols;
illustrative parabens include (also known as parahydroxybenzioc
acids) methyl, propyl and butyl parabens; illustrative quaternary
nitrogen containing compounds include benzalkonium chloride,
Quartenium 15; illustrative isothiazoles include
methylisothiazoline, methychlorolisothiazoline; illustrative
aldehyde releasing agents include DMDM hydantion, imiadolidinyl
urea and diazolidinyl urea; illustrative antioxidants include
butylated hydroxytoluene, tocopherol and illustrative halogenated
compounds include triclosan and chlorohexidene digluconate.
Examples of preservatives useful for the purpose of the present
disclosure can be found in Steinberg, D. "Frequency of Use of
Preservatives 2007". Cosmet. Toilet. 117, 41-44 (2002) and,
"Preservative Encyclopedia" Cosmet. Toilet. 117, 80-96 (2002). In
addition, enzyme preservative systems such as those described in
the article by Ciccognani D. Cosmetic Preservation Using Enzymes,
in "Cosmetic and Drug Microbiology", Orth D S ed., Francis &
Taylor, Boca Raton, Fla. (2006) can also be effective for use with
the composition of the present disclosure.
[0212] In one embodiment, the composition further comprises an
active ingredient. Suitable active ingredients include, but are not
limited to botanicals, nutraceuticals, cosmeceuticals,
therapeutics, pharmaceuticals, antimicrobials, steroidal hormones,
antidandruff agents, anti-acne components, sunscreens, antibiotics,
antivirals, antifungals, steroids, analgesics, antitumor drugs,
investigational drugs or any compounds which would result in a
complimentary or synergistic combination with the factors in the
metabolized conditioned media or metabolized cell extract.
[0213] The compositions may be in the form of tablets, capsules,
skin patches, inhalers, eye drops, nose drops, ear drops,
suppositories, creams, ointments, injectables, hydrogels and into
any other appropriate formulation known to one of skill in the art.
For oral administration the pharmaceutical compositions may take
the form of, for example, tablets or capsules prepared by
conventional means with acceptable excipients or carriers such as
binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolae); or
wetting agents (e.g., sodium lauryl sulphate). Tablets may be
coated using methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
acceptable excipients or carriers such as suspending agents (e.g.,
sorbitol syrup cellulose derivatives or hydrogenated edible fats);
emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles
(e.g., almond oil, oily esters, ethyl alcohol or fractionated
vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0214] In one embodiment, the compositions are in a form suitable
for cosmetic application including, but not limited to, lotions,
ointments, creams, sprays, spritzes, aqueous or aqueous-alcoholic
mixture gels, mousses, patches, pads, masks, moistened clothes,
wipes, solid sticks, clear sticks, lip sticks, aerosol creams,
anhydrous powders, talcs, tonics, oils, emulsions or bath
salts.
[0215] In one embodiment, the metabolized conditioned growth medium
or the metabolized cell extract in the composition is present in a
chemical delivery vehicle. Chemical delivery vehicles include, but
are not limited to, liposomes, niosomes, sub-micron emulsions,
polymeric encapsulates, gels, creams, lotions, and combinations
thereof. In some embodiments, the metabolized conditioned growth
medium or the metabolized cell extract is encapsulated within an
encapsulant.
[0216] The compositions of the disclosure may be delivered to a
subject via a variety of routes using standard procedures well
known to those of skill in the art. For example, such delivery may
be site-specific, oral, nasal, intravenous, subcutaneous,
intradermal, transdermal, intramuscular or intraperitoneal
administration. Also, they may be formulated to function as
controlled or slow release vehicles.
[0217] The metabolized conditioned growth medium and/or metabolized
cell extract may be used in any state, i.e., liquid or solid,
frozen lyophilized or dried into a powder, as a film for topical
wound treatments and anti-adhesion applications, as an injectable,
see PCT WO 96/39101, incorporated herein by reference it its
entirety.
[0218] Alternatively, the conditioned cell medium of the present
disclosure may be formulated with polymerizable or cross-linking
hydrogels as described in U.S. Pat. Nos. 5,709,854; 5,516,532;
5,654,381; and WO 98/52543, each of which is incorporated herein by
reference in its entirety. Examples of materials which can be used
to form a hydrogel include modified alginates. Alginate is a
carbohydrate polymer isolated from seaweed, which can be
cross-linked to form a hydrogel by exposure to a divalent cation
such as calcium, as described, for example in WO 94/25080, the
disclosure of which is incorporated herein by reference. Alginate
is ionically cross-linked in the presence of divalent cations, in
water, at room temperature, to form a hydrogel matrix. As used
herein, the term "modified alginates" refers to chemically modified
alginates with modified hydrogel properties.
[0219] Additionally, polysaccharides which gel by exposure to
monovalent cations, including bacterial polysaccharides, such as
gellan gum, and plant polysaccharides, such as carrageenans, may be
cross-linked to form a hydrogel using methods analogous to those
available for the cross-linking of alginates described above.
[0220] Modified hyaluronic acid derivatives are particularly
useful. As used herein, the term "hyaluronic acids" refers to
natural and chemically modified hyaluronic acids. Modified
hyaluronic acids may be designed and synthesized with preselected
chemical modifications to adjust the rate and degree of
cross-linking and biodegradation.
[0221] Covalently cross-linkable hydrogel precursors also are
useful. For example, a water soluble polyamine, such as chitosan,
can be cross-linked with a water soluble diisothiocyanate, such as
polyethylene glycol diisothiocyanate.
[0222] Alternatively, polymers may be utilized which include
substituents which are cross-linked by a radical reaction upon
contact with a radical initiator. For example, polymers including
ethylenically unsaturated groups which can be photochemically
cross-linked which may be utilized, as disclosed in WO 93/17669,
the disclosure of which is incorporated herein by reference. In
this embodiment, water soluble macromers that include at least one
water soluble region, a biodegradable region, and at least two free
radical-polymerizable regions, are provided. Examples of these
macromers are PEG-oligolactyl-acrylates, wherein the acrylate
groups are polymerized using radical initiating systems, such as an
eosin dye, or by brief exposure to ultraviolet or visible light.
Additionally, water soluble polymers which include cinnamoyl groups
which may be photochemically cross-linked may be utilized, as
disclosed in Matsuda et al., ASAID Trans., 38: 154-157 (1992).
[0223] The preferred polymerizable groups are acrylates,
diacrylates, oligoacrylates, dimethacrylates, oligomethacrylates,
and other biologically acceptable photopolymerizable groups.
Acrylates are the most preferred active species polymerizable
group.
[0224] Naturally occurring and synthetic polymers may be modified
using chemical reactions available in the art and described, for
example, in March, "Advanced Organic Chemistry", 4.sup.th Edition,
1992, Wiley-Interscience Publication, New York.
[0225] Polymerization is preferably initiated using photo
initiators. Useful photo initiators are those which can be used to
initiate polymerization of the macromers without cytotoxicity and
within a short time frame, minutes at most and most preferably
seconds.
[0226] Numerous dyes can be used for photopolymerization. Suitable
dyes are well known to those of skill in the art. Preferred dyes
include erythrosin, phloxime, rose bengal, thonine, camphorquinone,
ethyl eosin, eosin, methylene blue, riboflavin,
2,2-dimethyl-2-phenylacetophenone, 2-methoxy-2-phenylacetophenone,
2,2-dimethoxy-2-phenyl acetophenone, other acetophenone
derivatives, and camphorquinone. Suitable cocatalysts include
amines such as N-methyl diethanolamine, N, N-dimethyl benzylamine,
triethanol amine, trithylamine, dibenzyl amine,
N-benzylethanolamine, -isopropyl benzylamine. Triethanolamine is a
preferred cocatalyst.
[0227] In another embodiment, the metabolized conditioned growth
media and/or metabolized cell extract of the disclosure, or
alternatively particular metabolized extracellular matrix proteins
elaborated into the media, are used to provide an excellent
substance to coat sutures. The metabolized, naturally secreted
extracellular matrix provides the conditioned media with an
activity of type I and type III collagens, fibronectin, terascin,
glycosaminologycans, acid and basic FGF, TGF-.alpha. and
TGF-.beta., KGF, versican, decorin and various other secreted human
dermal matrix proteins. Similarly, the conditioned cell media of
the disclosure or the extracellular matrix proteins derived from
the conditioned media may be used to coat conventional implantation
devices, including vascular prosthesis, in surgical approaches to
correct defects in the body--resulting in superior implantation
devices. The implants should be made of biocompatible, inert
materials that replace or substitute for the defective function and
made of either non-biodegradable materials or biodegradable
materials. By coating implantation devices with the medium
containing these extracellular proteins, the implant invites proper
cellular attachments resulting in superior tissue at the
implantation site. Thus, sutures, bandages, and implants coated
with conditioned cell media, or proteins derived from the media,
enhance the recruitment of cells, such as leukocytes and
fibroblasts into the injured area and induce cell proliferation and
differentiation resulting in improved wound healing.
[0228] In another embodiment, the metabolized conditioned growth
media and/or metabolized cell extract of the disclosure may be
formulated with a pharmaceutically acceptable carrier as a vehicle
for internal administration. Also, the medium may be further
processed to concentrate or reduce one or more factor or component
contained within the medium, for example, enrichment of a growth
factor or other protein using immunoaffinity chromatography or,
conversely, removal of a less desirable component, for any given
application as described herein.
IV. Methods for Treating or Preventing Conditions
[0229] In one embodiment, the disclosure provides methods for
preventing or treating a condition in a subject comprising
administering to the subject a therapeutically effective amount of
a composition comprising: an acceptable carrier and a metabolized
conditioned growth medium.
[0230] In another embodiment, the disclosure provides methods for
preventing or treating a condition in a subject comprising
administering to the subject a therapeutically effective amount of
a composition comprising: an acceptable carrier and a metabolized
cell extract.
[0231] Conditions to be treated include, but are not limited to,
skin conditions, including cosmetic defects, congenital defects,
hair loss and acquired defects. In some embodiments, the conditions
to be treated include, but are not limited to, fine lines and
wrinkles; age spots and dyspigmentation; decreased skin texture,
tone and elasticity; roughness and photo damage; decreased ability
of skin to regenerate itself; environmental damage; decreased
smoothness and tightness of skin; age spots; fine and coarse lines
and wrinkles; fine and coarse periocular wrinkles; nasolabial
folds; facial fine and coarse lines; decreased skin radiance and
evenness; decreased skin firmness; hyperpigmentation; dark spots
and/or patches; decreased skin brightness and youthful appearance;
photoaged skin; intrinsically and extrinsically aged skin; abnormal
skin cellular turnover; decreased skin barrier; decrease of skin's
ability to retain moisture; brown and red blotchiness; redness;
abnormal skin epidermal thickness; reduction of dermal epidermal
junction; increased pore size and number of pores; or a combination
thereof.
A. Skin Conditions
[0232] In some embodiments, the condition to treat or prevent is a
skin condition. In other embodiments, the skin condition is a
cosmetic defect, a congenital defect, hair loss or an acquired
defect. Skin conditions also include, but are not limited to, fine
lines and wrinkles; age spots and dyspigmentation; decreased skin
texture, tone and elasticity; roughness and photo damage; decreased
ability of skin to regenerate itself; environmental damage;
decreased smoothness and tightness of skin; age spots; fine and
coarse lines and wrinkles; fine and coarse periocular wrinkles;
nasolabial folds; facial fine and coarse lines; decreased skin
radiance and evenness; decreased skin firmness; hyperpigmentation;
dark spots and/or patches; decreased skin brightness and youthful
appearance; photoaged skin; intrinsically and extrinsically aged
skin; abnormal skin cellular turnover; decreased skin barrier;
decrease of skin's ability to retain moisture; brown and red
blotchiness; redness; abnormal skin epidermal thickness; reduction
of dermal epidermal junction; increased pore size and number of
pores; or a combination thereof.
[0233] In one non-limiting example, a topical composition as
described herein is applied to the skin. Application of the
compositions disclosed herein rejuvenates sun damaged and aging
skin; improves the appearance of fine lines and wrinkles; promotes
cell renewal; diminishes the appearance of age spots and
dyspigmentation; improves skin tone, texture and elasticity;
reduces roughness and photo damage; prevents or reduces
environmental damage; plumps the skin; brightens the skin; lightens
the skin; strengthens the ability of skin to regenerate itself;
improves the appearance of age spots; brightens and lightens age
spots; improves skin firmness, elasticity, resiliency; smoothes,
tightens, or fills in fine lines on the skin; reduces the
appearance of dark circles under the eye; improves lip texture or
condition; enhances natural lip color; increases lip volume;
promotes epithelialization of post-procedure skin; restores the
skin's barrier or moisture balance; improves the appearance of age
spots; improves the appearance of skin pigmentation, or a
combination thereof.
[0234] In certain embodiments, administration of a composition
described herein may result in at least a 2-fold improvement of one
or more symptoms or conditions. Folds improvement of one or more
symptoms or conditions include, but are not limited to, 3-fold,
5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 75-fold, 100-fold or
more, or any number therebetween. In certain embodiments,
administration of a composition described herein may result in
improvement of about 1% to about 100%, about 10% to about 90%,
about 20% to about 80%, about 30% to about 70%, about 40% to about
60%, or about 50%. In other embodiments, administration of a
composition described herein may result in improvement of one or
more symptoms or conditions of about 5%, about 10%, about 15%,
about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 100%, about 125%, about 150%
or more of one or more symptoms or conditions.
[0235] The effect of the metabolized conditioned growth media
and/or metabolized cell extract described above to influence human
skin can be measured in a number of ways known to those skilled in
the art. In particular, the metabolized conditioned growth media
and/or metabolized cell extract described above can be screened for
their effects on skin by employing analytical techniques such as,
for example, human genomic microarrays on specific skin cells such
as keratinocytes or fibroblasts, or by protein expression analysis
on individual skin cells, tissue models or ex vivo or in vivo skin
models. In these testing models, specific genes and or proteins may
be up-regulated or down-regulated as a result of the extract
treatment. Genes and proteins that are capable of regulating skin
conditions are of particular interest in the screening. Of
particular interest for the purpose of the present disclosure are
genes and proteins related to inflammation, extracellular matrix
expression, melanin regulation, skin moisturization, exfoliation
and the like. Of particular interest are proteins related to
cyclooxygenase expression, in particular cyclooxygenase-1 and 2 and
also extracellular matrix protein expression, in particular, types,
I, IV and VI collagen expression, and elastin and fibronectin
expression. In addition, the influence of the extract on skin
melanin expression is also of considerable interest.
[0236] The effect of the metabolized conditioned growth media
and/or metabolized cell extract of the disclosure on expression of
these and other important cutaneous proteins can be monitored by
human genomic microarray analysis and protein expression as well as
by non-invasive test methods well-known to those in skilled in the
art, including, but not limited to, improved moisturization,
wrinkle reduction, reduced pigmentation, improved skin tone and the
like. Application of the metabolized conditioned growth media
and/or metabolized cell extract of the disclosure can manifest
itself by measured reductions in skin wrinkles, for example, as
measured by techniques such as SiIFIo silicone modeling, PRIMOS and
VISIO photographic systems and the like. In addition,
moisturization might be measured using transepidermal water loss
(TEWL) or cutometer or corneometric measurements. Likewise, skin
pigmentation could be measured using a chromometer. Such testing
technologies are well-known to those skilled in the art and can be
found in the "Handbook of Non-Invasive Methods and the Skin",
2.sup.nd edition, Serup J, Jemec G B E, Grove G L (ed.), Taylor and
Francis Boca Raton Fla. 2006.
[0237] Human gene microarray analysis on human epidermal
keratinocytes indicates that the metabolized conditioned growth
medium up-regulates several genes involved in various skin
functions. See Example 18. It is thus appreciated that the
yeast-metabolized conditioned growth media or metabolized cell
extract according to the present disclosure can be used in personal
care compositions for the treatment of skin conditions.
B. Wound Healing Applications
[0238] The metabolized conditioned growth media and/or metabolized
cell extract of the disclosure may be processed to promote wound
and burn healing. When tissue is injured, polypeptide growth
factors, which exhibit an array of biological activities, are
released into the wound to promote healing. Wound healing is a
complex process that involves several stages and is capable of
sealing breaches to the integument in a controlled manner to form
functionally competent tissue. The process begins with hemostasis
followed by an inflammatory phase involving neutrophils and
macrophages. The process continues with the development of
granulation tissue and re-epithelialization to close the wound.
Subsequently, scar tissue forms and is remodeled over the
succeeding months to an approximation of the original anatomical
structure. Ideally, scar tissue is minimal so that healthy tissue,
functionally competent tissue which histologically and
physiologically resembles the original normal tissue, may form.
[0239] Each stage of the healing process is controlled by cellular
interactions through regulatory proteins such as cytokines, growth
factors, and inflammatory mediators as well as cell contact
mechanisms. For example, inflammatory mediators such as IL-6, IL-8,
and G-CSF induce lymphocyte differentiation and acute phase
proteins, as well as neutrophil infiltration, maturation and
activation, processes that are important in the inflammatory stages
of wound healing. Other examples of regulatory proteins involved in
the wound healing process are VEGF that induces angiogenesis during
inflammation and granulation tissue formation, the BMP's which
induce bone formation, KGF that activates keratinocytes and
TGF-.beta.1 that induces deposition of extracellular matrix.
[0240] In chronic wounds, the healing process is interrupted at a
point subsequent to hemostasis and prior to re-epithelialization,
and is apparently unable to restart. Most of the inflammation seen
in the wound bed is related to infection, but the inflammation
gives rise to an environment rich in proteases that degrade
regulatory proteins and thus interfere with the wound healing
process.
[0241] The metabolized conditioned growth media and/or metabolized
cell extract of the disclosure contains activity thought to be
important in wound healing and which have been shown to be depleted
in in vivo models of wound healing. For example, the metabolized
conditioned growth media and/or metabolized cell extract of the
disclosure may contain an activity of one or more of a number of
growth factors that support healing process, such as VEGF, G-CSF,
IL-8, KGF, and TGF-.beta.. Furthermore, in some medical conditions,
such as diabetes, some of the regulatory proteins needed for wound
healing are in short supply. For example, it has been found in a
mouse model of non-insulin-dependent diabetes (e.g., the db/db
mouse) that secretion of VEGF and PDGF and expression of the PDGF
receptor are all depressed in wounds compared to the levels in
wounds of normal mice.
[0242] Also, the metabolized conditioned growth media and/or
metabolized cell extract of the disclosure is useful in the
treatment of other types of tissue damage, e.g., traumatic or
congenital, wherein the repair and/or regeneration of tissue
defects or damage is desired since an activity of many of these
growth factors are found in Applicants' metabolized conditioned
growth media and/or metabolized cell extract, including, for
example, fibroblast growth factors (FGFs), platelet derived growth
factors (PDGFs), epidermal growth factors (EGFs), bone
morphogenetic proteins (BMPs) and transforming growth factors
(TGFs); as well as those which modulate vascularization, such as
vascular endothelial growth factor (VEGF), keratinocyte growth
factor (KGF), and basic FGF; angiogenesis factors, and
antiangiogenesis factors. Stress proteins, such as GR 78 and MSP90
induce growth factors such as TGF-.beta.. TGF-.beta., including TGF
.beta.-1, TGF .beta.-2, TGF .beta.-3, TGF .beta.-4 and TGF
.beta.-5, regulate growth and differentiation and accelerate wound
healing (Noda et al. 1989, Endocrin. 124: 2991-2995; Goey et al.
1989, J. Immunol. 143: 877-880, Mutoe et al. 1987, Science 237:
1333-1335). Mitogens, such as PDGF increase the rate of cellularity
and granulation in tissue formation (Kohler et al. 1974, Exp. Cell.
Res. 87: 297-301). As previously mentioned, the cells are
preferably human to minimize immunogenicity problems.
[0243] Because the metabolized conditioned growth media and/or
metabolized cell extract of the disclosure contains an activity of
an array of wound healing factors, the conditioned media is
advantageously used in the treatment of wound and burn healing
including skin wounds, broken bones, gastric ulcers, pancreas,
liver, kidney, spleen, blood vessel injuries and other internal
wounds. Further, the metabolized conditioned growth media and/or
metabolized cell extract of the disclosure may be combined with
other medicinal ingredients such as antibiotics and analgesics.
Embodiments include formulations of the metabolized conditioned
growth media and/or metabolized cell extract of the disclosure with
a salve or ointment for topical applications.
[0244] Alternatively, the metabolized conditioned growth media
and/or metabolized cell extract of the disclosure may be combined
with a bandage (adhesive or non-adhesive) to promote and/or
accelerate wound healing.
[0245] Wounds at specialized tissues may require medium conditioned
by that specialized tissue. For example, injuries to neuronal
tissues may require proteins contained in medium conditioned by
neuronal cell cultures. Specific products may be derived, or
alternatively, the metabolized conditioned growth media and/or
metabolized cell extract may be enriched by immunoaffinity
chromatography or enhanced expression of a desired protein from the
specific medium such as, for example, NGF. NGF-controlled features
include, but are not limited to, the cholinergic neurotransmitter
function (acetylcholinesterase (AChE) and the
acetylcholine-synthesizing enzyme (ChAT)), neuronal cell size, and
expression of Type II NGF receptors; NGF is secreted into the
conditioned medium conditioned by glial and other neuronal cells
cultured on a three-dimensional stromal tissue, which can then be
used in a composition for nerve healing.
[0246] Deficits of endogenous NGF aggravate certain human
neurodegenerative disorders and there is an apparent inability of
injured adult CNS neurons to regenerate. Specifically, injury to a
nerve is followed by degeneration of the nerve fibers distal to the
injury, the result of isolation of the axon from the cell body. In
the central nervous system, there is no significant growth at the
site of injury typically leading to death of the damaged neuron.
NGF plays a crucial role in the regenerative capabilities of adult
CNS cholinergic neurons at the cell body level (e.g., septum), the
intervening tissue spaces (e.g., nerve bridge) and the reinervation
area (e.g., hippocampal formation). Additionally, NGF may be
beneficial in improving cognitive defects. Metabolized conditioned
growth media and/or metabolized cell extract conditioned with glial
cells for example, can supply an activity of exogenous NGF and
other nerve growth factors so that new axons can grow out from the
cut ends of the injured nerve (e.g., develop a growth cone)
elongating to the original site of the connection.
[0247] Further, injury to the brain and spinal cord is often
accompanied by a glial response to the concomitant axonal
degeneration, resulting in scar tissue. This scar tissue was
initially thought to be a physical barrier to nerve growth,
however, of greater significance is the presence or absence of
neuronotropic factors in the extra neuronal environment. Astrocytes
appear to be capable of synthesizing laminin in response to injury
(laminin can also be found in the metabolized conditioned growth
media). Collagen and fibronectin, and especially laminin, have been
found to promote the growth of neurities from cultured neurons or
neuronal explants in vitro. These extracellular matrix proteins
appear to provide an adhesive substratum which facilitates the
forward movement of the growth cone and elongation of the axon.
Thus, the presence of neuronotropic factors and a supportive
substratum are required for successful nerve regeneration since
regeneration appears to require that: the neuronal cell body be
capable of mounting the appropriate biosynthetic response; and the
environment surrounding the injury site be capable of supporting
the elongation and eventual functional reconnection of the axon.
Metabolized conditioned growth media and/or metabolized cell
extract conditioned by nerve cells such as astrocytes and glial
cells contains an activity of neuronotropic growth factors and
extracellular matrix proteins necessary for nerve regeneration in
brain and spinal cord injuries. Thus, in one embodiment, the
metabolized conditioned growth media and/or metabolized cell
extract is formulated for the treatment of such injuries.
[0248] In other embodiments, the treatment of skin, bones, liver,
pancreas, cartilage, and other specialized tissues may be treated
with media conditioned by their respective specialized cell types,
preferably cultured in three-dimensions, resulting in a conditioned
medium containing an activity of one or more characteristic
extracellular proteins and other metabolites of that tissue type
useful for treating wounds to that respective tissue type.
[0249] The metabolized conditioned growth media and/or metabolized
cell extract of the disclosure may also be added to devices used in
periodontal surgery in order to promote uniform tissue repair, to
provide biodegradable contact lenses, corneal shields or bone
grafts, to provide surgical space fillers, to promote soft tissue
augmentation, particularly in the skin for the purpose of reducing
skin wrinkles, and as urinary sphincter augmentation, for the
purpose of controlling incontinence.
[0250] In another embodiment, the compositions may be
lyophilized/freeze-dried and added as a wound filler (e.g., fill
holes left from hair plugs for implantation) or added to existing
wound filling compositions to accelerate wound healing. In another
embodiment, the medium is conditioned with genetically engineered
cells to increase the concentration of wound healing proteins in
the medium. For example, the cells may be engineered to express
gene products such as any of the growth factors listed above.
C. The Repair and Correction of Congenital Anomalies, Acquired
Defects and Cosmetic Defects
[0251] As disclosed above, the metabolized conditioned growth media
and/or metabolized cell extract of the disclosure may also be used
to repair and correct a variety of anomalies, both congenital and
acquired as well as cosmetic defects, both superficial and
invasive. In one embodiment the metabolized conditioned growth
media and/or metabolized cell extract of the disclosure may be used
to treat or prevent fine lines and wrinkles; age spots and
dyspigmentation; decreased skin texture, tone and elasticity;
roughness and photo damage; decreased ability of skin to regenerate
itself; environmental damage; decreased smoothness and tightness of
skin; age spots; fine and coarse lines and wrinkles; fine and
coarse periocular wrinkles; nasolabial folds; facial fine and
coarse lines; decreased skin radiance and evenness; decreased skin
firmness; hyperpigmentation; dark spots and/or patches; decreased
skin brightness and youthful appearance; photoaged skin;
intrinsically and extrinsically aged skin; abnormal skin cellular
turnover; decreased skin barrier; decrease of skin's ability to
retain moisture; brown and red blotchiness; redness; abnormal skin
epidermal thickness; reduction of dermal epidermal junction;
increased pore size and number of pores; or a combination thereof.
In another embodiment, the metabolized conditioned growth media
and/or metabolized cell extract of the disclosure may be used to
rejuvenate sun damaged and aging skin; improve the appearance of
fine lines and wrinkles; promote cell renewal; and/or improve skin
tone, texture and/or firmness. In another embodiment, the
metabolized conditioned growth media and/or metabolized cell
extract of the disclosure may be used to plump the skin and/or
brighten and/or lighten the skin. In another embodiment, the
metabolized conditioned growth media and/or metabolized cell
extract of the disclosure may be used to strengthen the skin's
ability to regenerate itself; improve the appearance of age spots;
and/or improve skin firmness, elasticity, and/or resiliency. In
another embodiment, the metabolized conditioned growth media and/or
metabolized cell extract of the disclosure may be used to smooth,
tighten, and/or fill in fine lines on the skin. In another
embodiment, the metabolized conditioned growth media and/or
metabolized cell extract of the disclosure may be used to reduce
the appearance of dark circles under the eye. In another
embodiment, the metabolized conditioned growth media and/or
metabolized cell extract of the disclosure may be used to improve
lip texture and/or condition, enhance natural lip color, and/or
increase lip volume. In another embodiment, the metabolized
conditioned growth media and/or metabolized cell extract of the
disclosure may be used to promote epithelialization of
post-procedure skin and/or restore the skin's barrier and/or
moisture balance. In another embodiment, the metabolized
conditioned growth media and/or metabolized cell extract of the
disclosure may be used to improve the appearance of age spots
and/or pigmentation.
[0252] For example, compositions comprising metabolized conditioned
growth media and/or metabolized cell extract of the disclosure may
be added in any form and may be used in a hydrogel, injectable,
cream, ointment, and may even be added to eye shadow, pancake
makeup, compacts or other cosmetics to fortify the skin
topically.
[0253] In another embodiment, topical or application by any known
method such as injection, oral, etc., of the metabolized
conditioned growth medium is made to reverse and/or prevent
wrinkles and a number of the deleterious effects induced by UV
light, exposure to a variety of pollutants and normal aging for
example.
[0254] Additionally, in another embodiment, the metabolized
conditioned growth media and/or metabolized cell extract of the
disclosure is used to reduce cell aging and inhibit the activity of
the factors which cause skin cancer. That the metabolized
conditioned growth media and/or metabolized cell extract has
antioxidant activity is shown in the examples. Again, application
to a mammal may be topical or application by any known method such
as injection, oral, etc. Applicants have discovered that a
statistically significant (p<0.003) reduction in intracellular
oxidation of approximately 50 percent was noted in human
keratinocytes exposed to Applicants' metabolized conditioned growth
medium.
[0255] Thus, in addition to inducing epidermal and dermal cell
proliferation and collagen secretion in vitro the metabolized
conditioned growth media and/or metabolized cell extract of the
disclosure has antioxidant activity. See Example 18.
[0256] This sterile enriched nutrient solution represents a
bioengineered cosmeceutical that is readily available in large
volumes and may be useful as an additive for a variety of skin,
cosmetic, and dermatologic products to supplement the levels of
growth factors and matrix molecules in human skin, hair, and nails.
Products are envisioned to use with Alpha Hydroxy Acids exfoliates
to potentially optimize penetration of the growth factors and other
biomolecules into the skin and with chemical peels to potentially
accelerate healing and reduce inflammation.
[0257] The metabolized conditioned growth media and/or metabolized
cell extract of the disclosure may be formulated for eliminating
wrinkles, frown lines, scarring and other skin conditions instead
of using silicone or other products to do so. The metabolized
conditioned growth media and/or metabolized cell extract contains
an activity of growth factors and inflammatory mediators such as,
for example, VEGF, HGF, IL-6, IL-8, G-CSF and TFG.beta., as well as
extracellular matrix proteins such as type I and type III
collagens, fibronectin, tenascin, glycosaminologycans, acid and
basic FGF, TGF-.alpha. and TGF-.beta., KGF, versican, decorin
betaglycens, syndean and various other secreted human dermal matrix
proteins which are useful in repairing physical anomalies and
cosmetic defects.
[0258] The metabolized conditioned growth media and/or metabolized
cell extract of the disclosure can be formulated into injectable
preparations. Alternatively, products derived from the conditioned
media can be formulated. For example, biologically active
substances, such as proteins and drugs, can be incorporated in the
compositions of the present disclosure for release or controlled
release of these active substances after injection of the
composition. Exemplary biologically active substances can include
tissue growth factors, such as TGF-.beta., and the like which
promote healing and tissue repair at the site of the injection.
Methods of product purification include, but are not limited to gel
chromatography using matrices such as SEPHADEX.RTM., ion exchange,
metal chelate affinity chromatography, with an insoluble matrix
such as cross-linked agarose, HPLC purification, hydrophobic
interaction chromatography of the conditioned media. Such
techniques are described in greater detail in Cell & Tissue
Culture; Laboratory Procedures, supra; Sanbrook et al., 1989,
Molecular Cloning: A Laboratory Manual, 2.sup.nd Ed., Cold Spring
Harbor Lab Press, Cold Spring Harbor, N.Y.
[0259] In the injectable embodiment, an aqueous suspension is used
and the formulation of the aqueous suspension may have a
physiological pH (i.e., about pH 6.8 to 7.5). Additionally, a local
anesthetic, such as lidocaine, (usually at a concentration of about
0.3% by weight) is usually added to reduce local pain upon
injection. The final formulation may also contain a fluid
lubricant, such as maltose, which must be tolerated by the body.
Exemplary lubricant components include glycerol, glycogen, maltose
and the like. Organic polymer base materials, such as polyethylene
glycol and hyaluronic acid as well as non-fibrillar collagen,
preferably succinylated collagen, can also act as lubricants. Such
lubricants are generally used to improve the injectability,
intrudability and dispersion of the injected biomaterial at the
site of injection and to decrease the amount of spiking by
modifying the viscosity of the compositions. The final formulation
is by definition the processed metabolized conditioned growth cell
media in a pharmaceutically acceptable carrier.
[0260] The processed metabolized conditioned growth media and/or
metabolized cell extract of the disclosure is subsequently placed
in a syringe or other injection apparatus for precise placement of
the metabolized conditioned growth media and/or metabolized cell
extract of the disclosure at the site of the tissue defect. In the
case of formulations for dermal augmentation, the term "injectable"
means the formulation can be dispensed from syringes having a gauge
as low as 25 under normal conditions under normal pressure without
substantial spiking. Spiking can cause the composition to ooze from
the syringe rather than be injected into the tissue. For this
precise placement, needles as fine as 27 gauges (200.mu. I.D.) or
even 30 gauges (150.mu. I.D.) are desirable. The maximum particle
size that can be extruded through such needles will be a complex
function of at least the following: particle maximum dimension,
particle aspect ratio (length:width), particle rigidity, surface
roughness of particles and related factors affecting
particle:particle adhesion, the viscoelastic properties of the
suspending fluid, and the rate of flow through the needle. Rigid
spherical beads suspended in a Newtonian fluid represent the
simplest case, while fibrous or branched particles in a
viscoelastic fluid are likely to be more complex.
[0261] The above described steps in the process for preparing
injectable secreted metabolized conditioned growth media and/or
metabolized cell extract of the disclosure are preferably carried
out under sterile conditions using sterile materials. The processed
metabolized conditioned growth media and/or metabolized cell
extract of the disclosure in a pharmaceutically acceptable carrier
can be injected intradermally or subcutaneously to augment soft
tissue, to repair or correct congenital anomalies, acquired defects
or cosmetic defects. Examples of such conditions are congenital
anomalies as hemifacial microsomia, malar and zygomatic hypoplasia,
unilateral mammary hypoplasia, pectus excavatum, pectoralis
agenesis (Poland's anomaly) and velopharyngeal incompetence
secondary to cleft palate repair or submucous cleft palate (as a
retropharyngeal implant); acquired defects (post-traumatic,
post-surgical, post-infectious) such as depressed scars,
subcutaneous atrophy (e.g., secondary to discoid lupis
erythematosus), keratotic lesions, enophthalmos in the unucleated
eye (also superior sulcus syndrome), acne pitting of the face,
linear scleroderma with subcutaneous atrophy, saddle-nose
deformity, Romberg's disease and unilateral vocal cord paralysis;
and cosmetic defects such as glabellar frown lines, deep nasolabial
creases, circum-oral geographical wrinkles, sunken cheeks and
mammary hypoplasia. The compositions of the present disclosure can
also be injected into internal tissues, such as the tissues
defining body sphincters to augment such tissues.
[0262] Other tissue types used to condition the media include but
are not limited to bone marrow, skin, epithelial cells, and
cartilage, however, it is expressly understood that the
three-dimensional culture system can be used with other types of
cells and tissues.
[0263] Alternatively, the metabolized conditioned growth media
and/or metabolized cell extract of the disclosure may be formulated
with polymerizable or cross-linking hydrogels as described in the
previous section on wound treatment.
D. Pharmaceutical Applications
[0264] The metabolized conditioned growth media and/or metabolized
cell extract of the disclosure contain a variety of useful
pharmaceutical factors and components such as growth factors,
regulatory factors, peptide hormones, antibodies, etc., as
described throughout the specification and are therefore useful for
a variety of pharmaceutical applications. Also, products which may
be added include, but are not limited to, antibiotics, antivirals,
antifungals, steroids, analgesics, antitumor drugs, investigational
drugs or any compounds which would result in a complimentary or
synergistic combination with the factors in the conditioned media.
As previously discussed, the cells are cultured, and the media
recovered under aseptic conditions. Additionally, the media can be
tested for pathogens. If sterilization is done, it must be done in
a manner which minimally affects the desired biological activity as
described, supra. The medium may be further processed to
concentrate or reduce one or more factor or component contained
within the medium, for example, enrichment of a growth factor using
immunoaffinity chromatography or, conversely, removal of a less
desirable component, for any given application as described
therein. In a preferred embodiment, formulations are made from
medium conditioned by a three-dimensional cell construct. The
three-dimensional cultures produce a multitude of growth factors
and proteins that are secreted into the medium at optimal
physiological ratios and concentrations. See for example, Table 2.
The medium, therefore, provides a unique combination of factors and
specified ratios that closely represent those found in vivo. Bovine
serum is generally not preferred in this application. It may be
preferable to remove cellular debris or other particular matter as
well as proteases, lactic acid and other components possibly
detrimental to cell growth.
[0265] Assays commonly employed by those of skill in the art may be
utilized to test the activity of the particular factor or factors,
thereby ensuring that an acceptable level of biological activity
(e.g., a therapeutically effective activity) is retained by the
attached molecule or encapsulated molecule.
[0266] Thus, the metabolized conditioned growth media and/or
metabolized cell extract of the disclosure and products derived
from the media may be used, for example, to provide insulin in the
treatment of diabetes, nerve growth factor for the treatment of
Alzheimer's disease, factor VIII and other clotting factors for the
treatment of hemophilia, dopamine for the treatment of Parkinson's
disease, enkaphalins via adrenal chromaffin cells for the treatment
of chronic pain, dystrophin for the treatment of muscular
dystrophy, and human growth hormone for the treatment of abnormal
growth.
[0267] Doses of such therapeutic protein agents are well known to
those of skill in the art and may be found in pharmaceutical
compedia such as the PHYSICIANS DESK REFERENCE, Medical Economics
Data Publishers; REMINGTON'S PHARMACEUTICAL SCIENCES, Mack
Publishing Co.; GOODMAN & GILMAN, THE PHARMACOLOGICAL BASIS OF
THERAPEUTICS, McGraw Hill Publ., THE CHEMOTHERAPY SOURCE BOOK,
Williams and Wilkens Publishers.
[0268] The therapeutically effective doses of any of the drugs or
agents described above may routinely be determined using techniques
well known to those of skill in the art. A "therapeutically
effective" dose refers to that amount of the compound sufficient to
result in amelioration of at least one symptom of the processes
and/or diseases being treated.
[0269] Toxicity and therapeutic efficacy of the drugs can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0270] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the disclosure, the
therapeutically effective dose can be estimated initially from cell
culture assays. A circulating plasma concentration range that
includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0271] Additionally, the cells and tissues may be genetically
engineered to enhance expression of a desired product such as
insulin, for example, and/or to express nucleotide sequences and/or
moieties which target the gene products listed above e.g. ribozyme,
antisense molecules and triple helices, which may have an
inhibitory effect on target gene expression and/or activity. This
might be advantageous when culturing tissues in which specialized
stromal cells in the medium may play particular
structural/functional roles, e.g., glial cells of neurological
tissue, Kupffer cells of liver, etc.
E. Stimulation of Hair Growth
[0272] The medium may be conditioned using, for example, human hair
papilla cells. Preferably, the medium conditioned by such cells is
grown in three-dimensions. Hair papilla cells are a type of
mesenchymal stem cell that plays a pivotal role in hair formation,
growth and restoration (Matsuzaki et al., Wound Repair Regen,
6:524-530 (1998)). The subsequently metabolized conditioned growth
medium is preferably concentrated and applied as a topical
formulation. The metabolized conditioned media compositions may be
formulated for topical applications using an agent that facilitates
penetration of the compound into the skin, for example, DMSO, and
applied as a topical application for stimulating hair growth.
[0273] The compositions of the disclosure promote or restore hair
growth when applied topically by providing factors that increase
epithelial cell migration to hair follicles. In addition to the
factors found in the metabolized conditioned media, other
compounds, such as minoxidil and antibiotics can be used. During
hair growth there is a reduction in blood supply during catagen
(the transitional phase of the hair follicle between growth and
resting phases) and telogen (the resting phase). Biologically
active molecules derived from the metabolized conditioned cell
medium can be determined and optimized for use during these phases
of hair growth using assays known in the art including the
stump-tailed macaque model for male-patterned baldness, see for
example, Brigham, P.a., A. Cappas, and H. Uno, The Stumptailed
Macaque as a Model for Androgenetic Alopecia: Effects of Topical
Minoxidil Analyzed by Use of the Folliculogram, Clin Dermatol,
1988, 6(4): p. 177-87; Diani, A. R. and C. J. Mills,
Immunocytochemical Localization of Androgen Receptors in the Scalp
of the Stumptail Macaque Monkey, a Model of Androgenetic Alopecia,
J Invest Dermatol, 1994, 102(4): p. 511-4; Holland, J. M., Animal
Models of Alopecia, Clin Dermatol, 1988, 6(4): p. 159-162; Pan, H.
J., et al., Evaluation of RU58841 as an Anti-Androgen in Prostate
PC3 Cells and a Topical Anti-Alopecia Agent in the Bald Scalp of
Stumptailed Macaques, Endocrine, 1998, 9(1): p. 39-43; Rittmaster,
R. S., et al., The Effects of N, N-diethyl-4-methyl-3-oxo-4-aza-5
alpha-androstane-17 beta-carboxamide, a 5 alpha-reductase Inhibitor
and Antiandrogen, on the Development of Baldness in the Stumptail
Macaque, J. Clin Endocrinol Metab, 1987, 65(1): p. 188-93 (each of
which is incorporated by reference in its entirety). Additional
models include measuring differences in hair follicle proliferation
from follicles cultured from bald and hairy areas, a newborn rat
model as well as a rat model of alopecia areata, see, Neste, D. V.,
The Growth of Human hair in Nude Mice, Dermatol Clin., 1996, 14(4):
p. 609-17; McElwee, K. J., E. M. Spiers, and R. F. Oliver, In Vivo
Depletion of CD8+T Cells Restores Hair Growth in the DEBR Model for
Alopecia Areata, Br J Dermatol, 1996, 135(2): p. 211-7; Hussein, A.
M., Protection Against Cytosine Arabinowide-Induced Alopecia by
Minoxidil in a Rat Animal Model, Int J Dermatol, 1995, 34(7): p.
470-3; Oliver, R. F., et al., The DEBR Rat Model for Alopecia
Areata, J Invest Dermatol, 1991, 96(5): p. 978; Michie, H. J., et
al., Immunobiological Studies on the Alopecic (DEBER) Rat, Br J
Dermatol, 1990, 123(5): p. 557-67 (each of which is incorporated by
reference in its entirety).
V. Other Uses
[0274] A. Food Additives and Dietary Supplements
[0275] The metabolized conditioned growth media and/or metabolized
cell extract of the disclosure may be used as food additives and
formulated into dietary supplements. The metabolized conditioned
growth media and/or metabolized cell extract of the disclosure may
contain many useful nutrients including essential amino acids,
minerals, and vitamins in an abundance and variety not found in
individual foods or good groups. The metabolized conditioned growth
media and/or metabolized cell extract of the disclosure can be used
as an inexpensive source for a balanced nutritional supplement for
weight loss or alternatively for enhancing the nutritional content
of food, particularly for third world countries. The metabolized
conditioned growth media and/or metabolized cell extract of the
disclosure is sterile and is free from contamination by human
pathogens (i.e., aseptic). The metabolized conditioned growth media
and/or metabolized cell extract of the disclosure may be
concentrated and/or lyophilized and preferably administered in
capsules or tablets for ingestion. Alternatively, the compositions
may be directly added to adult or baby food to enhance nutritional
content. This rich source of nutrients may be processed relatively
inexpensively and can be invaluable to undernourished elderly
people, and in particular, to children in underdeveloped countries
where increased mortality due to poor responses to infection have
been associated with malnutrition.
[0276] Additionally, many trace elements, such as iron and
magnesium, are critical for mammalian survival and reproduction,
and there is concern that marginal trace element deficiency may be
a public health problem. The intake of various essential
micronutrients has been suggested to decrease infection as well as
cancer risk by modifying specific phases of carcinogenesis.
Micronutrients also enhance the functional activities of the immune
system and its interacting mechanism of T cells and B cells, Mos,
and NK cells specifically by enhancing the production of various
cytokines to facilitate their phagocytic and cytotoxic action
against invading pathogens and/or to destroy emerging premalignant
cells in various vital organs. See, Chandra, R. K. ed. (1988),
Nutrition and Immunology. Contemporary Issues in Clinical
Nutrition, Alan R. Liss, New York. Thus, there is a need for a
relatively inexpensive source of balanced nutrients. Ideal food
products for enrichment with the conditioned media are breads,
cereals and other grain products such as pastas, crackers, etc.
Also, the metabolized medium may be further processed to
concentrate or reduce one or more factor or component contained
within the medium, for example, enrichment of a growth factor using
immunoaffinity chromatography or, conversely, removal of a less
desirable component, for any given application as described in this
section.
[0277] B. Animal Feed Supplement
[0278] The metabolized conditioned growth media and/or metabolized
cell extract of the disclosure may be used as a supplemental to
animal feed. In one embodiment, the metabolized conditioned growth
media and/or metabolized cell extract of the disclosure contains
bovine serum that provides a source of protein and other factors
that are beneficial for mammals such as cattle and other ruminant
animals, such as cows, deer and the like. The medium is screened
for pathogens and is free of bovine pathogens and mycoplasma. The
metabolized conditioned growth media and/or metabolized cell
extract of the disclosure is preferably obtained from cows raised
in the United States so that the likelihood of pathogens is
markedly diminished.
[0279] In one embodiment, provided are compositions comprising a
metabolized conditioned growth medium and an acceptable carrier,
wherein the metabolized conditioned growth medium is conditioned
growth medium metabolized by yeast cells. In some embodiments, the
conditioned growth medium is prepared by culturing cells in a
growth medium sufficient to meet the nutritional needs required to
grow the cells in vitro to form a conditioned growth medium. In
some embodiments, the composition is an injectable composition or a
topical composition. In some embodiments, the topical composition
is an ointment, a cream, a hydrogel, or a lotion. In other
embodiments, the composition is used to treat or prevent a cosmetic
defect, a congenital defect, hair loss or an acquired defect. In
other embodiments, the cosmetic defect is a glabellar frown line,
deep nasolabial crease, circum-oral geographical wrinkle, sunken
cheeks or mammary hypoplasia. In some embodiments, the acquired
defect is a medical condition that occurs post-trauma,
post-surgery, post-infection or post-medical procedure defect.
[0280] In other embodiments, provided are compositions comprising a
metabolized cell extract and an acceptable carrier, wherein the
metabolized cell extract is cell extract metabolized by yeast
cells. In some embodiments, the cell extract is derived from
fibroblasts. In other embodiments, the composition is an injectable
composition or a topical composition. In yet other embodiments, the
topical composition is an ointment, a cream, a hydrogel, or a
lotion. In still other embodiments, the composition is used to
treat or prevent a cosmetic defect, a congenital defect, hair loss
or an acquired defect. In some embodiments, the cosmetic defect is
a glabellar frown line, deep nasolabial crease, circum-oral
geographical wrinkle, sunken cheeks or mammary hypoplasia. In yet
other embodiments, the acquired defect is a medical condition that
occurs post-trauma, post-surgery, post-infection or post-medical
procedure defect.
[0281] In still other embodiments, provide are metabolized
conditioned growth medium prepared by a process comprising:
[0282] (a) culturing cells in a growth medium sufficient to meet
the nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells;
[0283] (b) culturing yeast cells;
[0284] (c) exposing the yeast cells to the conditioned growth
medium;
[0285] (d) culturing the yeast cells to metabolize at least a
portion of the conditioned growth medium; and
[0286] (e) collecting the metabolized conditioned growth
medium.
[0287] In some embodiments, the yeast is Pichia pastoris. In other
embodiments, the cells in step (a) are fibroblasts. In yet other
embodiments, the process further comprises (e) processing the
metabolized conditioned growth medium, wherein processing is
concentrating, filtering, purifying, or a combination thereof.
[0288] In yet other embodiments, provided are processes for
preparing the metabolized conditioned growth medium of any one of
claims 15 to 18, comprising:
[0289] (a) culturing cells in a growth medium sufficient to meet
the nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells;
[0290] (b) culturing yeast cells;
[0291] (c) exposing the yeast cells to the conditioned growth
medium;
[0292] (d) culturing the yeast cells to metabolize at least a
portion of the conditioned growth medium; and
[0293] (e) collecting the metabolized conditioned growth
medium.
[0294] In some embodiments, the metabolized conditioned growth
medium is combined with an acceptable carrier. In yet other
embodiments, the metabolized conditioned growth medium is present
in an amount of about 0.0001% to about 95% by weight of the
composition, about 0.01% to about 50% by weight of composition or
about 0.01% to about 10% by weight of composition. In still other
embodiments, the composition further comprises at least one of
water, surfactants, emulsifiers, conditioners, emollients, waxes,
oils, polymers, thickeners, fixatives, colorants, nutraceuticals,
cosmeceuticals, therapeutics, pharmaceuticals, antifungals,
antimicrobials, steroidal hormones, antidandruff agents, anti-acne
components, sunscreens, and preservatives. In still other
embodiments, the composition is an injectable composition or a
topical composition. In some embodiments, the topical composition
is an ointment, a cream, a hydrogel, or a lotion.
[0295] Also provided herein are metabolized cell extract prepared
by a process comprising:
[0296] (a) providing a cell extract;
[0297] (b) culturing yeast cells;
[0298] (c) exposing the yeast cells to the cell extract;
[0299] (d) culturing the yeast cells to metabolize at least a
portion of the cell extract; and
[0300] (e) collecting the metabolized cell extract.
[0301] In some embodiments, the yeast is Pichia pastoris. In other
embodiments, the cell extract is derived from fibroblasts. In some
embodiments, the process further comprises (e) processing the
metabolized cell extract, wherein processing is concentrating,
filtering, purifying, or a combination thereof.
[0302] In one embodiment, provided are compositions comprising: an
acceptable carrier and a metabolized conditioned growth medium
prepared by a process comprising:
[0303] (a) culturing cells in a growth medium sufficient to meet
the nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells;
[0304] (b) culturing yeast cells;
[0305] (c) exposing the yeast cells to the conditioned growth
medium;
[0306] (d) culturing the yeast cells to metabolize at least a
portion of the conditioned growth medium; and
[0307] (e) collecting the metabolized conditioned growth
medium.
[0308] In some embodiments, the yeast is Pichia pastoris. In other
embodiments, the cells in step (a) are fibroblasts. In some
embodiments, the composition further comprises (e) processing the
metabolized conditioned growth medium, wherein processing is
concentrating, filtering, purifying, or a combination thereof. In
some embodiments, the metabolized conditioned growth medium is
present in an amount of about 0.0001% to about 95% by weight of the
composition, about 0.01% to about 50% by weight of the composition,
or about 0.01% to about 10% by weight of the composition. In other
embodiments, the composition further comprises at least one of
water, surfactants, emulsifiers, conditioners, emollients, waxes,
oils, polymers, thickeners, fixatives, colorants, nutraceuticals,
cosmeceuticals, therapeutics, pharmaceuticals, antifungals,
antimicrobials, steroidal hormones, antidandruff agents, anti-acne
components, sunscreens, and preservatives. In some embodiments, the
composition is an injectable composition or a topical composition.
In yet other embodiments, the topical composition is an ointment, a
cream, a hydrogel, or a lotion.
[0309] Also provided herein are compositions comprising: an
acceptable carrier and a metabolized cell extract prepared by a
process comprising:
[0310] (a) providing a cell extract;
[0311] (b) culturing yeast cells;
[0312] (c) exposing the yeast cells to the cell extract;
[0313] (d) culturing the yeast cells to metabolize at least a
portion of the cell extract; and
[0314] (e) collecting the metabolized cell extract.
[0315] In one embodiment, the yeast is Pichia pastoris. In some
embodiments, the cell extract is derived from fibroblasts. In other
embodiments, the process further comprises (e) processing the
metabolized cell extract, wherein processing is concentrating,
filtering, purifying, or a combination thereof. In some
embodiments, the metabolized conditioned growth medium is present
in an amount of about 0.0001% to about 95% by weight of the
composition, about 0.01% to about 50% by weight of the composition,
or about 0.01% to about 10% by weight of the composition. In some
embodiments, the composition further comprises at least one of
water, surfactants, emulsifiers, conditioners, emollients, waxes,
oils, polymers, thickeners, fixatives, colorants, nutraceuticals,
cosmeceuticals, therapeutics, pharmaceuticals, antifungals,
antimicrobials, steroidal hormones, antidandruff agents, anti-acne
components, sunscreens, and preservatives. In other embodiments,
the composition is an injectable composition or a topical
composition. In still other embodiments, the topical composition is
an ointment, a cream, a hydrogel, or a lotion.
[0316] Also provided herein are methods for preventing or treating
a condition in a subject comprising administering to the subject a
therapeutically effective amount of a composition comprising: an
acceptable carrier and a metabolized conditioned growth medium
prepared by a process comprising:
[0317] (a) culturing cells in a growth medium sufficient to meet
the nutritional needs required to grow the cells in vitro to form a
conditioned growth medium and removing the conditioned growth
medium from the cultured cells;
[0318] (b) culturing yeast cells;
[0319] (c) exposing the yeast cells to the conditioned growth
medium;
[0320] (d) culturing the yeast cells to metabolize at least a
portion of the conditioned growth medium; and
[0321] (e) collecting the metabolized conditioned growth
medium.
[0322] In one embodiment, the condition is a cosmetic defect, hair
loss, a congenital defect or an acquired defect. In other
embodiments, the acquired defect is a medical condition that occurs
post-trauma, post-surgery, post-infection or a post-medical
procedure defect. In yet other embodiments, the cosmetic defect is
a glabellar frown line, deep nasolabial crease, circum-oral
geographical wrinkle, sunken cheeks or mammary hypoplasia. In still
other embodiments, the yeast is Pichia pastoris. In some
embodiments, the cells in step (a) are fibroblasts. In other
embodiments, the process further comprises (e) processing the
metabolized conditioned growth medium, wherein processing is
concentrating, filtering, purifying, or a combination thereof. In
some embodiments, the metabolized conditioned growth medium is
present in the composition in an amount of about 0.0001% to about
95% by weight of the composition, about 0.01% to about 50% by
weight of the composition, or about 0.01% to about 10% by weight of
the composition. In one non-limiting example, the metabolized
conditioned growth medium is present in the composition in an
amount of about 10%, about 15%, or about 20% by weight of the
composition. In still other embodiments, the composition further
comprises at least one of water, surfactants, emulsifiers,
conditioners, emollients, waxes, oils, polymers, thickeners,
fixatives, colorants, nutraceuticals, cosmeceuticals, therapeutics,
pharmaceuticals, antifungals, antimicrobials, steroidal hormones,
antidandruff agents, anti-acne components, sunscreens, and
preservatives. In yet other embodiments, the composition is an
injectable composition or a topical composition. In other
embodiments, the topical composition is an ointment, a cream, a
hydrogel, or a lotion. In still other embodiments, at least one
extracellular matrix protein is up-regulated by the administration
of the composition to the subject. In one embodiment, the
extracellular matrix protein is up-regulated by about 5% to about
100%, by about 10% to about 50%, or by about 60% to about 100%. In
another embodiment, the extracellular matrix protein is a collagen
protein or a lysyl hydroxylase protein. In still another
embodiment, the extracellular matrix protein is encoded by COL4A1
gene or PLOD1 gene. In one embodiment, at least one repair protein
is up-regulated by the administration of the composition to the
subject. In another embodiment, the repair protein is up-regulated
by about 10% to about 70%, by about 35% to about 50%, or by about
25% to about 50%. In another embodiment, the repair protein is a
fibronectin protein. In another embodiment, the repair protein is
encoded by FN1 gene. In still another embodiment, at least one
cellular connectivity protein is up-regulated by the administration
of the composition to the subject. In some embodiments, the
cellular connectivity protein is up-regulated by about 5% to about
200%, by about 10% to about 80%, or by about 30% to about 50%. In
some embodiments, the cellular connectivity protein is involucrin
protein. In still other embodiments, the cellular connectivity
protein is encoded by IVL gene. In one embodiment, at least one
antioxidant protein is up-regulated by the administration of the
composition to the subject. In another embodiment, the antioxidant
protein is up-regulated by about 5% to about 300%, by about 10% to
about 50%, or by about 200% to about 250%. In another embodiment,
the antioxidant protein is a superoxide dismutase protein. In still
other embodiments, the antioxidant protein is encoded by SOD2
gene.
[0323] Also provided herein are methods for preventing or treating
a condition in a subject comprising administering to the subject a
therapeutically effective amount of a composition comprising: an
acceptable carrier and a metabolized cell extract prepared by a
process comprising:
[0324] (a) providing a cell extract;
[0325] (b) culturing yeast cells;
[0326] (c) exposing the yeast cells to the cell extract;
[0327] (d) culturing the yeast cells to metabolize at least a
portion of the cell extract; and
[0328] (e) collecting the metabolized cell extract.
[0329] In some embodiments, the condition is a cosmetic defect,
hair loss, a congenital defect or an acquired defect. In some
embodiments, the acquired defect is a medical condition that occurs
post-trauma, post-surgery, post-infection, or post-medical
procedure defect. In other embodiments, the cosmetic defect is a
glabellar frown line, deep nasolabial crease, circum-oral
geographical wrinkle, sunken cheeks or mammary hypoplasia. In one
embodiment, the yeast is Pichia pastoris. In another embodiment,
the cell extract is derived from fibroblasts. In still another
embodiment, the process further comprises (e) processing the
metabolized cell extract, wherein processing is concentrating,
filtering, purifying, or a combination thereof. In still other
embodiments, the metabolized conditioned growth medium is present
in the composition in an amount of about 0.0001% to about 95% by
weight of the composition, by about 0.01% to about 50% by weight of
the composition, or by about 0.01% to about 10% by weight of the
composition. In one non-limiting example, the metabolized cell
extract is present in the composition in an amount of about 10%,
about 15%, or about 20% by weight of the composition. In still
other embodiments, the composition further comprises at least one
of water, surfactants, emulsifiers, conditioners, emollients,
waxes, oils, polymers, thickeners, fixatives, colorants,
nutraceuticals, cosmeceuticals, therapeutics, pharmaceuticals,
antifungals, antimicrobials, steroidal hormones, antidandruff
agents, anti-acne components, sunscreens, and preservatives. In one
embodiment, the composition is an injectable composition or a
topical composition. In another embodiment, the topical composition
is an ointment, a cream, a hydrogel, or a lotion. In one
embodiment, at least one extracellular matrix protein is
up-regulated by the administration of the composition to the
subject. In another embodiment, the extracellular matrix protein is
up-regulated by about 5% to about 100%, by about 10% to about 50%,
or by about 60% to about 100%. In yet another embodiment, the
extracellular matrix protein is a collagen protein or a lysyl
hydroxylase protein. In some embodiments, the extracellular matrix
protein is encoded by COL4A1 gene or PLOD1 gene. In some
embodiments, at least one repair protein is up-regulated by the
administration of the composition to the subject. In one
embodiment, the repair protein is up-regulated by about 10% to
about 70%, or by about 25% to about 50%. In another embodiment, the
repair protein is a fibronectin protein. In still another
embodiment, the repair protein is encoded by FN1 gene. In one
embodiment, at least one cellular connectivity protein is
up-regulated by the administration of the composition to the
subject. In another embodiment, the cellular connectivity protein
is up-regulated by about 5% to about 200%, by about 10% to about
80%, or by about 30% to about 50%. In still another embodiment, the
cellular connectivity protein is involucrin protein. In still
another embodiment, the cellular connectivity protein is encoded by
IVL gene. In one embodiment, at least one antioxidant protein is
up-regulated by the administration of the composition to the
subject. In another embodiment, the antioxidant protein is
up-regulated by about 5% to about 300%, by about 10% to about 50%,
or by about 200% to about 250%. In another embodiment, the
antioxidant protein is a superoxide dismutase protein. In yet
another embodiment, the antioxidant protein is encoded by SOD2
gene.
EXAMPLES
Example 1: Fibroblast Monolayer Cell Culture
[0330] Normal human dermal fibroblasts, isolated from a human
foreskin, were cultured in a 150 cm.sup.2 tissue culture flasks
(Corning, Corning, N.Y.) in monolayer culture using pre-conditioned
cell culture media (in this example, high-glucose Dulbecco's
Modified Eagle's Media (DMEM; GibcoBRL, Grand Island, N.Y.)
supplemented with 10% bovine calf serum (Hyclone Laboratories,
Logan, Utah), nonessential amino acids (GibcoBRL), and 100 U/ml
penicillin-streptomycin-250 ng/ml amphotericin B (GibcoBRL) ("DMEM
1") in a 37.degree. C., 5% CO.sub.2 incubator. Monolayer cultures
were fed twice weekly with fresh pre-conditioned media and passaged
weekly using a 1 to 10 split, as described. See generally, Pinney
et al., J. Cell. Physio., 183:74 82 (2000). The dermal fibroblasts
may also be expanded in roller bottles with DMEM 1. The conditioned
media from these monolayer cultures is collected and saved for
future use.
[0331] While fibroblast cells have been used for illustrative
purposes in this example, the skilled artisan will understand that
many other types of cells, for example, but not limited to, other
epithelial cell types, endothelial cells, smooth muscle cells,
myocytes, keratinocytes, chondrocytes, and the like, may be grown
in monolayer culture and in three-dimensional culture.
Example 2: Conditioning the Medium
[0332] Human dermal fibroblasts were seeded onto the substrate of
the apparatus described in the '766 patent and described in detail
above. The substrate is within the casing designed to facilitate
three-dimensional tissue growth on its surface and the cells were
cultured in a closed system in cultured in high glucose DMEM (10%
BCS supplemented with 2 mM L-glutamine and 50 mg/ml ascorbic acid)
at 37.degree. C. in a humidified, 5% CO.sub.2 atmosphere. After 10
days the cell culture was removed, fresh medium was added. The
cells were cultured for an additional 4 days as described above.
The resulting conditioned medium, having been exposed to the cell
and tissue culture for four days (days 10-14) was then removed from
the individual chambers and pooled. The conditioned medium
(approximately 5 to 10 liters/pool) was dispensed into 200 ml
aliquots and further concentrated 10- to 20-fold using a positive
pressure concentration device having a filter with a 10,000 MW
cut-off (Amicon, Beverly, Mass.). The resulting 10 to 20 ml of
concentrated conditioned medium was dispensed into 1 ml aliquots
and frozen at -20.degree. C. for analysis. A 1.times. concentration
of conditioned medium results from 10.times. conditioned medium
added to base medium as a 10% (vol/vol) solution. Likewise, a
1.times. concentration of "medium" or "serum free medium" results
from 10.times. medium (i.e., base medium) or 10.times. serum free
medium (base medium without serum) added to base medium as a 10%
(vol/vol) solution which are then used as controls.
Example 3: Preparation of Metabolized Conditioned Growth Media
[0333] a. Organism and Media
[0334] Pichia pastoris (wild-type strain) was used as the yeast
cell culture for fermentation work. Stock cultures were maintained
on yeast-peptone-dextrose (YPD) agar plates. The parent stock
culture was grown in YPD broth and maintained at -20.degree. C. The
fermentation was carried out in Yeast Nitrogen-Base (YNB) growth
media and supplemented with glycerol containing, 2.7%
H.sub.3PO.sub.4, 0.09% CaSO.sub.4, 1.8% K.sub.2SO.sub.4, 1.5%
MgSO.sub.4, 0.41% KOH, 4% glycerol (Sigma St. Louis, Mo.).
Condition Growth Media was supplied by SkinMedica (California).
[0335] b. Fermentor
[0336] The fermentation process was scaled up to 2 L and 15 L
fermentation stages (2 L New Brunswick Scientific, Edison N.J. and
15 L Applikon Biotechnology Foster City Calif.) using standard
growing conditions for the yeast.
[0337] c. Conditioned Growth Media Metabolization Conditions
[0338] Fed-batch cultures were grown at 29.degree. C., in Yeast
Nitrogen-Base (YNB) growth media and supplemented with glycerol
(2.7% H.sub.3PO.sub.4, 0.09% CaSO.sub.4, 1.8% K.sub.2SO.sub.4, 1.5%
MgSO.sub.4, 0.41% KOH, 4% glycerol). The pH was kept constant at
5.0.+-.0.5 by titration with 2M NH.sub.4OH. The dissolved oxygen
levels were measured by a sterilizable DO probe and the oxygen
saturation was kept at 30% by regulating the stirring velocity
between 100 and 600 rpm. Conditioned growth media was introduced to
the fermentation process as a nutritional supplement by inoculating
the liquid media into the fermentor during the active growth phase
of the yeast. The fed-batch process was initiated during the
logarithmic growth phase of Pichia pastoris. Growth was measured by
monitoring optical density of Pichia pastoris during fermentation.
The fermentation was carried out until such time that the
conditioned growth media was metabolized by Pichia pastoris, as
measured by the changes in the growth phase of Pichia pastoris from
logarithmic phase to stationary phase. The fermentation may be
carried out to completion wherein the conditioned growth media is
fully metabolized, or may be partially fermented, i.e., partially
metabolized.
[0339] The conditioned growth media may be concentrated, filtered
and/or purified prior to combining the metabolized conditioned
growth medium with a suitable vehicle.
Example 4: Preparation of Metabolized Cell Extract
[0340] a. Cell Extract
[0341] Whole cell extract is obtained from animal cells or plant
cells, for example, by any method known to the skilled artisan.
[0342] b. Organism and Media
[0343] Pichia pastoris (wild-type strain) is used as the yeast cell
culture for fermentation work. Stock cultures are maintained on
yeast-peptone-dextrose (YPD) agar plates. The parent stock culture
is grown in YPD broth and maintained at -20.degree. C. The
fermentation is carried out in Yeast Nitrogen-Base (YNB) growth
media and supplemented with glycerol containing, 2.7%
H.sub.3PO.sub.4, 0.09% CaSO.sub.4, 1.8% K.sub.2SO.sub.4, 1.5%
MgSO.sub.4, 0.41% KOH, 4% glycerol (Sigma St. Louis, Mo.).
[0344] b. Fermentor
[0345] The fermentation process is scaled up to 2 L and 15 L
fermentation stages (2 L New Brunswick Scientific, Edison N.J. and
15 L Applikon Biotechnology Foster City Calif.) using standard
growing conditions for the yeast.
[0346] c. Cell Extract Metabolization Conditions
[0347] Fed-batch cultures are grown at 29.degree. C., in Yeast
Nitrogen-Base (YNB) growth media and supplemented with glycerol
(2.7% H.sub.3PO.sub.4, 0.09% CaSO.sub.4, 1.8% K.sub.2SO.sub.4, 1.5%
MgSO.sub.4, 0.41% KOH, 4% glycerol). The pH is kept constant at
5.0.+-.0.5 by titration with 2M NH.sub.4OH. The dissolved oxygen
levels are measured by a sterilizable DO probe and the oxygen
saturation was kept at 30% by regulating the stirring velocity
between 100 and 600 rpm. Cell extract is introduced to the
fermentation process as a nutritional supplement by inoculating the
liquid media into the fermentor during the active growth phase of
the yeast. The fed-batch process is initiated during the
logarithmic growth phase of Pichia pastoris. Growth is measured by
monitoring optical density of Pichia pastoris during fermentation.
The fermentation is carried out until such time that the cell
extract is metabolized by Pichia pastoris, as measured by the
changes in the growth phase of Pichia pastoris from logarithmic
phase to stationary phase. The cell extract may be may be
concentrated, filtered and/or purified prior to combining the
metabolized cell extract with a suitable vehicle.
[0348] d. Proliferating Activity of Three-Dimensional-Conditioned
Medium
[0349] The conditioned medium and metabolized conditioned growth
medium and metabolized cell extract of Examples 2-4 may be
optionally examined for the ability to promote the proliferation of
human fibroblasts and keratinocytes. Human fibroblasts or human
basal keratinocytes are seeded into 96 well plates (.about.5,000
cells/well) and cultured in high glucose DMEM (10% BCS supplemented
with 2 mM L-glutamine and 1.times. antibiotic/antimycotic)
supplemented with 1.times. final concentration of
serum-free-medium, medium, or the three-dimensional conditioned
medium, metabolized conditioned growth medium or metabolized cell
extract as described above in Examples 2-4. The cultures are
maintained at 37.degree. C. in a humidified, 5% CO.sub.2 atmosphere
for 3 days.
[0350] Cellular proliferation is measured using a commercially
available, fluorescent-based dye assay that measures total nucleic
acid content as an estimation of cell proliferation (CyQuant Cell
Proliferation Assay Kit, Molecular Probes, Eugene, Or). All assays
are performed according to the manufacturer's instructions. Medium
is removed by blotting and the cells are lysed using lysis buffer
containing the green fluorescent dye, CyQuant GR dye. The dye
exhibits strong fluorescence enhancement when bound to cellular
nucleic acids and the amount of fluorescence is proportional to the
amount of nucleic acid present in the sample. Samples are incubated
for 5 minutes in the absence of light and sample fluorescence is
determined using a microtiter plate reader with filters appropriate
for .about.480 nm excitation and .about.520 nm emission maxima. The
amount of nucleic acid in each sample is calculated by comparing
the amount of observed fluorescence in each well against a standard
curve, derived using known concentrations of calf thymus DNA as a
standard.
[0351] An exemplary result is shown in FIG. 3, where the cells
cultured in the medium containing the conditioned medium resulted
in increased cellular proliferation of both fibroblast and
keratinocyte cells when compared to two controls.
Example 5: Modulation of Collagen Deposition into Tissues by
Metabolized Conditioned Growth Medium
[0352] a. Wound Healing Applications
[0353] The effect of metabolized conditioned growth medium on the
preparation and composition of three-dimensional tissues is
examined by measuring the amount of collagen secreted into the
extracellular matrix of tissues cultured in the presence of
serum-free medium, medium or three dimensional conditioned
medium.
[0354] Nylon scaffolds are laser-cut into 11 mm.times.11 mm
squares, washed in 0.5M acetic acid, rinsed extensively in FBS, and
seeded with 12F clinical fibroblasts at passage 8
(.about.38,000/cm.sup.2). Cultures are grown in 1 ml of DMEM (10%
BCS supplemented with 2 mM L-glutamine and 1.times.
antibiotic/antimycotic) supplemented with 1.times. final
concentration of serum-free-medium, medium, or the metabolized
conditioned growth medium as described above in with the addition
of 50 mg/ml ascorbate at each feeding. Copper sulfate is added to a
final concentration of 2.5 ng/ml, and high oxygen (40%, about twice
atmospheric) is maintained by regulated gassing of a standard
incubator. Cultures (n=3 or greater) are maintained at 37.degree.
C. in a humidified, 5% CO.sub.2 atmosphere for 10 days. A no
ascorbate control is also included.
[0355] b. Collagen Isolation
[0356] Collagen is isolated and purified to near homogeneity from
three-dimensional tissue cultures grown in the presence of base
medium supplemented with a 1.times. final concentration of
serum-free medium, medium or metabolized conditioned growth medium
described above. The purity of the final samples preparations is
estimated by subjecting the purified collagen samples to
electrophoresis on gradient SDS-polyacrylamide gels, visualizing
the separate protein bands using Coomassie blue, and estimating the
amount of collagen-specific alpha-, beta- and gamma-bands compared
to total protein (below). Purification methods should yield similar
patterns in all samples.
[0357] Samples are rinsed in PBS, then sterile water, followed by
2-6 hours in 0.5M acetic acid. The samples are then digested
overnight in 1 mg/ml pepsin (Worthington, Inc.) in 0.012N HCl at
4.degree. C. Samples are clarified by centrifugation at 13000 rpm
at 4.degree. C. Collagen is precipitated 30-60 minutes at 4.degree.
C. after addition of 5 M NaCl to a final concentration of 0.7M.
Precipitated collagen is separated by centrifugation at 13000 rpm
at 4.degree. C. for 30 to 60 minutes, and resuspended in 0.012N
HCl.
[0358] c. Analysis
[0359] Total protein is determined using a commercially available
colorimetric assay kit (Pierce, Inc. BCA assay kit) and assays are
performed according to the manufacturer's instructions. Bovine skin
collagens are used as a standard (InVitrogen, Carlsbad, Calif.;
Cohesion Technologies, Inc., Palo Alto, Calif.) for quantifying
total protein.
[0360] Samples (10 mg) are then subjected to SDS-PAGE analysis with
electrophoresis on 3-8% gradient gels. The samples of isolated
collagens are then heated to 95.degree. C. in reducing sample
buffer. Gels are stained with Coomassie Blue, destained, and
computer-scanned for visualization.
[0361] Enhanced deposition of collagen in vivo has a number of
applications, including wound healing, the treatment of wrinkles
and contour lines that appear with increased age as well as being
able to promote matrix deposition over bony-prominences susceptible
to pressure ulcers in paralyzed or bedridden patients.
Example 6: Occlusive Patch Test Assessing the In Vivo Effects of
Metabolized Conditioned Growth Medium
[0362] a. Experimental Design:
[0363] Six consenting adult females (30-60 yr) in good health are
enrolled. Exclusion criteria includes sensitivity to proteins, skin
diseases, damaged skin in or near test sites, diabetes, renal,
heart or immunological disorders, use of anti-inflammatory, immuno
suppressive, antihistamine or topical drugs or cosmetics and
pregnancy. Test articles are assigned to test sites (2 sites, 3.8
cm.sup.2) on the right or left forearm of each subject according to
a rotational scheme to minimize position or order bias. Site 1 is
designated for vehicle control and site 2 treatment (i.e.,
metabolized conditioned growth medium). Occlusion patches are of a
Webril nonwoven cotton pad with either 0.2 ml of vehicle or
treatment. Patches are covered and held by a 3M occlusive, plastic,
hypoallergenic tape. Occlusion patches are positioned daily on the
forearms of 3 subjects for 5 consecutive, 24-hour periods. The
remaining 3 subjects are patched daily for 12, consecutive 24-hour
periods. On the day following the last patch application, a 2-mm
biopsy is taken from each site. This protocol is approved by the
IRB for the investigative organization, the California Skin
Research Institute (San Diego, Ca.), and is in accordance with
Title 21 of the CFR, Parts 50 and 56.
[0364] b. Evaluations:
[0365] Gross observations are graded for glazing, peeling,
scabbing, fissuring, hyperpigmentation, and hypopigmentation.
Irritation is scored visually using a 5 point scale and graded
numerically for erythema, edema, papules and vesicles (>25%
patch site), and identifiable reactions (<25% patch site), i.e.,
bulla reaction with or without weeping, spreading, and induration.
The H & E histological assessment by a board certified
pathologist includes parameters for viable epidermal thickness,
epidermal hyperplasia (acanthosis), granular cell layer thickness,
inflammatory infiltrate, mitotic figures, appearance of collagen
and elastic fibers, and vasculature.
Example 7: Modulation of Human Endothelial Cell Behavior
[0366] The effects of metabolized conditioned growth medium on
angiogenesis and endothelial cell motility are determined.
Metabolized conditioned growth medium is either concentrated
(10.times.) or lyophilized.
[0367] a. Endothelial Cell Tubule Formation Assay
[0368] Endothelial cell tubule formation assay with human umbilical
vein endothelial cells (HUVEC) is used to assess angiogenesis.
[0369] b. Wounding Assay
[0370] A confluent layer of endothelial cells ere scratched and the
speed of closure of the resulting "wound" is measured used to
assess cell motility. The "wounding" assay is measured as speed of
closure in mm/h (millimeters/hour).
Example 8: Preparation of Yeast Extract
[0371] a. Organism and Media
[0372] Yeast cell culture was obtained from ATCC (Pichia pastoris
#60372). Stock cultures were maintained on yeast-peptone-dextrose
(YPD) agar plates. The parent stock culture was grown in YPD broth
and maintained at -20.degree. C. The fermentation was carried out
in Yeast Nitrogen-Base (YNB) growth media and supplemented with
glycerol containing, 2.7% H.sub.3PO.sub.4, 0.09% CaSO.sub.4, 1.8%
K.sub.2SO.sub.4, 1.5% MgSO.sub.4, 0.41% KOH, 4% glycerol (Sigma St.
Louis, Mo.). Antifoam sigma-emulsion B was used throughout the
process (Sigma, St. Louis, Mo.).
[0373] b. Fermentor
[0374] After optimization of treatment via the shake flask trials,
the process was scaled up to 2 L and 15 L fermentation stages (2 L
New Brunswick Scientific, Edison N.J. and 15 L Applikon
Biotechnology Foster City Calif.).
[0375] c. Stress Conditions
[0376] Fed-batch cultures were grown at 29.degree. C., in Yeast
Nitrogen-Base (YNB) growth media and supplemented with glycerol
(2.7% H.sub.3PO.sub.4, 0.09% CaSO.sub.4, 1.8% K.sub.2SO.sub.4, 1.5%
MgSO.sub.4, 0.41% KOH, 4% glycerol). The pH was kept constant at
5.0.+-.0.5 by titration with 2M NH.sub.4OH. The dissolved oxygen
levels were measured by a sterilizable DO probe and the oxygen
saturation was kept at 30% by regulating the stirring velocity
between 100 and 600 rpm. The aeration rate was set at IVVM.
Example 9: Full Thickness Evaluation
[0377] The metabolized cell extract disclosed above are tested on
the MatTek full thickness skin tissue model. This skin model
consists of: 1) normal human-derived epidermal keratinocytes that
have been cultured to form a multilayered, highly differentiated
model of the human epidermis and, 2) human fibroblasts that have
been seeded into a collagen matrix to form the dermis. Upon
arrival, the tissues are stored at 4.degree. C. until used. For
use, the tissues are removed from the agarose-shipping tray and
placed into a 6-well plate containing 4 ml of assay medium and
allowed to equilibrate overnight at 37.+-.2.degree. C. and 5.+-.1%
CO.sub.2. After the overnight equilibration, the media is replaced
with 4 ml of fresh media and 50 .mu.l of test material (i.e.
metabolized cell extract) is then applied topically to the tissues.
The tissues are then incubated for 24 hours at 37+2.degree. C. and
5+1% CO.sub.2 and physiological effects of the treatment measured
over time.
Example 10: Preparation of Liposomal Encapsulated Metabolized
Conditioned Growth Medium
[0378] Samples of metabolized conditioned growth medium are
incorporated into liposome comprising phospholipid and lecithin
layer obtained from soybeans. The lysate is slurried together with
liposome using a high-pressure homogenizer obtained from Hydraulic
Engineering Corporation (Brea, Calif.). The milky white mixture
contains the metabolized conditioned growth medium encapsulated
with the liposomal components.
Example 11: Preparation of Maltodextrin-Encapsulated Metabolized
Cell Extract
[0379] Samples of metabolized cell extract are encapsulated in
maltodextrin and spray-dried to essentially provide an anhydrous
powder of maltodextrin-encapsulated metabolized cell extract using
the methodologies in WO2003/068161.
Example 12: Water-in-Oil Emulsion
[0380] The example illustrates a high internal phase water-in-oil
emulsion incorporating the metabolized conditioned growth medium as
disclosed above.
TABLE-US-00003 Ingredient Wt % 1,3-dimethyl-2-imdazolidione 0.2
Polyoxylene (2) oleyl ether1 (Oleth-2) 5.0 Disteardimonium
Hectorite 0.5 MgSO.sub.4--7H.sub.2O 0.3 Preservative 0.01
Metabolized conditioned growth medium 10.0 Water To 100
Example 13: Water-in-Oil Cream
[0381] The example illustrates a water-in-oil cream incorporating
the metabolized conditioned growth medium prepared as disclosed
above.
TABLE-US-00004 Ingredient Wt % Mineral oil 4
1,3-dimethyl-2-imdazolidione 1 Ceteth-10 4 Cetyl alcohol 4
Triethanolamine 0.75 Butylene glycol 3 Xanthum gum 0.3 Methyl,
propyl and butyl paraben 0.01 Metabolized conditioned growth medium
10 Water To 100
Example 14: Alcoholic Lotion
[0382] The example illustrates an alcoholic lotion incorporating
the metabolized cell extract prepared as disclosed above.
TABLE-US-00005 Ingredient Wt % 1,3-dimethyl-2-imdazolidione 0.3
Ethyl alcohol 40.0 Metabolized cell extract 10.0 Water To 100
Example 15: Sub-Micron Emulsion Concentrate
[0383] The example illustrates a sub micron emulsion concentration
that contains the metabolized conditioned growth medium prepared as
disclosed above.
TABLE-US-00006 Ingredient Wt % Trimethylopropane
Tricaprylate/Tricaprate 18.0 Glycerin 8.0 Cetcaryl alcohol 2.0
Cetcareth 20 2.0 Glyceral stearate 2.0 Butylated hydroxytoluene
0.01 Metabolized conditioned growth medium 10 Water To 100
Example 16: Dilution of Metabolized Cell Extract
[0384] The example illustrates a dilution that contains the
metabolized cell extract prepared as disclosed above
TABLE-US-00007 Ingredient Wt % Water 89 Metabolized cell extract 10
Preservative 1.0
Example 17: Human DNA Micro Array Studies on Normal Human Dermal
Fibroblasts
[0385] Normal human dermal fibroblasts were treated for 24-hour
exposure as follows: (i) control 1: Nouricel (10.times.), i.e.,
conditioned growth medium (see, e.g., Example 2); (ii) control 2:
Pichia ferment filtrate (20.times.), i.e., yeast ferment extract
(see Example 8); (iii) partially fermented Nouricel (20.times.),
i.e., partially metabolized conditioned growth medium (see Example
3); and (iv) fully fermented Nouricel (20.times.), i.e., fully
metabolized conditioned growth medium (see Example 3).
[0386] The results are presented in the table below and are
represented as a ratio of median between treated and untreated
samples. A ratio of median >1.3 indicates strong up-regulation
and < indicates strong down-regulation of the gene.
TABLE-US-00008 Sample (ii) Pichia (iii) Partially (iv) Fully
Ferment fermented fermented Gene Name (i) Nouricel Filtrate
Nouricel Nouricel COL1A1 1.53 1.114 1.013 1.12 COL1A2 1.608 1.994
1.371 1.806 COL4A1 0.978 0.443 1.148 1.327 ELN 1.075 0.581 0.86
0.488 FN1 1.248 1.573 0.706 1.35 IVL 0.675 0.316 0.256 0.5 PLOD1
0.981 1.196 0.855 1.417 TIMP1 1.874 1.852 1.397 1.967 SOD1 1.184
1.396 1.215 0.965 SOD2 1.293 2.373 2.543 2.739 CAT 0.902 0.294
0.678 0.48
[0387] Results from a second assay are presented in the table below
and are as a ratio of median between treated and untreated samples.
A ratio of median >1.3 indicates strong up-regulation and <
indicates strong down-regulation of the gene.
TABLE-US-00009 Sample Gene Name (i) Nouricel (iv) Fully fermented
Nouricel COL1A1 2.914 1.388 COL1A2 3.2 2.102 COL4A1 0.957 0.714
VEGFB 1.223 1.339 TGF-Beta1 1.795 1.615 FGF3 2.284 1.867 PDGFRB
2.194 1.773 TJP1 2.302 1.804 HSP90AB1 1.965 1.921 HSP90A1 2.871
2.05 HSPB1 1.947 1.881 ELN 1.554 0.526 FN1 2.667 1.903 PLOD1 0.709
0.463 TIMP1 2.832 1.547 SOD1 1.313 1.613 MMP3 0.601 0.660 MMP12
0.364 0.428 SOD2 1.293 2.594 CAT 0.696 0.291
[0388] In one embodiment, the administration of the metabolized
conditioned growth medium or the metabolized cell extract to a
subject results in the up-regulation of at least one extracellular
matrix protein. In some embodiments, the extracellular matrix
protein is up-regulated by about 5% to about 100%, about 10% to
about 90%, about 20% to about 80%, about 30% to about 70%, about
40% to about 60%, or about 25% to about 50%. In other embodiments,
the extracellular matrix protein is up-regulated by about 10% to
about 50% or about 60% to about 100%. In yet another embodiment,
the extracellular matrix protein is up-regulated by about 5%, by
about 10%, by about 15%, by about 20%, by about 25%, by about 30%,
by about 35%, by about 40%, by about 45%, by about 50%, by about
55%, by about 60%, by about 65%, by about 70%, by about 75%, by
about 80%, by about 85%, by about 90%, by about 95% or by about
100%. In some embodiments, the extracellular matrix protein that is
up-regulated is a collagen or lysyl hydroxylase protein. In some
embodiments, the extracellular matrix protein is encoded by COL4A1
or PLOD1.
[0389] In one embodiment, the administration of the metabolized
conditioned growth medium or the metabolized cell extract to a
subject results in the up-regulation of at least one repair
protein. In some embodiments, the repair protein is up-regulated by
about 5% to about 100%, about 10% to about 90%, about 20% to about
80%, about 30% to about 70%, about 40% to about 60%, or about 25%
to about 50%. In other embodiments, the repair protein is
up-regulated by about 10% to about 70%. In some embodiments, the
repair-protein is up-regulated by about 5%, by about 10%, by about
15%, by about 20%, by about 25%, by about 30%, by about 35%, by
about 40%, by about 45%, by about 50%, by about 55%, by about 60%,
by about 65%, by about 70%, by about 75%, by about 80%, by about
85%, by about 90%, by about 95% or by about 100%. In some
embodiments, the repair protein is fibronectin. In some
embodiments, the repair protein is encoded by FN1.
[0390] In one embodiment, the administration of the metabolized
conditioned growth medium or the metabolized cell extract to a
subject results in the up-regulation of at least one cellular
connectivity protein. In one embodiment, the cellular connectivity
protein is up-regulated by about 5% to about 200%. In one
embodiment, the cellular connectivity protein is up-regulated by
about 10% to about 80%. In some embodiments, the cellular
connectivity protein is up-regulated by about 30% to about 50%. In
one embodiment, the cellular connectivity protein is up-regulated
by about 5%, by about 10%, by about 15%, by about 20%, by about
25%, by about 30%, by about 35%, by about 40%, by about 45%, by
about 50%, by about 55%, by about 60%, by about 65%, by about 70%,
by about 75%, by about 80%, by about 85%, by about 90%, by about
95%, by about 100%, by about 110%, by about 120%, by about 130%, by
about 140%, by about 150%, by about 160%, by about 170%, by about
180%, by about 190% or by about 200%. In other embodiments, the
cellular connectivity protein is involucrin protein. In some
embodiments, the cellular connectivity protein is encoded by IVL
gene.
[0391] In one embodiment, the administration of the metabolized
conditioned growth medium or the metabolized cell extract to a
subject results in the up-regulation of at least one antioxidant
protein. In some embodiments, the antioxidant protein is
up-regulated by about 5% to about 300%, about 5% to about 200%,
about 5% to about 150%, about 5% to about 100%, about 10% to about
90%, about 20% to about 80%, about 30% to about 70%, about 40% to
about 60%, or about 25% to about 50%. In other embodiments, the
antioxidant protein is up-regulated by about 10% to about 50% or
about 200% to about 250%. In some embodiments, the antioxidant
protein is up-regulated by about 5%, by about 10%, by about 15%, by
about 20%, by about 25%, by about 30%, by about 35%, by about 40%,
by about 45%, by about 50%, by about 55%, by about 60%, by about
65%, by about 70%, by about 75%, by about 80%, by about 85%, by
about 90%, by about 95%, by about 100%, by about 125%, by about
150%, by about 175%, by about 200%, by about 250% or by about 300%.
In some embodiments, the antioxidant protein that is up-regulated
is superoxide dismutase. In some embodiments, the antioxidant
protein that is up-regulated is encoded by SOD2.
Example 18: In Vitro and Clinical Studies
Patients and Methods
Methods: In Vitro Study
Gene Array Analysis
[0392] Human dermal fibroblasts were seeded into either a 96-well
plate (cytotoxicity assay) or into T-25 flasks (cytokine array) and
grown at 37.+-.2.degree. C. and 5.+-.1% CO.sub.2 using fibroblast
growth media (FGM). Safe working concentration for each test
material was determined using an MTT assay. Upon reaching
confluence, the cells were treated with the test materials for 24
hours. After the 24 hour treatment, total RNA was isolated using an
RNAqueous.RTM. Kit (Ambion) per the manufacturer's instructions.
After purification, the total RNA was prepared for array use by
first amplifying the RNA using a MessageAmp.TM. aRNA Kit (Ambion),
and then fluorescently labeling the aRNA with Cy3 or Cy5 using an
ASAP Labeling Kit (Perkin Elmer), both per the manufacturer's
instructions. The fluorescently labeled aRNA was applied to the DNA
microarray chips (Agilent Technologies) and the chips were
hybridized overnight and washed per the manufacturer's recommended
protocol. After washing, the microarrays were scanned with an Axon
GenePix 4100A Scanner and analyzed with GenePix Pro software.
Fluorescence intensities for the microarrays were subjected to
global normalization. The total fluorescent signal for both dyes
was normalized with a correction factor that would make the ratio
of total intensities for both dyes equal to one. For this study a
Cy3/Cy5 (untreated/treated) fluorescence intensity ratio greater
than 1.3 or less than 0.7 (this relates to a change in gene
expression of at least 30%) was used as the cutoff for up- and
down-regulated genes, respectively. In addition, the fluorescence
intensity of the gene marker had to be greater than the background
intensity.
Protein Array Analysis
[0393] Human dermal fibroblasts were seeded into either a 96-well
plate (cytotoxicity assay) or into T-25 flasks (cytokine array) and
grown at 37.+-.2.degree. C. and 5.+-.1% CO2 using fibroblast growth
media (FGM). Safe working concentration for each test material was
determined using an MTT assay. Upon reaching confluence the cells
were treated with the test materials for 24 hours, after which
cytokine release into the culture media was assessed using cytokine
arrays (Ray Biotechnology). The microarrays were scanned with an
Axon GenePix 4100A Scanner analyzed with GenePix Pro software.
Methods: 12-Week, Clinical-Usage Study (Combination Product)
[0394] A 12-week open-label clinical study examined the efficacy
and tolerability of the combination treatment serum in subjects
with mild to severe facial photodamage. The criteria for study
participation included female subjects aged 30 to 60 years (having
a Fitzpatrick Skin Phototype of I-IV) with clinically-determined
mild to severe, fine and coarse wrinkles in the periocular area.
Subjects were excluded from the study if they had used topical
retinoids within 3 months of the study start, received injections
of dermal fillers or botulinum toxin or had facial peels or had
facial resurfacing procedures within 6 months or used other forms
of anti-aging products on the face within 30 days of study start.
Subjects with known allergies to the facial product regimen were
also excluded.
[0395] Subjects were instructed to apply the treatment serum on
their entire facial skin, twice daily (morning and evening) for
twelve weeks. In addition to the treatment serum, subjects agreed
to the use of a basic skincare regimen, including a cleanser, light
moisturizer and sunscreen. Subjects also agreed not to begin the
usage of any new facial products other than the provided materials
for the duration of the study.
[0396] IRB approval was obtained for this open-label, single-center
study from IntegReview of Austin, Tex. The study was conducted
according to ethical and regulatory principals from the
International Conference on Harmonization and good clinical
practices.
[0397] Clinical evaluations were conducted at baseline (Visit 1),
week 4 (Visit 2), week 8 (Visit 3) and week 12 (Visit 4). The
following procedures were conducted at each visit.
[0398] Efficacy Assessments: Fine and coarse wrinkles were
clinically graded using a ten point scale, on each subject's right
and left periocular area (where 0=none, 0.5-3.5=mild,
4-6.5=moderate and 7-9=severe). Ten point scales were also used to
assess skin tactile roughness (where 0=smooth, 9=rough), skin tone
(0=blotchy/uneven, 9=clear/even), skin firmness (where 0=skin
appears loose and sags, 9=skin appears firm) and skin radiance
(0=dull/flat matte, 9=bright/luminous/radiant). All grading
assessments were performed by the same investigator at each visit
to ensure grading consistency.
[0399] Safety Assessments: Tolerability of the treatment product
was assessed at all visits by the reporting of adverse events and
by objective and subjective assessments. Objective irritation
(erythema, edema and scaling) was assessed by the investigator
whereas subjective irritation (burning/stinging, itching and
tingling) was assessed by the subject, all using a 4-point scale
(where 0=none, 1=mild, 2=moderate and 3=severe).
[0400] Standardized Photography: Standardized, digital images were
taken of the subject's left, right and frontal facial views using
raking light (standard color).
[0401] Self-Assessment Questionnaires: Subjects completed a
Self-Assessment Questionnaire at Week 12, where they rated their
facial skin condition and the treatment serum's efficacy on a four
point scale (Strongly Agree, Agree, Disagree, Strongly Disagree).
Subjects rated their overall satisfaction with the treatment serum
on a four point scale where 1=Excellent, 2=Good, 3=Fair and
4=Poor.
[0402] Statistical Analysis: Mean clinical grading scores at weeks
4, 8 and 12 were compared to mean baseline scores using a student's
paired t-test. Changes from baseline were considered significant at
the p<0.05 level. For the subject Self-Assessment
Questionnaires, the percent incidence of positive responders was
reported for the treatment serum's efficacy and aesthetic
attributes sections.
Results
Results: In Vitro Study
[0403] After treatment of human dermal fibroblasts with Biometa
Complex (yeast metabolized conditioned growth media), the gene
expression profiled was analyzed with a special emphasis on genes
implicated in the anti-aging cascade. The following Table lists
some of the key genes regulated by Biometa complex: Biometa complex
stimulates a number of genes involved in the extracellular matrix
regeneration.
TABLE-US-00010 Function Genes Brief Description Extracellular
Matrix Col 1A1, Genes encoding collagen Proteins COL12A1, COL15A1,
Growth Factors and TGF-Beta Induces synthesis and secretion of
Cytokines involved major extracellular matrix proteins, in repair
and ECM collagen and elastin production TIMP1/ TIMPs play an
important role in the TIMP3 regulation of MMP activity VEGF-A/ Key
role in angiogenesis VEGF-B FGF3 Enhanced hyaluronan production and
implications in tissue remodeling PDGFRB Implicated in tissue
remodeling IGF Enhanced hyaluronan production and implications in
tissue remodeling IL-10RB, Involved in several signal IL-11,
transduction pathways that include IL-8, etc. tissue remodeling,
etc Antioxidant SOD1 involved in regulation of ROS- mediated tissue
damage; usually found in the extracellular matrix and is ideally
situated to prevent cell and tissue damage initiated by reactive
oxygen species (ROS) Stress Related HSP Heat shock proteins are
part of cellular defense against stress Skin Barrier TJP1/ZO-1 Help
improve skin barrier function
[0404] Additionally we saw a down-regulation of MMP (enzyme
responsible for breaking down collagen and elastin). This
down-regulation of MMP correlated well with an upregulation of
TIMPS (a natural inhibitor of MMPs). Biometa complex has also
upregulated other genes necessary to maintain the overall health of
the skin including stress proteins (HSP), antioxidant proteins
(SOD1), and proteins important for maintaining skin barrier
(TJ1).
[0405] Biometa Complex also stimulated fibroblasts to express and
synthesize key proteins (cytokines, growth factors, etc.) known to
be involved in skin repair and remodeling including bFGF
(Fibroblast Growth Factor), TIMP-1 (Tissue inhibitor of
Metalloproteinases), and VEGF (Vascular endothelial growth factor)
based on analysis of proteins released into medium after treatment.
This data shows a good correlation between gene expression and
protein synthesis. These results demonstrate that Biometa complex
has the ability to stimulate fibroblasts, thereby reducing signs of
aging.
Results: 12 Week Clinical-Usage Study
[0406] Thirty-three female subjects aged 35-56 years with mild to
severe, fine and coarse periocular wrinkles were enrolled and
completed the twelve week study.
[0407] At weeks 4, 8 and 12, statistically significant improvements
in mean scores for all photoaging parameters were achieved after
twice-daily topical use of the combination treatment serum.
Investigator assessments demonstrated significant reductions in
mean scores for fine and coarse periocular wrinkles at week 4 with
continued significant reductions through week 12 (all P<0.0001)
as shown in FIG. 4. Most notably, mean scores for coarse periocular
wrinkles decreased by 30% at week 12. In addition, mean scores for
skin firmness, skin tone, radiance and tactile roughness reflected
similar significant and progressive improvements from baseline and
at all subsequent follow-up visits (all P.ltoreq.0.02) as depicted
in FIG. 5 and FIG. 6. Especially prominent was the improvement in
the texture of the skin achieved as early as week 4, shown by a
significant reduction (79.5%) in mean tactile roughness scores
(P<0.0001).
[0408] The subject responses in the Self-Assessment Questionnaire
at week 12 strongly support the improvements observed in the
investigator assessments, with over 85% of subjects responding
favorably to all questions. The percentage of subjects who selected
Strongly Agree or Agree in response to the questionnaire at Week 12
are presented in FIG. 7.
[0409] Standardized digital photographs (data not shown) represent
examples of clinical responses after twice-daily use of the
combination treatment serum. Briefly, improvements in peri-ocular
fine and coarse wrinkles and skin tone were demonstrated in a 49
year old Caucasian female subject after only four weeks of product
use. In addition, a 46 year old Hispanic female subject showed
visible improvements in fine peri-ocular wrinkles, skin tone,
tactile roughness and radiance at week 4. A 46 year old
Korean/Caucasian female subject presenting with fine and coarse
peri-ocular wrinkles and uneven skin tone visibly improved after
four weeks of product use.
[0410] The combination treatment serum was well-tolerated and there
were no treatment-related adverse events reported during the course
of the study. Mean scores for edema, scaling, burning/stinging,
itching and tingling remained less than mild throughout the study
(all scores <0.09 on the 0-4 scale). Notably, mean scores for
erythema decreased from baseline at all follow-up visits, with a
statistically significant decrease observed at week 8
(P<0.03).
CONCLUSION
[0411] This treatment serum provides physicians and patients with a
unique and effective topical combination product that improves
signs of skin aging.
[0412] The present disclosure is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications in addition to those described will become apparent
to those skilled in the art from the foregoing description and
accompanying figures. Such modifications are intended to fall
within the scope of the appended claims.
[0413] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
* * * * *