U.S. patent application number 10/680370 was filed with the patent office on 2004-04-15 for treatment of skin with adenosine or adenosine analog.
This patent application is currently assigned to University of Massachusetts, a Massachusetts corporation. Invention is credited to Dobson, James G. JR., Ethier, Michael F..
Application Number | 20040071749 10/680370 |
Document ID | / |
Family ID | 22654844 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040071749 |
Kind Code |
A1 |
Dobson, James G. JR. ; et
al. |
April 15, 2004 |
Treatment of skin with adenosine or adenosine analog
Abstract
Methods for enhancing the condition of non-diseased skin by
application of compositions containing adenosine or an adenosine
analog are disclosed. Also disclosed are methods for increasing DNA
synthesis or protein synthesis in dermal cells, and methods for
increasing dermal cell size, by application of compositions
containing adenosine.
Inventors: |
Dobson, James G. JR.;
(Auburn, MA) ; Ethier, Michael F.; (Grafton,
MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
University of Massachusetts, a
Massachusetts corporation
|
Family ID: |
22654844 |
Appl. No.: |
10/680370 |
Filed: |
October 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10680370 |
Oct 7, 2003 |
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10184810 |
Jun 28, 2002 |
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6645513 |
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10184810 |
Jun 28, 2002 |
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09672348 |
Sep 28, 2000 |
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6423327 |
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09672348 |
Sep 28, 2000 |
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09179006 |
Oct 26, 1998 |
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Current U.S.
Class: |
424/401 ;
514/46 |
Current CPC
Class: |
A61K 2800/70 20130101;
A61Q 19/08 20130101; A61K 8/606 20130101; A61P 17/16 20180101; A61K
8/0208 20130101; A61K 31/70 20130101; A61P 43/00 20180101; A61Q
19/00 20130101; A61Q 19/007 20130101 |
Class at
Publication: |
424/401 ;
514/046 |
International
Class: |
A61K 031/7076; A61K
007/00 |
Goverment Interests
[0001] Work on this invention was supported by funds from the
United States government (Public Health Service Grants HL-22828 and
AG-11491). The government therefore has certain rights in this
invention.
Claims
We claim:
1. A method for enhancing the condition of non-diseased skin of a
mammal, comprising topically applying a therapeutically effective
amount of a composition comprising adenosine or an adenosine
agonist to non-diseased skin of said mammal.
2. The method of claim 1, wherein said composition further
comprises an angiogenic factor.
3. The method of claim 1, wherein the therapeutically effective
amount of adenosine is an adenosine concentration of 10.sup.-3 M to
10.sup.-7 M.
4. The method of claim 3, wherein said adenosine concentration is
10.sup.-4 M to 10.sup.-6 M.
5. The method of claim 4, wherein said adenosine concentration is
about 10.sup.-4 M.
6. The method of claim 1, wherein said composition further
comprises a conditioning agent.
7. The method of claim 6, wherein said conditioning agent is
selected from the group consisting of a humectant, an emollient,
and occlusive agent.
8. The method of claim 1, wherein addition of adenosine does not
affect skin cell proliferation.
9. The method of claim 1, wherein said skin comprises a skin
graft.
10. The method of claim 1, wherein said mammal is a human.
11. A method for promoting healing of broken, non-diseased skin in
a mammal, comprising topically administering a composition
comprising a therapeutically effective amount of adenosine or an
adenosine agonist to said mammal.
12. The method of claim 11, wherein said composition further
comprises an angiogenic factor.
13. The method of claim 11, wherein the therapeutically effective
amount of adenosine is an adenosine concentration of 10.sup.-3 M to
10.sup.-7 M.
14. The method of claim 13, wherein said adenosine concentration is
10.sup.-4 M to 10.sup.-6 M.
15. The method of claim 14, wherein said adenosine concentration is
about 1.times.10.sup.-4 M.
16. The method of claim 11, wherein said composition further
comprises a conditioning agent.
17. The method of claim 16, wherein said conditioning agent is
selected from the group consisting of a humectant, an emollient,
and occlusive agent.
18. The method of claim 11, wherein addition of adenosine does not
affect skin cell proliferation.
19. The method of claim 11, wherein said region of skin comprises a
skin graft.
20. The method of claim 11, wherein said mammal is a human.
21. A method for increasing DNA synthesis in a dermal cell of
non-diseased skin of a mammal, comprising topically administering a
therapeutically effective amount of adenosine to a region of
non-diseased skin of said mammal containing said dermal cell,
wherein addition of said adenosine does not cause proliferation of
said dermal cell.
22. The method of claim 21, wherein said composition further
comprises an angiogenic factor.
23. The method of claim 21, wherein the therapeutically effective
amount of adenosine is an adenosine concentration of 10.sup.-3 M to
10.sup.-7 M.
24. The method of claim 23, wherein said adenosine concentration is
10.sup.-4 M to 10.sup.-6 M.
25. The method of claim 24, wherein said adenosine concentration is
about 10.sup.-4 M.
26. The method of claim 21, wherein said composition further
comprises a conditioning agent.
27. The method of claim 26, wherein said conditioning agent is
selected from the group consisting of a humectant, an emollient,
and occlusive agent.
28. The method of claim 21, wherein said region of skin comprises a
skin graft.
29. The method of claim 21, wherein said mammal is a human.
30. A method of increasing protein synthesis in a dermal cell of
non-diseased skin of a mammal, comprising topically administering a
composition comprising a therapeutically effective amount of
adenosine to a region of skin of said mammal containing said dermal
cell, wherein addition of said adenosine does not cause
proliferation of said dermal cell.
31. The method of claim 30, wherein said composition further
comprises an angiogenic factor.
32. The method of claim 30, wherein the therapeutically effective
amount of adenosine is an adenosine concentration of 10.sup.-3 M to
10.sup.-7 M.
33. The method of claim 32, wherein said adenosine concentration is
10.sup.-4 M to 10.sup.-6 M.
34. The method of claim 33, wherein said adenosine concentration is
about 10.sup.-4 M.
35. The method of claim 30, wherein said composition further
comprises a conditioning agent.
36. The method of claim 35, wherein said conditioning agent is
selected from the group consisting of a humectant, an emollient,
and occlusive agent.
37. The method of claim 30, wherein said region of skin comprises a
skin graft.
38. The method of claim 30, wherein said mammal is a human.
39. A method of increasing cell size in a dermal cell in
non-diseased skin of a mammal, comprising topically administering a
composition comprising a therapeutically effective amount of
adenosine to a region of skin of said mammal containing said dermal
cell, wherein addition of said adenosine does not cause
proliferation of said dermal cell, wherein addition of said
adenosine does not cause proliferation of said dermal cell.
40. The method of claim 39, wherein said composition further
comprises an angiogenic factor.
41. The method of claim 39, wherein the therapeutically effective
amount of adenosine is an adenosine concentration of 10.sup.-3 M to
10.sup.-7 M.
42. The method of claim 41, wherein said adenosine concentration is
10.sup.-4 M to 10.sup.-6 M.
43. The method of claim 42, wherein said adenosine concentration is
about 10.sup.-6 M.
44. The method of claim 39, wherein said composition further
comprises a conditioning agent.
45. The method of claim 44, wherein said conditioning agent is
selected from the group consisting of a humectant, an emollient,
and occlusive agent.
46. The method of claim 39, wherein said region of skin comprises a
skin graft.
47. The method of claim 39, wherein said mammal is a human.
48. A method for enhancing skin condition in a mammal, comprising
providing fibroblasts from said mammal ex vivo, culturing said
fibroblasts in the presence of adenosine; and reintroducing said
fibroblasts into said mammal.
49. The method of claim 48, wherein the adenosine concentration in
said culturing step is from about 10.sup.-3 M to about 10.sup.-7
M.
50. A method for increasing protein synthesis in a cultured skin
fibroblast, comprising culturing said fibroblast in a culture
medium comprising about 10.sup.-3 M to about 10.sup.-7 M
adenosine.
51. The method of claim 50, wherein the adenosine concentration is
about 10.sup.-4 M.
52. A composition comprising 10.sup.-3 M to 10.sup.-7 M adenosine
and an angiogenesis factor.
53. The composition of claim 52, wherein the concentration of said
adenosine is about 10.sup.-4 M.
Description
FIELD OF THE INVENTION
[0002] This invention relates to dermatology and cell biology.
BACKGROUND OF THE INVENTION
[0003] Skin includes a surface layer, known as the epidermis, and a
deeper connective tissue layer, known as the dermis. The epidermis
undergoes continuous turnover as the outermost cells are exfoliated
and replaced by cells that arise from inner dermal layers. The
dermis is composed of a variety of cell types, including
fibroblasts.
[0004] Skin thickness begins to decline in humans after the age of
20 as the dermis becomes thinner and the number of skin fibroblasts
declines. As skin ages, or is exposed to UV light and other
environmental insults, changes in the underlying dermis can lead to
the functional and morphological changes associated with damaged
skin.
[0005] Decreases in the abundance and function of products of the
fibroblasts, which include collagen and proteoglycans, are believed
to play major roles in wrinkled and damaged skin.
SUMMARY OF THE INVENTION
[0006] We have discovered that adenosine stimulates DNA synthesis,
increases protein synthesis, and increases cell size in cultures of
human skin fibroblasts. Based on this discovery, the invention
provides methods and compositions for enhancing the condition of
skin.
[0007] In general, the invention provides a method for enhancing
the condition of non-diseased skin of a mammal, e.g., a human. The
method includes topically applying a therapeutically effective
amount of a composition including adenosine or an adenosine analog
to non-diseased skin of the mammal.
[0008] The invention also provides a method for promoting healing
of broken, non-diseased skin in a mammal by topically administering
a composition including a therapeutically effective amount of
adenosine or an adenosine analog to the mammal.
[0009] Also included in the invention is a method for increasing
DNA synthesis in a dermal cell of non-diseased skin of a mammal.
The method includes topically administering a therapeutically
effective amount of adenosine or an adenosine analog to a region of
non-diseased skin of the mammal containing dermal cell. The
adenosine is added so that it does not cause proliferation of the
dermal cell.
[0010] The invention also features a method of increasing protein
synthesis in a dermal cell of non-diseased skin of a mammal. The
method includes topically administering a composition including a
therapeutically effective amount of adenosine or an adenosine
analog to a region of skin of the mammal containing the dermal
cell. The adenosine or adenosine analog does not cause
proliferation of the dermal cell.
[0011] Also provided in the invention is a method of increasing
cell size in a dermal cell in non-diseased skin of a mammal, e.g.,
a human. The method includes topically administering a composition
including a therapeutically effective amount of adenosine to a
region of skin of the mammal containing the dermal cell, wherein
addition of the adenosine does not cause proliferation of the
dermal cell, wherein addition of the adenosine does not cause
proliferation of the dermal cell. The invention also includes a
method for enhancing skin condition in a mammal, e.g., a human. The
method includes providing fibroblasts from the mammal ex vivo,
culturing the fibroblasts in the presence of adenosine, and
reintroducing the fibroblasts into the mammal. The therapeutically
effective amount of adenosine used in the above-described methods
is preferably 10.sup.-3 M to 10.sup.-7 M, more preferably 10.sup.-4
M to 10.sup.-6 M, and most preferably about 10.sup.-4 M.
[0012] The composition used in the above-described methods can
include a second agent in addition to adenosine. The second agent
can be, e.g. an agent that promotes binding of adenosine or an
adenosine analog to an adenosine receptor, an angiogenic factor
such as vascular endothelial cell growth factor (VEGF), basic
fibroblast growth factor (BFGF), an agent that itself enhances skin
condition, such as tretoinin or another known conditioning agent
such as an emollient, a humectant, or an occlusive agent.
[0013] In preferred embodiments of the invention, the adenosine or
an adenosine analog does not promote skin cell proliferation.
[0014] The invention also provides a composition including about
10.sup.-3 M to about 10.sup.-7 M adenosine and a therapeutically
effective amount of an angiogenesis factor. In some embodiments,
the composition of the adenosine is about 10.sup.-4 M.
[0015] As used herein, "enhancement of skin condition" means a
noticeable decrease in the amount of wrinkling, roughness, dryness,
laxity, sallowness, or pigmentary mottling in skin.
[0016] As used herein, a "therapeutically effective amount" of
adenosine or an adenosine analog means an amount that enhances skin
condition when applied to skin. As used herein, "non-diseased skin"
means skin free of any proliferative disorder observable by visual
inspection.
[0017] The present invention advantageously allows for enhancement
of skin condition. This results in skin that shows a less wrinkled,
rough, or dry complexion. For example, the invention provides for
enhancing the condition of skin damaged due to exposure to the sun
or skin whose condition has deteriorated due to normal aging.
[0018] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0019] Other features and advantages of this invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A and 1B are histograms showing the effect of
adenosine on [.sup.3H]thymidine incorporation in cultures of normal
human skin (FIG. 1A) and lung fibroblasts (FIG. 1B). After
incubation in serum-free medium for 24 hours, cells were exposed to
10.sup.-4 M adenosine for 18 hours. Medium was replaced with
serum-free medium without adenosine, and [.sup.3H]thymidine was
added. Results are expressed as percent [.sup.3H]thymidine
incorporation compared to control cultures without adenosine and
are means.+-.SEM for 4-5 experiments. "*" denotes value was
significantly different from control value without adenosine.
[0021] FIGS. 2A and 2B are histograms showing concentration
responses of adenosine-stimulated protein synthesis in human skin
fibroblasts from a young (FIG. 2A) and aged (FIG. 2B) donor. Cells
were grown to 75% confluence. Medium was then replaced with
serum-free medium with or without adenosine. After 48 hours,
[.sup.3H]phenylalanine incorporation was determined as described.
Results are expressed as % [.sup.3H]phenylalanine incorporation
compared to control cultures without adenosine and are means.+-.SEM
for 6-25 experiments. "*" denotes value was significantly different
from control value without adenosine.
DETAILED DESCRIPTION
[0022] The invention is suitable for treating skin of a mammal,
e.g., a human, for which promotion of fibroblast-associated dermal
functions is desired. For example, promotion of
fibroblast-associated functions is desirable in enhancing the
condition of aged skin, which is associated with a decrease in
dermal cell function and is characterized by increased dryness or
roughness, or both. The method can also be used on subjects having
otherwise damaged skin, e.g., wrinkled skin and skin with a
non-proliferative disorder. The method can may further be used
prophylactically on a subject to minimize deterioration of skin
condition associated with aging or environmental factors, such as
photodamage.
[0023] Adenosine and suitable adenosine analogs are suitable for
use in enhancing skin condition. Adenosine analogs such as
adenosine agonists, adenosine receptor agonists, and compounds that
increase intracellular or extracellular adenosine levels are
suitable for use in the invention.
[0024] Agonists of adenosine include 2'-deoxyadenosine;
2',3'-isopropoylidene adenosine; toyocamycin; 1-methyladenosine;
N-6-methyladenosine; adenosine N-oxide; 6-methylmercaptopurine
riboside; 6-chloropurine riboside, 5'-adenosine monophosphate,
5'-adenosine diphosphate, or 5'-adenosine triphosphate. Adenosine
receptor agonists include phenylisopropyl-adenosine ("PIA"),
1-Methylisoguanosine, ENBA (S(-), N.sup.6-Cyclohexyladenosine
(CHA), N.sup.6-cyclopentyladenosine (CPA),
2-Chloro-N.sub.6-cyclopentyladenosine, 2-chloroadenosine, and
adenosine amine congener (ADAC), all of which are agonists for the
adenosine A.sub.1 receptor. Other receptor agonists include
2-p-(2-carboxy-ethyl)phenethyl-amino-5'-N-ethylcarboxamido-adenosine
(CGS-21680), N-ethylcarboxamido-adenosine (NECA) and
napthyl-substituted aralkoxyadenosine (SHA-082),
5'(N-Cyclopropyl)-carboxamidoadenosine, DPMA (PD 129,944),
Metrifudil, which are agonists for the adenosine A.sub.2 receptor.
Other adenosine receptor agonists include those which
preferentially bind the A.sub.1 receptor relative to the A.sub.2
receptor, such as 2-Chloroadenosine, N.sup.6-Phenyladenosine, and
N.sup.6-Phenylethyladenosine; and those which preferentially bind
the A.sub.2 receptor relative to the A.sub.1 receptor, such as
2-Phenylaminoadenosine and MECA.
[0025] Also suitable for use are compounds that increase
intracellular adenosine concentration by inhibiting the cellular
uptake of adenosine or the breakdown of adenosine. One pathway of
adenosine metabolism is the conversion of adenosine to inosine by
adenosine deaminase. An example of an adenosine deaminase inhibitor
is erythro-9-(2-hydroxy-3-nonyl) adenine ("EHNA"). Adenosine kinase
inhibitors can also be used. Adenosine kinase converts adenosine to
adenosine monophosphate by adenosine kinase. An example of an
adenosine kinase inhibitor is iodotubercidin. Other suitable
compounds include those that inhibit the dipyridamole-sensitive
nucleoside transporter, which exports adenosine from the cytoplasm,
and agents that promote the activity of a 5'-nucleotidase, e.g.,
the ATP-activated 5'-nucleotidase, which forms adenosine. Compounds
that increase tissue adenosine and ATP levels include acadesine
(AICA-riboside), which is described in Gruber et al., Circulation
80:1400-1411 (1989).
[0026] Adenosine can be also be administered with a second
compound. The second compound can enhance the action of adenosine
or the adenosine analog, e.g., by enhancing binding of adenosine or
an adenosine analog to an adenosine receptor. An example of such a
compound is PD 81,728, which is described in Kollias-Baker et al.
J. Pharmacol. Exp. Ther. 281:761-68. Alternatively, the second
agent can itself act to enhance skin condition. Examples of these
types of agents include tretinoin, a recognized skin conditioning
agent (see, e.g., Olsen et al., J. Amer. Acad. Dermatol. 37:217-26,
1997), an angiogenic factor such as vascular endothelial cell
growth factor (VEGF) or basic fibroblast growth factor (BFGF), or a
conditioning agent.
[0027] The second compound can also be a conditioning agent such as
an emollient, humectant, or occlusive agent. Numerous examples of
particular conditioning agents are provided in the CTFA Cosmetic
Ingredient Handbook (Cosmetic Toiletries and Fragrances
Association, Washington, D.D., 1988). Emollients help to maintain
the soft, smooth, and pliable appearance of skin and function by
remaining on the skin surface or in the stratum corneum to act as
lubricants, to reduce flaking, and to improve the skin's
appearance. Examples of emollients include acetyl trioctyl citrate,
cetyl alcohol, butyl myristate, cetyl alcohol, and mineral oil.
[0028] Humectants act to increase the water content of the top
layers of the skin. Humectants include, e.g., acetamide MEA,
fructose, and xylitol. Occlusive agents inhibit the evaporation of
water from skin, thereby increasing the water contend of the skin.
Acetylated castor oil, mineral oil, and lauryl stearate are
examples of occlusive agents.
[0029] A subject can be treated by applying adenosine or an
adenosine analog in a pharmaceutical composition in an effective
amount and for a period of time sufficient to improve the condition
of the skin.
[0030] The pharmaceutical composition may be formulated using
conventional methods to prepare pharmaceutically useful
compositions. Such compositions preferably include at least one
pharmaceutically acceptable carrier, such as those described in
Remington's Pharmaceutical Sciences (E. W. Martin). In addition,
the compositions preferably include a pharmaceutically acceptable
buffer, preferably phosphate buffered saline, together with a
pharmaceutically acceptable compound for adjusting isotonic
pressure, such as, for example, sodium chloride, mannitol, or
sorbitol.
[0031] Adenosine or an adenosine agonist can also be provided in
carriers and adjuvants such as ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum albumin,
buffer substances, such as phosphates, glycine, sorbic acid,
potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances and
polyethylene glycol. Adjuvants for topical or gel base forms of
adenosine or adenosine analogs may, for example, be selected from
the group consisting of sodium carboxymethylcellulose,
polyacrylates, polyoxythylene-polyoxypropylene-block polymers,
polyethylene glycol and wood wax alcohols. For all administrations,
conventional depot forms may be used.
[0032] The adenosine or adenosine analog-containing compositions
may be in any pharmaceutically acceptable dosage form. They are
preferably applied by topical routes to exert local therapeutic
effects. For topical application, the penetration of the adenosine
into skin tissue may be enhanced by a variety of methods known to
those of ordinary skill in the art. For example, adenosine may be
applied directly and mechanically rubbed into the skin.
Alternatively, adenosine or adenosine analogs may be incorporated
into a transdermal patch that is applied to the skin. Preferably,
the penetration resulting from these methods is enhanced with a
chemical transdermal delivery agent such as dimethyl sulfoxide
(DMSO) or the nonionic surfactant, n-decylmethyl sulfoxide (NDMS),
as described in Choi et al., Pharmaceutical Res., 7(11):1099,
1990.
[0033] Other modes of administration include, e.g., oral,
subdermal, intradermal, or intravenous. When oral administration is
used, it is critical that the adenosine or adenosine analog be
delivered to that it is not degraded prior to exiting the digestive
system.
[0034] The most effective mode of administration and dosage regimen
of adenosine or the adenosine analog will depend upon the skin
condition, previous therapy, the subject's health status, response
to the adenosine, the judgment of the treating physician and the
mode in which the adenosine is applied. For example, dosages for a
therapeutically effective amount for topical application would be
in the range of 100 ng to 10 mg per treated surface area per day.
The adenosine may be administered to the patient at one time or
over a series of treatments. When adenosine or the adenosine analog
is administered in conjunction with a second agent, they can be
administered either concurrently or sequentially, and can be
administered in the same mode or a different mode, e.g., topical or
oral.
[0035] Adenosine or an adenosine analog enhances skin condition
when there is a noticeable decrease in noticeable decrease in the
amount of wrinkling, roughness, dryness, laxity, sallowness, or
pigmentary mottling of the treated skin. Methods of measuring
improvements in skin condition are well known in the art (see,
e.g., Olsen et al., J. Amer. Acad. Dermatol. 26:215-24, 1992), and
can include subjective evaluations by the patient or a second
party, e.g., a treating physician. Objective methods can include
skin topography measurements, such as those described in Grove et
al., J. Amer. Acad. Dermatol. 21:631-37 (1989). In skin topography
measurements, silicone rubber replicas are made of a small area of
skin, e.g., a 1 cm diameter circular area. The silicone rubber
replicas capture fine lines and wrinkles on the skin. These
specimens are then analyzed using computerized digital image
processing to provide an objective measurement of the skin's
topography. Skin topography measurements generated following
digital-image processing can be measured using the values R.sub.a
and R.sub.z as described in Olsen et al., J. Amer. Acad. Dermatol.
37:21726, 1997, where R.sub.a represents the area of deviation of
skin surface features above and below an average central line, and
R.sub.z represents the difference between the maximum and minimum
heights in five equal segments of the skin surface profile. A
statistically significant decline (e.g., P<0.05) in R.sub.a and
R.sub.z values in skin treated with adenosine or an adenosine
analog compared to untreated skin indicates an enhancement of skin
condition.
[0036] Fibroblasts treated with adenosine or adenosine analogs can
also be incorporated into a matrix and implanted in the body, e.g.,
as part of a skin graft. In addition, fibroblasts can be
genetically engineered ex vivo to increase the amount of
intracellular adenosine levels and then re-introduced into a human
patient. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each
of which is incorporated by reference herein in its entirety)
Experimental Information
[0037] Cell Culture
[0038] Human skin fibroblasts and human lung fibroblasts were
supplied by the N.I.A. Aging Culture Repository Center (Camden,
N.J.). For skin fibroblasts, primary cultures had been initiated
from explants obtained from a 3 mm punch biopsy of the mesial
aspect of the upper left arm. Human lung fibroblasts (IMR-90) were
established from a 16-week normal female fetus. All cells displayed
a normal diploid karyotype and all cells tested negative for
bacteria, fungi and mycoplasma contamination.
[0039] Cells were grown in Eagle's minimal essential medium (MEM)
supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin
and 100 mg/ml streptomycin in a 37.degree. C., 5% CO.sub.2/95% air
environment. After reaching confluence, cells were subcultivated
with 0.25% trypsin in MEM with no added Ca.sup.2+ or Mg.sup.2+.
[0040] Incorporation of [.sup.3H]Thymidine
[0041] As an index of DNA synthesis incorporation of
[.sup.3H]thymidine was measured as described in Ethier et al., Am.
J. Physiol. 272:H1470-79 (1997). Confluent monolayers of human skin
fibroblasts in MEM plus 10% FBS were seeded into 16 mm diameter
culture wells (24-well plates) at a density of 1.times.10.sup.4
cells/cm.sup.2. Cells were grown at 37.degree. C. under standard
culture conditions (5% C02-95% air) until they were approximately
75% confluent. Medium was then removed and the cells were made
"serum-free by incubation in MEM with no FBS for 24 hours.
Adenosine or vehicle (MEM) was added for an additional 18 hours.
This medium was then replaced with fresh MEM, and the cells were
pulsed with 1 mCi/ml [3H] thymidine (6.7 Ci/mmol). After a 2 hour
incubation period, the medium was discarded and the cells were
rinsed twice with cold (4.degree. C.) Hank's balanced salt solution
(HBSS) and incubated for 5 minutes with 0.5 ml cold 10% (w/v)
trichloroacetic acid (TCA). The wells were then rinsed with 8% TCA
and the TCA-insoluble material was solubilized with 0.5 ml of a
solution of 0.2M NaOH and 0.2% sodium decyl sulfate (SDS). The
radioactivity of this fraction was determined by standard liquid
scintillation spectrometric techniques.
[0042] Incorporation of [.sup.3H]thymidine was expressed as counts
per minute (cpm) of .sup.3H per culture. Data in each experiment
was derived from 4 identically treated wells. Since the cpm/well
exhibited variation between experiments, data representing combined
experiments are expressed herein as a percent of their respective
mean control value.
[0043] Incorporation of [.sup.3H]phenylalanine
[0044] Incorporation of [.sup.3H]phenylalanine was measured as an
index of protein synthesis. Human skin fibroblasts were seeded into
24-well culture plates in MEM containing 10% FBS. When cells had
grown to approximately 75% confluence the culture medium was
replaced with serum-free MEM with or without adenosine. After 48
hours, 2 .mu.Ci/ml [3H]phenylalanine was added to the cultures.
Unlabeled phenylalanine (0.36 mM) was also added to equalize
concentrations of intracellular and extracellular phenylalanine.
After 8 hours, medium was removed and the cells were washed twice
with cold (4.degree. C.) HBSS and incubated for 20 minutes in cold
10% (w/v) TCA. Cells were then incubated 5 minutes in 95% ethanol
(4.degree. C.) and the TCA-insoluble material was solubilized with
a solution of 0.2M NaOH and 0.2% SDS. The radioactivity of this
fraction was determined by standard liquid scintillation
spectrometric techniques.
[0045] Incorporation of [.sup.3H]phenylalanine was expressed as cpm
of .sup.3H per culture well and data in each experiment were
derived from six identically treated wells. Since the cpm/well
exhibited variation between experiments, data representing combined
experiments are expressed as a percent of their respective mean
control value.
[0046] Determination of Cell Size
[0047] Human fibroblasts in MEM 10% FBS were seeded into 25
cm.sup.2 culture flasks at a density of 1.times.10.sup.4
cells/cm.sup.2. When the cells had grown to approximately 80%
confluence the culture medium was removed and the cells were
incubated in serum-free MEM for 24 hours. Adenosine or vehicle
(MEM) was added for 18 hours and cells were then washed twice with
cold (4.degree. C.) HBSS. Cells were removed with 0.25% trypsin in
calcium-and magnesium-free MEM and diluted in cold (4.degree. C.)
HBSS for measurement of relative cell size with a
fluorescence-activated cell sorter (FACS; Becton Dickinson
Vantage). Cell size was determined by forward light scatter on a
minimum of 1.times.10.sup.4 cells per experiment.
[0048] Experimental Materials
[0049] MEM, FBS, penicillin, streptomycin, trypsin, and HBSS were
obtained from GIBCO (Grand Island, N.Y.), [.sup.3H]thymidine (6.7
Ci/mmol) and phenylalanine, L-ring-2,3,4,5,6.sup.3H] (92 Ci/mmol)
were obtained from Dupont NEN (Boston, Mass.) Adenosine was from
Boehringer Mannheim, SDS was from National Diagnostics, (Highland
Park, N.J.) and TCA and ethanol were obtained from Fisher
Scientific (Pittsburgh, Pa.).
[0050] Data Analysis
[0051] Analysis of variance (ANOVA) was used to determine
statistical differences between means. The Dunett's test was
applied for multiple comparisons as described in Zar, J. H.,
Biostatistical Analysis. Englewood Cliffs, N.J., Prentice Hall,
Inc. pp. 150-153, 1984. In addition, the Wilcoxon test was employed
to verify differences between values expressed as a percentage.
Differences were considered statistically different when
P<0.05.
[0052] DNA Synthesis
[0053] Exposure to 10.sup.-4M adenosine increased
[.sup.3H]thymidine incorporation by 43.+-.9% in five studies on
cultures of human fibroblasts (AG607720B) made quiescent by serum
removal. These results are summarized in FIG. 1A. In contrast,
adenosine (10.sup.-4M) had no effect on [.sup.3H]thymidine
incorporation in cultures of human lung fibroblasts (IMR-90) (FIG.
1B). Concentrations of adenosine ranging from 10-7 M to 10.sup.-3M
also failed to stimulate [.sup.3H]thymidine incorporation in IMR-90
lung fibroblasts (data not shown).
[0054] The effect of adenosine on DNA synthesis was additionally
determined on skin fibroblast cultures from six different human
donors. Adenosine (10.sup.-4M) stimulated DNA synthesis in all
three cultures derived from young human donors (Table 1). Values
shown are means.+-.SEM, where n is number of experiments. Exposure
to adenosine and determination of [.sup.3H]thymidine incorporation
were as described above. The asterisk denotes a value significantly
different from the corresponding control (100%).
1TABLE 1 Effect of adenosine on [.sup.3H] thymidine incorporation
into cultured human skin fibroblasts derived from young donors
[.sup.3H] thymidine Cell Adenosine Donor incorporation Strain
(10.sup.-4 M) Age Sex (% of control) n AG07720B - 24 F 100 24 + 124
.+-. 7* 24 AG07306A - 28 F 100 6 + 193 .+-. 20* 6 AG09605 - 30 M
100 12 + 133 .+-. 15* 12
[0055] Peak stimulation of [.sup.3H]thymidine incorporation
(93.+-.20%, n=6) was achieved in human skin fibroblast cultures
derived from a 28 year old female (AG07306A).
[0056] Adenosine (10.sup.-4M) stimulated DNA synthesis in 2 of 3
cultures derived from aged human donors (Table 2). As in Table 1,
values are means.+-.SEM, and n is the number of experiments
performed. The asterisk denotes a measurement significantly
different from the corresponding control (10.0%). Adenosine
exposure increased [.sup.3H]thymidine incorporation by 53.+-.31%
and 54.+-.22% in human skin fibroblast cultures derived from a 70
year-old male and a 84 year-old male, respectively. Adenosine had
no effect on cultures derived from a 67-year old female.
2TABLE 2 Effect of adenosine on [.sup.3H] thymidine incorporation
into cultured human skin fibroblasts derived from aged donors
[.sup.3H] thymidine Cell Adenosine Donor incorporation Strain
(10.sup.-4 M) Age Sex (% of control) n AG11728 - 67 F 100 6 + 91
.+-. 6 6 AG12949 - 70 M 100 11 + 150 .+-. 31* 11 AG11730 - 84 M 100
10 + 154 .+-. 22* 10
[0057] Protein Synthesis
[0058] The effect of adenosine on protein synthesis was determined
by measuring [.sup.3H]phenylalanine incorporation into cultures of
human fibroblasts from a young and aged donor.
[0059] Cultures made quiescent by serum removal were exposed to
adenosine (10.sup.-6M to 10.sup.-4M) for 48 hours and then pulsed
with phenylalanine. In skin fibroblast cultures derived from a
28-year old female (AG073060A) and an 84-year old male (AG11730),
adenosine (10.sup.-4M) increased protein synthesis by 13.+-.4%
(n=25) and 13.+-.6% (n=17), respectively (FIG. 2).
[0060] Cell Size
[0061] The effect of adenosine on cell size was determined on human
skin fibroblasts from young and aged donors by measuring forward
light scatter in a FACS analyzer. Cultures made quiescent by serum
removal were exposed to adenosine for 18 hours, removed by
trypsinization, and diluted in 4.degree. C. HBSS. A minimum of
1.times.10.sup.4 cells were measured for each experiment. The
results are shown in Table 2. Values are mean.+-.SEM for relative
cell size determined by forward light scatter (FLS) in a
fluorescence-activated cell sorter, and n=number of cells measured.
The asterisk denotes the measurement is significantly different
from corresponding control.
[0062] In skin fibroblast cultures from a 28 year old female
(AG073060A) adenosine (10.sup.-4M) significantly increased cell
size by 1.8 and 2.2% in two of three experiments (Table 3).
[0063] The effect of adenosine on cell size was also measured on
skin fibroblasts from an aged donor. The results are shown in Table
IV. Values are mean.+-.SEM for relative cell size determined by
forward light scatter (FLS) in a fluorescence-activated cell
sorter, where n is the number of cells measured. An asterisk
indicates a value significantly different from corresponding
control.
[0064] In cultures derived from an 84-year old male (AG11730),
adenosine (10.sup.-4M) significantly increased cell size by
2.7-4.9% in 3 of 3 experiments (Table 4).
3TABLE 3 Effect of adenosine on cell size in cultured human skin
fibroblasts derived from young donors Relative Experiment Adenosine
Size % Number (10.sup.-4 M) (FLS) increase n 1 - 524 .+-. 0.55 --
1.5 .times. 10.sup.4 + 526 .+-. 0.55 0.4 1.5 .times. 10.sup.4 2 -
319 .+-. 1.24 -- 1.0 .times. 10.sup.4 + 326 .+-. 1.16* 2.2* 1.0
.times. 10.sup.4 3 - 342 .+-. 0.94 -- 1.0 .times. 10.sup.4 + 348
.+-. 0.95* 1.8* 1.0 .times. 10.sup.4
[0065]
4TABLE 4 Effect of adenosine on cell size in cultured human skin
fibroblasts derived from aged donors Relative Experiment Adenosine
Size % Number (10.sup.-4 M) (FLS) increase n 1 - 333 .+-. 0.79 --
1.0 .times. 10.sup.4 + 342 .+-. 0.75* 2.7* 1.0 .times. 10.sup.4 2 -
323 .+-. 1.01 -- 1.0 .times. 10.sup.4 + 337 .+-. 0.96* 4.3* 1.0
.times. 10.sup.4 3 - 306 .+-. 0.81 -- 1.0 .times. 10.sup.4 + 321
.+-. 0.81* 4.9* 1.0 .times. 10.sup.4
Other Embodiments
[0066] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention.
For example, while the invention has been described using adenosine
and adenosine agonists, other compounds structurally similar to
adenosine can also be used, e.g., purine-containing compounds and
compounds having a ribosyl moiety. Other aspects, advantages, and
modifications of the invention are within the scope of the
following claims.
* * * * *