U.S. patent application number 08/963831 was filed with the patent office on 2002-01-17 for antimutagenic compositions for treatment and prevention of photodamage to skin.
Invention is credited to ROMANTSEV, FEDOR, VON BORSTEL, REID W..
Application Number | 20020006913 08/963831 |
Document ID | / |
Family ID | 25507775 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020006913 |
Kind Code |
A1 |
VON BORSTEL, REID W. ; et
al. |
January 17, 2002 |
ANTIMUTAGENIC COMPOSITIONS FOR TREATMENT AND PREVENTION OF
PHOTODAMAGE TO SKIN
Abstract
A method of improving DNA repair and reducing DNA damage and for
reducing mutation frequency in skin for the purpose of reducing
consequences of exposure to solar or ultraviolet radiation is
disclosed. The methods comprise administering to the skin a
composition containing deoxyribonucleosides in concentrations
sufficient to enhance DNA repair or reduce mutation frequency in a
vehicle capable of delivering effective amounts of
deoxyribonucleosides to the necessary skin cells.
Inventors: |
VON BORSTEL, REID W.;
(POTOMAC, MD) ; ROMANTSEV, FEDOR; (GAITHERSBURG,
MD) |
Correspondence
Address: |
NIXON & VANDERHYE
1100 NORTH GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201
|
Family ID: |
25507775 |
Appl. No.: |
08/963831 |
Filed: |
November 4, 1997 |
Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 8/606 20130101;
Y10S 514/944 20130101; A61P 1/02 20180101; A61P 35/00 20180101;
Y10S 514/969 20130101; A61P 17/04 20180101; A61K 31/70 20130101;
Y10S 514/848 20130101; A61Q 19/08 20130101; A61Q 19/004 20130101;
Y10S 514/847 20130101; A61P 17/00 20180101; A61P 17/06 20180101;
A61P 17/16 20180101; Y10S 514/844 20130101; A61P 1/04 20180101 |
Class at
Publication: |
514/44 |
International
Class: |
A61K 031/70; A01N
043/04 |
Claims
What is claimed is:
1. A method of improving DNA repair in the skin or mucosa of a
mammal comprising administering to said mammal at least one
deoxyribonucleoside, deoxyribonucleotide, or
oligodeoxyribonucleotide.
2. A method as in claim 1 wherein said at least one
deoxyribonucleoside is administered topically.
3. A method as in claim 1 wherein said at least one
deoxyribonucleoside is selected from the group consisting of
deoxycytidine, deoxyadenosine, deoxyguanosine, and thymidine.
4. A method as in claim 3 wherein said at least one
deoxyribonucleoside is deoxycytidine.
5. A method as in claim 3 where said at least one
deoxyribonucleoside is deoxyadenosine.
6. A method as in claim 3 wherein said at least deoxyribonucleoside
is deoxyguanosine.
7. A method as in claim 3 wherein said at least deoxyribonucleoside
is thymidine.
8. A method as in claim 3 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 10 milligrams per milliliter of each
deoxyribonucleoside.
9. A method as in claim 3 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
1.0 to 5 milligrams per milliliter of each deoxyribonucleoside.
10. A method as in claim 3 wherein said at least one
deoxyribonucleoside is a mixture of deoxycytidine, deoxyadenosine,
deoxyguanosine, and thymidine.
11. A method as in claim 10 wherein said deoxyribonucleoside is
administered in a vehicle containing from 0.1 to 10 milligrams per
milliliter of each deoxyribonucleoside.
12. A method as in claim 10 wherein aid deoxyribonucleoside is
administered in a vehicle containing from 1 to 5 milligrams per
milliliter of each deoxyribonucleoside.
13. A method for reducing mutation frequency in skin of a mammal
exposed to a mutagen comprising administering to said skin a source
of at least one deoxyribonucleoside.
14. A method as in claim 13 wherein siad mutagen is selected from
the group consisting of ultraviolet or solar radiation, a chemical
mutagen, a free radcial, or ionizing radiation.
15. A method as in claim 13 wherein said source is selected from
the group consisting of at least one free or acyl
deoxyribonucleoside.
16. A method as in claim 13 wherein said source is selected from
the group consisting of a deoxyribonucleotide, an
oligodeoxyribonucleotide, and a polydeoxyribonucleotide.
17. A method as in claim 13 wherein said source of at least one
deoxyribonucleoside is administered in a vehicle at a concentration
of from 1.0 to 20 milligrams per milliliter.
18. A method as in claim 15 wherein said at least one
deoxyribonucleoside is free or acyl deoxycytidine.
19. A method as in claim 15 wherein said at least one
deoxyribonucleoside is free or acyl deoxyadenosine.
20. A method as in claim 15 wherein said at least one
deoxyribonucleoside is free or acyl deoxyguanosine.
21. A method as in claim 15 wherein said at least one
deoxyribonucleoside is free or acyl thymidine.
22. A method as in claim 15 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 10 milligrams per milliliter of each
deoxyribonucleoside.
23. A method as in claim 15 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
1.0 to 5 milligrams per milliliter of each deoxyribonucleoside.
24. A method as in claim 15 wherein said at least one
deoxyribonucleoside is a mixture of free or acyl deoxycytidine,
deoxyadenosine, deocquanodsione and thymidine.
25. A method as in claim 24 where said at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 10 milligrams per milliliter of each
deoxyribonucleoside.
26. A method as in claim 24 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from 1
to 5 milligrams per milliliter of each deoxyribonucleoside.
27. A method for reducing the chance of developing skin cancer in a
mammal due to exposure to a mutagen comprising administering to the
skin of said mammal a source of at least one deoxyribonucleoside
wherein said source is administered such that said at least one
deoxyribonucleoside is present on skin of said mammal during or
after exposure to said mutagen in an amount sufficient to reduce
the deleterious consequences of said exposure.
28. A method as in claim 27 wherein said source is administered
within 3 days after exposure to a mutagen.
29. A method as in claim 28 wherein said mutagen is selected from
the group consisting of solar, ultraviolet, or ionizing
radiation.
30. A method as in claim 27 wherein said source is selected from
the group consisting of at least one free or acyl
deoxyribonucleoside.
31. A method as in claim 27 wherein said source is selected from
the group consisting of a deoxyribonucleotide, an
oligodeoxyribonucleotide, and a polydeoxyribonucleotide.
32. A method as in claim 27 wherein said source of at least one
deoxyribonucleoside is administered in a vehicle at a concentration
of from 1.0 to 20 millegrams per milliliter.
33. A method as in claim 30 wherein said at least one
deoxyribonucleoside is free or acyl deoxycytidine.
34. A method as in claim 30 wherein said at least one
deoxyribonucleoside is free or acyl deoxyadenosine.
35. A method as in claim 30 wherein said at least one
deoxyribonucleoside is free or acyl deoxyguanosine.
36. A method as in claim 30 wherein said at least one
deoxyribonucleoside is free or acyl thymidine.
37. A method as in claim 30 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 10 milligrams per milliliter of each
deoxyribonucleoside.
38. A method as in claim 30 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
1.0 to 5 milligrams per milliliter of each deoxyribonucleoside.
39. A method as in claim 30 wherein said at least one
deoxyribonucleoside is a mixture of free or acyl deoxycytidine,
deoxyadenosine, deoxyguanosine, and thymidine.
40. A method as in claim 39 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 10 milligrams per milliliter of each
deoxyribonucleoside.
41. A method as in claim 39 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from 1
to 5 milligrams per milliliter of each deoxyribonucleoside.
42. A method for reducing the rate of skin photoaging in a mammal
comprising administering to the skin of said mammal at least one
deoxyribonucleoside.
43. A method as in claim 42 wherein said at least one
deoxyribonucleoside is deoxycytidine.
44. A method as in claim 42 wherein said at least one
deoxyribonucleoside is deoxyadenosine.
45. A method as in claim 42 wherein said at least one
deoxyribonucleoside is deoxyguanosine.
46. A method as in claim 42 wherein said at least one
deoxyribonucleoside is thymidine.
47. A method as in claim 42 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 10 milligrams per milliliter of each
deoxyribonucleoside.
48. A method as in claim 42 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
1.0 to 5 milligrams per milliliter of each deoxyribonucleoside.
49. A method as in claim 42 wherein said at least one
deoxyribonucleoside is a mixture of deoxycytidine, deoxyadenosine,
deoxyguanosine, and thymidine.
50. A method as in claim 49 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 10 milligrams per milliliter of each
deoxyribonucleoside.
51. A method as in claim 49 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from 1
to 5 milligrams per milliliter of each deoxyribonucleoside.
52. A method for reducing the chance of developing actinic
keratoses in at mammal comprising administering to the skin of said
mammal a source of a least one deoxyribonucleoside.
53. A method as in claim 52 wherein said source is selected from
the group consisting of a deoxyribonucleoside, a
deoxyribonucleotide, an oligodeoxyribonucleotide, and an acyl
derivatives of deoxyribonucleoside.
54. A method of reducing the deleterious consequences of
photosensitization or photodynamic sensitization on the skin of a
mammal caused by an endogenous or exogenous photochemically active
chromophore comprising administering to said skin an energy
scavenging agent with a lowest triplet state energy less than or
equal to that of nucleobases in DNA.
55. A method as in claim 54 wherein said exogenous chromophore is a
sunscreen agent.
56. A method as in claim 55 wherein said sunscreen agent is
selected from the group consisting of avobenzone (t-butyl
dimethoxydibenzoylmethane), oxybenzone (benzophenone-3),
dioxybenzone (benzophenone-8), sulisobenzone (benzophenone-4;
2-hydroxy-4-methoxybenzophenone-5-sulfonic acid), octocrylene
(2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octyl methoxycinnamate
(2-ethylhexyl p-methoxycinnamate), octyl salicylate
(2-ethylhexylsalicylate), homosalate (homomenthyl salicylate),
trolamine salicylate (triethanolamine salicylate),
phenylbenzimidazole sulfonic acid, PABA (para-aminobenzoic acid),
roxadimate (ethyl 4-bis hydroxypropyl aminobenzoate), lisadimate
(glyceryl PABA), Padimate O (octyldimethyl PABA), menthyl
anthranilate, or Parsol 1789 (butyl methoxydibenzoylmethane)
57. A method as in claim 54 wherein said energy scavenging agent is
selected from the group consisting of DNA, an
oligodeoxyribonucleotide, a ribonucleoside, a deoxyribonucleoside,
a ribonucleotide, a deoxyribonucleotide, an acyl
deoxyribonucleoside, and an acyl ribonucleoside.
58. A method as in claim 57 wherein said energy scavenging agent is
administered in a vehicle containing 0.1 to 20 mg/ml.
59. A method as in claim 57 wherein said energy scavenging agent is
a mixture comprising free or acylated deoxycytidine,
deoxyadenosine, deoxyguanosine, and thymidine.
60. A method as in claim 59 wherein said mixture is administered in
a vehicle containing from 0.1 to 10 milligrams per milliliter of
each deoxyribonucleoside.
61. A method for reducing the deleterious consequences of exposure
of a mutagen to the skin of a mammal comprising administering to
said mammal at least one deoxyribonucleoside, deoxyribonucleotide,
or oligodeoxyribonucleotide.
62. A method as in claim 61 wherein said mutagen is solar or
ultraviolet radiation.
63. A method as in claim 61 wherein said mutagen is ionizing
radiation.
64. A method as in claim 61 wherein said mutagen is a free
radical.
65. A method as in claim 61 wherein said at least one
deoxyribonucleoside is administered topically.
66. A method as in claim 61 wherein said at least one
deoxyribonucleoside is selected from the group consisting of
deoxycytidine deoxyadenosine, deoxyguanosine, and thymidine.
67. A method as in claim 66 wherein said at least one
deoxyribonucleoside is deoxycytidine.
68. A method as in claim 66 wherein said at least one
deoxyribonucleoside is deoxyadenosine.
69. A method as in claim 66 wherein said at least one
deoxyribonucleoside is deoxyguanosine.
70. A method as in claim 66 wherein said at least one
deoxyribonucleoside is thymidine.
71. A method as in claim 66 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 10 milligrams per milliliter of each
deoxyribonucleoside.
72. A method as in claim 66 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
1.0 to 5 milligrams per milliliter of each deoxyribonucleoside.
73. A method as in claim 66 wherein said at least on
deoxyribonucleoside is a mixture deoxycytidine, deoxyadenosine,
deoxyguanosine, and thymidine.
74. A method as in claim 73 wherein said deoxyribonucleoside is
administered in a vehicle containing from 0.1 to 10 milligrams per
milliliter of each deoxyribonucleoside.
75. A method as in claim 73 wherein said deoxyribonucleoside is
administered in a vehicle containing from 1 to 5 milligrams per
milliliter of each deoxyribonucleoside.
76. A method of reducing the deleterious consequences of exposure
of skin to endogenously produced nitric oxide comprising
administering to said skin a source of at least one
deoxyribonucleoside.
77. A method as in claim 76 wherein said source of at least one
deoxyribonucleoside is selected from the group consisting of a
deoxyribonucleoside, a deoxyribonucleotide, as
oligodeoxyribonucleotide, or an acyl deoxyribonucleoside.
78. A method as in claim 76 wherein said source of at least one
deoxyribonucleoside is administered in a vehicle at a concentration
of from 1.0 to 20 milligrams per milliliter.
79. A method as in claim 77 wherein said at least one
deoxyribonucleoside is free or acyl deoxycytidine.
80. A method as in claim 77 wherein said at least one
deoxyribonucleoside is free or acyl deoxyadenosine.
81. A method as in claim 77 wherein said at least one
deoxyribonucleoside is free or acyl deoxyguanosine.
82. A method as in claim 77 wherein said at least one
deoxyribonucleoside is free or acyl thymidine.
83. A method as in claim 77 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 10 milligrams per milliliter of each
deoxyribonucleoside.
84. A method as in claim 77 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
1.0 to 5 milligrams per milliliter of each deoxyribonucleoside.
85. A method as in claim 77 wherein said at least one
deoxyribonucleoside is a mixture of free or acyl deoxycytidine,
deoxyadenosine, deoxyguanosine, and thymidine.
86. A method as in claim 85 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from
1.0 to 10 milligrams per milliliter of each
deoxyribonucleoside.
87. A method as in claim 85 wherein said at least one
deoxyribonucleoside is administered in a vehicle containing from 1
to 5 milligrams per of each deoxyribonucleoside.
88. A method of treating skin inflammation in a mammal comprising
administering to inflamed skin a source of at least one
deoxyribonucleoside or ribonucleoside.
89. A method as in claim 88 wherein said skin inflammation is
selected from the group consisting of dermatitis, psoriasis,
eczema, and acne.
90. A method as in claim 88 wherein said skin inflammation is due
to exposure to solar or ultraviolet radiation.
91. A method as in claim 88 wherein said source of at least one
deoxyribonucleoside or ribonucleoside is selected from the group
consisting of DNA, an oligodeoxyribonucleotide, a ribonucleoside, a
deoxyribonucleoside, a ribonucleotide, a deoxyribonucleotide, an
acyl deoxyribonucleoside, and an acyl ribonucleoside.
92. A method as in claim 9 wherein said source of at least one
deoxyribonucleoside or ribonucleoside is administered in a vehicle
containing from 0.1 to 20 mg/ml.
93. A method as in claim 91 wherein said source of at least one
deoxyribonucleoside is a mixture comprising free or acylated
deoxycytidine, deoxyadenosine, deoxyguanosine, and thymidine.
94. A method as in claim 88 wherein said source of at least one
deoxyribonucleoside is deoxycytidine.
95. A method as in claim 88 wherein said source of at least one
ribonucleoside is adenosine.
96. A method as in claim 88 wherein said adenosine is administered
in a vehicle containing from 0.1 to 10 mg/ml.
97. A method of treating mucosal inflammation in a mammal
comprising administering to said mucosal inflammation a source of
at least one deoxyribonucleoside or ribonucleoside.
98. A method as in claim 97 wherein said mucosal skin inflammation
is selected from the group consisting of inflammatory bowel
disease, ulcerative colitis, Crohn's disease, stomatitis or
mucositis.
99. A method as in claim 97 wherein said source of at least one
deoxyribonucleoside or ribonucleoside is selected from the group
consisting of DNA, an oligodeoxyribonucleotides, a ribonucleoside,
a deoxyribonucleoside, a ribonucleotide, a deoxyribonucleotide, an
acyl deoxyribonucleoside, and an acyl ribonucleoside.
100. A method as in claim 99 wherein said source of at least one
deoxyribonucleoside is administered in a vehicle containing from
0.1 to 20 mg/ml.
101. A method as in claim 99 wherein said source of at least one
deoxyribonucleoside is a mixture comprising free or acylated
deoxycytidine, deoxyadenosine, deoxyguanosine, and thymidine.
102. A method as in claim 97 wherein said source of at least one
deoxyribonucleoside is deoxycytidine.
103. A method of reducing the chance of developing skin cancer in a
mammal due to exposure to solar or ultraviolet radiation comprising
topically administering a composition comprising a sunscreen agent
and an energy scavenging agent.
104. A method as in claim 103 wherein said sunscreen agent is
selected from the group consisting of avobenzone (t-butyl
dimethoxydibenzoylmethan- e), oxybenzone (benzophenone-3),
dioxybenzone (benzophenone-8), sulisobenzone (benzophenone-4;
2-hydroxy-4-methoxybenzophenone-5-sulfonic acid), octocrylene
(2-ethylexyl-2-cyano-3,3-diphenylacrylate), octyl methoxycinnamate
(2-ethylhexyl p-methoxycinnamate), octyl salicylate
(2-ethylhexylsalicylate), homosalate (homomenthyl salicylate),
trolamine salicylate (triethanolamine salicylate),
phenylbenzimidazole sulfonic acid, PABA (para-aminobenzoic acid),
roxadimate (ethyl 4-bis hydroxypropyl aminobenzoate), lisadimate
(glyceryl PABA), Padimate O (octyldimethyl PABA), menthyl
anthranilate, or Parsol 1789 (butyl methoxydibenzoylmethane)
105. A method as in claim 103 wherein said energy scavenging agent
is selected from the group consisting of DNA, an
oligodeoxyribonucleotide, a ribonucleoside, a deoxyribonucleoside,
a ribonucleotide, a deoxyribonucleotide, an acyl
deoxyribonucleoside, and an acyl ribonucleoside.
106. A method as in claim 105 wherein said energy scavenging agent
is administered in a vehicle containing from 0.1 to 20 mg/ml.
107. A method as in claim 105 wherein said energy scavenging agent
is a mixture comprising free or acylated deoxycytidine,
deoxyadenosine, deoxyguanosine, and thymidine.
108. A composition comprising a) at least one deoxyribonucleoside
and b) an agent that enhances penetration of said at least one
deoxyribonucleoside into the skin.
109. A composition as in claim 108 wherein said skin is human
skin.
110. A composition as in claim 108 wherein said at least one
deoxyribonucleoside is deoxycytidine.
111. A composition as in claim 108 wherein said source of at least
one deoxyribonucleoside is deoxyadenosine.
112. A composition as in claim 108 wherein said source of at least
one deoxyribonucleoside is deoxyguanosine.
113. A composition as in claim 108 wherein said source of at least
one deoxyribonucleoside is thymidine.
114. A composition as in claim 108 wherein said at least one
deoxyribonucleoside is present in a concentration of from 0.1 to 10
milligrams per milliliter of each deoxyribonucleoside.
115. A composition as in claim 108 wherein said at least one
deoxyribonucleoside is present in a concentration of from 1.0 to 5
milligrams per milliliter of each deoxyribonucleoside.
116. A composition as in claim 108 wherein said at least one
deoxyribonucleoside is a mixture comprising free deoxycytidine,
deoxyadenosine, deoxyguanosine, and thymidine.
117. A composition as in claim 116 wherein each deoxyribonucleoside
is present in a concentration of 0.1 to 10 milligrams per
milliliter.
118. A composition as in claim 116 wherein each deoxyribonucleoside
is present in a concentration of 1 to 5 milligrams per
milliliter.
119. A composition as in claim 108 wherein said agent that enhances
penetration is selected from the group consisting of ethanol,
isopropanol, azone (1-dodecylazacycloheptan-2-one), oleic acid,
linoleic acid, propylene glycol, hypertonic glycerol, lactic acid,
glycolic acid, citric acid, and malic acid.
120. A composition as in claim 108 wherein said composition is a
hydrogel.
121. A composition as in claim 120 wherein the gelling agent for
said hydrogel is selected from the group consisting of
methylcellulose, carboxymethylcellulose,
hydroxypropylmethylcellulose, carbomer, Hypan, polyacrylate, and
glycerine polyacrylate.
122. A composition comprising a) deoxyguanosine and deoxyadenosine
and b) benzyl alcohol.
123. A composition as in claim 122 wherein the concentration of
said benzyl alcohol is between 0.1 and 5%.
124. A composition as in claim 122 wherein the concentrations of
deoxyguanosine and deoxyadenosine are between 0.1 and 10 mg/ml.
125. A composition as in claim 122 further containing deoxycytidine
and thymidine.
126. A composition as in claim 125 wherein the concentrations of
deoxycytidine and thymidine are between 0.1 and 10 mg/ml.
127. A composition comprising a methylxanthine and source of at
least one deoxyribonucleoside.
128. A composition as in claim 127 wherein said source of at least
one deoxyribonucleoside is selected from the group consisting of at
least one deoxyribonucleoside and at least one acyl derivative of a
deoxyribonucleoside.
129. A composition comprising a) a sunscreen agent b) an energy
scavenging agent.
130. A composition as in claim 129 wherein said sunscreen agent is
selected from the group consisting of avobenzone (t-butyl
dimethoxydibenzoylmethane), oxybenzone (benzophenone-3),
dioxybenzone (benzophenone-8), sulisobenzone (benzophenone-4;
2-hydroxy-4-methoxybenzo- phenone-5-sulfonic acid), octocrylene
(2-ethylhexyl-2-cyano-3,3-diphenylac- rylate), octyl
methoxycinnamate (2-ethylhexyl p-methoxycinnamate), octyl
salicylate (2-ethylhexylsalicylate), homosalate (homomenthyl
salicylate), trolamine salicylate (triethanolamine salicylate),
phenylbenzimidazole sulfonic acid, PABA (para-aminobenzoic acid),
roxadimate (ethyl 4-bis hydroxypropyl aminobenzoate), lisadimate
(glyceryl PABA), Padimate O (octyldimethyl PABA), menthyl
anthranilate, and Parsol 1789 (butyl methoxydibenzoylmethane).
131. A composition as in claim 129 wherein said energy scavenging
agent is selected from the group consisting of a source of at least
one deoxyribonucleoside.
132. A composition as in claim 131 wherein said source of at least
one deoxyribonucleoside is selected from the group consisting of a
deoxyribonucleoside, a deoxyribonucleotide, an
oligodeoxyribonucleotide, or an acyl deoxyribonucleoside.
133. A composition as in claim 132 wherein said at least one
deoxyribonucleoside is a mixture comprising free or acylated
deoxycytidine, deoxyadenosine, deoxyguanosine, and thymidine.
134. A composition as in claim 132 wherein each deoxyribonucleoside
is present in a concentration of from 0.1 to 10 milligrams per
milliliter.
135. A composition in claim 132 wherein said at least one
deoxyribonucleoside is free or acyl deoxycytidine.
136. A composition as in claim 135 wherein said free or acyl
deoxycytidine is present in a concentration of from 0.1 to 100
milligrams per milliliter.
137. A composition as in claim 129 wherein said energy scavenging
agent is selected from the group consisting of a source of at least
one ribonucleoside.
138. A composition as in claim 137 wherein said source of at least
one deoxyribonucleoside is selected from the group consisting of a
ribonucleoside, a ribonucleotide, an oligoribonucleotide, or an
acyl ribonucleoside.
139. A method for reducing the rate of development of skin
photodamage in a mammal exposed to solar or ultraviolet radiation
comprising administering to the skin of said mammal a source of at
least one deoxyribonucleoside wherein said source is administered
such that said deoxyribonucleoside is present on skin of said
mammal during or after exposure to said radiation in an amount
sufficient to reduce the deleterious consequences of said
exposure.
140. A method as in claim 139 wherein said source is selected from
the group consisting of at least one free or acyl
deoxyribonucleoside.
141. A method as in claim 139 wherein said source is selected from
the group consisting of a deoxyribonucleoside, an
oligodeoxyribonucleotide, and a polydeoxyribonucleotide.
142. A method of reducing the rate of development of skin
photodamage in a mammal due to exposure to solar or ultraviolet
radiation comprising topically administering a composition
comprising a sunscreen agent and an energy scavenging agent.
143. A method as in claim 142 wherein said energy scavenging agent
is selected from the group consisting of DNA, an
oligodeoxyribonucleotide, a ribonucleoside, a deoxyribonucleoside,
a ribonucleotide, a deoxyribonucleotide, an acyl
deoxyribonucleoside, and an acyl ribonucleoside.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to treatment and prevention
of photodamage, genetic damage, and tumorigenesis in skin and other
tissues caused by exposure to solar or ultraviolet radiation or
other mutagens, comprising administration of deoxyribonucleosides
or esters of deoxyribonucleosides to a mammal such as a human.
These compounds are capable of reducing DNA damage, mutation
frequency, and tumorigenesis when applied topically before, during,
or after exposure to mutagenic radiation or chemical mutagens.
BACKGROUND OF THE INVENTION
[0002] Exposure of skin to ultraviolet (or ionizing) radiation
damages DNA, which if unrepaired or improperly repaired, can lead
to carcinogenesis as well as contribute to acceleration of the
aging process. DNA damage and consequent genomic instability are
defining characteristics of both carcinogenesis and biological
aging. Patients with defective DNA repair capabilities in diseases
like xeroderma pigmentosa display premature skin aging and a very
high incidence of skin cancers (Robbins and Moshell, J. Inv.
Dermatol., 73:102-107, 1979) on sun-exposed areas of the skin.
Pharmacological intervention in damage to skin due to solar or
ultraviolet radiation has heretofore been largely restricted to
agents like sunscreens or free-radical scavengers intended to
prevent damage, or agents like retinoic acid or glycolic acid which
are intended to remodel the surface of radiation-damaged skin
without necessarily addressing the most fundamental mechanisms of
cell or tissue damage and repair at the level of genomic
integrity.
[0003] In practice, preventive measures like sunscreen use are not
completely effective, and exposure to sunlight is not always
anticipated. The incidence of skin cancers in the United States
approaches 1,000,000 cases per year. Therefore, there exists a need
for a therapeutic agent which will reduce the risk of development
of skin cancer or other consequences of skin photodamage even when
applied after exposure to sunlight has already occurred. Sunscreens
and agents which induce or improve tanning are not useful in such
situations, since they are only useful if applied prior to exposure
to UV radiation. Moreover, there are situations wherein sunscreens
and even endogenous melanin can actually enhance UV-induced DNA
damage through photodynamic sensitization.
[0004] There have been several attempts to improve or accelerate
DNA repair and to reduce the consequences of DNA damage in skin
cells after damage has already occurred. The first major step in
DNA repair is detection and excision of damaged portions of DNA.
The viral enzyme T4 endonuclease V can accomplish this step with
some forms of DNA damage. T4 endonuclease V, when packaged in
epidermis-penetrating liposomes, has been shown to accelerate the
rate of excision of pyrimidine dimers, the most common form of
photolesion, in photodamaged skin of mice in vivo (Yarosh et al.,
Cancer Res. 52:4227-31, 1992). A bacterial extract has been
reported to increase the rate of unscheduled DNA synthesis, which
is often used as an index of DNA repair activity (Kludas and Heise,
U.S. Pat. No. 4,464,362), in UV-exposed skin; however, this effect
was not confirmed in a subsequent study (Natarajan et al., Mutation
Research 206:47-54, 1988).
[0005] The key issue in DNA repair, however, is not necessarily the
rate of lesion excision, but the fidelity of repair. Agents which
accelerate the excision step of DNA repair can actually exacerbate
damage if the cells are incapable of accurate repair synthesis at a
rate that matches the rate of excision of damaged segments of DNA
(Collins and Johnson, J. Cell Physiol. 99:125-137, 1979).
[0006] Deoxyribonucleosides or deoxyribonucleotides have been added
to cells in culture with variable or divergent effects on DNA
damage or mutagenesis in response to irradiation of the cells. In
some cell types, e.g. lymphocytes, which have limited capabilities
for de novo deoxyribonucleotide synthesis, exogenous
deoxynucleosides are reported to improve cell survival after
exposure to UV radiation (Yew and Johnson, Biochim. Biophys. Acta,
562:240-251, 1979; Green et al., Mutation Research, 350:239-246,
1996) or ionizing radiation (Petrovic et al., Int. J. Radiat.
Biol., 18:243, 1970); no significant improvement in survival was
seen after addition of deoxyribonucleosides to UV-irradiated normal
human fibroblasts (Green et al., Mutation Research, 350:239-246,
1996). A crucial point is that increasing cell survival after
genomic damage caused by UV radiation or other mutagens is not
necessarily desirable. The process of programmed cell death, or
apoptosis, is integrated with cellular mechanisms for detecting DNA
damage. Thus, genomic damage which by itself is not sufficient to
cause cell death, can trigger apoptosis, an active cellular suicide
process, so that the DNA damage in the cell is not perpetuated in
subsequent cell generations, with tumorigenesis as a possible
outcome as genomic damage accumulates. The mechanisms for detecting
genomic damage and inducing apoptosis involve cell-cycle regulating
proteins such as the tumor-suppressor protein p53. Therefore,
agents which promote cell survival (e.g. by inhibiting apoptosis)
after irradiation are not necessarily anticarcinogenic, and may
actually enhance mutation frequency and risk of malignant
transformation by permitting survival of damaged cells that would
otherwise be eliminated by apoptosis. A significant illustration of
this principle is the demonstration that embryonic p53 knockout
mice exposed to ionizing radiation in utero have a higher survival
rate (live birth) than wild-type controls, but also have a much
higher frequency of congenital defects (Norimura et al., Nature
Medicine, 2:577-580).
[0007] In studies where the effect of exogenous
deoxyribonucleosides on mutation frequency in UV-irradiated cells
has been explicitly studied, variable results have been obtained.
Bianchi and Celotti (Mutation Research 146:277-284, 1985) reported
that thymidine or deoxycytidine at high concentrations increased
the mutation frequency in UV-irradiated V79 Chinese hamster cells;
no reduction in mutation frequency was observed at any
concentrations of added nucleosides. Musk et al. (Mutation Research
227:25-30, 1989) reported that a mixture of deoxyribonucleosides
which included excess deoxycytidine relative to the other
nucleosides, reduced the mutation frequency in response to UV-C
(254 nm) irradiation in MM96L melanoma cells, a cell line with a
known constitutive excess of purine deoxyribonucleotides. In the
same study, exogenous deoxyribonucleosides had no effect on
mutation frequency in another neoplastic cell line, human HeLa
cells, after exposure to UV-C radiation. It is important to note
that UV-C radiation is not a component of solar radiation at the
surface of the earth, since it is blocked effectively by the
atmosphere (Pathak, 1974, in Sunlight and Man, ed. by T. B.
Fitzpatrick, University of Tokyo Press, Tokyo, Japan, p. 815). The
effect of deoxyribonucleosides on mutation frequency in cells
exposed to solar radiation or UV radiation at wavelengths that are
present in solar radiation was not tested, and the authors
explicitly conclude their discussion with the statement ". . . the
lower [mutation] frequency in sun-[irradiated] compared with
UVC-irradiated MM96L cells suggests that sunlight either does not
perturb the deoxynucleoside pools or it induces a cellular response
that is insensitive to nucleoside levels."
[0008] In addition to agents which inhibit apoptosis or improve DNA
repair sufficiently to permit cell survival but not necessarily for
correction of potentially tumorigenic mutations, growth factors in
general (including those that are involved in normal wound healing
responses like TGF-.beta. or PDGF) act as tumor promoters.
[0009] U.S. Pat. No. 5,246,708 discloses the methods and
compositions involving the use of mixtures of deoxyribonucleosides
for promotion of the healing of wounds, ulcers, and burns,
including those caused by ultraviolet or solar radiation.
[0010] Acyl derivatives of deoxyribonucleosides have been taught as
delivery molecules for promoting entry of deoxyribonucleosides into
the skin, as disclosed in U.S. patent application Ser. No. 466,379.
It is disclosed that acyl derivatives of deoxyribonucleosides can
improve cellular repair and cell survival after damage to skin
caused by radiation.
[0011] Oligodeoxyribonucleotides have been proposed as melanogenic
stimuli, based on the idea that DNA damage, or excision products of
DNA damage, might be cellular signals for increasing melanin
production in the skin to help protect against subsequent damage.
Gilchrest et al. (U.S. Pat. No. 5,470,577; WO Application Serial
No. 95/01773) proposed that exogenous DNA photodamage products may
stimulate melanogenesis without actual damage to cellular DNA as a
necessary intermediate step. The stated intention was to mimic the
presence of cyclobutane pyrimidine dimers or other DNA photodamage
products ill order to provide the cell with false DNA damage
signals that might trigger induction of melanogenesis in the
absence of actual DNA damage. Treatment of melanoma cells in vitro
and guinea pig skin in vivo with thymidine dinucleotide resulted in
increases in melanin production. The authors stated that they
believed that DNA fragments entered the cells, and even their
nuclei, intact. They proposed that sunless tanning accomplished
over a period of weeks by topical administration of
oligodeoxyribonucleotides, especially thymidine dinucleotide, could
protect skin by inducing melanin synthesis, with consequent
reduction of passage of UV radiation into and through the skin.
[0012] Wiskemann (1974; in Sunlight and Man, ed. by T. B.
Fizpatrick, University of Tokyo Press, Tokyo, Japan, p. 51)
reported that systemic (intraperitoneal) administration the
deoxyribonucleoside thymidine or the ribonucleosides and congeners
adenosine, cyclic-AMP, uridine, cytidine increased the period of
latency for extravasation of systemically administered Evan's Blue
dye in the skin in the first few hours after UV exposure,
indicating a reduction in acute UV-induced edema. In this system,
DNA administered after irradiation had no effect on extravasation
of dye. The author also explicitly states that nucleobases
incorporated into ointments do not penetrate the horny layer (the
stratum corneum, the outer layer of enucleated keratinocytes
comprising the main moisture barrier of skin) of human
epidermis.
OBJECTS OF THE INVENTION
[0013] It is an object of the invention to provide compositions and
methods for reducing mutation frequency, photaging, and
tumorigenesis in skin, thereby attenuating consequences of exposure
to solar and ultraviolet radiation and to other mutagens including
endogenous oxidants.
[0014] It is an object of the invention to provide a composition
that enhances DNA repair and prevents consequences of mutagenic
radiation even when administered after damage or exposure to
radiation or other mutagens has already occurred.
[0015] It is a primary object of this invention to provide
compositions and methods for effectively preventing or treating
consequences of exposure of the skin to solar and ultraviolet
radiation and other environmental mutagens.
[0016] It is a further object of the invention to provide
compositions and methods for improving the activity of chemical
sunscreens.
[0017] It is a further object of the invention to provide
compositions and methods for reducing deleterious effects of
sunscreens and other compounds, exogenous and endogenous, which act
as photosensitizing or photodynamic enhancers of UV-induced damage
to skin.
[0018] It is a further object of the invention to provide
compositions and methods for reducing some consequences of
inflammatory skin and mucosal conditions, including psoriasis,
dermatitis and inflammatory bowel disease.
SUMMARY OF THE INVENTION
[0019] The subject invention involves methods and compositions for
improving DNA repair (or genomic fidelity) and reducing photodamage
in skin exposed to ultraviolet radiation, ionizing radiation, or
chemical mutagens by topical administration of compositions
containing effective amounts of a source of deoxyribonucleosides.
The compositions are capable of delivering deoxyribonucleosides to
the necessary target cells. Such compositions are applied to the
skin before, during or after exposure to solar, ultraviolet, or
ionizing radiation, or chemical mutagens including but not limited
to endogenous or exogenous sources of free radicals or nitric
oxide. Treatment with compositions of the invention improves the
net fidelity of DNA repair and thereby reduces mutation frequency
and the risk of tumorigenesis in response to solar or ultraviolet
radiation or other mutagens.
[0020] The invention provides methods and compositions for
delivering deoxyribonucleosides to skin cells in concentrations
sufficient to support and improve repair of damaged DNA and to
reduce deleterious consequences of exposure of skin to radiation or
chemical mutagens.
[0021] The deoxyribonucleosides are administered either as free
deoxyribonucleosides, or as derivatives thereof which are converted
to deoxyribonucleosides after application to the skin. Such
derivatives include deoxyribonucleotides, oligonucleotides, DNA
itself, and acyl derivatives of deoxyribonucleosides or other
derivatives of deoxyribonucleosides which are converted to free
deoxyribonucleosides by endogenous enzymes.
[0022] Methods and compositions of the invention also improve
activity and reduce side effects of other agents used on skin for
prophylactic, therapeutic, or cosmetic purposes, including but not
limited to sunscreens, retinoids, alpha-hydroxy acids,
methylxanthines, and DNA repair enzymes.
[0023] The invention also relates to compositions and methods for
reducing deleterious consequences (e.g. cellular damage, especially
to DNA, which can result in increased likelihood of mutations or
other potentially carcinogenic damage to the genome) of endogenous
and exogenous photochemically-active compounds or chromophores
which act as photosensitizers or photodynamic enhancers of DNA
damage caused by solar or ultraviolet radiation.
[0024] The invention, as well as other objects, features and
advantages thereof will be understood more clearly and fully from
the following detailed description, when read with reference to the
accompanying results of the experiments discussed in the examples
below.
DETAILED DESCRIPTION OF THE INVENTION
[0025] DNA damage and repair is involved in the development of skin
cancer and photoaging. The subject invention provides compounds
which successfully improve the net fidelity of DNA repair. The
subject invention will have important consequences in health care
and will improve the cosmetic appearance of skin.
A. Definitions
[0026] The term "deoxyribonucleoside" refers to any one of the four
principle nucleoside constituents of DNA: deoxyadenosine,
deoxycytidine, deoxyguanosine, and thymidine. The term
"ribonucleoside" refers to any one of the four major nucleoside
constituents of RNA: adenosine, cytidine, guanosine, and
uridine.
[0027] The term "acyl derivative of a deoxyribonucleoside" refers
to deoxyribonucleosides bearing acyl substituents derived from
carboxylic acids (which modify the pharmacokinetics and
bioavailability of the free deoxyribonucleosides), as disclosed in
U.S. patent application Ser. No. 466,379, hereby incorporated by
reference in its entirety.
[0028] The term "source of at least one deoxyribonucleoside" or
"deoxyribonucleoside source" in the context of the subject
invention refers to deoxyribonucleosides themselves or derivatives
of deoxyribonucleosides which can be converted to
deoxyribonucleosides by endogenous enzymes, especially esterases.
Examples include acyl derivatives of deoxyribonucleosides
(carboxylic acid esters), deoxyribonucleotides (phosphate esters),
or oligodeoxyribonucleotides (phosphate diesters). Since esterase
activity (involving various enzymes capable of cleaving carboxylic
acid esters and phosphate esters) is ubiquitous in mammalian
tissues including skin, these esters of deoxyribonucleosides are
converted to deoxyribonucleosides when applied to skin. Similarly,
a "source of at least one ribonucleoside" refers to a
ribonucleoside or ribonucleoside ester, including a ribonucleotide,
an oligoribonucleotide, or an acyl derivative of a
ribonucleoside.
[0029] The term "ester of a deoxyribonucleoside" (or
deoxyribonucleoside ester) refers to either an acyl derivative of
deoxyribonucleosides as described above or to a phosphate ester of
a deoxyribonucleoside (or deoxyribonucleosides), e.g.
deoxyribonucleotides, oligodeoxyribonucleotid- es, or
polydeoxyribonucleotides.
[0030] The term "photosensitization" in the context of the subject
invention refers to the process whereby light-absorbing (UV or
visible light) molecules directly transfer the energy of an excited
state, generally a triplet state, to a target molecule, resulting
in damage to DNA and other cellular structures. The target molecule
can be DNA itself or another target which results in damage to DNA,
e.g. membranes components of lysosomes, which contain
deoxyribonuclease.
[0031] The term "photodynamic sensitization" herein refers to the
process whereby UV-absorbing molecules generate free radical
species or other diffusible reactive intermediates as a result of
excitation by UV or visible radiation.
[0032] The term "sunscreen agents" refers to a UV-absorbing
chemicals that are intended to be used in sunscreen products as
active ingredients for reducing exposure of the skin to the UV
component of solar radiation. Examples of sunscreen agents
currently used as such in commercial products include avobenzone
(t-butyl dimethoxydibenzoylmethane), oxybenzone (benzophenone-3),
dioxybenzone (benzophenone-8), sulisobenzone (benzophenone-4;
2-hydroxy-4-methoxybenzophenone-5-sulfonic acid), octocrylene
(2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octyl methoxycinnamate
(2-ethylhexyl p-methoxycinnamate), octyl salicylate
(2-ethylhexylsalicylate), homosalate (homomenthyl salicylate),
trolamine salicylate (triethanolamine salicylate),
phenylbenzimidazole sulfonic acid, PABA (para-aminobenzoic acid),
roxadimate (ethyl 4-bis hydroxypropyl aminobenzoate), lisadimate
(glyceryl PABA), Padimate O (octyldimethyl PABA), menthyl
anthranilate, and Parsol 1789 (butyl methoxydibenzoylmethane).
[0033] The term "energy scavenger" refers to a compound which
absorbs energy from the excited states of sunscreen agents or
endogenous photosensitizing or photodynamic enhancers of DNA
damage, reducing damage to target biological molecules like DNA. In
this context, energy scavengers should be nontoxic at effective
concentrations and should yield a net reduction in damage to DNA
when present during UV irradiation in the presence of a
photosensitizer or photodynamic enhancer of DNA damage. In the
context of this invention, "energy scavengers" refer particularly
to compounds with lowest triplet states with energies less than or
equal to those of the nucleobase constituents of genomic DNA. The
primary quality of energy scavengers of the invention is that, by
virtue of photochemical energy state properties, or mass action
(concentration), they prevent damage to genomic DNA that would
otherwise occur due to energy transfer for excited chromophores,
whether endogenous (e.g. melanin) or exogenous (e.g. sunscreens).
Energy scavengers with photochemical properties similar to those of
structural constituents of DNA are advantageous, and include
sources of at least one deoxyribonculeoside or ribonucleoside, like
a deoxyribonucleoside, an acyl deoxyribonucleoside, a
deoxyribonucleotide, an oligodeoxyribonucleotide, a ribonucleoside,
a ribonucleotide, an oligoribonucleotide, and an acyl
ribonucleoside.
[0034] The term "deleterious consequences" as used herein refers to
cellular damage in a mammal caused by a mutagen, especially damage
to the genome, resulting in an increased chance of developing skin
cancer or other skin lesions like solar lentigines, actinic
keratoses, or other signs of photoaging like skin wrinkles or "age
spots". Mutagens capable of causing such deleterious consequences
include solar radiation, ultraviolet radiation, ionizing radiation,
free radicals (whether produced as a result of irradiation of a
photochemically active chromophore or from some other source,
including normal metabolic processes), nitric oxide, and
environmental mutagens.
B. Compounds of the Invention
[0035] The compounds of the invention are primarily the major
deoxyribonucleoside constituents of DNA: deoxyadenosine,
deoxycytidine, deoxyguanosine, and thymidine. The invention also
includes the use of effective amounts of precursors of these
deoxyribonucleosides, e.g. oligodeoxyribonucleotides, DNA,
deoxyribonucleotides and acyl derivatives of deoxyribonucleosides,
and, particularly for minimization of effects of photodynamic
sensitizers and photosensitizing agents on DNA, ribonucleosides and
their congeners, e.g. oligoribonucleotides, ribonucleotides, and
acyl derivatives of ribonucleosides.
[0036] While not wishing to be bound by a theory, it is believed
that the active agents of the invention that pass into cells are
the deoxyribonucleosides or acyl derivatives of
deoxyribonucleosides, since the anionic phosphate moiety on
deoxyribonucleotides or oligodeoxyribonucleotides impedes passage
across cell membranes. Phosphorylated deoxyribonucleoside
precursors are converted to free deoxyribonucleosides by enzymatic
and nonenzymatic degradation before or after application to the
skin, prior to their entry into cells.
[0037] The deoxyribonucleosides are produced by any of several
methods. They are produced by degradation of DNA from biological
sources, e.g. fish sperm, by chemical synthesis, or by fermentation
technology.
[0038] Also encompassed by the invention are pharmaceutically
acceptable salts of the above-noted compounds.
C. Compositions of the Invention
[0039] The invention includes pharmaceutical compositions for
improving the net fidelity of DNA repair and for protecting the
skin against mutagens. The composition comprises 1) an effective
amount of a source of one or more deoxyribonucleosides, and
optionally 2) an effective amount of a pharmaceutically acceptable
topical carrier capable of delivering the deoxyribonucleosides or
their precursors to appropriate target cells in the skin under in
vivo conditions.
[0040] While individual deoxyribonucleosides, especially
deoxycytidine (see Example 7) have some activity in attenuating
UV-induced tumorigenesis, two or more deoxyribonucleosides, or
preferably all four, are typically included in a formulation of the
invention. Encompassed by the invention are compositions containing
deoxyadenosine, deoxycytidine, deoxyguanosine, or thymidine, either
as single agents, or in all possible combinations of two, three, or
all four of these compounds. Compositions containing deoxycytidine
are particularly advantageous. The concentrations of individual
deoxyribonucleosides in compositions encompassed by the invention,
whether present individually or in combination with other
deoxyribonucleosides or deoxyribonucleoside precursors (such as
deoxyribonucleoside esters), and normalized to the amount of free
nucleoside or nucleoside moiety in the case of deoxyribonucleoside
phosphates or oligonucleotides or prodrugs like acylated
deoxyribonucleoside derivatives, range from 0.1 to 10 mg/ml,
advantageously 1 to 5 mg/ml.
[0041] In order to permit access of the deoxyribonucleosides and
related compounds of the invention to deeper-lying skin cells,
vehicles which improve their penetration through the outer layers
of the skin, e.g. the stratum corneum, are useful. Vehicle
constituents which improve the penetration of compounds of the
invention into the skin include but are not limited to: ethanol,
isopropanol, azone (1-dodecylazacycloheptan-2-on- e), oleic acid,
linoleic acid, propylene glycol, hypertonic concentrations of
glycerol, lactic acid, glycolic acid, citric acid, and malic
acid.
[0042] In addition to promoting absorption of agents into the skin,
use of topical alpha-hydroxy acids (AHA), e.g. lactic acid and
glycolic acid, can affect the ability of the skin to reduce the
penetration of ultraviolet light into the vulnerable basal layers
of the epidermis. Thus, there is also an increased need for agents
which reduce the consequences of exposure to solar or ultraviolet
radiation in people using AHA's, e.g. for promoting exfoliation of
epidermal cells. Since penetration of UV-absorbing sunscreen agents
into the skin is undesirable because of possible photosensitization
and photodynamic enhancement of UV-induced damage to cells, the
compounds of the invention are uniquely suitable for combination
with AHA's, either in the same formulation or a separate one, for
improving skin resistance to damaging effects of solar or
ultraviolet radiation.
[0043] One embodiment of the invention is a hydrogel formulation,
comprising an aqueous or aqueous-alcoholic medium and a gelling
agent, and a deoxyribonucleoside source. Suitable gelling agents
include but are not limited to methylcellulose,
carboxymethylcellulose, hydroxypropylmethylcellulose, carbomer
(carbopol), hypan, polyacrylate, and glycerol polyacrylate.
[0044] Liposomes are microscopic lipid vesicles which can contain
pharmacologically active agents either enclosed in the aqueous
space within the vesicle or in the lipid membrane itself, depending
on the lipophilicity of the agent. Liposomes are capable of
delivering a pharmacologic agent through the stratum corneum into
deeper layers of the skin, and are therefore suitable vehicles for
compounds and compositions of the invention.
[0045] Niosomes are lipid vesicles similar to liposomes with
membranes consisting largely of non-ionic lipids, some forms of
which are effective for transporting compounds across the stratum
corneum.
[0046] In one embodiment of the invention, lipophilic acyl
derivatives of deoxyribonucleosides, e.g. oleic or palmitic acid
esters of deoxyribonucleosides are incorporated into membranes of
niosome or liposome membranes, in addition to or instead of being
enclosed within the vesicular membranes.
[0047] Other agents which are advantageous for incorporation into a
composition of the invention include corticosteroids, especially
hydrocortisone in concentrations of 0.05 to 1% other
anti-inflammatory corticosteroids at therapeutically effective
concentrations, topical anesthetics including but not limited to
benzocaine, lidocaine, and benzyl alcohol, aloe vera and aloe
barbadensis, retinoids, antioxidants like Vitamins C and E,
flavins, polyphenols (e.g. extracted from green tea or black tea),
allantoin, liposomal DNA repair enzymes, antibacterial agents (e.g.
quaternary ammonium compounds, bacitracin, neomycin, polymyxin),
zinc salts, and methylxanthines. All of these listed agents have
some utility in treating or attenuating various aspects of skin
injury or discomfort caused by ultraviolet radiation or
inflammatory skin conditions, and are therefore complementary to
the unique actions of the deoxyribonucleosides of the
invention.
[0048] Benzyl alcohol, which is known to have anesthetic and
preservative properties, has the unexpected effect of improving
aqueous solubility of the relatively insoluble purine
deoxyribonucleosides, deoxyadenosine and deoxyguanosine; preferred
concentrations of benzyl alcohol in topical formulations of
deoxyribonucleosides are 0.5 to 5%. This is very important in
permitting high concentrations of the deoxyribonucleosides of the
invention to be stably incorporated into aqueous vehicles.
Sunscreens
[0049] The compounds of the invention are advantageously
incorporated into the same formulation as a UV-absorbing chemical
sunscreen agent such as: avobenzone (t-butyl
dimethoxydibenzoylmethane), oxybenzone (benzophenone-3),
dioxybenzone (benzophenone-8), sulisobenzone (benzophenone-4;
2-hydroxy-4-methoxybenzophenone-5-sulfonic acid), octocrylene
(2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octyl methoxycinnamate
(2-ethylhexyl p-methoxycinnamate), octyl
salicylate(2-ethylhexylsalicylate), homosalate (homomenthyl
salicylate), trolamine salicylate (triethanolamine salicylate),
phenylbenzimidazole sulfonic acid, PABA (para-aminobenzoic acid),
roxadimate (ethyl 4-bis hydroxypropyl aminobenzoate), lisadimate
(glyceryl PABA), Padimate O (octyldimethyl PABA), menthyl
anthranilate, or Parsol 1789 (butyl methoxydibenzoylmethane).
[0050] Alternatively, the compounds of the invention are formulated
in a base which is suitable for application to the skin prior to,
or after, application of a sunscreen. This embodiment of the
invention permits the benefits of deoxyribonucleosides, in reducing
consequences of UV damage and in attenuating photodynamic
enhancement of cellular damage caused by sunscreen agents
themselves, to be obtained in conjunction with use of a wide
variety of commercial sunscreen products.
D. Therapeutic Uses of the Compounds and Compositions of the
Invention
Reduction of Deleterious Consequences of Exposure of Skin to
Ultraviolet or Solar Radiation
[0051] The compounds and composition of the invention, when applied
or administered before, during, or after exposure of the skin of a
mammal to mutagenic radiation, have the unexpected activity of
improving the net fidelity DNA repair, thereby reducing mutation
frequency, photoaging, and the chance of developing skin
cancers.
[0052] DNA repair proceeds by several steps. A chemical lesion in
DNA, which can be caused by ultraviolet radiation, ionizing
radiation, free radicals, or chemical mutagens, is detected by the
DNA repair system. A segment of nucleotides including the damaged
region is excised, generally along with a number of surrounding
nucleotides. The excised strand is then resynthesized from free
deoxyribonucleotides, using the intact DNA strand as a
template.
[0053] It is generally believed that improvement of repair of DNA
in skin cells (e.g. keratinocytes, melanocytes, fibroblasts) would
require alteration in the activity of enzymes or other proteins
involved in the detection and excision of DNA lesions. Thus, there
have been attempts to improve DNA repair in skin by delivering DNA
repair enzymes via topically-applied liposomes (US patents (Yarosh
et al., Cancer Res. 52:4227-31, 1992).
[0054] Far more important than the initial rate of excision of
lesions is the fidelity or accuracy of repair. Agents which
accelerate the excision step of DNA repair can actually exacerbate
damage if the cells are incapable of accurate repair synthesis at a
rate that matches the rate of excision of damaged segments of DNA
(Collins and Johnson, J. Cell Physiol. 99:125-137, 1979).
[0055] Compounds and compositions of the invention, when applied to
skin before, during or after exposure to solar or ultraviolet
radiation or other environmental mutagens, reduces the mutation
frequency otherwise induced by the mutagen (Example 1). This
reduces the damage to cells caused by the mutagen, and reduces the
chance of development of skin cancers, as shown in Examples 2 and
7. The effect on genomic fidelity is in principle mediated by
actual improvement in the fidelity of repair in an individual cell
or by improvement of the elimination of cells with irreparable DNA
damage. Either mechanism results in a net improvement in genomic
fidelity in skin exposed to mutagens.
[0056] The compounds of the invention have unanticipated benefits
when used in combination with other agents known to be useful in
various aspects of skin care, including but not limited to
sunscreens, methylxanthines, retinoids, DNA repair enzymes,
exfoliants, and protease inhibitors, corticosteroids and
nonsteroidal anti-inflammatory agents.
[0057] The compounds and compositions of the invention, when
applied soon enough, e.g. within about 3 days after exposure to
ultraviolet or solar radiation, improve the repair of cellular and
macromolecular damage and improve net genomic fidelity, thereby
reducing the chance of development and severity of macroscopically
visible deleterious consequences of such exposure, including but
not limited to photoaging, sunburn symptoms, actinic keratoses,
solar lentigines, "age spots", and skin cancer. The compounds and
compositions of the invention are also optionally applied before or
during exposure to solar or ultraviolet radiation to shorten the
time gap between damage and onset of repair enhancement by the
compounds of the invention.
[0058] Treatment of skin with compounds and compositions of the
invention results in a reduced chance of development of skin
cancers and other deleterious consequences of exposure to solar or
UV radiation like photaging even when the compounds of the
invention are applied even after irradiation, e.g. after unintended
exposure to potentially-damaging doses of solar radiation. This
type of activity is not shared by conventional sunscreens or agents
which might act by enhancing melanogenesis, which are useful only
if applied before irradiation.
Improvement of Sunscreen Activity and Attenuation of Photodynamic
Enhancement of UV Damage by Sunscreens
[0059] Sunscreens are typically designed and tested on the basis of
prevention of sunlight-induced erythema. While erythema and its
attenuation by sunscreens is an important short-term effect,
reduction of erythema and inflammation by sunscreens does not
necessarily mean that they produce a proportionate protection of
DNA (or prevention of skin cancers and some features of photoaging
secondary to DNA damage). Sunscreens are certainly useful in
preventing some manifestations of photoaging and UV-related
carcinogenesis, but do not provide complete protection, and in some
situations may actually exacerbate photoinjury by acting as
photodynamic sensitizers (see Example 3).
[0060] Chemical sunscreens are intended to act by absorbing photons
at mutagenic (or erythmogenic) wavelengths, thus producing a
short-lived excited singlet state; return to the ground state is
accompanied by photon emission at longer wavelengths that are
supposed to be less harmful than the incident radiation. Photon
emission during the rapid return of a molecule from an excited
singlet state to a ground state is known as "fluorescence".
However, sunscreens or other exogenous or endogenous UV-absorbing
molecules can also be excited to longer-lived triplet states which
can facilitate further reactions (the energy-emission that occurs
during return of a molecule from an excited triplet state to a
ground state is known as "phosphorescence", and typically occurs
over a much longer time span than fluorescence). The consequence is
that some UV-absorbing agents, especially those with a lowest
triplet state that has a higher energy level than the lowest
triplet state of genomic DNA constituents, can absorb photons and
actually exacerbate damage to DNA by direct or indirect energy
transfer (e.g. from a triplet excited state) rather than by simple
fluorescence, or photon emission at harmless wavelengths.
[0061] Benzophenone, a close structural analog of oxybenzone,
increases the yield of strand breaks and pyrimidine dimers in
UV-irradiated DNA, and is known to produce free radicals upon
irradiation with UV light (Charlier et al., Photochemistry and
Photobiology, 15:527-536, 1972). The results presented in Examples
3, 4, 5 and 6 indicate that a similar phenomenon also occurs with
approved sunscreen ingredients, and that the deoxyribonucleosides
of the invention attenuate this deleterious consequence of
sunscreen use.
[0062] When present in concentrations sufficient to block access of
UV radiation to cells, sunscreen agents are protective. However, if
present in very low concentrations, insufficient to adequately
block UV transmission, photon-absorbing agents, including common
sunscreen ingredients, can operate as energy-transfer molecules,
efficiently trapping UV energy and transferring it to cell
components, either directly (photosensitization) or by catalyzing
formation of reactive oxygen radicals (photodynamic sensitization).
Thus, low concentrations of oxybenzone, for example, enhance DNA
damage induced by ultraviolet radiation, whereas higher
concentrations, sufficient to block UV access to the cells or their
immediate microenvironment altogether, protect against DNA damage
(see Example 3).
[0063] The expected activity in vivo is that an
oxybenzone-containing sunscreen would protect cells from damage if
present in a layer sufficient to block access of light to the
target cells altogether. However, at lower concentrations,
insufficient to prevent penetration of UV radiation to target
cells, and especially if some oxybenzone has been absorbed into the
critical cell layers, either via passage through the stratum
corneum or through hair follicles, there may be potentiation of
damage to DNA in vivo. In mice treated topically with commercial
sunscreen containing oxybenzone, effects consistent with this
hypothesis are in fact observed after exposure to UV (see Example
6). Classes of sunscreens other than benzophenone derivatives also
exacerbate UV-induced damage to DNA when present at low
concentrations during irradiation.
[0064] A strain of transgenic mice (v-HA-ras transgenic TG.AC mice)
which is very susceptible to a variety of carcinogens, including UV
radiation has been developed recently (Leder et al., Proc. Nat.
Acad. Sci. USA, 87:9178-9182, 1990). In response to a relatively
small exposure to UV radiation, these mice reliably develop
cutaneous papillomas within a few weeks. When a circular patch of
commercial sunscreen is applied to the back of such a mouse, the
center of the protected region does in fact have lower incidence of
UV-induced papillomas than the unprotected side, but often, along
the margin of the applied sunscreen, there is a very high incidence
(sometimes higher than in unprotected areas) of papillomas (See
Example 6). A layer of sunscreen sufficient to block UV access to
target cells is protective, but low concentrations, e.g. at the
margin of a patch of sunscreen) can act as photosensitizers
increasing the incidence of a UV-induced skin cancer beyond that
seen in completely "unprotected" skin. Exposure of relevant skin
cells to low photosensitizing (rather than protective)
concentrations of sunscreens clearly must occur during ordinary
usage, e.g. at the margin of an applied patch, or as a protective
layer is washed or worn off.
[0065] A benefit of deoxyribonucleosides or related compounds added
to conventional sunscreen formulations (or other cosmetics
containing sunscreens), beyond the support of DNA repair, is to
synergize with conventional sunscreen compounds by acting as energy
scavengers which trap energy emitted by (or radicals produced by)
sunscreen agents that is chemically similar to the cellular target,
DNA. Exogenous deoxyribonucleosides (in addition to their direct
absorbance of UV energy) serve as "decoys" for energy captured by
sunscreen agents or other photosensitizers that would otherwise be
transferred to cellular targets, including DNA. Thus,
deoxyribonucleosides provide a dose-dependent reduction in damage
caused to cellular DNA by UV radiation in the presence of low
concentrations of photosensitizing agents like oxybenzone or other
sunscreen agents (Example 4).
[0066] A defining characteristic of suitable energy scavenging
agents is that their lowest triplet energy state is equal to or
lower than that of DNA constituents in situ. Because of the
similarity of physicochemical properties of deoxyribonucleosides
(or deoxyribonucleotides, acyl deoxyribonucleosides, or
oligodeoxyribonucleotides) and genomic DNA, the compounds of the
invention are particularly suitable as energy-scavenging agents to
protect genomic DNA from damage due to energy transfer from
photosensitizers or photodynamic sensitizers. In this embodiment,
ribonucleosides, ribonucleotides, oligoribonucleotides and acyl
derivatives of ribonucleosides are within the scope of the
invention. Appropriate concentrations of such scavengers in a
composition for topical application range from 0.1 to 100
milligrams per milliliter (normalized to the amount of free
nucleoside present in the case of nucleotides or oligonucleotides
or acyl derivatives of nucleosides). Advantageously, such
scavengers are present in concentrations ranging from 0.1 to 20
mg/ml or especially 1 to 5 mg/ml.
[0067] The problem of photodynamic enhancement of damage to DNA
extends beyond sunscreens. Other compounds including endogenous
molecules in the skin, can absorb UV radiation at wavelengths that
do not necessarily directly damage DNA significantly, and transfer
that energy to cellular targets including DNA, or generate free
radicals that damage cellular DNA. Examples of endogenous
photosensitizing or photodynamically active skin constituents
(photochemically active chromophores) include but are not limited
to porphyrins, tryptophan, riboflavin, and melanin. Exogenous
photodynamically active compounds include psoralens, which are
present in some perfume oils (bergamot), and which are in fact used
to enhance sunlight-induced tanning and UV phototherapy of
psoriasis through exacerbation of cellular injury. Many therapeutic
drugs or their metabolites are photochemically active chromophores
which can produce skin adverse reactions when a patient is exposed
to solar, visible, or ultraviolet radiation. Pigments and other
light-absorbing constituents of cosmetics are also photochemically
active chromophores which can exacerbate cellular photodamage.
[0068] The deoxribonucleosides and related compounds of the
invention (e.g. deoxyribo-nucleotides, oligonucleotides, or DNA
itself) are useful for attenuating cellular damage caused by
excited light-absorbing molecules, including exogenous
photochemically active chromophores like sunscreens and cosmetic
pigments, and also from endogenous chromophores like tryptophan,
porphyrins, urocanic acid and melanin.
[0069] Furthermore, since the photodynamic enhancement of DNA
damage caused by benzophenone derivatives is in part mediated by
production of free radicals (Charlier et al., Photochemistry and
Photobiology, 15:527-536, 1972), compounds and compositions of the
invention are useful for protecting the skin and mucosa from free
radical damage, whether or not the free radicals (e.g. hydroxyl
radicals, peroxide radicals, or lipoperoxide radicals) are
initiated or produced by photodynamic phenomena. Examples 3,4,5 and
6 provide evidence that the deoxyribonucleosides of the invention
protect against DNA caused by free radicals.
[0070] The deoxyribonucleosides and related compounds of the
invention are advantageously incorporated into the same formulation
as chemical sunscreen agents, which include but are not limited to:
avobenzone (t-butyl dimethoxydibenzoylmethane), oxybenzone
(benzophenone-3), dioxybenzone (benzophenone-8), sulisobenzone
(benzophenone-4), octocrylene
(2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octyl methoxycinnamate
(2-ethylhexyl p-methoxycinnamate) octyl salicylate
(2-ethylhexylsalicylate), homosalate (homomenthyl salicylate),
trolamine salicylate (triethanolamine salicylate),
phenylbenzimidazole sulfonic acid, PABA (para-aminobenzoic acid),
roxadimate (ethyl 4-bis hydroxypropyl aminobenzoate), lisadimate
(glyceryl PABA), Padimate O (octyldimethyl PABA), menthyl
anthranilate, or Parsol 1789 (butyl methoxydibenzoylmethane). Such
sunscreen agents are present in formulations at concentrations that
are in accord with regulatory guidelines and standard use.
[0071] Similarly, in another embodiment of the invention, compounds
of the invention are incorporated into cosmetics containing
photochemically active chromophores to minimize deleterious
consequences of combined exposure of skin to solar or ultraviolet
radiation and such cosmetic ingredients.
[0072] Alternatively, the compounds of the invention are applied to
the skin in a separate composition, e.g. a spray, lotion, roll-on,
stick, or gel, before or after a sunscreen product or cosmetic is
applied.
Methylxanthines
[0073] Methylxanthines, such as caffeine, theophylline,
aminophylline or isobutylmethylxanthine, have been proposed as
"sunless" tanning agents, which act by modulating activity of the
biochemical pathways involved in melanogenesis. Their proposed
mechanism involves inhibition of cyclic-AMP phosphodiesterase, thus
enhancing the biological activity of cyclic AMP in the pathways
regulating melanogenesis. Methylxanthines can enhance the
production of melanin in melanocytes, acting either alone or in
combination with tanning stimulants like ultraviolet or solar
radiation. However, methylxanthines are also known to exacerbate
DNA damage caused by ultraviolet radiation, which has been
attributed to an impairment of DNA repair or disruption of cell
cycle control mechanisms (Kastan et al., Cancer Research,
51:6304-6311, 1991).
[0074] Compounds of the invention are useful for reducing the
deleterious effect of methylxanthines on DNA damage caused by
exposure of skin to ultraviolet or solar radiation. The compounds
of the invention thus improve the safety of skin tanning products
that contain methylxanthines as active ingredients. Compounds of
the invention are applied either separately or in the same
formulation as the methylxanthines.
Exfoliants
[0075] Cosmetics containing alpha-hydroxy acids (AHA) such as
lactic acid, glycolic acid, citric acid, or malic acid are widely
used. They have moisturizing and exfoliant properties. Products
containing high concentrations of AHA are also used to produce more
extreme "skin peels", in which the outer layers of the epidermis
are removed, essentially by means of a chemical burn. New epidermis
growing in is often softer and smoother than the skin layers that
were removed. Beta-hydroxy acids like salicylic acid are also
useful exfoliants. Retinoic acid is used for similar purposes
through its exfoliant actions and through stimulation of epidermal
cell turnover and alteration of epidermal metabolism.
[0076] Exfoliants are reported to reduce the sun-blocking
capabilities of the stratum corneum, and furthermore increase the
permeability of the skin to other agents. Thus, there is a need for
use of UV protection in conjunction with AHA's, yet the increased
skin permeability produced by AHA requires caution in the selection
of sun protection agents, since absorbed sunscreen agents can
produce photodynamic enhancement of DNA damage. The compounds of
the invention are effective in reducing deleterious consequences of
exposure to sunlight or ultraviolet radiation or other
environmental mutagens in subjects using exfoliants, including but
not limited to alpha-hydroxy acids, beta-hydroxy acids, and
retinoids.
Nonsteroidal A anti-inflammatory Agents
[0077] Nonsteroidal anti-inflammatory agents are commonly used for
treatment of arthritis and other anti-inflammatory agents.
Moreover, some members of this class, e.g. diclofenac
(2,6-dichloro-phenyl-amino-phenyla- cetate) are under investigation
as topical agents for reducing some aspects of skin
photodamage.
[0078] One of the prototypical members of this class of drugs,
acetaminophen, inhibits DNA repair after damage caused by UV
radiation by inhibiting the enzyme ribonucleotide reductase, which
converts ribonucleoside diphosphates to deoxyribonucleoside
diphosphates (Hongslo et al., Mutagenesis, 8:423-429, 1993). By
supplying deoxyribonucleosides to skin, especially to areas of the
skin that are generally exposed to sunlight, of patients receiving
either oral or topical treatment with nonsteroidal
anti-inflammatory agents, compositions of the invention overcome a
deleterious consequence associated with this widely-used class of
drugs.
Ornithine Decarboxylase Inhibitors
[0079] One consequence of UV irradiation of skin is an increase in
cell proliferation rate and in the activity of enzymes necessary
for cell proliferation, one of which is ornithine decarboxylase
(ODC). Difluoromethylornithine (DFMO) is an inhibitor of ODC, which
is necessary for polyamine synthesis, which in turn is necessary
for DNA replication. DFMO is useful for reducing the incidence of
skin cancer and precancerous actinic lesions. Inhibition of cell
cycling after exposure to solar or UV radiation may give cells more
time to repair DNA before mutagenic lesions are fixed by cell
division, but cell cycle stasis also generally results in reduced
levels of deoxyribonucleosides, which are necessary for repair of
DNA damage. Deoxyribonucleosides are therefore useful in
conjunction with DFMO and other antiproliferative agents which act
via mechanisms not directly involving induced depletion or
imbalance of deoxyribonucleotide pools. Deoxyribonucleosides of the
invention are optionally incorporated into the same formulation as
DFMO (or other inhibitors of skin cell proliferation that act
through mechanisms other than impairment of DNA precursor
synthesis), or are applied in a separate formulation.
Treatment of Skin During Exposure to Endogenous Nitric Oxide
[0080] Nitric oxide (NO) is a biologically active mediator released
by endothelial cells, macrophages and other cell types, especially
during inflammatory episodes. Nitric oxide is also an important
mediator of erythema associated with UV exposure. Inhibitors of NO
synthetase attenuate the increase in skin blood flow following
exposure (Deliconstantinos et al., J Cardiovasc Pharmacol 20 Suppl
12:S63-5, 1992; Deliconstantinos et al., Br J Pharmacol
114(6):1257-65, 1995; Warren, FASEB Journal, 8(2):247-51,
1995).
[0081] Nitric oxide is a potent inhibitor of the enzyme
ribonucleotide reductase (RR), which is the key enzyme for de novo
synthesis of deoxyribonucleotides (Kwon et al., J Exp Med
174(4):761-7, 1991; Lepoivre et al., J Biol Chem 269(34):21891-7,
1994) The antiproliferative effects of nitric oxide are in part
attributable to inhibition of ribonucleotide reductase. This can be
beneficial in an inflammatory response to an infectious
microorganism or a neoplasm, but is deleterious in cells in need of
capability for DNA repair, e.g. skin exposed to inflammatory
mediators elicited by UV exposure. The amounts of NO released from
macrophages are sufficient to inhibit ribonucleotide reductase and
thereby induce cytostasis in neighboring cells.
[0082] The best-known inhibitor of RR, hydroxyurea (HU), has
structural similarities to N-omega-hydroxy-l-arginine, a
physiological intermediate in NO production. Hydroxyurea can act as
an NO-like nitrosating reactant (LePoivre et al., J Biol Chem
269(34):21891-7, 1994). Both NO and hydroxyurea inhibit RR by
quenching a tyrosyl radical in the active site of the enzyme.
[0083] A discovery first disclosed herein is that NO sensitizes
cells to UV-induced DNA damage via mechanisms that are reversible
with exogenous deoxyribonucleosides (see Example 8). Moreover, NO
by itself, in the absence of UV exposure, causes DNA damage that is
prevented or reversed by compounds of the invention (Example
8).
[0084] The inflammatory response to UV exposure (which also plays
an important role in UV-associated immunosuppression), via
inhibition of ribonucleotide reductase by NO, exacerbates the
mutagenic and cytotoxic effects of UV exposure. The subject
invention provides a means for compensating for this deleterious
effect of NO. Exogenous deoxyribonucleosides, which enter into DNA
metabolism downstream of ribonucleotide reductase, compensate for
reduced RR activity. NO, although deleterious in cells needing
deoxyribonucleotides, has some beneficial effects in sun-exposed
skin. Inhibitors of NO production also impair the healing of skin
damaged by exposure to excessive UV radiation, i.e., UV doses that
produce a sufficiently severe sunburn for wound healing processes
to be called into action (Benrath et al., Neurosci Lett, 1995).
Thus, NO is useful for tissue repair (probably by improving blood
flow and perhaps also by stimulating growth factor release from
macrophages or keratinocytes), but may simultaneously exacerbate
UV-related damage to DNA by reducing cellular capacities for
production of deoxyribonucleotides for DNA repair.
[0085] NO-mediated inhibition of RR provides a mechanism for a
disproportionate increase in DNA damage without a corresponding
increase in other symptoms of sun exposure during repeated exposure
to strong sunlight. Topical application of the compounds and
compositions of the invention provides a method for ameliorating
this form of conditional hypersensitivity.
[0086] NO participates in skin inflammatory reactions that do not
necessarily involve exposure to solar or ultraviolet radiation. NO,
which is released from activated macrophages, is component of most
inflammatory reactions. The compounds and methods of the invention
provide a means of overcoming some deleterious effects of NO in
inflammatory skin conditions, including but not limited to
psoriasis, dermatitis, allergic dermatitis, eczema and acne. In
these conditions, NO sensitizes some cell types to UV-induced DNA
damage by inhibiting deoxyribonucleotide synthesis. Compounds and
compositions of the invention ameliorate this deleterious
consequence of combined UV exposure and inflammatory skin
conditions. Since NO and other endogenous oxidants that cause DNA
damage are present in inflammatory skin conditions like psoriasis
or dermatitis even in the absence of significant exposure to UV
radiation, the deoxyribonucleosides of the invention are useful for
protecting genetic integrity of skin cells in inflammatory
conditions. Compounds of the invention prevent or repair DNA damage
caused by NO alone, without exposure to UV radiation (Example
8).
[0087] Ribonucleosides are also useful for treating inflammatory
skin an mucosal conditions, in part by providing higher
concentrations of the ribonucleotide substrates for ribonucleoside
reductase. Ribonucleosides (or ribonucleoside esters) in this
context are used in the same ways that deoxyribonucleosides,
advantageously in topically-applied compositions, with
ribonucleosides (or ribonucleoside esters) incorporated in
concentrations ranging from 0.1 to 20 mg/ml, advantageously 1 to 5
mg/ml. Adenosine is particularly useful for treatment of
inflammatory conditions.
[0088] Inhibition of DNA precursor synthesis by hydroxyurea leads
to enhanced activity and leakage of hydrolytic lysosomal enzymes
which participate in extracellular damage, e.g. in inflammatory
skin conditions or photodamage (Malec et al., Chem. Biol.
Interact., 57:315-324, 1986). The compounds of the invention
prevent this component of inflammatory tissue injury, especially
when such inhibition of DNA precursor synthesis is mediated by
endogenously-produced NO, which is functionally similar to
hydroxyurea.
[0089] A further contribution to genomic damage is that exposure of
cells to ultraviolet radiation results in the release of enzymes,
including deoxyribonuclease, from lysosomes. Deoxyribonuclease II,
which is present in lysosomes, is an endonuclease that can produce
strand breaks in nuclear DNA following lysosome disruption. Leakage
of enzymes from lysosomes also occurs in inflammatory conditions in
general. The compounds and compositions of the invention are useful
for preserving genomic integrity after chromosomal damage caused by
deoxyribonuclease released from lysosomes in inflammatory skin
conditions and after exposure of skin to solar or ultraviolet
radiation.
[0090] By limiting some of the deleterious consequences of skin
inflammation, compounds and compositions of the invention are
useful as anti-inflammatory agents, and are optionally administered
(either as separate compositions or, advantageously, in the same
formulation) in conjunction with other topical or systemic
anti-inflammatory agents including but not limited to
corticosteroids like hydrocortisone and its congeners.
[0091] Similarly, compounds and compositions encompassed by the
invention are useful for treatment of mucosal inflammatory
conditions, including but not limited to inflammatory bowel
disease, ulcerative colitis, or Crohn's disease, or mucositis
anywhere in the gastrointestinal tract. The preferred mode of
treatment is by topical administration, in this case via enema or
suppository, for which purposes deoxyribonucleosides or other
compounds of the invention are incorporated into suitable
vehicles.
Treatment of Skin and Mucosal Tissues Exposed to Ionizing
Radiation
[0092] Patients receiving therapeutic treatment (e.g. for cancer)
with ionizing (X-Ray or gamma) radiation can suffer damage to skin
overlying an internal tumor, leading to desquamation and poor
healing. Compounds and compositions of the invention are useful for
treating damage to skin and mucosal surfaces caused by intentional
or accidental exposure to ionizing radiation. For treatment of
skin, compositions of the invention are applied topically before or
after radiation treatment. For treatment of mucosal surfaces, e.g.
in the mouth, gastrointestinal tract, urethra or vagina,
appropriate compositions of the invention are also applied
topically. Suitable compositions for treatment of mucosal surfaces
include gels, lotions, ointments, suppositories,
orally-administered capsules, pills or dragees, or solutions.
E. Administration and Formulation of Compounds and Compositions of
the Invention
[0093] Compounds of the invention are formulated in
pharmaceutically acceptable vehicles that deliver the compounds to
the necessary cell populations in skin at concentrations adequate
to accomplish the objectives of reducing mutation frequency and
chance of developing cancer.
[0094] Compositions of the invention are applied before, during, or
after exposure to sunlight or other mutagens. A lotion or hydrogel
containing deoxyribonucleosides (0.1 to 10 mg/ml, advantageously 1
to 5 mg/ml) is applied to skin as a thin film. The composition
should be applied within about 48 or 72 hours after exposure to
damaging doses of sunlight or ultraviolet radiation, in order to
provide support for DNA repair prior to the first cell divisions
after irradiation, although application before, during, or within
12 hours after exposure to intense sunlight is advantageous.
Compositions of the invention are also effective when applied
before exposure to radiation or other mutagens, as long as the
deoxyribonucleosides so provided, or their anabolites, are
available to cells in need of their beneficial effects at the time
of exposure to a mutagen. Advantageously, compositions of the
invention are applied within about 12 hours before exposure of the
skin to a solar radiation or other mutagens.
[0095] Compositions of the invention are advantageously applied as
a daily-use skin treatment, once to several times per day,
especially on sun-exposed parts of the body, or sites of
inflammatory skin conditions. Exposure to solar radiation leading
to skin photodamage and photoaging is generally a cumulative
process, involving repeated exposure to sunlight, even daily, over
a period of years. In this context, use of the compounds and
compositions of the invention to prevent or treat photodamage to
the skin involves treatment of existing lesions due to prior sun
exposure, as well as prevention of, or attenuation of the severity
of, damage due to present and future exposure to sunlight or other
mutagens.
[0096] By improving repair of molecular damage to DNA as it occurs
or before it is permanently established in the genome by cell
division, or by preventing initial damage through energy
scavenging, compositions of the invention prevent or delay the
manifestation of deleterious consequences of radiation, free
radicals, or chemical mutagens, such as grossly visible skin
damage, photoaging, actinic keratoses, and skin cancer. Thus,
compositions and methods of the invention reduce the rate of
appearance and the incidence of signs of skin photodamage,
especially when administered regularly, e.g. daily, or especially
before, during, or after exposure to solar radiation.
[0097] In one embodiment of the invention, a composition containing
deoxyribonucleosides is applied to skin prior to application of a
sunscreen, as an alternative to use of formulations containing both
conventional sunscreens and deoxyribonucleosides or related
compounds of the invention.
[0098] For treatment of colon mucosal inflammation, e.g.
inflammatory bowel disease, compositions of the invention are
administered as a suppository or enema, approximately once per day
according to clinical need. Volumes of 10 to 500 ml of a suitable
enema composition containing deoxyribonucleosides are suitable for
treatment of inflammatory bowel disease, e.g. ulcerative colitis or
Crohn's disease. For treatment of mucositis in other parts of the
gastrointestinal tract, such as the mouth, standard
pharmaceutically acceptable vehicles for that route of
administration are used, e.g. mouthwashes or adherent
hydrocolloids.
F. Synthesis of the Compounds of the Invention
[0099] Deoxyribonucleosides, being constituents of DNA, are present
in all living organisms, and can therefore in principle be
extracted from a variety of sources. In practice, the most
convenient biological sources at present are fish milt, which
contains a relatively high concentration of DNA. Fish milt sacs are
homogenized, and the DNA therein is partially purified and treated
with deoxyribonucleases and phosphatases to degrade it to the level
of nucleosides, which are then purified by chromatography and
recrystallization.
[0100] Since mixtures of deoxyribonucleosides are used in some
embodiments of the invention, purified deoxyribonucleosides are
recombined in appropriate proportions. Alternatively,
deoxyribonuclosides are not separated from each other during
purification from other fish milt components (or other contaminants
if the DNA is derived from other biological sources);
appropropriate quantities of individual deoxyribonucleosides are
added to such a mixture, if necessary, to adjust the relative
proportions of deoxyribonucleosides.
[0101] Deoxyribonucleosides can also be synthesized chemically from
simpler precursors.
[0102] Acyl derivatives of deoxyribonucleosides, as disclosed in
U.S. patent application Ser. No. 466,379, are useful for 1)
providing sustained availability of deoxyribonucleosides due to
gradual deacylation by nonspecific esterases in the skin, and 2)
improved penetration through hydrophobic biological membranes or
extracellular media, e.g. the intercellular lipids in the stratum
corneum of the epidermis.
[0103] It will be obvious to the person skilled in the art that
other methods of synthesis may be used to prepare the compounds of
the invention.
[0104] The following examples are illustrative, but not limiting of
the methods and compositions of the present invention. Other
suitable modifications and adaptations of a variety of conditions
and parameters normally encountered in clinical therapy which are
obvious to those skilled in the art are within the spirit and scope
of this invention.
EXAMPLE 1
Post-irradiation Topical Deoxyribonucleosides Improve DNA Repair in
Mouse Skin after UVB Exposure
[0105] The incidence of mutations in skin in response to
ultraviolet radiation was determined using the "Big Blue"
transgenic mouse test system. These mice carry approximately 40
copies per cell of a lambda phage shuttle vector containing a lad
gene as a target for mutagenesis, as well as the lad promoter, the
lac operator, and the .alpha.lacZ reporter gene.
[0106] Following exposure of the mice to ultraviolet radiation and
treatment with compounds of the invention or vehicle, genomic DNA
from skin samples is extracted and the shuttle vectors are packaged
in lambda virus heads. The phage lambda viruses containing the
shuttle vectors are plated on E.Coli with the color reagent X-Gal,
which turns blue when enzymatically altered by galactosidase, the
product of the .alpha.lacZ gene. Viruses containing nonmutated lacI
genes produce white plaques; mutation of the lacI gene results in
blue plaques. The mutation frequency is determined by counting the
relative numbers of white and blue plaques.
[0107] Stratagene BigBlue.TM. transgenic mice (n=7/group) were
shaved and then irradiated the next day with UVB radiation (85% of
energy at 313 nm), 1.25 kJ/m2.
[0108] Deoxyribonucleosides ("dNsides"; 4 mg/ml each of
deoxyadenosine, deoxycytidine, deoxyguanosine, and thymidine) or
vehicle (propylene glycol) were applied topically 30 minutes after
irradiation and again each day for 5 days.
[0109] Mice were sacrificed on day 5 after irradiation. DNA was
extracted from dorsal (irradiated) and ventral (non-irradiated)
skin, packaged into lambda phage and plated on E.Coli along with
X-Gal. Colonies with mutations were blue. >200,000 colonies were
counted in each group.
1TABLE 1 Topical deoxyribonucleosides reduce mutation frequency in
UV-irradiated skin Spontaneous mutation frequency (nonirradiated
skin): Average: 4.5 .times. 10.sup.-5 Mutation frequency in
UVB-irradiated skin: Total Increment due to UV Control 34.9 .times.
10.sup.-5 30.4 .times. 10.sup.-5 dNsides 7.8 .times. 10.sup.-5 3.3
.times. 10.sup.-5
[0110] As shown in Table 1, post-irradiation treatment with topical
deoxyribonucleos ides reduced the incidence of mutations caused by
UV-B radiation by a factor of nearly 10 in this experiment
(30.4.times.10.sup.-5 versus 3.3.times.10.sup.-1)
EXAMPLE 2:
Post-irradiation Treatment with Topical Deoxyribonucleosides
Prevents Development of UV-induced Papillomas in v-Ha-ras
Transgenic TG.AC Mice
[0111] Example 1 demonstrated that post-irradiation topical
treatment can reduce the frequency of UV-induced mutations in a
reporter gene in "Blue" transgenic mice, through support and
improvement of DNA repair processes. One of the consequences of
reduced mutation frequency in response to a carcinogen like UV
radiation should be a reduction of UV-induced tumorigenesis.
[0112] A strain of transgenic mice has been developed which is
extremely sensitive to carcinogens. It permits rapid determination
of carcinogenic potential of various chemical agents and other
treatments. Normal mice require repeated exposure to ultraviolet
radiation over a number of weeks in order to reliably develop skin
tumors. In contrast, v-Ha-ras TG.AC transgenic mice can develop
tumors rapidly after a single exposure, or small number of
exposures to UV radiation.
[0113] In 9 mice exposed to UV-B radiation (0.3-1.25 kJ/m.sup.2 x3
q2d) and treated after irradiation with vehicle (propylene glycol),
a total of 35 papillomas were found 4 weeks after exposure.
[0114] Among 7 mice exposed to the same doses of UV-B radiation and
treated with deoxyribonucleosides (4 mg/ml of each major
deoxyribonucleoside in propylene glycol) after irradiation, only 1
papilloma was observed (Table 2).
2TABLE 2 Topical deoxyribonucleosides reduce UV-induced
tumorigenesis Control: 3.89 tumors/mouse dNside-treated: 0.14
tumors/mouse
[0115] The beneficial effect of deoxyribonucleosides in reducing
UV-induced tumorigenesis must be due to improvement of repair
phenomena or cellular proofreading, or to inhibition of tumor
promotion, and not to prevention of initial damage, since the
deoxyribonucleosides were applied after irradiation. This
observation indicates that deoxyribonucleoside treatment after (or
presumably also during) exposure to UV-B radiation has important
inhibitory effects on tumorigenesis, as a consequence of improved
maintenance of genomic fidelity.
[0116] This antitumorigenic effect of topical deoxyribonucleosides
even when applied after irradiation is particularly surprising in
view of the reported efficacy of deoxyribonucleosides in
accelerating wound healing (U.S. Pat. No. 5,246,708), since other
growth factors known to accelerate wound healing, like
platelet-derived growth factor (PDGF) or transforming growth factor
beta (TGF-.beta.), act as tumor promoters.
[0117] The antitumorigenic effect of deoxyribonucleosides in this
experiment is also unexpected in view of the beneficial effect of
deoxyribonucleosides on survival of cells in culture when the
deoxyribonucleosides are applied after exposure of the cells to
ultraviolet or ionizing radiation. Prevention of apoptosis of
damaged cells would be expected to increase the likelihood of tumor
development, as occurs in animals with defective p53-related
mechanisms.
EXAMPLE 3
Low Concentrations of Oxybenzone Exacerbate UV-induced Damage to
DNA
[0118] Confluent human fibroblasts in T25 flasks were washed 3
times with HBSS (Hank's Balanced Salt Solution) and incubated with
vehicle or with various concentrations of oxybenzone (OB) for 2
hours. Media was aspirated and cells were covered with a 1 mm layer
of HBSS and irradiated from above with UV-B (50 J/m.sup.2). The
medium was aspirated and cells were incubated for three hours with
2 mM hydroxyurea. Medium was again aspirated, and cells were
trypsinized with 0.25% trypsin/EDTA. Cells were centrifuged at
4.degree. C., resuspended in 50 microliters of HBSS and incubated
at room temperature with 200 microliters of 1N NaOH for 15 minutes.
DNA damage (single strand breaks) was assessed by alkaline sucrose
gradient centrifugation. "Nucleoid position" in the sucrose
gradient is proportional to the number of DNA single strand
breaks.
[0119] UV irradiation without oxybenzone results in a three to
four-fold increase in the nucleoid position over baseline (Table
3). Oxybenzone at the higher concentrations tested, 20 and 200
micromolar, protected cellular DNA against damage from the UV
irradiation, as the nucleoid position for these groups is close to
that of non-irradiated cells. However, cells exposed to 2
micromolar OB display substantially more damage than cells
irradiated with no OB at all; nucleoid position values are 10-fold
greater than those of non-irradiated cells. Thus, oxybenzone
strongly enhances DNA damage when present at low concentrations,
whereas it protects cells at higher concentrations. In practice,
even under conditions of proper sunscreen use, there will always be
areas of skin exposed to low, potentially deleterious
concentrations of sunscreen, either at the edge of a patch of
applied sunscreen, or as an applied layer wears off over the course
of a day.
3TABLE 3 Low concentrations of oxybenzone enhance UV-induced DNA
damage Group Nucleoid Position (mm) No UV 3 UV 50 J/m.sup.2 11 UV
50 J/m.sup.2 + 2 .mu.M OB 32 UV 50 J/m.sup.2 + 20 .mu.M OB 4 UV 50
J/m.sup.2 + 200 .mu.M OB 3
EXAMPLE 4
Deoxribonucleosides Attenuate Photodynamic Enhancement of DNA
Damage Caused by Oxybenzone
[0120] Confluent human fibroblasts were exposed to 2 micromolar
oxybenzone (OB), as in Example 3, prior to exposure to UV-B
radiation (50 J/m.sup.2). Different flasks of cells also were
exposed to increasing concentrations of deoxyribonucleosides. Cells
were processed for determination of nucleoid position in a sucrose
density gradient, a measure of DNA single strand breaks.
[0121] As shown in Table 4, deoxyribonucleosides produce a
dose-dependent reduction in the yield of DNA single strand breaks
induced by UV exposure plus 2 .mu.M OB. Deoxyribonucleosides at 2
.mu.M slightly reduce DNA damage; at 200 micromolar, the
deoxyribonucleosides almost completely abrogate the DNA damage.
4TABLE 4 Deoxyribonucleosides attenuate photodynamic enhancement of
DNA damage caused by UV plus oxybenzone Group Nucleoid Position
(mm) UV + 2 .mu.M OB 56 UV + 2 .mu.M OB + 2 .mu.M dNsides 47 UV + 2
.mu.M OB + 20 .mu.M dNsides 23 UV + 2 .mu.M OB + 100 .mu.M dNsides
22 UV + 2 .mu.M OB + 200 .mu.M dNsides 7
EXAMPLE 5
Effect of Individual Versus Combined Deoxribonucleosides on
Photodynamically-enhanced, UV-Induced DNA Damage
[0122] Human fibroblasts were prepared and treated as in Example 4,
and the effects of individual deoxyribonucleosides on
photodynamically enhanced DNA damage (2 .mu.M oxybenzone) were
determined. Individual deoxyribonucleosides were tested at
concentrations of 20 .mu.M, and the combination of all four
deoxyribonucleosides contained thymidine, deoxycytidine,
deoxyadenosine and deoxyguanosine at 20 .mu.M each.
5TABLE 5 Effect of individual versus combined deoxyribonucleosides
on UV-induced DNA damage DNA Strand Breaks Group (Breaks/47 MDa)
Control (no UV) .0 UV + vehicle .325 UV + thymidine .13 UV +
deoxycytidine .12 UV + deoxyadenosine .17 UV + deoxyguanosine .17
UV + dNsides (20 .mu.M each) .01
[0123] As shown in Table 5, each of the individual
deoxyribonucleosides attenuated DNA damage; the combination of all
four deoxyribonucleosides was substantially more effective than any
individual compound.
EXAMPLE 6
Sunscreen-induced Exacerbation of UV-induced Tumorigenesis and its
Prevention with Deoxyribonucleosides
[0124] A circular patch of commercial sunscreen (Coppertone SPF 8,
which includes oxybenzone) was applied to the backs of TG.AC mice
prior to exposure to 125 J/m.sup.2 UV-B radiation.
[0125] Around the circular margin of the area that was covered with
sunscreen during irradiation, 8 papillomas per mouse were present
at 10 days. 3 tumors per mouse were observed in animals not treated
with sunscreen. In mice exposed to UV radiation after application
of the same commercial sunscreen to which deoxyribonucleosides had
been added, no papillomas were elicited (Table 6).
6TABLE 6 Deoxyribonucleosides attenuate tumorigenesis in in mice
treated with UV plus sunscreen UV Radiation 3 tumors/mouse
Sunscreen + UV radiation: 8 tumors/mouse Sunscreen containing
dNsides + UV radiation: 0 tumors/mouse
[0126] Thus, at the margin of the applied patch of sunscreen where
the sunscreen concentration diminishes in a rapid gradient toward
zero, tumorigenic UV damage in fact appears to be enhanced rather
than reduced by the sunscreen agents, leading to formation of more
premalignant papillomas than on skin not treated with sunscreen at
all.
[0127] Addition of deoxyribonucleosides to a commercial sunscreen
abrogated the deleterious effect of the sunscreen on
tumorigenesis.
EXAMPLE 7
Effect of Individual Deoxyribonucleosides Versus a Combination on
UV-induced Tumorigenesis
[0128] Thirty v-Ha-ras TG.AC mice aged twelve weeks were shaved and
subjected to ultraviolet radiation, 1.25 kJ/m.sup.2 on days 0, 6, 8
and 11, at a dose rate of 12.5 W/m2.
[0129] Mice were divided into groups of five animals each and
treated, beginning 30 minutes after irradiation, with:
[0130] 1. Vehicle (propylene glycol)
[0131] 2. dNsides--Deoxyribonucleosides (4 mg/ml each of
deoxyadenosine, deoxycytidine, deoxyguanosine, and thymidine)
[0132] 3. dC--Deoxycytidine (4 mg/ml)
[0133] 4. dG--Deoxyguanosine (4 mg/ml)
[0134] 5. dA--Deoxyadenosine (4 mg/ml)
[0135] 6. dT--Thymidine (4 mg/ml)
[0136] Animals were observed for 8 weeks, during which time the
development of papillomas was observed.
7TABLE 7 Effect of individual versus combined deoxyribonucleosides
on UV-induced tumorigenesis Papillomas/mouse at the end of 8 weeks:
Vehicle 1.8 dNsides 0.0 dC 0.25 dG 1.0 dA 0.67 dT 1.6
[0137] As shown in Table 7, the mixture of all four
deoxyribonucleosides provided the best activity in terms of
prevention of tumor development. Deoxycytidine also provided
protection; deoxyadenosine and deoxyguanosine were less protective
but nonetheless had activity. Thymidine did not have significant
protective actions.
EXAMPLE 8
Nitric Oxide Causes and Enhances DNA Damage by a
Deoxyribonucleoside-rever- sible Mechanism
[0138] Nitric oxide is a mediator of UV-induced erythema, and is
also present in inflammatory skin conditions. NO is mutagenic, and
may exacerbate UV-induced DNA damage, in part by inhibiting
ribonucleotide reductase, the enzyme responsible for conversion of
ribonucleotides to deoxyribonucleotides.
[0139] Human melanocytes were exposed to 160 .mu.M DETA NONOate
(Alexis Corporation, Cat# 430-014-M005), a reagent which
spontaneously produces nitric oxide when exposed to water. Cells
were exposed to NO alone, or NO plus increasing doses of UV
radiation (50 and 300 J/m.sup.2). Groups of cells were also exposed
to deoxyribonucleosides of the invention before, or before and
after, exposure to NO or NO+UV radiation; control groups were
treated identically except for addition of
deoxyribonucleosides.
[0140] DNA was extracted from cells and subjected to pulsed field
gel electrophoresis to determine the incidence of DNA strand
breaks.
[0141] As shown in Table 8, NO alone produced a significant
incidence of DNA strand breaks, which were further increased by
exposure to UV radiation. The deoxyribonucleosides of the invention
strongly reduced the incidence of DNA strand breaks caused by NO,
as well as those caused by the combination of NO plus UV
radiation.
8TABLE 8 Deoxyribonucleosides attenuate Nitric Oxide-induced DNA
damage DNA Strand Breaks Groups (breaks/105 MDa) Control 0.00 NO
0.13 NO + UV 50 J/m.sup.2 0.21 NO + UV 300 J/m.sup.2 0.51 NO +
dNsides before and after UV 0.00 NO + UV 50 J/m.sup.2 + dNsides
before/after UV 0.03 NO + UV 300 J/m.sup.2 + dNsides before/after
UV 0.03 NO + dNsides after UV 0.06 NO + UV 50 J/m.sup.2 + dNsides
after UV 0.06 NO + UV 300 J/m.sup.2 + dNsides after UV 0.03
* * * * *