Method for alleviating skin irritation by formulations containing superoxide dismutase

This invention discloses use of superoxide dismutase (SOD) as an agent for the protection of the skin against inflammatory reactions associated with chemical irritation and acne. Topical application of SOD in a formulation attenuates dermal injury associated with the induction of reactive oxygen species by a wide variety of chemical irritants, associated with inflammation, and associated with acne.

BACKGROUND OF THE INVENTION 
Adverse skin reactions caused by chemical irritants are common among 
humans. Many of these irritants are constituents of cosmetic products 
while others are noxious industrial chemicals including organic solvents 
and caustic materials. In view of this wide spectrum of irritants, several 
physiologically injurious mechanisms have been postulated as underlying 
the adverse reactions. To deal with these possibilities, researchers have 
developed several classes of antiirritants. 
According to Goldemberg, (In "Principles of Cosmetics for the 
Dermatologist", Phillip Frost and Stephen N. Horwitz, Eds., The C. V. 
Mosby Company, St. Louis, Mo. p.34; Goldemberg, R. M., 1979, J. Soc. 
Cosmet. Chem., 30:415) an antiirritant could be reacted with the irritant 
chemical to complex the irritant or change its nature so that it no longer 
irritates. Alternatively, the reactive skin sites can be blocked so that 
they no longer react with irritants. Additionally, a heavy layer of grease 
which is positioned between the skin and chemical irritants may also be 
used. Finally, the problem of irritation may be attenuated by extreme 
dilution of the irritant. 
In general it is possible that chemical irritant injury of cells may be 
associated with the generation of toxic, free radicals. For example, when 
insulin secreting cells are exposed in vitro to solutions containing 
alloxan, hydroxyl radical is produced extracellularly with ensuing 
manifestations of plasma membrane damage. (Fischer, L. J. and Harmon, A. 
W. 1982, In "Pathology of Oxygen," Anne P. Auter. Ed. Academic Press. N.Y. 
p. 261). Paraquat, a pyrazine derivative which is easily reduced to a 
relatively stable free radical, is believed to augment the production of 
superoxide by chloroplasts and lung microsomes, and this is probably one 
reason for paraquats lethality in both plants and animals. (Hassan, H. M. 
and Fridovich, I., 1977, J. Bacteriol, 130:805; J. Bacteriol, 132:505). 
Also, carrageenan-induced and kaolin-induced inflammation in the rat has 
been suppressed by the use of the oxygen free radical scavenger, 
superoxide dismutase (Huber, W. and Saifer, M. G. 1977, In "Superoxide and 
Superoxide Dismutases", A. M. Michelson, J. M. McCord, and I. Fridovich, 
Eds. Academic Press, N.Y., 1977, p. 517; Oyanagui, Y., 1976, Biochem., 
Pharmacol, 25:1465). 
It is known that biological reduction of molecular oxygen is accompanied by 
the production of reactive free-radical intermediates. The complete 
reduction of a molecule of oxygen to water requires four electrons, and in 
a sequential univalent process several intermediates are encountered. 
These are the superoxide anion-radical, hydrogen peroxide, and the 
hydroxyl radical, and such intermediates are too reactive to be well 
tolerated within living systems (Czapski, G. 1971, Annu. Rev. Phys. Chem., 
22:171). 
The superoxide anion radical, hydrogen peroxide and the hydroxyl radical 
which may be generated enzymatically or photochemically, are known to 
inactivate microorganisms, induce lipid peroxides, damage membranes and 
kill cells (Fridovich, I., 1982, In "Pathology of Oxygen," Anne P. Autor., 
Ed., Academic Press, Inc., New York.,p. 1). A primary defense against 
toxic oxygen radicals is provided by enzymes that catalytically scavenge 
the intermediates of oxygen reduction. For example, the superoxide anion 
radical may be eliminated by superoxide dismutases, which catalyze its 
conversion to hydrogen peroxide and oxygen. 
There are indications that superoxide is not itself the species that causes 
injury to cells but may be the precursor of a more potent oxidant, the 
hydroxyl radical, the generation of which depends on the simultaneous 
presence of hydrogen peroxide. (Haber, R. and Weiss, 1934, J. Proc. Roy. 
Soc. London, Ser. A., 147:332). Thus, it seems that the greatest danger 
posed by superoxide is its interaction with hydrogen peroxide or with 
organic peroxides, which can generate a highly reactive entity like 
hydroxyl radical that can then attack essential cell components. 
Several reports have appeared presenting data that the enzyme, superoxide 
dismutase (SOD), may function at the level of the skin. For example, two 
groups of researchers hav shown that systemic administration of SOD to 
rats will inhibit the reverse passive Arthus reaction in the skin (Petrone 
et al., 1980, Proc. Natl. Acad. Sci., 77:1159; Parellada, P. and Planas, 
J. M., 1978, Biochem. Pharm., 27:535). Further, treatment of facial 
lesions in patients with Crohn's disease with a topical application of 
liposomes containing superoxide dismutase was followed by marked 
improvement with a diminution of swelling (Michelson, A. M., 1982. In 
"Pathology of Oxygen", Anne P. Autor, Ed., Academic Press, N.Y., p. 277). 
Kalopissis et al. (U.S. Pat. No. 4,129,644 "Protecting Skin and Hair with 
Cosmetic Compositions Containing Superoxide Dismutase") claim that 
representative superoxide dismutase extracts of marine bacteria protect 
the keratinic structure of the skin of rats from the effects of 
intraperitoneally injected testosterone propionate. They additionally 
claim that topical application of a cream containing SOD would protect the 
skin from the harmful effects of ultra-violet rays produced by irradiation 
of human subjects with a Xenon, U.V. Solar Simulator. Other workers have 
shown that topical administration of a low molecular weight lipophilic 
copper coordination complex with superoxide dismutase-mimetic activity 
inhibits certain phorbol ester-induced biochemical and biological 
responses associated with carcinogenesis as well as the number of 
developing papillomas (Kensler, T. W., Bush, D. M., Kozumbo, W. J., 1983, 
Science, 221:75). Superoxide radical scavenging agents have also been used 
to protect rabbit cornea against alkali injury. (Nirakari et al., 1981, 
Arch. Ophthalmol., 99; 886). The unique avascularity of the cornea 
suggests that such oxygen radicals participate directly in the promotion 
of the corneal ulceration. 
Until the present invention, however, agents for scavenging the 
intermediates of the biological reduction of oxygen have not been used to 
protect the skin from the deleterious action of diverse classes of 
chemical irritants. Moreover, there is no evidence indicating that the 
toxic dermal manifestations induced by a wide array of chemical irritants 
of the skin can be abrogated through the use of a single biological agent. 
Current knowledge of skin irritation requires use of divergent protective 
approaches as explained above. Finally, scavenger agents have not been 
suggested for use in the control of dermal inflammatory reactions 
including acne. 
Accordingly, it is an object of the invention to provide a general method 
for prevention or alleviation of chemical irritation of the skin through 
the topical use of a single biological agent in combination with a 
suitable carrier or vehicle. Another object is the use of such a 
biological agent which scavenges free radical intermediates of biological 
oxidation-reduction reactions. It is a further object to use such an agent 
for the general control of dermal inflammation and acne. Other objects 
include the formulation of creme, gel, lotion and liquid preparations for 
the skin which will protect it from injury by diverse chemical irritants 
and will prevent or alleviate inflammation and acne. 
SUMMARY OF THE INVENTION 
These and other objects are achieved by the present invention which is 
directed to a topical formulation for the protection of the skin against 
chemical irritation and a method for preventing or alleviating skin 
irritation, general skin inflammation and acne by employing such a topical 
formulation. 
The formulation is a suitable, cosmetic or dermatologicly acceptable, 
non-toxic, non-allergenic carrier containing such an amount of SOD that 
with one application of the formulation at least about 1 CIU, preferably 
at least about 2.5 CIU, more preferably about 10 to 1000 CIU's of purified 
superoxide dismutase (hereinafter SOD) are applied per cm.sup.2 of treated 
skin. It appears that this minimum amount of about 1 CIU of SOD, 
preferably at least about 2.5 CIU of SOD, and more preferably about 10 to 
1000 CIU of SOD per cm.sup.2 of skin is important for the achievement of 
the protection against or alleviation of the injury to skin caused by 
diverse classes chemical irritants of the skin. This minimum amount of SOD 
also prevents or alleviates skin inflammation and acne. The SOD used for 
cosmetic or dermatologic formulation can be in the form of tissue or cell 
extracts containing preparations provided that the appropriate adjustments 
in SOD concentration are made. 
The method for alleviation or treatment of skin irritation, inflammation 
and acne is topical administration to the skin of a cosmetic or 
dermatologic formulation containing at least about 0.026 percent purified 
SOD with a specific activity of about 3800 CIU/mg protein relative to the 
total weight of the formulation. It appears that this percentage 
corresonding to about 1 CIU/cm.sup.2 of skin is the minimum effective 
amount which will protect against or alleviate many deleterious 
manifestations of the skin. In particular, the method of the invention can 
be used to protect from or alleviate the pathological effects to the skin 
which are caused by many chemical irritants. The method may also be used 
to prevent or alleviate inflammation of the skin caused by an 
indeterminant vector. Further, the method may be used to prevent or 
alleviate acne. 
DETAILED DESCRIPTION OF THE INVENTION 
This present invention is inter alia based upon the belief that chemical 
initiation of dermal injury, concomitant inflammatory responses and acne 
vulgaris are associated with the production of oxygen-derived metabolites 
which can be detoxified by topical application of superoxide dismutase. 
However, this belief is not meant to be limiting of the invention which is 
set forth in the text herein. 
Tissue injury, whether it be mechanical, chemical, toxic or thermal, 
produces a local reaction which is usually referred to as inflammation. In 
association with this, changes occur in the plasma and cells of the blood 
as part of a general reaction to injury. These changes affect the local 
reaction and are often concerned with defense mechanisms such as 
phagocytosis, hemostasis, and repair. 
Tissue injury may result from either the direct effects of the pathologic 
chemical agent or as a consequence of an inflammatory cell influx. In this 
regard, it has been found that superoxide dismutase can either prevent 
direct chemical injury as well as subsequent deleterious manifestations 
caused by inflammatory consequences. 
Accordingly, the present invention employing a topical formulation of SOD 
can function as a single biological agent for the protection of the skin 
against diverse classes of chemical irritants. These classes of chemical 
irritants include inorganic and organic peroxides, inorganic and organic 
acids including for example long chain fatty acids and acnegenic fatty 
acids, inorganic and organic bases, agents which produce free radicals 
including nitroso compounds, heterocyclic compounds organic peroxides and 
the like, chlorine, astringents, keratinizing agents, skin sloughing 
agents, para-aminobenzoic acid derivatives and the like. 
It is believed that superoxide plays a key role in the initiation and 
perpetuation of granulocyte-mediated inflammation. The mechanisms appear 
to involve the reaction of superoxide with a plasma protein to form a 
potent chemotactic factor responsible for the initial accumulation of 
granulocytes at the site of the developing lesion. Further, the ability of 
macrophages and neutrophils to injure cells and host tissues appears to be 
dependent upon the production of oxygen-derived free radicals and their 
metabolites and the ability of the target cells and tissues to detoxify 
the reactive species. The balance between the production and catabolism of 
oxidants by cells and tissues is important for the maintenance of their 
biologic integrity. In this regard, release of superoxide intermediates 
into extracellular fluids would be largely unopposed by SOD since SOD is 
ordinarily not present in such fluids. Thus, inflammation appears to be 
correlated with oxygen metabolite induced cellular injury. Superoxide 
dismutase plays an important role in the defense of superoxide mediated 
toxicity. Accordingly, it is believed that topical application of SOD can 
protect skin against direct chemical injury and subsequent deleterious 
inflammatory reactions associated with superoxide generating systems. 
Surface lipids of man contain appreciable amounts of free fatty acids 
formed by the action of bacterial lipases on the triglycerides of sebum. 
Since these free fatty acids are very irritating when injected 
intracutaneously, they are thought to be implicated in acnegenesis, and a 
major hypothesis of comedogenesis has evolved concerning their role. The 
free fatty acid hypothesis proposes that under the driving stimulation of 
androgenic hormones, human sebaceous glands enlarge with resulting 
increased production of sebaceous lipids. The major sebaceous lipid 
component, triglycerides is hydrolyzed in the sebaceous follicle by 
bacterial esterases and lipases. Certain free fatty acids of chain lengths 
between C.sub.12 and C.sub.18 act as irritants and/or comedogenic agents 
to damage the wall of the sebaceous follicle leading to subsequent 
follicular rupture and extrusion of keratinous debris, lipids and bacteria 
into the surrounding dermis producing the initial inflammatory events 
associated with clinical acne. Accordingly, the present invention protects 
the dermis against the changes induced by acnegenic fatty acids produced 
by bacterial lipolysis of the follicle content which induces the 
production of toxic oxygen intermediates. 
Based on these principles, the formulation of the present invention 
protects the skin against injury caused by chemical irritants, against 
inflammation and can substantially prevent or alleviate the untoward 
conditions of acne vulgarus and associated dermal abcesses such as boils, 
carbuncles, pustules and the like. 
According to the invention, the topical formulation can be prepared with 
common cosmetic, non-toxic, non-allergenic carriers for use in skin 
cremes, lotions, sprays, liquids, emulsions, cleansing preparations and 
the like. For the purposes of achieving protection from or alleviation of 
the irritation and inflammation of the skin by the foregoing classes of 
chemical irritants, and for achieving protection from, or alleviation of, 
dermal inflammation and acne the minimum concentration of SOD present in 
the topical formulation preferably should be at least about 0.026 weight 
percent of a SOD preparation with a specific activity of about 3800 CIU/mg 
protein relative to the total weight of the composition. It appears that 
protection against some classes of irritants is afforded below this 
concentration, but attenuation of injury induced by other chemicals is 
diminished. Thus when the minimum concentration of SOD is used in the 
formulation of the invention, skin wheal and reddening as well as more 
potent indications of dermal inflammation such as swelling, edema and 
leukocyte infiltration are lessened compared with the intensities of these 
pathological manifestations which appear in controls or when less than the 
minimum concentration of SOD is employed. 
A typical formulation prepared according to the present invention will 
contain from about 50 to 100% water, from about 0 to 20% organic polyol, 
from about 0 to 30% C.sub.12 to C.sub.18 fatty acid ester, from about 0 to 
30% of an oil or paraffin, from about 0 to 20% of a suitable ionic or 
nonionic emulsifier base and from at least about 0.026% SOD with a 
specific activity of about 3800 CIU/mg protein wherein the weight 
percentages are relative to the total weight of the composition. 
Additional ingredients may be added according to the understanding of 
those familiar with the cosmetic art in order to vary the texture, 
consistency, viscosity, appearance, weight, etc. of the formulation. Its 
physical character may be that of a semi-solid paste, gel, solution, 
emulsion, lotion, liquid, spray, creme and the like and these ingredients 
may be employed to produce such adjustment of the physical character. 
These additional ingredients include inter alia emulsifying agents such as 
nonionic ethoxylated and or nonethexylated surfactants, fatty alcohols, 
fatty acids, organic or inorganic bases, preserving agents, wax esters, 
steroid alcohols, triglyceride esters, phospholipids such as lecithin and 
cephalin, polyhydric alcohol esters, fatty alcohol ethers, hydrophilic 
lanolin derivatives, hydrophilic beeswax derivatives, hydrocarbon oils and 
such as palm oil, coconut oil, mineral oil, carnuba wax, cocoa butter 
waxes, silicone oils, pH balancers such as borax, gum thickeners such as 
acacia, tragacanth, guar, alginate and the like, cellulose derivatives 
such as methyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl- or 
hydroxypropyl-cellulose and the like, perfume and the like. 
Use of the formulation of the invention will generally be effective under 
the following conditions of application; however, the use may be varied on 
an individual basis in order to establish the most effective conditions 
for the individual. 
Generally, the formulation may be applied to the skin at least 5 to 10 
minutes before potential exposure to a chemical irritant. The application 
may be made over the entire area of the skin which could be exposed and 
the amount of formulation applied should be calculated to deliver at least 
about 1 CIU (CIU is defined as cytochrome inhibition unit), preferably at 
least about 2.5 CIU of SOD, and more preferably about 10 to 1000 CIU of 
SOD or its equivalent per square cm of skin. In this fashion, the minimum 
amount of SOD will be applied to the skin. 
Post exposure to irritant treatment will follow the same procedures. The 
course of control of inflammation and acne will be alleviated in this 
fashion but application also will be made concommitant with the onset of 
inflammatory symptoms. 
In a typical application, a skin creme, gel or lotion containing at least 
about 0.026 weight percent of SOD with a specific activity of about 3800 
CIU/mg protein can be applied at an area concentration of about 2 to 20 mg 
per square cm of skin. In practical terms, this area concentrations of 
creme, gel or lotion may be achieved by application of about 0.4 to 4 
drops per 10 sq cm on the basis that each drop is about 0.05 ml or 50 mg 
of formulation.

The invention will now be illustrated by the following examples which 
delineate some of the foregoing features of the invention. The examples 
are not meant as limiting, however. 
EXPERIMENTAL PROTOCOLS FOR THE EXAMPLES 
The following examples illustrate the protective effects of topical 
application to the skin of either purified superoxide dismutase 
concentrate or partially purified SOD isolated as extract from blood, 
liver or other suitable organ of animals, from microorganisms or suitable 
cell cultures. 
The used purified commercial preparation of SOD was assayed by its ability 
to inhibit the reduction of cytochrome C (cytochrome inhibition unit), 
hereafter CIU, pursuant to the method of McCord, J. M. and Fridovich, I., 
1968, J. Biol. Chem., 243:5753. Cytochrome c is readily reduced by 
superoxide and serves as a useful indicator. SOD acts to inhibit 
cytochrome c reduction because the enzyme catalyzes the dismutation of 
superoxide and competes with cytochrome c for the available superoxide 
generated by the xanthine, xanthine oxidase system. 
Superoxide dismutase in extracts was assayed by its ability to inhibit the 
solution autooxidation of an aqueous pyrogallol solution at pH 8 
(pyrogallol inhibition unit) hereafter PIU in which the superoxide anion 
radical acts as a chain propagating species. In such situations, SOD will, 
by scavenging the superoxide anion radical, shorten the reaction chains 
and so decrease the overall rate of that autooxidation of pyrogallol. This 
assay is used pursuant to the method of Marklund, S. and Marklund, G., 
1974, Eur. J. Biochem., 47:469. For these determinations, autooxidation 
activity was converted to cytochrome C inhibition units (1 CIU corresponds 
to about 2.5 PIU). 
For each experiment, 0.05 ml of either an aqueous solution of commercial 
SOD (purified) with an activity of about 5000 CIU/ml or of bovine SOD 
extract (semi-pure) with an activity of about 5000 PIU/ml was applied to 
the lower third of the shaven ear of either New Zealand White or French 
lop rabbits. The enzyme preparation was allowed to set for approximately 5 
minutes and 0.05 ml to 0.1 ml of the chemical irritant was added to the 
same area of approximately 1.5 cm=2.25 sq cm in size. After 5 minutes, 
0.05 ml of SOD solution or extract was reapplied to the same site. As a 
oontrol, the other ear was utilized substituting phosphate buffered saline 
(PBS) for SOD. 
Erythema was recorded two hours after application of the chemical irritant 
and scored as follows: 1+slight/minimal but definite redness; 2+moderate 
redness; 3+considerable redness; 4+intense redness; 5+maximal redness. 
Results are expressed as erythema initiated by irritant chemical in 
animals treated with superoxide dismutase and erythema initiated by same 
irritant chemical but treated with PBS as control. The protective index 
represents the diminution in erythema and is expressed as the difference 
between the scores of the erythematous reaction of the PBS-treated control 
side minus that of the SOD, treated side. For further verification, 
descriptions are presented of other pertinent inflammatory and pathologic 
manifestations which develop over a four day period. 
In the Examples 1-9 PSOD indicates purified, aqueous superoxide dismutase 
solution with an activity of about 5000 CIU/ml while SPSOD denotes 
semi-pure SOD tissue extract preparation with an activity of 5000 PIU/ml 
used for testing. All irritants were tested on at least five different 
rabbits and each experiment on any given animal was repeated four times. 
The data presented represent the average of these experiments. 
Experimentation with dose responses reveals that effective quantities as 
low as from about 1 CIU of SOD per sq. cm. of skin initiates dermal 
protection against chemical irritation. 
EXAMPLE 1 
______________________________________ 
Protective 
Irritant Chemical 
Experiment 
SPSOD/Control 
Index 
______________________________________ 
Sodium Lauryl 
1 2+/3+ 1 
Sulfate 20% (w/v) 
2 1+/2+ 1 
aqueous solution 
3 0/3+ 3 
4 3+/4+ 1 
5 0/2+ 2 
6 0/1+ 1 
7 1+/3+ 2 
______________________________________ 
After 30 minutes, erythema is markedly greater in the PBS control ear when 
contrasted with the SP SOD treated site. At 24 hours, the SPSOD ear 
displays minimal erythema (0-1+) with tiny scab-like formations around 
small follicles which cover approximately 1/5 the SPSOD application area. 
In four days, one occasionally obsreves a small raised scab covering 
approximately 1/10 the application diameter. In addition to erythema 
observed at 30 min in the control ear, there is considerable edema at 80 
min. Edema and erythema persist and continue to spread beyond the area of 
SPSOD application with the development of discoloration surrounding the 
follicles at this interval. After 24 hours, erythema remains while edema 
begins to subside. Brown scab-like formations around the follicles 
covering 75% of the application area become evident. After four days, 
raised scab formation persists over 75% of the area of application in the 
control ear. 
EXAMPLE 2 
______________________________________ 
Protective 
Irritant Chemical 
Experiment 
PSOD/Control Index 
______________________________________ 
Sodium Lauryl 
1 0/3+ 3 
Sulfate 20% (w/v) 
2 0/2+ 2 
aqueous solution 
3 2+/3+ 1 
4 3+/4+ 1 
5 0/2+ 2 
______________________________________ 
Thirty minutes after applicaion of PSODS, erythema was markedly greater in 
the PBS-control ears when contrasted with either the PSOD or SPSOD treated 
sites. At 24 hours, the SOD ears displayed minimal erythema with tiny 
scab-like formations around small follicles which covered approximately 
1/5 the SDS application area. After four days, one occasionally observed a 
small raised scab covering approximately 1/10 the application diameter. In 
addition to the erythema observed at 30 min in the control ears, some 
animals exhibited considerable edema at 80 min. Edema and erythema 
persisted and continued to spread beyond the area of PSOD application with 
the development of discoloration surrounding the follicles at this 
interval. After 24 hours, erythema remained while edema began to subside. 
In some rabbits, brown scab-like formations around the follicles covering 
75% of the application area became evident. After four days, raised scab 
formation persisted over 75% of the area of PSOD-application in the 
control ears. 
EXAMPLE 3 
______________________________________ 
Protective 
Irritant Chemical 
Experiment 
PSOD/Control Index 
______________________________________ 
Lauric Acid 1 2+/3+ 1 
50% (w/v) in 
2 1+/2+ 1 
n-propanol 3 1+/3+ 2 
4 2+/3+ 1 
5 1+/2+ 1 
6 3+/3+ 0 
7 4+/4+ 0 
8 1+/3+ 2 
9 0/2+ 2 
10 2+/2+ 0 
11 2+/2+ 1 
12 2+/4+ 2 
13 2+/4+ 2 
______________________________________ 
Experiments with lauric acid indicated on the PSOD-ear a minimal erythema 
after 10 minutes and a 2+ erythema after 30 minutes which subsided at 24 
hours. However, skin was scaling over approximately 1/3 the area of 
application with reddening surrounding folicles and visible under 
10.times. magnification. In contrast, the control ear revealed a 2+ 
erythema reaction covering twice the area of application already after ten 
minutes and after 30 minutes, the erythematous reaction was considerable 
(3+). Although erythema was minimal after 24 hours, skin was scaling over 
1/2 the area of application. Surrounding the region of scaling were large 
numbers of follicles with red scab-like formations. Additionally, in 
contrast to the PSOD treated ear, a darker and larger area of erythema was 
observed surrounding individual follicles. These observations are the 
expression for a much more severe inflammatory reaction of the control 
sites compared to the PSOD treated skin. 
EXAMPLE 4 
______________________________________ 
Protective 
Irritant Chemical 
Experiment 
SPSOD/Control 
Index 
______________________________________ 
Cinnamaldehyde 
1 2+/3+ 1 
0.1% (v/v) in 
2 2+/4+ 2 
methanol 3 2+/4+ 2 
4 2+/2+ 0 
______________________________________ 
The prodominant difference observed between the SOD ears and the 
PBS-treated control ears was the more extensive erythematous reaction in 
the latter which persisted to a larger extent than on the SPSOD-treated 
ears. 
EXAMPLE 5 
______________________________________ 
Protective 
Irritant Chemical 
Experiment 
SPSOD/Control 
Index 
______________________________________ 
Benzoyl Peroxide 
1 1+/2+ 1 
5% (w/v) in 2 3+/4+ 1 
ethyl ether 
______________________________________ 
EXAMPLE 6 
______________________________________ 
Protective 
Irritant Chemical 
Experiment 
PSOD/Control Index 
______________________________________ 
Benzoyl Peroxide 
1 3+/4+ 1 
5% (w/v) in 2 2+/4+ 1 
ethyl ether 
______________________________________ 
In SOD-(SPSOD) treated ears, the inflammatory response developed slowly 
with considerable erythema eveloping 75 and 100% of the application 
diameter after 24 and 48 hours respectively. In marked contrast, the 
PBS-treated control ears displayed intense redness at 24 hours with 
erythema spreading to twice the diameter of the application site at 48 
hours. 
EXAMPLE 7 
______________________________________ 
Protective 
Irritant Chemical 
Experiment 
PSOD/Control Index 
______________________________________ 
Sodium Hydroxide 
(aqueous solution) 
1% (w/v) 1 1+/2+ 1 
4% (w/v) 2 0/1 1 
5% (w/v) 3 3+/4+ 1 
______________________________________ 
EXAMPLE 8 
______________________________________ 
Protective 
Irritant Chemical 
Experiment 
SPSOD/Control 
Index 
______________________________________ 
Sodium Hydroxide 
(aqueous solution) 
1% (w/v) 1 4+/5+ 1 
4% (w/v) 2 2+/2+ 0 
5% (w/v) 3 2+/3+ 1 
______________________________________ 
In examples 7 and 8, erythematous reactions developed more rapidly and with 
greater intensity in the PBS-treated controls. Following application of 
sodium hydroxide, SOD-treated animals revealed slight edema 45 minutes 
after application with inflammatory manifestations subsiding at 90 
minutes. There was generally no visible reaction after 24 hours. In 
contrast, the PBS-treated controls showed a rapidly developing and 
spreading erythematous reaction extending to twice the application 
diameter after 30 minutes. There was more intensive edema in the area of 
application which progressively intensified at 90 minutes. After 24 hours 
moderate erythema, scab-like eruptions with occasional dermal necrosis 
surrounding the perimeter of the application site was observed with the 
controls. 
EXAMPLE 9 
______________________________________ 
Protective 
Irritant Chemical 
Experiment 
PSOD/Control Index 
______________________________________ 
Eugenol 1 1+/1+ 0 
75% (v/v) 2 1+/2+ 1 
in methanol 3 2+/4+ 2 
______________________________________ 
The predominant difference observed between the PSOD ears and the 
PBS-treated controls were the intensity and size of erythema. The controls 
generally revealed more erythema covering a larger area of the application 
diameter, than the PSOD-treated ears. 
Additional tests were performed in the same manner as described before, 
using formaldehyde solution, retinoic acid and squalene as chemical 
irritant which showed similar results of reduced erythematous reactions on 
those ears treated with SOD-preparations in comparison to the PBS-treated 
controls. 
For skin care products, SOD can be dispersed in non-toxic, non-allergenic 
topical cremes such as the cremes or lotions described below or their 
equivalents. 
EXAMPLE 10 
A Creme Formulation 
A protective skin care SOD formulation of the type of an oil in water (o/w) 
emulsion cream having the following composition can be prepared. 
3.0% of polyoxyethyleneglyceryl monostearate 
2.0% of glyceryl distearate 
3.0% of cetyl alcohol 
6.0% of stearic acid 
10.0% of isopropyl myristate 
5.0% of fatty acid triglyceride ("Miglycol" 812) 
0.2% of p-hydroxybenzoic acid ester as preserving agent ("Nipagin") 
2.0% of glycerol 
2.0% of propylene glycol 
0.3% of preserving agent ("Germall" 115R) 
5.0% of SOD-containing tissue extract with 20,000 PIU per ml 
61.5% of demineralized water 
EXAMPLE 11 
A Lotion Formulation 
A skin care lotion of the o/w type with addition of SOD-concentrate can be 
prepared in the following fashion 
3.0% of polyoxyethyleneglyceryl monostearate 
2.0% of sorbitan fatty acid ester 
2.0% of cetyl alcohol 
8.0% of isopropyl myristate 
0.2% of p-hydroxybenzoic acid ester as preserving agent ("Nipagin") 
2.0% of propylene glycol 
0.3% of preserving agent ("Germall" 115R) 
78.2% of demineralized water 
3.0% of SOD-concentrate with 1,000,000 PIU/ml 0.5% of perfume. 
EXAMPLE 12 
A Gel 
A fat and oil free skin care gel with addition of a SOD containing tissue 
extract can be prepared as follows. 
1.4% w/w of Na-carboxymethyl cellulose as thickener 
3.0% w/w of propyleneglycol 
20.0% w/w of SOD-tissue extract with 20,000 PIU/ml 
0.2% w/w of p-hydroxybenzoic acid as preservative 
0.5% w/w of benzyl alcohol as preservative 
74.9% w/w of water 
EXAMPLE 13 
A Solution 
An aqueous, fat and oil free skin care solution with addition of a SOD 
containing cell extract can be prepared as follows. 
71.7% w/w of demineralized water 
10.0% w/w of propyleneglycol 
0.3% w/w of preserving agent ("Phenonip" R) 
0.5% w/w of Na-carboxymethyl cellulose 
2.5% w/w of Na-citrate as buffer substance 
10.0% w/w of native, soluble collagen as moisturizer 
5.0% w/w of SOD-cell extract with 100,000 PIU/ml.