United States Patent: 4,296,104


( 11 of 13 )

United States Patent 4,296,104
Herschler October 20, 1981




Therapeutic dimethyl sulfoxide composition and methods of use


Abstract


The use of urea and/or ethanol with pharmaceutical compositions containing DMSO
is disclosed. The result is a reduction in the undesirable side-effects normally
associated with the application of DMSO compositions, enhancement of the desired
physiological effects produced by DMSO compositions, and other benefits.
Specific compositions contain DMSO with urea and/or alcohol and may also contain
NaCl, KCl and/or acetamide. The use of DMSO to reduce the mortality rate of fish
treated by hyperosmotic delivery is also disclosed.


Inventors: Herschler; Robert J. (3080 NW. 8th St., Camas, WA 98607)
Appl. No.: 071072
Filed: August 30, 1979

Current U.S. Class:424/679; 424/680; 514/588
Intern'l Class: A61K 031/10; A61K 031/17; A61K 033/14
Field of Search: 424/322,153,337




References Cited [Referenced By]



U.S. Patent Documents
3334012Aug., 1967Herschler424/337.
3361555Jan., 1968Herschler71/103.
3499961Mar., 1970Dobson et al.424/68.
3549770Dec., 1970Herschler424/337.
3549771Dec., 1970Herschler424/337.
3551554Dec., 1970Herschler424/7.
3558434Jan., 1971Herschler195/81.
3711602Jan., 1973Herschler424/45.
3711606Jan., 1973Herschler424/45.
3740420Jan., 1973Herschler424/45.
3743727Jul., 1973Herschler424/181.
4112946Sep., 1978Herschler128/253.
4177267Dec., 1979Herschler424/238.



Other References
Mallach, 141, Annals New York Academy of Sciences, pp. 457-462.

Primary Examiner: Friedman; Stanley J.
Attorney, Agent or Firm: Blakely, Sokoloff, Taylor & Zafman



Claims




I claim:

1. In a method for administrating dimethyl sulfoxide to a host, the improvement
comprising reducing the side effects of malodorous breath and skin reactions
normally associated with such dimethyl sulfoxide administration, comprising the
step of:

administrating to said host a composition comprising dimethyl sulfoxide and a
side-effect reducing agent taken from the group consisting of urea, or ethanol
or mixtures thereof in an amount which is effective for said purpose.

2. A method according to claim 1 wherein said composition further includes
acetamide.

3. The method according to claim 1 wherein said composition further includes
sodium chloride.

4. The method according to claim 1 wherein said composition further includes
potassium chloride.

5. The method according to claim 1 wherein said composition further includes
water.

6. The method according to claim 1 wherein the weight ratio of urea to dimethyl
sulfoxide is greater than 1:99.

7. The method according to claim 1 wherein the ratio of urea to dimethyl
sulfoxide is greater than 1:9.

8. The method according to claim 1 wherein said composition has the following
formulation:


______________________________________
dimethyl sulfoxide
about 19 grams;
urea about 9 grams;
sodium chloride about 1 gram; and
water at least about 6 grams;
______________________________________


whereby the aforementioned composition formulation is provided with the weight
ratios noted.


Description




BACKGROUND OF THE INVENTION

The present invention relates to pharmaceutical compositions containing DMSO for
adminstration to human or other animal subjects. More specifically, it relates
to new DMSO formulations containing substances which enhance the effectiveness
of DMSO, reduce undesirable side-effects sometimes created by the use of DMSO
and make DMSO compositions more appealing to users.

Dimethyl sulfoxide (DMSO) is a versatile substance that has numerous
pharmaceutical and nonpharmaceutical uses. It is widely used throughout the
world for treating humans and other animal subjects.

As Described in U.S. Pat. No. 3,549,770, No. 3,740,420, and No. 3,790,682,
incorporated herein by the reference, DMSO is an active agent in relieving the
signs and symptoms of numerous body disorders, including accelerating the
healing of certain injured body tissues and in relieving the signs and symptoms
of anxiety.

U.S. Pat. No. 3,551,554, No. 3,711,606 and No. 3,743,727, incorporated herein by
reference, describe how DMSO is effective to enhance tissue penetration of other
substances, especially other physiologically active agents. DMSO can thus be
added to a variety of pharmaceutical compositions to accelerate assimilation
into body tissue. In some instances this means that smaller doses can be
administered when DMSO is used.

Yet, despite their many benefits, DMSO compositions are sometimes passed over in
favor of other pharmaceutical compositions even in instances where DMSO would be
the most effective pharmaceutical agent. This is because many subjects suffer
from one or more side-effects when treated with DMSO. In some cases, the
side-effects are so pronounced that subject or physician will forego the use of
DMSO in favor of a less effective therapeutic agent.

A variety of undesirable side-effects have been observed to result from
administration of DMSO. The most frequently occurring are adverse skin
reactions, malodorous breath and foul taste.

The adverse reactions caused by DMSO are well documented. At page 356 of the
standard reference Contact Dermatitis by Alexander A. Fisher, M.D. (2nd Ed.,
1973), dimethyl sulfoxide (DMSO) is listed as a primary urticariogen. Volume 141
of Annals New York Academy of Sciences includes several articles describing the
undesirable side-effects attributed to DMSO. These include articles by Goldman,
et al. at pages 429, 433-35; Sulzberger, et al. at pages 439-40; Brown at pages
500-501; and several others.

The magnitude of the malodorous breath problem is so large that, in some
instances, hospitals have had to isolate wards where DMSO is administered from
the central air conditioning system. Skin irritations from topically applied
DMSO have been so great that a substantial number of patients refuse treatment.

Another, potentially more serious side-effect is sometimes observed when DMSO is
administered intravenously. This is red blood cell lysis. Intravenous
administration of DMSO is crucial if the substance is to be used for treating
the brain and spinal cord, for cancer therapy or to treat organ hypoxia, heart
attack and other internal conditions. Large intravenous doses of DMSO can have a
therapeutic effect for such purposes but red blood cell lysis which results from
intravenous DMSO administration can be injurious or even fatal to the subject.

Furthermore, it is generally advisable to minimize the dosage of any
pharmaceutical substance administered to a human or other animal subject to the
smallest effective amount. Although DMSO is one of the most penetrating of
pharmaceutical substances and is known to be effective in minute doses, it would
be desirable to further reduce the minimum dosage of DMSO needed to achieve a
desired physiological effect.

A related problem is observed when fish are treated with hyperosmotic
concentrations of a membrane permeability altering agent as discussed in U.S.
Pat. No. 4,112,946, incorporated herein by reference. Such agents include urea,
NaCl and acetamide.

These substances, with the possible exception of acetamide, are substantially
nontoxic when applied externally to mammalian subjects. But, when incorporated
in a solution in contact with an epithelial membrane of fish, such substances
can be lethal at moderate concentrations (as low as about 3 weight percent).

To optimize delivery of therapeutic agents to fish, solutions containing greater
than 3 weight percent of the above listed solutes are required. Fish mortality
is thus a substantial barrier to the effective use of hyperosmotic treatments.

SUMMARY OF THE INVENTION

Specific DMSO compositions and methods of application have now been discovered.
Use of such compositions expands the acceptability of DMSO by eliminating or
reducing undesirable side-effects. And, the new DMSO compositions and methods of
use are observed to provide new therapeutic effects and beneficial uses.

More specifically, it is found that when DMSO and urea are both administered to
epithelial regions of a human or other animal subject, expected adverse skin
reactions, malodorous breath and foul taste are substantially reduced. In most
cases they are entirely eliminated.

Furthermore, urea is discovered to potentiate DMSO in certain instances. It
appears that a DMSO composition containing urea and/or ethanol is more rapidly
absorbed into tissue than are similar compositions containing no urea or
ethanol. If the DMSO composition includes another pharmaceutical substance, such
other substance is also absorbed more rapidly when urea and/or ethanol are
present. The presence of NaCl, KCl and/or acetamide reduces discomfort resulting
from topical application of DMSO compositions and appears to further enhance
penetration.

It has also been discovered that certain DMSO formulations, which contain urea,
are effective in treating diseased finger and toe nails, for softening cuticle
to be removed from finger and toe nails, and for softening epidermal thickenings
to ease removal.

When DMSO is administered intravenously with ethanol, red blood cell lysis is
reduced and continues to decline with repeated administrations.

When DMSO is added to solutions containing urea, NaCl, and/or acetamide, such
solutions are less toxic to fish than similar solutions without DMSO.

It is therefore an object of this invention to provide pharmaceutical
compositions and methods to allow the application of DMSO to a human or other
animal subject without creating adverse skin reactions, malodorous breath or
foul taste.

A further object is to provide compositions and methods of application whereby
the lysis of red blood cells, resulting from intravenous administration of DMSO,
is reduced.

A further object is to provide compositions and methods of treatments for
diseases of the fingers and/or toe nails.

Another object is to provide formulations and methods of application to enhance
penetration of DMSO and DMSO based pharmaceutical compositions into animal
tissue.

An additional object is to provide safe and effective skin softening agents
which can be used to soften cuticle for removal from finger and/or toe nails and
for softening epidermal thickenings to facilitate removal.

Yet a further object is to provide means for protecting fish from the toxicity
of solutions used for hyperosmotic treatment of fish.

These and other objects, advantages and features of the present invention will
be apparent from the following detailed description.

DESCRIPTION OF PREFERRED EMBODIMENTS

The side-effects which have long hindered the use of DMSO as a therapeutic agent
are quite surprisingly eliminated when urea is administered with formulations
containing DMSO. Even small amounts of urea are beneficial in reducing histamine
release, burning and itching, localized dermatitis, drying, cracking and
blistering of skin, upleasant breath odor, after-taste, headache and nausea in
subjects receiving dermally administered DMSO compositions.

Urea is an especially excellent inhibitor of the side-effects, because it is a
naturally occurring substance in human and other animal subjects. Urea is
substantially non-toxic and animal subjects are quite tolerant to its presence.
It thus can be used with almost any pharmaceutical composition containing DMSO,
without fear of toxic effects.

The reasons why urea is effective from reducing and eliminating the above listed
undesired side-effects is not fully understood. It appears, however, that the
urea inhibits or prevents the production of undesirable DMSO metabolites.

In the case of the breath odor problem, it is known that dimethyl sulfide is a
minor metabolite of DMSO and that it is expired through the lungs causing
malodorous breath and foul taste. Urea possibly prevents the breakdown of DMSO
to dimethyl sulfide. Supporting laboratory experiments show that DMSO will
decompose to dimethyl sulfide when heated in a test tube. But, when urea is
added and the experiment repeated, no dimethyl sulfide is detected.

Similarly, the adverse skin reaction, such as histamine wheal and flare may be
the result of an attack by the dimethyl sulfide molecule against mast cells in
the subcutaneous layer of skin to which DMSO is applied topically. Urea's
blocking of DMSO breakdown to dimethyl sulfide would thus account for the
observed antiurticariogenic effectiveness in reducing adverse skin reactions
caused by DMSO.

The enhanced penetration of DMSO solutions containing urea is possibly related
to the fact that urea has a weight per volume percentage concentration at
isotonicity less than that of DMSO and most other pharmaceutical agents so that,
at a given weight percent of solute, compositions containing urea in combination
with DMSO, have an enhanced penetrating ability. The effect is even greater if
the amount of solute is increased by addition of urea to the DMSO composition.

It appears that the only suitable substances for enhancing the penetration of
DMSO compositions are substances having a weight per volume percentage
concentration at isotonicity less than that of DMSO, but only a few of such
substances produce the desired increase in penetration.

Further criteria of substances which enhance DMSO penetration include water
solubility at ambient temperatures and a molecular weight no greater than 78.
Substances which tend to polymerize are unsuitable. If the substance is to be
used with DMSO-urea compositions applied to living animals, it must also be
relatively non-toxic to the intended animal subject.

Specific substances of value in promoting penetration are found to include urea,
NaCl, KCl, ethanol and acetamide. Of these substances urea is the most
effective. It appears to act by "opening" membranes to allow increased
penetration.

The fact that urea, NaCl, KCl, acetamide and/or ethanol may be combined to
enhance DMSO penetration is important in several respects. Although urea and
ethanol reduce or eliminate the side-effects created by the administration of
DMSO, some subjects will continue to have at least mild reactions to the
administration of DMSO. By administering urea, NaCl, KCl, acetamide and/or
ethanol, it should be possible to reduce the amount of DMSO administered as an
active therapeutic agent since greater tissue penetration of the applied DMSO
can be obtained. The ability to reduce the amount of DMSO administered, without
reducing the amount which is absorbed, should further diminish adverse
side-effects of the DMSO.

A similar benefit results when urea, NaCl, KCl, acetamide and/or ethanol are
added to compositions which contain DMSO as a carrier and penetration enhancer
for other pharmaceutical substances such as antineoplastic agents, analgesics,
anti-inflammatory agents, anticoagulants, vasodilators, anti-microbial agents,
ultra-violet screening agents, diagnostic dyes, diagnostic radiopaque agents,
dietary supplements, nutrients, physiologically active steroids and protein
modifying agents.

Protein modifying agents include those substances which modify collagen and,
possibly, other substances found in connective tissue such as hyaluronic acid,
elastin, and fibrinogen. One such protein modifying agent is
methylsulfonylmethane (MSM) as described in my simultaneously filed U.S. patent
application entitled Preparations Containing Methylsulfonylmethane and Methods
of Use and Purification.

DMSO is a known penetration enhancer for chemical agents having a molecular
weight less than about 8,000, administered to intact body membranes. It is also
found to enhance the penetration of higher molecular weight substances, such as
enzymes, that are administered to stressed membranes including membranes that
are inflamed, are scarified or have been subjected to severe osmotic stress. In
both situations, the inclusion of urea, NaCl, KCl, ethanol and/or acetamide can
further assist the penetration of DMSO and the pharmaceutical agent. Increased
penetration can improve pharmaceutical effectiveness and, in some instances,
make it possible to reduce the dosage administered.

Penetration enhancing substances, such as urea, are particularly useful in DMSO
solutions containing diagnostic dyes. In the staining of cells for diagnostic
examination, it is desirable to use the least possible amount of dye and other
foreign chemicals to minimize alterations in the cells being dyed. the enhanced
cell penetration that results from the addition of urea, makes it possible to
reduce the amount of dye and DMSO in staining compositions. The urea added has
no adverse effect on the structure of cells to be dyed.

An additional benefit discovered is that human subjects treated with the
previously described compositions preferred those compositions containing a
salt, most notably NaCl, over similar compositions without salt. Subjects
interviewed indicated that topically applied DMSO compositions containing salt
are more comfortable.

Urea can be administered with DMSO to produce the same physiological effects
attributed to DMSO compositions administered without urea. For example, urea can
be administered with DMSO to an area of tissue inflammation in an amount
effective for relieving signs and symptoms of inflammation, to a subject
suffering from pain in an amount effective to relieve pain, to a subject
suffering from abnormal muscle contractions in an amount effective to promote
muscle relaxation, to a subject suffering from symptoms of vascular
insufficiency in the blood and lymph circulatory system in an amount effective
to relieve symptoms of vascular insufficiency.

Treatment with effective amounts of DMSO and urea can also relieve signs and
symptoms of a burn, can promote healing of a skin graft area following a
transplant, and can relieve signs and symptoms of respiratory distress. When
DMSO is given with urea to subjects having joints with arthritic signs and
symptoms in an amount effective to relieve signs and symptoms of arthritis, to
subjects suffering from tissue damage in an amount effective to promote the
repair of tissue damage, or to mammalian subjects suffering from signs and
symptoms of anxiety in an amount effective to relieve signs and symptoms of
anxiety, improvement in subjects' conditions are observed.

It has also been found that certain compositions, containing both DMSO and urea,
have medical benefits not produced by compositions containing DMSO or urea
alone. As one example, it is found that DMSO, administered with urea, will
repair or remove abnormal, dead, or diseased tissue. DMSO-urea compositions can
thus be used to treat interstitial cystitis or connective tissue diseases such
as progressive systemic sclerosis.

DMSO-urea, compositions can also be administered to benefit diseased finger
and/or toe nails of human or other animal subjects. Administering DMSO and urea
to the diseased portion of a nail will soften the diseased portion. After
several days of treatment, the diseased portion of the nail can be removed
painlessly by gentle urging using forceps. In most instances, the undiseased
portion of the nail is not adversely affected by treatment with a DMSO-urea
composition.

When used to treat diseased or damaged tissue, DMSO-urea compositions are most
effective when applied at a temperature above 37.degree. C., preferably as hot
as the subject will tolerate. Warm applications are especially beneficial when
treating musculoskeletal disorders such as arthritis, sprains, strains, soft
tissue injury and the like.

DMSO-urea compositions are also well suited for use with physical therapy
techniques, particularly the use of energy such as ultra sound, in treating
musculoskeletal disorders.

As illustrated by several examples below, DMSO-urea compositions soften and
moisturize the skin of subjects receiving dermal applications. Certain DMSO-urea
compositions accordingly make excellent cosmetic skin softening lotions or gels.
Also DMSO-urea compositions are excellent as vehicles for other skin treating
cosmetic agents. When the phrase "pharmaceutical compositions" is used herein,
it thus includes cosmetic preparations.

Urea is known to have some beneficial effect on skin, but in most standard
cosmetic compositions it "washes off". When DMSO and urea are used together,
skin permeation of each is enhanced so the skin-softening benefits of urea are
increased and sustained even after washings.

As will be described below, the use of ethanol with DMSO in intravenous
administrations proved to be independently effective in reducing malodorous
breath and red blood cell lysis.

Formulations

As with any multi-purpose pharmaceutical composition, some experimentation is
necessary to determine the optimum dosage of DMSO and urea to be applied for a
particular purpose. For example, when it is a goal to reduce a side-effect
produced by the application of DMSO, the amount of urea used should be an amount
effective to obtain the desired reduction. Likewise, if the goal is to enhance
penetration, the amount of urea or other penetration enhancing substance should
be an amount sufficient to enhance penetration.

As described in the prior patents listed above, DMSO compositions for topical
application should contain at least 10 weight percent DMSO to have any
beneficial effect. Compositions for clinical use should have at least about 40
weight percent DMSO; and for greatest effect, a composition should contain at
least about 50 weight percent DMSO. To be effective in reducing DMSO-induced
side-effects and/or to enhance the penetration of DMSO compositions, urea should
be present in a weight ratio to DMSO of greater than 1:99. Most significant
results are achieved when the weight ratio of urea to DMSO is greater than 1:9.
Normally, DMSO-urea compositions should contain no more than about 60 weight
percent urea since larger concentrations could only be obtained at the cost of
reduced effectiveness due to diminished DMSO amounts.

In special circumstances the desired DMSO concentration for a pharmaceutical
composition might be substantially below 10 weight percent or above 50 weight
percent. The above urea ratios will still apply in such circumstances. For
example, if a low concentration (3 to 4 weight percent) DMSO composition is
prepared for application to the eye, a suitable urea amount would be one weight
percent.

Compositions containing one weight percent or less of DMSO are effective for
treating membranes that have been stressed, e.g. membranes that are inflamed,
are scarified or have been submitted to severe osmotic stress. In such
instances, the abovestated DMSO-urea ratios would still apply.

Pharmaceutical compositions for treating the skin, oral cavity and rectal
epithelium may contain DMSO and urea as their sole components. In any such
composition, the weight ratio of DMSO to urea should not be less than about
100:35. Even at this ratio, the solution is best applied while heated to prevent
precipitation of the urea.

When NaCl, KCl, ethanol and/or acetamide are present in DMSO-urea compositions,
to effectively increase penetration or comfort, they should be present in a
certain minimum amount. The combined weight of these agents, in ratio to the
weight of urea present in the composition, should be greater than 1:99.
Significant benefits are observed when the ratio is greater than 1:9.

The most effective formulations, at least for topical application, includes
about six grams of urea, one gram of NaCl and at least six grams of water for
every nineteen grams of DMSO. An aqueous medium is usually required for any
DMSO-urea composition containing NaCl or KCl.

When included in DMSO compositions, urea, NaCl, and KCl may complex with DMSO
molecules. As a result, such DMSO compositions may include DMSO-urea complexes,
DMSO-salt complexes and/or DMSO-urea-salt complexes of various types. The exact
effect that complexing has on the activity of the compositions containing DMSO
and urea is not known. It is believed, however, that compositions containing the
complexes listed above are effective in obtaining desirable reductions in
side-effects and penetration increases

When DMSO is used in the same composition with urea for the removal of cuticle,
for treating diseased finger and toe nails, or for the remover of epidermal
thickenings the composition should include a hydrophobic ointment base and at
least 10 weight percent each of DMSO and urea to be effective. It is also
advantageous to apply a salt such as NaCl or Na.sub.2 S. Use of such a salt
increases the rate at which the nail or thickened skin are softened for removal.
Specifically it appears that DMSO-urea compositions containing NaCl and/or
Na.sub.2 S penetrate more deeply into the area between the nail and plate than
do compositions without such a salt component.

Such solutions for treating diseased nails should include between 0.25 and 10
weight percent of a salt selected from NaCl, Na.sub.2 S and mixtures thereof,
along with effective amounts of urea and DMSO. Less than 5 weight percent salt
is found to be fully adequate in most instances.

To facilitate topical applications, any of the above compositions may include a
pharmaceutically acceptable thickening agent to increase the viscosity of a
composition. Such thickeners may be used to form creams, lotions, gels, pastes,
ointments and suppositories.

Methods of Application

Urea may be administered with DMSO compositions by any route previously known
for DMSO administration. The most dramatic reductions in side-effects are
observed when urea is added to DMSO compositions for topical application.
Subjects using topically applied DMSO suffer from substantially less malodorous
breath, foul taste, and adverse skin reaction when urea is used along with the
DMSO.

Topical applications of DMSO and urea may be by any standard technique. They may
be painted or spread on and allowed to dry or applied with saturated pads.

One advantageous method for treating limbs or digits is to place an appropriate
DMSO-urea liquid composition in a plastic bag and insert the limb so that the
bag forms an overwrap. Heat can be applied to the exterior of the bag to
accelerate treatment.

Similary, spinal injuries can be treated by saturating a fabric with a DMSO-urea
solution and then spreading the fabric along the spinal column. The fabric may
be covered with a nonporous plastic sheet and hot water bottles applied to speed
penetration of the DMSO-urea solution.

In most instances, it is preferred that DMSO and urea be combined in a common
composition for administration together or otherwise be administered
concurrently. In the specific case of topically applied DMSO composition, some
reduction in side-effects is also observed if patients are treated with DMSO
without urea after a preceeding treatment at the same site, with both DMSO and
urea. For topical administrations at least, it is thus possible to use treatment
regimens such as alternating applications of DMSO compositions with and without
urea.

If a DMSO composition, for topical application, includes substances which would
react adversely or be deactivated when combined with urea or would react with
urea to form macromolecules which would retard tissue penetration, a urea
composition can first be applied to the treatment site and allowed to dry. The
DMSO composition could thereafter be applied at the same site with less chance
that the urea-sensitive substances would be adversely affected. As desired NaCl,
KCl, ethanol and/or acetamide can be included in the urea composition or, if
compatible, in the DMSO composition.

As a penetration enhancing agent, urea can be administered with DMSO by topical
administration, intravenous administration, subepidermal injection or oral
ingestion. It can also be administered intrathecally, intravesically, rectally,
or by instillation into eye, ear, nose or abnormal sinuses of the body.

The following examples describe a few of many clinical tests which show that the
presence of urea can benefit pharmaceutical compositions containing DMSO. In
each of the listed examples, DMSO and urea were applied in a common composition.


As previously mentioned, alternating applications of a DMSO composition and a
urea composition may be advisable under some circumstances. For example, in the
unusual circumstance that a preferrred DMSO composition contains a chemical
agent that is unstable in the presence of urea, it would be preferable to
alternate applications of the DMSO composition and a urea composition or to mix
the two compositions immediately before administration. Sequential applications
of DMSO and urea compositions or mixing immediately before application might
also be preferred if the separate DMSO and urea compositions have a
substantially longer shelf life than some combined formulation. Whether to
combine urea with a particular DMSO composition is best determined by
experimentation.

1. Cutaneous Administrations

a. dermal administrations

EXAMPLE 1


______________________________________
Wt. Percent
______________________________________
DMSO 61.7
water 26.5
urea 8.8
NaCl 3.0
______________________________________


A control solution containing 62 wt. percent DMSO in water was also prepared.
Both formulations were dermally applied to each of five human subjects known to
be sensitive to compositions containing DMSO at a concentration of 50 wt.
percent.

In a first test, the two solutions were topically applied concurrently but at
different sites on skin of the subjects. Each subject complained of itching and
burning at sites where the control solution was administered. And each had bad
breath odor. At sites where the solution containing urea was applied, there was
no discomfort or adverse skin reaction, even when the solution was applied to
particularly sensitive areas of the neck, below the chin.

Several days later, the same subjects were treated with only the solution
containing urea. There was no discomfort or adverse skin reaction. Neither bad
breath nor foul taste were observed. Each of the subjects volunteered that their
skin was softened when the compositions containing urea was applied.

After several more days, the subjects were again treated with the control
solution. The subjects experienced a return of malodorous breath, foul taste,
and adverse skin reactions.

EXAMPLE 2

A test was conducted to determine whether it would be advantageous to use DMSO
compositions containing relatively large amounts of pharmaceutically acceptable
agents having a weight per volume percentage concentration at isotonicity less
than that of DMSO. Urea is such an agent. So, for comparison purposes, the
composition of Example 1 was tested against the following composition which
contains more urea and less water:


______________________________________
Wt. Percent
______________________________________
DMSO 61.7
water 17.6
urea 17.6
NaCl 3.0
______________________________________


Human subjects, suffering from a variety of ailments treatable with DMSO
compositions, received each of the two formulations at separate times during the
course of treatment. In every instance, the subject expressed a preference for
the reformulated composition containing a greater amount of urea.

Specific benefits observed include prompter medicinal response and increased
comfort (less tissue irritation). Effected skin area, of subjects suffering from
scleroderma were softened when the high urea comcentration composition was
applied topically. Such subjects receive no substantial relief when urea
compositions without DMSO are applied.

EXAMPLE 3

The following composition was used to treat subjects suffering from contact
dermatitis, from poison oak, insect bites and other conditions characterized by
subdermal histamine release and consequent discomfort:


______________________________________
Wt. Percent
______________________________________
DMSO 47.6
water 33.3
NaCl 4.8
urea 14.3
______________________________________


Topical applications of this composition provided excellent results. Irritations
and itching of the skin stopped promptly and were followed by rapid healing in
all cases. Minor cuts were also treated with the above solution; and infection
free healing resulted in each case.

EXAMPLE 4

DMSO compositions have been used successfully to treat a variety of athletic
injuries such as sprains, muscle cramps, and the like. In this example, the
following formulation was used:


______________________________________
DMSO 200 g.
urea 100 g.
ethanol (absolute) 50 g.
water 25 g.
methyl salicylate
(as odorant) 10 g.
3% Carbopol 934
(carbomer-934) 80 g.
triethanolamine 2 g.
______________________________________


Topical application of this composition has proved effective in test treatments
for sprains, muscle cramps and other discomforts. Athletes receiving the above
composition had no abnormal irritations.

One subject having very fair complexion and red hair was unable to tolerate a 50
wt. percent aqueous DMSO composition without urea. When a gel containing the
above listed ingredients was applied to the skin of the subject, good medical
relief was observed. Furthermore, there was no indication of the undesirable
side-effects previously experienced from the application of DMSO without urea.

EXAMPLE 5

A methyl salicylate ointment was prepared for use for treating human subjects
suffering from whiplash. The ointment included urea and ethanol to enhance
penetration of methyl salicylate and DMSO. Specifically, it included:


______________________________________
DMSO 200 g.
urea 100 g.
water 100 g.
ethanol 50 g.
methyl salicylate 10 g.
Carbopol 940
(carbomer-940) 2.4 g.
triethanolamine 2 g.
______________________________________


This ointment was applied to the necks of whiplash victims who previously had
experienced discomfort upon treatment with other DMSO substances. Methyl
salicylate in the ointment penetrated extremely rapidly such that the subjects
reported immediate relief from pain. The ointment was soothing when applied
topically to low sensitivity skin areas and no malodorous breath or foul taste
resulted.

EXAMPLE 6

The paste of Example 13 was used to impregnate polyolefin foam pads. The
impregnated pads were applied to unpared calluses and corns on the foot of a
subject. After daily treatments for four to eight days, the epidermal
thickenings were sufficiently softened for easy removal.

EXAMPLE 7

Persons with more severe epidermal thickenings were treated according to the
procedure of Example 13 using the composition of Example 6. Good success in the
softening of tissue for removal was achieved.

EXAMPLE 8

The DMSO-urea composition of Example 1 was administered to patients suffering
from a hardening of the skin as a result of incurable, progressive systemic
sclerosis. Previously, these patients had each been treated for their condition
with solutions of DMSO and water without urea, over a period of years.

After treatment with the DMSO-urea composition of Example 1, each of the
patients reported a strong preference for that composition. Specifically, they
reported relief from bad breath and a soothing effect upon application. Most
significantly, skin of the subjects was softer and more flexible after several
weeks of administration.

Also, blood circulation of the skin was improved. Systemic sclerosis typically
impairs blood circulation to the skin as evidenced by a minimum of color change
when finger pressure is applied to the effected area. After the patients were
treated with the DMSO-urea composition of Example 1, once daily over the entire
body for several weeks, vascular blanching of the skin was observed when finger
pressure was applied. Blushing occurred upon release.

EXAMPLE 9

The vascular effect of DMSO compositions containing urea was observed in the
dramatic recovery of one subject with ischemic ulceration of a finger. At the
beginning of treatment, the finger was cyanotic, ulceration was progressing, and
surgical amputation had been recommended.

In an attempt to improve this condition, the finger was treated by dipping in an
aqueous DMSO solution. But the treatments were soon abandoned because the pain
of treatment was too great.

In a second attempt at treatment, the finger was dipped in the following
preparation over a period of several weeks:


______________________________________
Wt. Percent
______________________________________
DMSO 50
water 35
urea 10
NaCl 5
______________________________________


Twenty-five to fifty milligrams of indomethacin were added to each 500 grams of
this solution to serve as an analgesic and anti-inflammatory agent.

After about 12 weeks of treatment, there was apparently full recovery of the
finger with ulcer healing and excellent general appearance of the entire finger.
At the end of the period amputation was counter-indicated; and treatment was
stopped except for occasional applications to relieve discomfort.

EXAMPLE 10

DMSO and urea may be used effectively together, even at very low concentrations,
when applied to animal membranes which have been stressed.

In one laboratory experiment hair was removed from both flanks of rabbits of
about three kilogram body weight. The flanks were then blade shaved to fully
expose the epidermis. Under anesthesia, the both flanks of five rabbits were
severely scarified using a scalpel to expose subcutaneous tissues.

The right flank of each rabbit was kept continuously moistened with a 0.75
weight percent aqueous solution of DMSO containing 1.0 mg. of superoxide
dismutase enzyme (m.w. 30,000-40,000) per 10 ml. of solution. The left flanks
were treated identically except that the solution applied was free of DMSO.

It was observed that healing was greatly accelerated on the right flanks as
compared to the left. Specifically, strong tension resistant healing of the
right flanks occurred in about half the time.

The addition of urea to such low concentrated DMSO compositions can further
improve healing rate, enhance enzyme penetration and eliminate side effects
resulting from DMSO application. When applied to stressed tissue, not more than
about 1.0 weight percent urea is required. For use in preparing the DMSO
formulation of this example, it would be sufficient for the aqueous solution to
contain about 0.75 weight percent urea.

EXAMPLE 11

Compositions containing methylsulfonylmetane (MSM) and urea improve the softness
and pliability of skin even of persons suffering from adverse skin conditions.
In one test, two human subjects suffering from "hide bound disease" or
progressive systemic sclerosis were treated with a solution containing 20 weight
percent MSM, 20 weight percent urea, 30 weight percent dimethyl sulfoxide and 30
weight percent water.

The subjects were treated by placing 15 milliliters of the solution in a plastic
bag, placing a hand or foot to be treated in the bag. The hand or foot with
plastic bag overwrap was then immersed in a heated water bath maintained at a
temperature as warm as the subject would tolerate, taking care not to dilute the
solution.

The hands and feet were thus immersed for 30 minutes, three times daily, for a
period of two weeks. The result was a reduction in discomfort and increased skin
softness and pliancy.

Dimethyl sulfoxide and urea in the solution enhanced penetration of MSM into the
effected tissue. No adverse side-effects resulted from administration of the
DMSO.

EXAMPLE 12

DMSO is a useful substance in many dermally applied cosmetic preparations. But,
cosmetics containing DMSO are yet to be widely marketed because many test users
experience malodorous breath and skin irritation. Such cosmetic preparations
include the long-lasting antiperspirants described in U.S. Pat. No. 3,499,961.

To test whether such antiperspirants would be improved by the addition of urea,
a comparison test was conducted.

A first paste was prepared containing, by weight, 10 parts of a 6:1
DMSO-aluminum chloride complex, 10 parts urea, 10 parts ethanol, 2 parts NaCl.
Sufficient colloidal silica (Cab-O-Sil) was added to the other ingredients so
that a proper paste consistency was achieved.

When applied to the left axilla of human subjects no breath odor or skin
irritation resulted.

A second, similar paste was formulated without urea. This applied one hour later
to the right axilla of the same subjects. The subjects experienced burning and
itching of the right axilla within 15 minutes after appplication of the second
paste. Malodorous breath was detected 30-35 minutes after application of the
second paste.

EXAMPLE 13

An anti-mosquito lotion was prepared by combining the following ingredients,
with heating, to form a solution:


______________________________________
DMSO 30 g.
ethanol 30 g.
urea 15 g.
N,N-diethyl-toluamide 10 g.
______________________________________


This preparation was applied to the skin of three human subjects, known to be
sensitive to DMSO compositions. None of the subjects had adverse skin reactions,
urticaria or bad breath odor.

b. nail administrations

EXAMPLE 14

A paste was prepared containing:


______________________________________
Wt. Percent
______________________________________
DMSO 50
urea 40
lanolin 10
______________________________________


By heating the mixture to 60.degree. and milled until a smooth, uniform paste
was formed.

This paste was applied to a trauma injured toenail and underlying plate. After
application, the toe was covered by a protective overwrap. At the seventh day
after application, the nail was easily removed with gentle forcep urging.

EXAMPLE 15

Another paste was formed according to the composition:


______________________________________
Wt. Percent
______________________________________
DMSO 45
urea 45
NaCl 5
lanolin 5
______________________________________


A mixture of these ingredients was heated to 60.degree. C. and milled until a
smooth, uniform paste was formed.

This paste was applied to a chronically defective fingernail of a human subject
and also to a healthy nail on an adjacent finger. Both the fingers were covered
by a protective overwrap. By the fifth day after application, the defective nail
was easily removed by gentle urging with forceps. The normal nail was uneffected
although cuticle of both fingernails was removable by gentle rubbing.

EXAMPLE 16

Another paste was formed having the composition:


______________________________________
Wt. Percent
______________________________________
DMSO 45
urea 45
sodium sulfide 5
lanolin 5
______________________________________


This paste was prepared by heating to 60.degree. C. amd milling to a smooth,
uniform paste. This paste was applied to subjects having defective fingernails
in the manner described in Example 15.

The paste of this example softened the defective nail and debrided the
underlying nail plate more rapidly than did the paste of Example 15. A slight
etching of the normal nail surface also occurred.

EXAMPLE 17

The following substances were mixed to form a gel:


______________________________________
DMSO 20 g.
urea 40 g.
ethanol 18 g.
water 9 g.
isopropyl palmitate 8 g.
Carbopol 940
(carbomer-940) 2 g.
di(2-ethylhexyl)amine 2 g.
griseofulvin 1 g.
______________________________________


Various subjects having defective fingernails with undergrowing fungal infection
were treated using this gel composition. Gauze or polyolefin foam pads were
impregnated with the gel, applied to the defective fingernails and covered by an
occlusive overwrap. After 10 days, the overwrap and pads were removed. The
diseased nails were then easily separated from the plate by forcep urging. After
removal of the nail, a single painting of the preparation on the plate achieved
both debridement of diseased and dead tissue and control of the infection.

EXAMPLE 18

In a related test, the composition of Example 16 was heated and then painted on
diseased nails of human subjects. Twenty to thirty minutes after application,
the nails were treated with an appropriate pharmaceutical composition, e.g. one
containing an antimicrobial agent.

After several days of treatment, the diseased nail portions were removable by
forcep urging and infections were under control.

EXAMPLE 19

Two liquid preparations were prepared for comparison purposes. These
compositions were as follows:


______________________________________
Preparation 1: Wt. Percent
______________________________________
urea 50
water 25
DMSO 25
______________________________________


The ingredients were mixed, heated to 50.degree. C. and thereafter milled to a
uniform dispersion.


______________________________________
Preparation 2: Wt. Percent
______________________________________
urea 50
water 50
______________________________________


The ingredients were mixed, heated to 50.degree. C. and thereafter stirred with
cooling to form a uniform solution/dispersion.

Each of the preparations was used to impregnate cellulose base foam sheets. The
sheets, in turn, were used to treat subjects having two adjacent digits with
defective toe or fingernails. One digit of each subject was wrapped with a foam
sheet containing Preparation 1 and the adjacent digit wrapped in a foam sheet
containing Preparation 2. It was observed that Preparation 1 was about one-third
more effective in softening defective, diseased nails for removal.

EXAMPLE 20

A nail conditioner was formulated from the following ingredients:


______________________________________
Wt. Percent
______________________________________
water 50
dimethyl sulfoxide 20
methylsulfonylmethane (MSM)
10
urea 10
glycerine 5
glyoxal (30% aqueous)
5
______________________________________


The formulation was applied with cotton pads to healthy nails of human subjects
and allowed to remain for at least 15 minutes. At the end of that time, the
nails were toughened, i.e. less brittle, and the cuticle was softened such that
it could be removed by gentle rubbing.

It is not fully understood how the brittleness of the nail is reduced by
application of the formulation. It appears, however, that the MSM is the
principal active agent. The DMSO and urea appear to accelerate penetration of
the MSM into the tough nail material.

Subjects using this formulation has no reddening or irritation of the skin
surrounding the nail and did not suffer from malodorous breath.

2. Mucosal and Urogenital Administrations

EXAMPLE 21

The composition of Example 9 was slightly reformulated to include:


______________________________________
Wt. Percent
______________________________________
DMSO 50
water 35
urea 12
NaCl 3
______________________________________


This formulation was administered to patients suffering from urological problems
such as prostatitis, through a catheter extending to the neck of the bladder.
Clinical tests showed that patients receiving the DMSO-urea composition
experienced significantly less discomfort and less malodorous breath than when a
50 wt. percent solution of DMSO in water was administered without urea. The
DMSO-urea composition was equally effective in reducing the signs and symptoms
of prostatitis.

EXAMPLE 22

The composition of Example 21 was administered to human subjects who suffered
from interstitial cystitis, and who previously showed little improvement when
treated with a 50 wt. percent solution of DMSO in water. In each case, thiry to
one hundred milliliters of the composition was administered daily through a
catheter inserted to the bladder.

As compared to treatment with the 50 wt. percent DMSO solution, the DMSO-urea
formulation produced less patient discomfort. Also, improved therapeutic results
were observed. Specifically, frequency of urination, pain and discomfort with
bladder palpitation were reduced. Visual observation with a a cystoscope and
biopsies of bladder tissue confirmed that there was a reduction in inflammatory
lesions. After changing from treatment with aqueous DMSO to treatment with the
DMSO-urea formulation, subjects' bad breath was significantly reduced.

EXAMPLE 23

For some time, DMSO has been used in concentrations of up to 100 percent for
treating hypersensitive teeth recovering from extensive restorative oral
surgery. Substantial pain and discomfort as well as trauma and localized tissue
injury to the teeth and gums frequently results from such surgical procedures.
DMSO is known to promote general tissue repair and reduce pain so that after
about 2 weeks a patient can comfortably chew solid food again.

A young female subject recovering from oral surgery was treated with the
following composition:


______________________________________
Wt. Percent
______________________________________
DMSO 71.4
urea 23.8
water 4.8
______________________________________


Cotton pledgets were saturated in the solution and applied to the gums. After
two days of treatment both the relief of pain and the extent of healing were
greater than observed for typical subjects receiving two weeks of treatment with
high concentrations of DMSO solutions without urea.

Oral surgery patients treated with DMSO typically experience a sulfurous breath
and bad aftertaste which can last for a full day after treatment. These adverse
side-effects were greatly reduced in the subject treated with the above
composition containing urea.

EXAMPLE 24

Another composition suitable for treating dental patients includes:


______________________________________
Wt. Percent
______________________________________
DMSO 70
urea 20
water 8
NaCl 2
______________________________________


When applied in the manner described in Example 23, this composition produces
superior results in reducing dental pain and swelling associated with procedural
trauma.

EXAMPLE 25

Elderly persons and subjects having connective tissue diseases, frequently have
a problem with drying, painful gums. To treat this condition, the following
composition was prepared:


______________________________________
DMSO 200 g.
urea 50 g.
water 50 g.
3% Carbopol 934
(carbomer-934) 10 g.
triethanolamine 0.4 g.
______________________________________


The Carbopol 934, a polymer of acrylic acid manufactured by B. F. Goodrich
Chemical Co. of Cleveland, Ohio, in combination with triethanolamine as a
neutralizer, caused the composition to be a soft gel. This was flavored with
small amounts of spirits of peppermint to mask the somewhat bitter taste of
DMSO.

When applied topically to the gums of subjects, pain was reduced and gum tissue
was softened.

3. Intravenous Administrations

In current practice, DMSO is administered intravenously for a variety of
therapeutic purposes, at a rate of 0.1 to 2.0 grams per kilogram body weight.
Typically, intravenously administered DMSO is in an aqueous solution.

Subjects receiving DMSO intravenously have suffered from not only malodorous
breath, but also from red blood cell lysis which could lead to renal failure. It
is now found that urea, intravenously administered at the same time as DMSO,
substantially reduces both red blood cell lysis and malodorous breath.

To effectively neutralize the hemolytic activity of DMSO and at the same time
reduce expired dimethyl sulfide to acceptable levels, the amount of ethanol
administered should be at least about 0.05 grams per kilogram body weight, up to
about 0.5 grams ethanol per kilogram body weight. The ethanol should be
administered in a weight ratio to DMSO of between about 1:40 and 5:1.

Superior results are achieved when the DMSO and urea are administered together
in a common composition with the amount of urea being selected to be effective
in reducing the undesirable side-effects of malodorous breath and/or hemolysis
which can be expected from administration of the DMSO.

To enhance penetrating activity of DMSO administered intravenously, such
solutions can contain substances to enhance penetration of the DMSO. Such
substances include urea, NaCl, KCl and/or acetamide. In most instances, such
solutions will contain water as a diluent.

Although several possible mechanisms can be postulated to explain the
improvements resulting from intravenously administered ethanol, the actual
mechanism whereby malodorous breath and red blood cell lysis are reduced, is not
understood. The following examples illustrate the effectiveness of this
treatment:

EXAMPLE 26

In a first test, nine volumes of whole, heparinized human blood were combined
with one volume of a 50 volume percent aqueous solution of DMSO. The extent of
red cell lysis was great and there was a pronounced evolution of dimethyl
sulfide.

The test was repeated with the DMSO solution replaced by a 50 volume percent
aqueous solution of ethanol. After the solution was combined with the blood, no
odor was evolved but there was some red blood cell lysis.

In a third test, the procedure was repeated again, only the additive solution
contained 66.6 volume percent DMSO and 33.3 volume percent ethanol. After one
volume of solution was added to nine volumes of blood, neither red blood cell
lysis nor dimethyl sulfide odor was observed. A sample of the atmosphere over
the blood was collected and analyzed with a gas chromatograph. There was no
detectable dimethyl sulfide peak.

EXAMPLE 27

Dog blood was drawn and heparinized to prevent clotting. After tubing, samples
of the blood were mixed with a 20 wt. percent aqueous dimethyl sulfoxide
solution in such an amount that the DMSO-blood mixture contained 12 grams of
DMSO to every 100 g. of blood.

Upon mixing, there was almost instant red blood cell lysis with a predominant
odor of dimethyl sulfide. The presence of dimethyl sulfide was confirmed by gas
chromatography.

In a separate test, a sample of the heparinized blood from the same dog was
combined with an aqueous solution containing 20 wt. percent DMSO and 10 wt.
percent ethanol. The solution and blood were again combined so that 12 grams of
DMSO were present for each 100 grams of blood in the resulting mixture. After
mixing, only a small amount of red blood cell lysis occurred. The odor of
dimethyl sulfide was absent, but gas chromatography analysis showed a small peak
for dimethyl sulfide.

EXAMPLE 28

Two intravenous administration bottles were prepared. The first contained a 20
wt. percent aqueous DMSO solution. The second contained an aqueous solution
having 20 wt. percent DMSO and 10 wt. percent ethanol. Two mongrel dogs of about
15 kg. body weight were catheterized. Then, the two solutions were delivered
intravenously to the respective dogs so that each dog received 15 g. of DMSO (1
g. per kg. body weight).

When the first solution (DMSO-water) was administered to one dog, the laboratory
rapidly filled with dimethyl sulfide odor. This odor was detected within 30-45
seconds after administration. Urine collected from the dog during the first
hours after administration was a deep red color, indicating severe red blood
cell lysis.

When the second solution was administered to the other dog, no dimethyl sulfide
odor was detected by nose. Expired air from the animal was captured for a period
of time in a toluene liquid trap. The trap was operated for 0.25 hours and the
toluene was then analyzed by gas chromatography. There was a small peak showing
that a trace of dimethyl sulfide had been collected.

Urine collected from the second dog was clean and on close observation was free
of evidence of any red blood cell lysis. A unit of urine from the second dog was
centrifuged and the sediment collected and observed. There were traces of a red
precipitate, presumably hemoglobin. This may have been due in part to
traumatization during catheterization or might represent a very slight amount of
red blood cell lysis.

4. Hyperosmotic Administrations to Fish

As mentioned above, urea, NaCl, and acetamide may be beneficial in compositions
for treating mammals. But, these same substances may be toxic to fish at
concentrations of as little as 3 wt. percent in an aqueous solution applied to
an epithelial membrane of the fish.

Because these substances are the driving force behind hyperosmotic treatment of
fish, the toxicity places an upper limit on the extent to which hyperosmotic
treatments can succeed.

It is now found that the lethality of certain solutions containing hyperosmotic
concentrations of a membrane permeability altering agent can be reduced by
adding DMSO to the composition. Such reformulated compositions can then be
administered, in conjunction with health and/or welfare modifying agents, to an
epithelial membrane, such as the gill membrane, of a water-living animal. The
risk of death from osmotic stress is greatly reduced when the DMSO is used.

Suitable procedures for administration are described in U.S. Pat. No. 4,112,946.
Other procedures for administration to epithelial membranes, particularly gill
membrane, may also be used. A reduction in mortality rate will result in any
instance where membranes is subjected to a composition having a moderately high
hyperosmotic concentration of urea, NaCl and/or acetamide.

That DMSO would have this beneficial effect is quite surprising in view of the
fact that DMSO is not greatly effective as a membrane permeability altering
agent for treating the membranes of water living animals, particularly gilled
animals.

Other dipolar, aprotic solutes, such as dimethyl acetamide (DMAC) and dimethyl
formamide (DMF) are also beneficial in reducing mortality rate. But, these are
less effective than DMSO.

The amount of dipolar, aprotic solute to use with a particular solution
containing a hyperosmotic concentration of urea, NaCl and/or acetamide, is best
determined by experimentation. The amount should be sufficient to produce a
desired reduction in mortality rate or cell damage, but should not be so high as
to expose the water-living animal subjects to toxic amounts of the solute. Also,
if the solution includes a health and/or welfare modifying agent along with the
permeability altering agent, the amount of the dipolar, aprotic substance
administered should not be so great as to inhibit transport of the health and/or
welfare modifying agent into the animals.

The following examples illustrate how DMSO is effective in reducing mortality
due to osmotic stress.

EXAMPLE 29

A series of tests were made using twelve small goldfish (Carassius auratus). In
each test, two fish were exposed, by imersion, to an aqueous test solution
containing a hyperosmotic concentration of NaCl. The fish were exposed for five
minutes each and thereafter were transferred to fresh water and observed. The
results are summarized in Table I:


TABLE I
______________________________________
Test Solute Conc. (wt. %)
Lethality
______________________________________
1 NaCl 3 1 of 2 dead
2 NaCl 4 2 of 2 dead
3 NaCl 5 2 of 2 dead
4 NaCl 3
DMSO 3 none
5 NaCl 4
DMSO 3 none
6 NaCl 5
DMSO 3 1 of 2 dead
______________________________________


Clearly, lethality was reduced when DMSO was used in combination with the NaCl.

It was also observed that the gills of all fish tested turned whitish upon
exposure to the hyperosmotic solution. The degree of whiteness, a possible
indicator of osmotic stress, corresponded to the concentration of NaCl. Less
whiteness, at a given NaCl concentration, was observed in fish treated with the
solutions containing DMSO.

EXAMPLE 30

The procedure of Example 29 was repeated, except that after exposure to the
hyperosmotic solutions, the goldfish were transfered to fresh water containing
0.5 wt. % trypan blue dye. The results appear in Table II:


TABLE II
______________________________________
Test Solute Conc. (wt. %)
Lethality
______________________________________
7 NaCl 3 none
8 NaCl 4 2 of 2 dead
9 NaCl 5 2 of 2 dead
10 NaCl 3
DMSO 3 none
11 NaCl 4
DMSO 3 none
12 NaCl 5
DMSO 3 none
13 urea 8 none
14 urea 12 none
15 urea 8
DMSO 5 none
16 urea 12
DMSO 5 none
______________________________________


Again, decreased lethality was observed for fish exposed to a NaCl solution,
when DMSO was added.

The fish were observed for dye uptake. Trypan blue, being a vital exclusion type
dye, only colors non-living cells. Fish exposed to the saline solutions without
DMSO demonstrated an increased dye uptake, pronounced when the NaCl
concentration was 5 wt. percent. From the dye uptake patterns observed, it
appears that NaCl without DMSO is highly lethal to epithelial cells, especially
of the gills, fins and tail.

Similar results were observed in tests 13-16 where the fish were exposed to
hyperosmotic concentrations of urea. Although none of the test animals died, dye
uptake was greater when DMSO was absent.

These results are contrary to expectations because DMSO is known to facilitate
tissue penetration in most instances. If DMSO behaved in an expected manner, it
would enhance NaCl and urea penetration. Such increased penetration should
logically increase cell damage and, consequently, trypan blue uptake. But, the
results of tests 7-16 show that the opposite is true. DMSO protects cells from
attack by hyperosmotic NaCl and urea solutions, and does not aid in the attack.
Furthermore, the results of tests 7-16 indicate that DMSO not only reduces the
lethality of hypersomotic solutions, but also retards injury of cells exposed to
extreme osmotic challenge.

EXAMPLE 31

Seven fingerling salmonids of 4-5 inch length were netted in the Washougal River
(State of Washington). The species was not identified; but most likely the
fingerlings were wild, coho salmon (Oncorhynchus kisutch).

Four of the fingerlings were immersed in an aqueous solution containing 5 wt.
percent NaCl and 3 wt. percent DMSO. After five minutes, the fingerlings were
returned, for observation, to a holding pond containing river water. The
procedure was repeated with the remaining three fingerlings being immersed in an
aqueous solution containing only 5 wt. percent NaCl.

All of the fingerlings lost their friskiness when exposed to a hyperosmotic
solution, but all were alive when transferred to the holding ponds. After three
hours, the three fingerlings exposed to the NaCl solution without DMSO had died.
The other four fingerlings were alive and were released into the river.

While I have described and given examples of preferred embodiments of my
invention, it will be apparent to those skilled in the art that changes and
modifications may be made without departing from my invention in its broader
aspects. I therefore intend the appended claims to cover all such changes and
modifications as fall within the true spirit and scope of my invention.


* * * * *