Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 93/10776 PCr/US92/08787
212367g
wa~ND ED~L~ o~osmoNs CO~AlN~G A PYRWATE, AN
ANllOXIDAt~ AND A MIXll~E OF FAl~Y A~IDS
BACKGROUND OF q~HE INVENTION
This application is a continuation-in-part of
application serial no. 663,500, filed 1 March 1991.
1. Field of the Inventio~
This invention pertains to tharapeutic wound
healing compositions useful for increasing the
proliferation and resuscitation rate of mammalian cells.
Nore particularly, the wound healing compositio~S
comprise (a) pyruvate, (b) an antioxidant, ~and (cj a
- mixture of saturated and unsaturated fatty acids. This
invention also pertains to methods for preparing and
us~ng the wound healing compositions and the topical
pharmaceutical products in which the therapeutic
compositions may be used.
-~ 2. D-s~ription of the B~ckground
; 35 ~
Wounds are bodily injuries caused by phy ical
means which disrupt the normal continuity of structures.
Such bodily injuries include contusions, wounds in which
WO93/10776 PCT/US92/08787
~l2~678 ' ,.
the skin is unbroken, incisions, wounds in which the skin
is broken by a cutting instrument, and lacerations,
wounds in which the skin is broken by a dull or blunt
instrument. Wounds may be caused ~y accidents or by
surgical procedures~ Patients who suffer major wounds
could benefit from an enhancement in the wound healing
process.
Wound healing consists of a series of processes
whereby injured tissue is repaired, specialized tissue is
regenerated, and new tissue is reorganized into a ~car.
Wound healing consists of three major phases: a) an
inflammation phase (0-3 days), b) a cellular
proliferation phase (3-12 days), and (c) a remodeling
phase 13 days-6 months).
During the inflammation phase, platelet
aggregation and clotting form a matrix which traps plasma
proteins and blood cells to induce the influx of various
types of cells. During the cellular proliferation phase,
new connective or granulation tissue and blood vessels
are formed. During the remodeling phase, granulation
tissue is replaced by a network of collagen and elastin
fibers leading to the formation of scar tissue.
~
When cells are injured or killed as a result of
a wound, a wound healin~ step is desirable to resuscitate
the injured cells and produce new cells to replace the
dead cells. The healing process requires thç reversal of
cytotoxicity, the suppression of inflammation, and the
stimulation of cellular viability and proliferation.
Wounds require low levels of oxygen in the initial stages
of healing to suppress oxidative damage and higher levels
of oxygen in the later stages of healing to promote
collagen formation by fibroblasts.
Stressed and injured mammalian cells are
exposed to activated oxygen species such as superoxide
(2 ), hydrogen peroxide (H2O~), hydroxyl radical ~OH-),
W093/10776 2 1 2 3 6 7 3 PCT/I 592/08787
. and singlet oxygen (102). In vivo these reactive oxygen
intermediates are generated by cells in response to
aerobic metabolism, catabolism of drugs and other
xenobiotics, ultraviolet and x-ray radiation, and the
respiratory burst of phagocy~ic cells (such as white
blood cells) to kill invading bacteria such as those
introduced through wounds. Hydrogen peroxide, for
example, is produced during respiration of most living
organisms especially by stressed and injured cells.
These active oxygen species can injure cells.
An important example of such damage is lipid peroxidation
which involves the oxidative degradation of unsaturated
lipids. Lipid peroxidation is highly detrimental to
membrane structure and function and can cause numerous
cytopathological effects. Cells defend against lipid
peroxidation by producing radical scavengers such as
superoxide dismutase, catalase, and peroxidase. ~njured
cells have a decreased ability to produ~e radical
scavengers. Excess hydrogen peroxide can react with DNA
to cause backbone breakage, produce mutations, and alter
and liberate bases. Hydrogen peroxide can also react
with pyrimidines to open the 5, 6-double bond, which
reaction inhibits the ability of pyrimidines to hydrogen
bond to complementary bases, Hallaender et al (197~.
Such oxidative biochemical injury can result in the loss
of cellular membrane integrity, reduced enzyme activity,
changes ~n transport kinetics, changes in membrane lipid
content, and leakage of potassium ions, amino acids, and
other cellular material.
Antioxidants have been shown to inhibit damage
associated with active oxygen species. For example,
pyruvate and other alpha-ketoacids have been reported to
react rapidly and stoichiometrically with hydrogen
peroxide to protect cells from cytolytic effects,
O'Donnell-Tormey et al., J. EXP. Med., 165, pp. 500-514
(1987).
W O 93/10776 PC~r/US92/087X7
2 ~2~78
7United States pat~nts nos. 3,920,835,
3,984,556, and 3,988,470, all issued to Van Scott et al.,
disclose methods for treating acne, dandruff, and palmar
keratosis, respectively, which consist of applying to the
affected area a topical composition comprising from about
1% to about 20% of a lower aliphatic compound containing
from two to six carbon atoms selected from the group
consisting o~ alpha-hydroxyacids, alpha-ketoacids and
esters thereof, and 3-nydroxybutryic acid in a
pharmaceutically acceptable carrier. The aliphatic
compounds include pyruvic acid and lactic acid.
United States patents nos. 4,105,783 and
4,197,316, both issued to Yu et al., disclose a method
and composition, respectively, for treating dry skin
which consists of applying to the affected area a topical
composition comprising from about 1~ to about 20% of a
compound selected from the group consisting of amides and
ammonium salts of alpha-hydroxyacids, beta-hydroxyacids,
and alpha-ketoacids in a pharmaceutically acceptable
carrier. The compounds include the amides and ammonium
salts of pyruvic acid and lactic acid.
United States patent no. 4,234,599, issued to
Van Scott et al., discloses a method for treating acti~i~c
and nonactinic skin keratoses which consists of applying
to the affected area a topical composition comprising an
~ffective amount of a compound selected from the group
con8isting of alpha-hydroxyacids, beta-hydroxyacids, and
alpha-ketoacids in a pharmaceutically acceptable carrier.
The acidic compounds include pyruvic acid and lactic
acid.
United States patent no. 4,294,852, issued to
Wildnauer et al., discloses a composition for treating
skin which comprises the alpha-hydroxyacids, beta-
hydroxyacids, and alpha-ketoacids disclosed above by
Van Scott et al. in combination with C3-C8 aliphatic
-
alcohols.
W O 93/10776 Pc~r/uss2/o8787
2~2~678
United States patent no. 4,663,166, issued to
Veech, discloses an electrolyte solution which comprises
a mixture of L-lactate and pyruvate in a ratio from 20:1
to 1:1, respectively, or a mixture of D-beta-
hydroxybutyrate and acetoacetate, in a ratio from 6:1 to
0.5:1, respectively.
Sodium pyruvate has been reported to reduce the
number of erosions, ulcers, and hemorrhages on the
gastric mucosa in guinea pigs and rats caused by
acetylsalicylic acid. The analgesic and antipyretic
properties of acetylsalicylic acid were not impaired by
sodium pyruvate, Puschmann, Arzneimittelforschunq, 33,
pp. 410 415 and 415-416 (1983).
Pyruvate has been reported to exert a positive
inotropic effect in stunned myocardum, which is a
prolonged ventricular dysfunction following brief periods
of coronary artery occlusions which does not produce
irreversible damage, Mentzer et al., Ann. Sura., ~Q~,
pp. 62g-633 (1989).
Pyruvate has been reported to produce a
relative stabilization of left ventricular pressure .~n~
work parameter and to reduce the si2e of infarctions.
Pyr~vate improves resumption of spontaneous beating of
the heart and restoration of normal rates and pressure
development, Bunaer et al., J. Mol. Cell. C3~iol., 18,
pp. 423-438 (1986~, Mochizuki et alO, J. Physiol.
(Paris)~ 76,pp. 805-812 (1980), Regi~z et al.,
Cardiovaso. Res.,15, pp~ 652-658 (1981),
Giannelli e.t al., Ann. Thorac. Surq., 21, pp. 386-396
(1976).
Sodium pyruvate has been reported to act as an
antagonist to cyanide intoxification (presumably through
the formation of a cyanohydrin) and to protect against
the lethal effects of sodium sulfide and to retard the
WO93/10776 PCT/US92/Q8787
2 l?~fi~8
onset and development of functional, morphological, and
biochemical measures of acrylamide neuropathy of axons,
Schwartz et al., Toxicol. APpl. Pharmacol., So, pp. 437-
442 (1979), Sabri et al., Brain Res., 483, pp. 1-11
S (19~9).
A chemotherapeutic cure of advanced L1210
leukemia has been reported using sodium pyruvate to
restore abnormally deformed red blood cells to normal.
The deformed red blood cells prevented adequate drug
delivery to tumor cells, Cohen, Cancer Chemother.
Pharmacol., 5, pp. 175-179 (1981).
Primary cultures of heterotopic tracheal
transplant exposed in vivo to 7, 12-dimethyl-
benz(a)anthracene were reported to be successfully
maintained in enrichment medium supplemented with sodium
pyruvate along with cultures of interleukin-2 stimulated
peripheral blood lymphocytes, and plasmacytomas and
hybridomas, pig embryos, and human blastocysts, Shacter,
J. Immunol. Methods, 99, pp. 25g-270 (1987),
Marchok et al., Cancer Res., 37, pp. 1811-1821 (~977),
Davis J. Reprod. Fertil. Suppl., 33, pp. 115-124 ~1985),
Okamoto et al., No To Shi~kei, 38, pp. 593-598 (1986),
Ççhçn_ç5_311, J. In Vitro Fert. Embrvo Transfer, .-~,
pp. 59-64 (1985).
United States patents nos. 4,158,057,
4,351,835, 4,415,576, and 4,645,764, all issued to
~n~, disclose methods for preventing the accumulation
of fat in the liver of a mammal due to the ingestion of
alcohol, for controlling weight in a mammal,~ for
inhibiting body fat while increasing protein
concentration in a mammal, and for controlling the
deposition of body fat in a living being, respectively.
The methods comprise administering to the mammal a
therapeutic mixture of pyruvate and dihydroxyacetone, and
optionally riboflavin. United States patent
no. 4,548,937, issued to Stanko, discloses a method for
WOg3/10776 pcT/uss2/o87B7
2~23~7~
controlling the weight gain of a mammal which comprises
administering to the mammal a therapeutically effective
amount of pyruvate, and optionally riboflavin. United
States patent no. 4,812,~79, issued to Stanko, discloses
a method for controlling the weight gain of a mammal
which comprises administering to the mammal a
therapeutically effective amount of dihydroxyacetone, and
optionally riboflavin and pyruvate.
Rats fed a calcium-oxalate lithogenic diet
including sodium pyruvate were reported to develop fewer
urinary calculi (stones) than control rats not given
sodium pyruvate, Oqawa et al., Hinyokika KiYo, 32,
pp. 1341-13~7 (1986).
United States patent no. 4,521,375, issued to
Houlsby, discloses a method for sterilizing surfaces
which come into contact with living tissue~ The method
comprises sterilizing the surface with aqueous hydrogen
peroxide and then neutralizing the surface with pyruvic
acid.
United States patent no. 4,416,982, issued to
Tauda et al., discloses a method for decomposing hydrogen
peroxide by reacting the hydrogen peroxide with a phe~l
or aniline derivative in the presence of peroxidase.
United States patent no. 4,696,917, issued to
Lindstrom et al., discloses an eye irrigation solution
which comprises Eagle's Minimum Essential Medi~m with
Earle's salts, chondroitin sulfate, a buf f er solution, 2-
mercaptoethanol, and a pyruvate. The irrigation solution
may optionally contain ascorbic acid and alpha-
tocopherol. United States patent no. 4,725,586, issued
to Lindstrom et al., discloses an irrigation solution
which comprises a balanced salt solution, chondroitin
sulfate, a buffer solution, 2-mercaptoe~hanol, sodium
bicarbonate or dextrose, a pyruvate, a sodium phosphate
W093/10776 PCT/US92~8787
2 ~ 2 ~ ~ 7 8
buffer system, and cystine. The irrigation soluti~n may
optionally contain ascorbic acid and gamma-tocopherol.
United States patent no. 3,887,702 issued to
Baldwin, discloses a composition for treating fingernails
and toenails which consists essentially of soybean oil or
sunflower oil in combination with Vitamin E.
United States patent no. 4,847,069, issued to
Bissett et al., discloses a photoprotective composition
comprising (a) a sorbohydroxamic acid, (b) an anti-
inflammatory agent selected from steroidal anti-
inflammatory agents and a natural anti~inflammatory
agent, and (c) a topical carrier. Fatty acids may be
present as an emollient. United States patent
no. 4,847,071, issued to Bissett et al., discloses a
photoprotective composition comprising (a) a tocopherol
or tocopherol ester radical scavenger, (b) an anti-
inflammatory agent selected from steroidal anti-
inflammatory agents and a natural anti-inflammatory
agent, and (c) a topical carrier. United States patent
no. 4,847,072, issued to Bissett et al., discloses a
topical composition comprising not more than 25%
tocopherol sorbate in a topical carrier.
._--
United States patent no. 4,533,637, issued to
Yamane et al., discloses a culture medium which comprises
a carbon source, a nucleic acid source precursor, amino
acids, vitamins, minerals, a lipophilic nutrient, and
~erum albumin, and cyclodextrins. The lipophilic
substances include unsaturated fatty acids and lipophilic
vitamins such as Vitamin A, D, and E. Ascorbic acid may
also be present.
United Kingdom patent application
no. 2,196,348A, to Kovar et al., discloses a synthetic
culture medium which comprises inorganic salts,
monosaccharides, amino acids, vitamins, buffering agents,
and optionally sodium pyruvate adding magnesium hydroxide
WO 93/10776 212 3 6 7 ~;) PCI/US92/087B7
or magnesium oxide to the emulsion. The oil phase may
inc lude ch i cken f at .
United States patent no. 4,284,630, issued to
Yu et al., discloses a method for stabilizing 3 water-in-
oil emulsion which comprises adding magnesium hydroxide
or magnesium oxide to the emulsion. The oil phase may
include chicken fat.
Preparation-HTM has been reported to increase
the rate of wound healing in artificially created rectal
ulcers. The active ingredients in Preparation-HT~ are
skin respiratory factor and shark liver oil,
Subramanyam et al., Diaestive Diseases and Sciences, 29,
pp. 829-832 (1984).
The addition of sodium pyruvate to bacterial
and yeast systems has been reported to inhibit hydrogen
peroxide production, enhance growth, and protect the
systems against the toxicity of reactive oxygen
intermediates. The unsaturated fatty acids and saturated
fatty acids contained within chicken fat enhanced
membrane repair and ~educed cytotoxicity. The
antîoxidants glutathione and thioglycollate reduced the
injury induced by oxygen radical species, Martin, P~
thesis, (1~87-89).
United States patent no. 4,615,697, issued to
Robinson, discloses a controlled release treatment
composition comprising a treating agent and a bioadhesive
agent comprising a water-swellable but water-insoluble,
fibrous cross-linked carboxy-functional polymer.
European patent application no. 0410696A~, to
Kellaway et al., discloses a mucoadhesive delivery system
comprising a treating agent and a polyacrylic acid cross-
linked with from about 1% to about 20% by weight of a
polyhydroxy compound such as a sugar, cyclitol, or lower
polyhydric alcohol.
W093/10776 PCT/US92/087X7
212~678
~o
While the above therapeutic compositions are
reported to innibit the production of reactive oxygen
intermediates, none of the above compositions are
entirely satisfactory wound healing compositions. None
of the compositions has the ability to simultaneously
decrease cellular levels of hydrogen peroxide production,
increase cellular resistance to cytotoxic agents,
increase rates of cellular proliferation, and increase
cellular viability to protect and resuscitate mammalian
cells. The present invention provides such improved
therapeutic wound healing compositions without the
disadvantages characteristic of previously known
compositions.
8UNMARY OF THE INVENTION
The present invention pertains to therapeutic
wound healing compositions. The compositions comprise
(a) pyruvate selected from the group consisting of
pyruvic acid, pharmaceutically acceptable salts of
pyruvic acid, and mixtures thereof, (b~ an antioxidant,
and (c) a mixture of saturated and unsaturated fa~t~
acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resu8citation of mammalian cells. The therapeutic
compositions may be utilized in a wide variety of topical
and ingestible pharmaceutical products. This invention
also relates to method for preparing and using the
therapeutic compositions and the topical pharmaceutical
products in which the therapeutic compositions may be
used.
WO93/10776 2 1 2 3 6 ~ 3 PCT/USg2/OX787
~RIEF DESCRIPTION OF THE FIGURES
FIGURE 1 i~ a photograph of wounded mice after
4 days of treatment with: preparation HT~ ~Example A); a
petrolatum base formulation containing live yeast cell
derivative, shark oil, and a mixture of sodium pyru~ate,
vitamin E, and chicken fat (Example B); a petrolatum base
formulation containing live yeast cell derivative and
shark oil (Example C); and no composition (Example E,
control).
FIGURE 2 is a photograph of a wounded mouse
after 4 days of treatment with a petrolatum base
formulation only (Example D).
DETAILED DESCRIPTION OF THE INVENTION
Applicant has discovered therapeutic wound
healing compositions for increasing the resuscitation
rate of injured mammalian cells and the proliferation
rate of new mammalian cells to replace dead cells. Cellæ
treated with the therapeutic compositions of the pres~nt
inv~ntion show decreased levels of hydrogen peroxide
;~ production, increased resistance to cytotoxic agents,
increased rates of proliferation, and increased
viability. Wounded mammals treated with the therapeutic
compositions show æignificantly~ improved wound closing
and healing over untreate~ mammals and mammals treated
` ~ith conventional healing composit~ions.
The term "injured ~ell" as used herein means a
cell which has ~a) injured~membranes so that transport
through the membranes is diminished resulting in an
increase` in toxins and normal cellular wastes inside the
cell ~and a decrease in nutrients and other componen~s
neoessary for cellular repair inside the cell, (b) an
WO93/10776 PCT/US92/08787
2123678
increase in concentration of oxygen radicals inside the
cell because of the decreased ability of the cell to
produce antioxidants and enzymes, and (c) damaged DNA,
RNA, and ribosomes which must be repaired or replaced
S before normal cellular functions can be resumed. The
term "resuscitation" of injured mammalian cells as used
h~rein means the reversal of cytotoxicity, the
stabilization of the cellular membrane, an increase in
the proliferation rate of the cell, and/or the
normalization of cellular functions such as the secretion
of growth factors, hormones, and the like. The term
"cytotoxicity" as used herein means a condition caused by
a cytotoxic agent that injures the cell. Injured cells
do not proliferate because injured cells expend all
energy on cellular repair. Aiding cellular repair
promotes cellular proliferation.
Epidermal keratinocytic cells and monocytic
cells have multiple oxygen generating mechanisms and the
degree to which each type of mechanism functions differs
in each type of cell. In monocytes, for example, the
respiratory bursting process is more pronounced than in
epidermal keratinocytes. Hence, the components in the
therapeutic compositions of the present invention may
vary depending upon the types of cells involved in ~h~
condition being treated.
In a first embodiment, the therapeutic wound
healing composition for treating mammalian cells,
preferably epidermal keratinocytes, comprises (a)
pyruvate, (b) an antioxidant, and (c) a mixture of
saturated and unsaturated fatty acids.
While not wishing to be bound by theory,
applicant believes that pyruvate (or pyruvic acid) can be
transported inside a cell where it can act as an
antioxidant to neutralize oxygen radicals in the cell.
Pyruvate can also be used inside the cell in the citric
acid cycle to provide energy to increase cellular
W O 93/10776 21 2 3 6 7 8 PC~r/US92/08787
viability, and as a precursor in the synthesis of
important biomolecules to promote cellular proliferation.
In addition, pyruvate can be used in the multifunction
oxidase system to reverse cytotoxicity. Antioxidants,
especially lipid-soluble antioxidants, can be absorbed
into the cell membrane to neutralize oxygen radicals and
thereby protect the membrane. The combination of
pyruvate inside the cell and an antioxidant in the
cellular membrane functions i~ a synergistic manner to
reduce hydrogen peroxide production in the cell to levels
lower than can be achieved by use of either type of
component alone.
The saturated and unsaturated fatty acids in
the present invention are those fatty acids required for
the repair of cellular membranes and resuscitation of
mammalian cells. Hence, the fatty acids in the
~herapeutic composition, which may be in the form of
mono-, di-, and/or triglycerides or free fatty acids, are
readily available for the repair of injured cells and the
production of new cells to replace dead cells. Cells
injured by oxygen radicals need to produce unsaturated
fatty acids to repair cellular membranes. However, the
production of unsaturated fatty acids by cells requires
oxygen. Thus, the injured cell needs high levels._cf
oxygen to produce unsaturated fatty acids and at the same
time needs to reduce the level of oxygen within the cell
to reduce oxidative injury. By providing the cell with
the un~aturated fatty acids needed for xepair, the need
of the cell to produce unsaturated fatty acids i~ reduced
and the need for high oxygen levels is also reduced. The
presence of mixtures of saturated and unsaturatedifatty
acids in the therapeutic composition significantly
enhances the ability of pyruvate and the antioxidant to
inhibit reactive oxygen production. By stabilizing the
cellular membrane, unsaturated fatty acids also improve
membrane function and enhance pyruvate transport into the
cell. By improving the viability of the cells,
unsaturated fatty acids also improve the repair of
W O 93/10776 PC~r/US92/08787
2.l~23678 ~Cf' ' ` ~
cellular membranes rate of the cells. ~ence~ the three
components in the therapeutic composition function
together in a synergis~ic manner to increase the
resuscitation rate of injured mammalian cells and the
production of new cells.
In a second embodiment, the therapeutic wound
healing composition for treating mammalian cells,
preferably epidermal keratinocytes, comprises (a)
pyruvate, (b) lactate, and ~c) a mixture of saturated and
unsaturated fatty acids. In this embodiment, lactate is
employed instead of an antioxidant. Antioxidants react
with, and neutralize, oxygen radicals after the radicals
are already forméd. Lactate, on the other hand, is a
component in the cellular feedback mechanism and inhibits
the respiratory bursting process to suppress the
production of active oxygen species. The combination of
pyruvate to neutralize active oxygen species and lactate
to suppress the respiratory bursting process functions in
a synergistic manner to reduce hydrogen peroxide
production in the cell to levels lower than can be
achieved by use of either type of component alone. The
presence of mixtures of saturated and unsaturated fatty
acids in the therapeutic composition significantly
enhances the ability of pyruvate and lactate to inhi~it
reactive oxygen production. Hence, the three components
in the therapeutic composit-ion in this embodiment
function together in a synergistic manner ~o increase the
proliferation and resuscitation rate of mammalian cells.
In a third embodiment, the therapeutic wound
healing composition for treating mammalian cells,
preferably epidermal keratinocytes, comprises (a) an
antioxidant, and (b) a mixture of saturated and
unsaturated fatty acids. The presence of mixtures of
saturated and unsaturated fatty acids in the therapeutic
composition in this embodiment significantly enhances the
ability of the antioxidant to inhibit reactive oxygen
production. The combination of an antioxidant to
W O 93~10776 2 12 3 6 ~ g PC~r/US9~/08787
~S
neutralize active oxygen species and fatty acids to
rebuild cellular membranes and reduce the need of t-he
cell for oxygen functions in a synergistic manner to
reduce hydrogen peroxide production in the cell to levels
lower than can be achieved by either type of component
alone. Hence, the components in the therapeutic
composition in this embodiment function together in a
synergistic manner to increase the proliferation and
resuscitation rate of mammalian cells.
In a fourth embodiment, the therapeutic wound
healing composition for treating mammalian cells,
preferably monocytes, comprises (a) lactate, (b) an
antioxidant, and (c) a mixture of saturated and
unsaturated fatty acids. In this embodiment, lactate is
employed because the respiratory bursting process is more
pronounced in monocytes than in epidermal keratinocytes.
The combination of lactate to suppress the respiratory
bursting process and an antioxidant to neutralize active
oxygen species functions in a synergistic manner to
reduce hydrogen peroxide production in the cell to levels
lower than can be achieved by either component alone.
The presence of mixtures of saturated and unsaturated
fatty acids in the therapeutic composition in this
embodiment significantly enhances the ability of lacta*e
and the antioxidant to inhibit reactive oxygen
production. Hence, the three components in the
therapeutic composition in this embodiment function
together in a synergistic manner to increase the
proliferation and resuscitation rate of mammalian cells.
i Accordingly, the combination of ingredients set
out in the above embodiments functions together in an
enhanced manner to increase the proliferation and
resuscitation rate of mammalian cells. The therapeutic
effect of the combination of the components in each of
the above embodiments is markedly greater than that
expected by the mere addition of the individual
therapeutic components. Hence, applicant's therapeutic
W093/10776 PCT/U~92/087X7
2l'~36~8
wound healing compositions have the ability to decrease
intracellular levels of hydrogen peroxide production,
increase cellular resistance ko cytotoxic agents,
increase rates of cellular proliferation, and increase
S cellular viability.
The cells which may be treated with the
therapeutic compositions in the present invention are
mammalian cells. Although applicant will describe the
present therapeutic compositions as useful for treating
mammalian epidermal keratinocytes and ma~malian
monocytes, applicant contemplates that all mammalian
cells which may be protected or resuscitated by
applicant~s therapeutic compositions may be used in the
lS present invention. Keratinocytes are representative of
normal mammalian cells and are the fastest proliferating
cells in the ~ody. The correlation between the reaction
of keratinocytes to injury and therapy and that of
mammalian cells in general is very high. Monocytes are
representative of specialized ma~malian cells such as the
white blood cells in the immune system and the organ
cell8 in liver, kidney, heart, and brain. The mammalian
cells may be treated in vivo and in vitro.
Epidermal keratinocytes are the specialized
~pithelial cells of the epidermis which synthesize
keratin, a scleroprotein whîch is the principal
constituent of epidermis, hair, nails, horny tissue, and
the organic matrix of the enamel of teeth~ Mammalian
epidermal keratinocytes constitute about 95% of the
epidermal cells and together with melanocytes form the
binary system of the epidermis. In its various
successive stages, epidermal keratinocytes are also known
as basal cells, prickle cells, and granular cells.
Monocytes are mononuclear phagocytic leukocytes
which undergo respiratory bursting and are involved in
reactive oxygen mediated damage within the epidermis.
Leukocytes are white blood cells or corpuscles which may
W093~10776 PCT/~S92/08787
21~ ,78
,~
,be classified into two main groups; granular leuk~cytes
(granulocytes) which are leukocytes with abundant
granules in the cytoplasm and nongranular leukocytes
(nongranulocytes) which are leukocytes without specific
granules in the cytoplasm and which include the
lymphocytes and monocytes. Phagocyte cells are cells
which ingest microorganisms or other cells and foreign
particles. Monocytes are also known as large
mononuclear leukocytes, and hyaline or transitional
leukocytes~
Pyruvic acid (2-oxopropanoic acid, alpha-
ketopropionic acid, CH3COCOOH) or pyruvate (at
physiological pHj is a fundamental intermediate in
protein and carbohydrate metabolism and in the citric
acid cycle. The citric acid cycle (tricarboxylic acid
cycle, Kreb's cycle) is the major reaction sequenceiwhich
executes the reduction of oxygen to generate adenosine
triphosphate (ATP) by oxidizing organic compounds in
respiring tissues to provide electrons to the transport
syste~. Acetyl coenzyme A ~"active acetyl") is oxidized
in this process and is thereafter utilized in a variety
of biological processes and is a precursor in the
biosynthesis of many fatty acids and sterols. The two
major sources of acetyl coenzyme A are derived from .the
metabolism of glucose and fatty acids. Glycolysis
consists of a series of transformations wherein each
glucose molecule is transformed in the cellular cytoplasm
into two molecules of pyruvic acid. Pyruvic acid may
then enter the mitochondria where it is oxidized by
coenzyme A in the presence of enzymes and cofactors to
acetyl coenzyme A. Acetyl coenzyme A can then enter he
~itric acid cycle.
In muscle, pyruvic acid ~derived from glycogen)
is reduced to lactic acid during exertion. Lactic acid
is reoxidized and partially retransformed to glycogen
during rest. Pyruvate can also act as an antioxidant to
W O 93/10776 PC~r/US92/08787
2 ~ 2 3 ~ ~utralize oxyg~n radicals in the cell and can be used in
the multifunction oxidase system to reverse cytotoxicity.
The pyruvate in the present invention may be
selected from the group consisting of pyruvic acid,
pharmaceutically acceptable salts of pyruvic acid, and
mixtures thereof. In general, the pharmaceutically
acceptable salts of pyruvic acid may be alkali salts and
alkaline earth salts. Preferably, the pyruvate is
selected from the group consisting of pyruvic acid,
sodium pyruvate, potassium pyruvate, magnesium pyruvate,
calcium pyruvate, zinc pyruvate, manganese pyruvate, and
mixtures thereof. More preferably, the pyruvate is
selected from the group of salts consisting of sodium
pyruvate, potassium pyruvate, magnesium pyruvate, calcium
pyruvate, zinc pyruvate, manganese pyruvate, and mixtures
thereof. Most preferably, the pyruvate is sodium
pyruvate.
The amount of pyruvate present in the
therapeutic compositions of the present invention is a
therapeutically effective amount. A therapeutically
effective amount of pyruvate is that amount of pyruvate
necessary to increase the proliferation and resuscitation
rate of mammalian cells. The exact amount of pyruvate ~g
a matter of preference subject to such factors as the
type of condition being treated as well as the other
ingredients in the composition. In a preferred
embodiment, pyruvate is present in the therapeutic
composition in an amount from about 10% to about S0%,
preferably from about 20% to about 45%, and more
preferably from about 25% to about 40%, by weight of the
therapeutic composition.
L-Lactic acid t(S)-2-hydroxypropanoic acid~
(+) alpha-hydroxypropionic acid, CH3CHOHCOOH) or lactate
occurs in small quantities in the blood and muscle fluid
of mammals. Lactic acid concentration increases in
muscle and blood after vigorous activity. Lactate is a
WO93/10776 2 ~ PCT/US92/08787
component in the cellular feedback mechanism and inhibits
the natural respiratory bursting process of cells thereby
suppressing the production of oxygen radicals.
The lactate in the present invention may be
selected from the group consisting of lactic acid,
pharmaceutically acceptable salts of lactic acid, and
mixtures thereof. In general, the pharmaceutically
acceptable salts of lactic acid may be alkali salts and
alkaline earth salts. Preferably, the lactate is
~elected from the group consisting of lactic acid, sodium
lactate, potassium lactate, magnesium lactate, calcium
lactate, zinc lactàte, manganese lactate, and mixtures
thereof. More preferably, the lactate is selected from
the group consisting of lactic acid, sodium lactate,
potassium lactate, magnesium lactate, calcium lactate,
zinc lactate, manganese lactate, and mixtures thèreof.
Most preferably, the lactate is lactic acid.
The amount of lactate present in the
therapeutic compositions of the present invention is a
therapeutically effective amount. A therapeutically
effective amount of lactate is that amount of lactate
necessary to increase the proliferation and resuscitation
rate of mammalian cells. For a composition,-~'a
therapeutically effective amount of lactate is that
amount necessary to suppress the respiratory bursting
process of white blood cells to protect and resuscitate
the mammalian cells. In general, a therapeutically
effective amount of lactate in a composition is from
about 5 to about lO times the amount of lactate normally
found in serum. The exact amount of lactate is a matter
of preference subject to such factors as the type of
condition being treated as well as the other ingredients
in the composition. In a p~eferred embodiment, lactate
is present in the therapeutic composition in an amount
from aboùt 10% to about 50%, preferably from about 20% to
about 45~, and more preferably from about 25% to about
40~, by weight of the therapeutic composition.
WO93/10776 PCT/US92/087~7
?,l23fi78
Antioxidants are substances which inhibit
oxidation or suppress reactions promoted by oxygen or
peroxides. Antioxidants, especially lipid-soluble
antioxidants, can be absorbed into the cellular membrane
to neutralize oxygen radicals and thereby protect the
membrane. The antioxidants useful in the present
invention may be selected from the group consisting of
Vitamin A (retinol), Vitamin A2 (3, 4-didehydroretinol),
all forms of carotene such as alpha-carotene, beta-
carotene (beta, ~eta-carotene~, gamma-carotene, del ta-
carotene, Vitamin C (ascorbic acid, L-ascorbic acid), all
forms of tocopherol such as Vitamin E (alpha-tocopherol,
3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri-
decyl)-2H-1-benzopyran-6-ol), beta-tocopherol, gamma-
tocopherol, and delta-tocopherol, and mixtures thereof.
Preferably, the antioxidant i5 selected from the group of
lipid-soluble antioxidants consisti~g of Vitamin A, b~ta-
carotene, Vitamin E, and mixtures thereof. More
preferably, the antioxidant is Vitamin E.
The amount of antioxidant present in the
therspeutic compositions of the present invention is a
therapeutically effective amount. A therapeutically
effective amount of antioxidant is that amount-~f
antioxidant necessary to increase thé proliferation and
resuscitation rate of mammalian cells. The exact amount
of antioxidant is a matter of preference subject to ~uch
factors as the type of condition being treated as well as
the other ingredients in the composition. In a preferred
embodiment, the antioxidant is present in the therapeutic
compositidn in an amount from about 10~ to about 50~,
preferably from about 20% to about 45%, and more
preferably from about 25% to about 40%, by weight of the
therapeutic composition.
The mixture of saturated and unsaturated fatty
acids in the present invention are those fatty acids
required for the repair of mammalian cellular membranes
W O 93/10776 2 1 2 ~ ~ r 3 PC~r/US92/08787
and the production of new cells. Hence, the fatty acids
are readily incorporated into the cell and are
immediately available for the repair of injured cells and
the proliferation of new cells. By providing the cell
with the unsaturated fatty acids needed for repair, the
need of the cell for unsaturated fatty acids is reduced
and the need for high oxygen levels is also reduced.
Accordingly, the presence of the mixtures of saturated
and unsaturated fatty acids in the therapeutic
compositions slgnificantly enhances the ability of
pyruvate, lactate, and the antioxidant to inhibit
reactive oxygen production.
Fatty acids are carboxylic acid compounds found
lS in animal and vegetable fat and oil. Fatty acids are
classified as lipids and are composed of chains of alkyl
groups containing from 4 to 22 carbon atoms and 0-3
double bonds and characterized by a terminal carboxyl
group, -COOH. Fatty acids may be saturated or
unsaturated and may be solid, semisolid, or liquid. The
most common saturated fatty acids are butyric acid (C4),
lauric acid (C12), palmitic acid (C16)~ and stearic acid
(C18). Unsaturated fatty acids are usually derived from
vegetables and consist of alkyl chains containing from 16
to 22 carbon atoms and 0-3 double bonds with ~He
characteristic terminal carboxyl group. The most common
unsaturated fatty acids are oleic acid, linoleic acid,
and linolenic acid (all C18 acids). ~
In general, the mixture of saturated and
unsaturated fatty acids required for the repair of
mammalian cellular membranes in the present invention may
be derived from animal fats and waxes. Cells produce the
ahemical components and the energy r~quired for cellular
viability and store excess energy in the form of fat.
Fat is adipose tissue stored between organs of the body
to furnish a reserve supply of energy. The preferred
animal fats and waxes have a fatty acid composition
similar to that of human fat and the fat contained in
WO93/10776 PCT/US92/08787
21 2 human breast milk. The preferred animal fats and waxes
may be selected from the group consisting of human fat,
chicken fat, cow fat (defined herein as a bovine domestic
animal regardless of sex or age), sheep fat, horse fat,
5 pig fat, and whale fat. The more preferred animal fats
and waxes may be selected from the group consisting of
human fat and chicken fat. The most preferred animal fat
is human fat. Mixtures of other fats and waxes, such as
vegetable waxes, marine oils (especially shark liver
oil), and synthetic waxes and oils, which have a fatty
acid composition similar to that of animal fats and
waxes, and preferably to that of human fats and waxes,
may also be employed. The mixture of saturated and
unsaturated fatty acids may also be derived from animal
and vegetable fats and waxes, and mixtures thereof.
In a preferred embodiment, the mixture of
saturated and unsaturated fatty acids has a composition
similar to that of human fat and comprises the following
fatty acids: butyric acid, caproic acid, caprylic acid,
capric acid, lauric acid, myristic acid, myristoleic
acid, palmitic acid, palmitoleic acid, stearic, oleic
acid, linoleic acid, linolenic acid, arachidic acid, and
gaddoleic acid. Preferably, butyric acid, caproic acid,
caprylic acid, capric ac.id, lauric acid, myristic ac~!
myristoleic acid, palmitic acid, palmitoleic acid,
8tearic, oleic acid, linoleic acid, linolenic acid,
arachidic acid, and gaddoleic acid are present in the
mixture in about the following percentages ~y weight,
respectively (carbon chain number and number of
unsaturations are shown parenthetically, respectively):
d.2%-0.4% (C4), 0.1% (C6), 0.3%-0.8% (C8), 2.2~-3.5%
~ClO), 0.9~-5.5% (Cl2), 2.8%-8.~% (Cl4), 0.1%-0 6%
~Cl4 l), 23.2%-24.6% ~Cl6), l.8%-3.0~ (Cl6 l), 6.9~-9.9%
~18~' 36.0~-36.5% (Cl~ l), 20%-20-6% (Cl8 2), 7.5-7.8%
(Cl8:3)~ l-1%-4-9% (C20), and 3.3%-6.4~ (C20 l).
In another preferred embodiment, the mixture of
saturated and unsaturated fatty acids is typically
WO93/10776 2 1 2 3 ~ r ~ PCT/US92/08787
~3
chicken fat comprising the following fatty acids: laurîc
acid, myristic acid, myristoleic acid, pentadecanoic
acid, palmitic acid, palmitoleic acid, margaric acid,
margaroleic acid, stearic, oleic acid, linoleic acid,
S linolenic acid, arachidic acid, and gaddoleic acid.
Preferably, lauric acid, myristic acid, myristoleic acid,
pentadecanoic acid, palmitic acid, palmitoleic acid,
margaric acid, margaroleic acid, stearic, oleic acid,
linoleic acid, linolenic acid, arachidic acid, and
gaddoleic acid are present in the mixture in about the
following percentages by weight, respectively: 0.1%
(C12)~ 0-8% (C14), 0-2% (Cl4 1), 0-1% (Cls), 25-3% (C16),
7-2% (C16~ 0-1~ (C17), 0-1% (C17 1), 6.5% (C18), 37.7%
(C18 1)~ 20-6% (C18 2), 0-8% (Clg 3), 0-2% ( 20)
0.3~ (C20 l), all percentages +/- 10%.
The above fatty acids and percentages thereof
present in the fatty acid mixture are given ~s an
example. The exact type of fatty acid present in the
fatty acid mixture and the exact amount of fatty acid
employed in the fatty acid mixture may be varied in order
to obtain the result desired in the final product and
such variations are now within the capabilities of those
skilled in the art without the need for undue
experimentation. .~-
~
The amount of fatty acids present in thetherapeutic compositions of the present invention is a
therapeutically effective amount. A therapeutically
effective amount of fatty acids is that amount of fatty
acids necessary to increase the repair of cellular
membranes and resuscitation rate of mammalian cells.! The
exact amount of fatty acids employed is subject to such
factors as the type and distribution of fatty acids
employed in the mixture, the type of condition being
treated, and the other ingredients in the composition.
In a preferred embodiment, the fatty acids are present in
the therapeutic composition in an amount from about 10~
to about 50%, preferably from about 20% to about 45%, and
W O 93/10776 P ~ /~S92/08787
more preferably from about 25% to about 40~, by weight of
the therapeutic composition.
In accord with the present invention, the
S therapeutic wound healing compositions for treating
mammalian cells may be selected from the group consisting
of:
(1) (a) pyruvate selected from t~e group
consisting of pyruvic acid, pharmaceutically acceptable
salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and
(c) a mix~ure of saturated and unsaturated
fatty acids whereln the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells;
(2) (a) pyruvate selected from the group
consisting of pyruvic acid, pharmaceutically acceptable
salts of pyruvic acid, and mixtures thereof;
(b) lactate selected from the group
consisting of lactic acid, pharmaceutically acceptable
salts of lactic acid, and mixtures thereof; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells; .
(3) (a) an antioxidant; and
(b) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells;
(4) ~a) lactate selected from the group
consisting of lactic acid, pharmaceutically acceptabl`e
salts of lactic acid, and mixtures thereof;
(b) an antioxidant; and
(c) a:mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells.
W093/10776 PCT/US92/08787
21231~78
In a preferred embodiment, the wound healing
compositions for treating mammalian cells, preferably
epidermal keratinocytes, may be selected from the group
consisting of:
(1) (a) pyruvate selected from the group
consisting of pyruvic acid, pharmaceutically acceptable
salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells;
(2) (a) pyruvate selected from the group
consisting of pyruvic acid, pharmaceutically acceptable
salts of pyruvic acid, and mixtures thereof;
(b) lactate selected from the group
consisting of lactic acid, pharmaceutically acceptable
salts of lactic acid, and mixtures thereof; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells; and
(3) (a) an antioxidant; and
(b) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty aci~
required for the repair of cellular membranes and
resuscitation of mammalian cells.
In a more preferred embodiment, the wound
healing compositions for treating mammalian cells,
preferably epidermal keratinocytes, may be selected from
` I the group consisting of:
(1) (a) pyruvate selected from the group
consisting of pyruvic acid, pharmaceutically acceptable
salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
WO93/10776 - PCT/US92/08787
2123678
required for the repair of cellular membranes~ and
resuscitation of mammalian cells; and
(2) (a) pyruvate selected from the group
consisting of pyruvic acid, pharmaceutically acceptable
salts of pyruvic acid, and mixtures thereof;
(b) lactate selected from the group
consisting of lactic acid, pharmaceutically acceptable
salts of lactic acid, and mixtures thereof; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells.
In a most preferred embodiment, the wound
healing compositions for treating mammalian cells,
preferably epidermal keratinocytes, comprise:
(a) pyruvate selected from the group
consisting of pyruvic acid, pharmaceutically acceptable
salts of pyruvic acid, and mixtures thereof;
(b) an antioxidant; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells.
_~
In another preferred embodiment, the wound
healing compositions for treatîng mammalian cells,
preferably monocytes, comprise:
(a) lactate selected from the group
consisting of lactic acid, pharmaceutically acceptable
salts of lactic acid, and mixtures thereof;
) an antioxidant; and
(c) a mixture of saturated and unsaturated
fatty a~ids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells.
The present invention extends to methods for
making the therapeutic wound healing compositions. In
W093/10776 2 1 2 ~ PCT/~Sg2/08787
general, a therapeutic composition is made by formlng an
admixture of the components of the composition. In one
embodiment, a therapeutic composition is made by forming
an admixture of (a) a pyruvate, (b) an antioxidant, and
(c) a mixture of saturated and unsaturated fatty acids.
In a second embodiment, a therapeutic composition is made
by forming an admixture of (a) a pyruvate, (b) a lacta~e,
and (c) a mixturP of saturated and unsaturated fatty
acids. In a third embodiment, a therapeutic composition
is made by forming an admixture of (a) an antioxidant,
and (b) a mixture of saturated and unsaturated fatty
acids. In a fourth embodiment, a therapeutic composition
i~ made by forming an admixture of (a) a lactate, ~b) an
antioxidant, and (c) a mixture of saturated and
unsaturated fatty acids.
For some applications, the admixture may be
formed in a solvent such as water. If necessary, the pH
of the solvent is adjusted to a range from about 3.5 to
abcut 8.0, and preferably from about 4.5 to about 7.5,
and more preferably about 6.0 to about 7.4. The
admixture is then sterile filtered. Other ingredients
may also ~e incorporated into the therapeutîc composition
as dictated by the nature of the desired composition a~s
well known by those having ordinary skill in the a~.
The ultimate therapeutic compositions are readily
prepared using methods generally known in the
pharmaceutical arts.
The present invention extends to methods for
employing the therapeutic wound healing compositions. In
general, ~a therapeutic composition is employed by
contacting the therapeutic composition with a wound. In
a specific embodiment, the invention is directed at a
method for healing a wound in a mammal which comprises
the steps of:
(A) providing a therapeutic wound healing
composition which co~prises:
W093/10776 PCT/USg2/08787
2 1 2-167 ~ (a) pyruvate selected from the group consisting
of pyruvic acid, pharmaceutically acceptable salts of
pyruvic acid, and mixtures thereof;
(b) an antioxidant; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
r~quired for the repair of cellular membranes and
resuscitation of mammalîan cells; and
(B) contacting the wound healing composition with
the wound.
In a first embodiment, the therapeutic
compositions may be utilized by themselves to increase
the proliferation and resuscitation rate of mammalian
cells. In a second embodiment, the therapeutic
compositions may ~e combined with a medicament useful for
treating wounds to form augmented wound healing
compositions having an enhanced ability to further
increase the proliferation and resuscitation rate of
mammalian cells.
In the first embodiment, the wound healing
therapeutic compositions may be utilized by themselves in
topical products to increase the proliferation and
resuscitation rate of mammalian cells. For example,.~e
therapeutic compositions may be used in topical skin care
products to treat wounds such as incisions and
lacerations.
In the second embodiment, the therapeutic wound
healing compositions of the present invention may be
combined with medicaments useful for treating wounds to
form augmented wound healing compositions. In this
embodiment, the combination of the therapeutic
ccmpositions of the present invention and the medicaments
useful for treating wounds provides an augmented wound
healing composition having an enhanced ability to
increase the proliferation and resuscitation rate of
mammalian cells. For example, the therapeutic
WO93/10776 2 1 2 3 ~ ~ PCT/US92/08787
compositions of the present invention may be used in
combination with medicaments useful for treating wounds
such as anti-inflammatory agents, respiratory bursting
inhibitors (lactic acid, adenosine), inhibitors of
prostaglandin synthesis (ibuprofen, aspirin,
indomethacin, meclofenomic acid, retinoic acid,
padimate O, meclomen, oxybenzone), steroidal anti-
inflammatory agents (corticosteroids including synthetic
analogs), antibacterial agents, antimicrobial agents
(neosporin ointment, silvadine)~ antiseptic agents,
anesthetic agents (pramoxine hydrochloride, lidocaine,
benzocaine), cell nutrient media, burn relief
medications, sun burn medications, acne preparations,
insect bite and sting medications, wound cleansers, wound
dressings, scar reducing agents (vitamin E),
immunostimulators (betafectin), and mixtures thereof, to
further enhance the proliferation and resuscitation rate
of mammalian cells. Preferably, the medicaments useful
for treating wounds are selected from the group
consisting of respiratory bursting inhibitors, inhibitors
of prostaglandin synthesis, antimicrobial agents, cell
nutrient media, scar reducing agents, and mixtures
thereof. More preferably, the medicament useful for
treating wounds is a cell nutrient medium.
A cell nutrient medium provides a complete diet
of nutrients necessary for wound healing. The cell
nutrient medium may be derived from animal, plant, and
yeast sources. Typical cell nutrient media includes live
yeast cell derivative, Eagles medium, and artificial
serum. A preferred cell nutrient medium is live yeast
cell deri~ative. Live yeast cell derivative supplies
skin respiratory factor which acts by increasing the
oxygen uptake o~ dermal tissues and facilitates collagen
formation. Live yeast cell derivative generally contains
numerous amino acids for collagen formation, mono- and
disaccharides as carbon sources, vitamins, minerals,
phosphorous containing compounds, nucleosides,
nucleotides, and salts. In general, the amino acids
WO93/10776 PCT/US92/08787
2123~78
present in live yeasit cell derivative include aspartic
acid, glutamic acid. histidine, serine, glycine, alanine,
arginine, tyrosine, valine, methionine, isoleucine,
leucine, phenylalanine, and lysine. The coenzymes
present in live yeast cell derivative include vitamin A,
vitamin E, vitamin D3, folic acid, pantothenic acid,
niacinamide, vitamin B1, vitamin B2, vitamin B6, and
vitamin B12. The cofactor type minerals present in live
yeast cell derivative include calcium, copper, iron,
magnesium, zinc, and phosphorus. A preferred tissue
respiratory factor is BiodynesR TRF, commercially
available from Brooks Industries, Inc., South Plainfield,
New Jersey. In general, the cell nutrient medium will be
present in the therapeutic composition in an amount from
about 0.01% to about 5%, preferably from about 0.1~ to
about 1%, and more preferably from about 0.2~ to about
0.4%, by weight of the therapeutic composition.
In a specific embodiment, the invention is
directed at an augmented wound healing composition which
comprises:
~ A) a therapeutic wound healing composition which
comprises:
(a) pyruvate selected from the group consisting
of pyruvic acid, pharmaceutically acceptable salts.iaf
pyruvic acid, and mixtures thereof;
(b) an antioxidant; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
re~uscitation of mammalian cells; and
(B) a medicament useful for treating wounds.
The present invention extends to methods for
making the augmented wound healing compositions. In
general, the augmented compositions are made by admixing
the therapeutic wound healing composition with the
medicament useful for treating wounds to prepare the
augmented wound healing composition.
W093/10776 2 1 2 3 ~ 7 8 PCT/US92/0~7X7
The present invention also extends to methods
for employing the augmented wound healing compositions~
In general, an augmented wound healing composition is
employed by contacting the composition with a wound. In
a specific embodiment, the invention is directed at a
method for healing a wound in a mammal which comprises
the steps of:
(A) providing a therapeutic wound healing
composition which comprises:
(a) pyruvate selected from the group consisting
of pyruvic acid, pharmaceutically acceptable salts of
pyruvic acid, and mixtures thereof;
(b) an antioxidant; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells;
(B) providing a medicament useful for treating
wounds; and
(C) contacting the wound healing composition from
~tep (~) and the medicament useful for treating wounds
from step (B) concurrently with the wound.
The types of wounds which may be healed using
the wound healing compositions and the augmented wound
healing compositions of the present invention are those
which result from an injury which causes epidermal damage
such as incisions, wounds in which the skin is broken by
a ~utting instrument, and lacerations, wounds in which
the skin is broken by a dull or blunt instrument. The
therapeutic compositions may also be used to treat
various dermatological disorders such as hyperkeratosis,
photo-aging, burns, donor sit~ wounds from skin
transplants, ulcers (cutaneous, decubitis, venous stasis,
and diabetic), psoriasis, skin rashes, and sunburn
photoreactive processes. The topical therapeutic
compositions may also be used orally in the form of a
mouth wash or spray to protect and accelerate the healing
WO93/10776 PCT/US92/08787
212~678
of injured oral tissue such as mouth sores and burns.
The topical therapeutic com~ositions may further be used
in ophthalmological preparations to treat wounds such as
those which result from corneal ulcers, radialkeratotomy,
corneal transplants, epikeratophakia and other surgically
induced wounds in the eye. The topical therapeutic
compositions may in addition be used in anorectal creams
and suppositories to treat such conditions as pruritus
ani, proctitis, anal fissures, and hemorrhoids. In a
preferred embodiment, the therapeutic compositions are
used to treat wounds such as incisions and lacerations.
Methods for healing a wound comprise topically
administering thé compositions of the present invention
directly to a wound site to increase the healing rate of
the wound. The composition is maintained in contact with
the wound for a period of time sufficient to increase the
proliferation and resuscitation rate of the cells.
Once prepared, the inventive therapeutic wound
healing compositions and augmented wound healing
compositions may be stored for future use or may be
formulated in effective amounts with pharmaceutically
acceptable carriers such as pharmaceutical appliances and
topical vehicles (non-oral and oral) to prepare a ~de
variety of pharmaceutical compositions.
Examples of pharmaceutical appliances are
sutures, staples, gauze, bandages, burn dressings,
artificial skins, liposome or micell formulations,
microcapsules, aqueous vehicles for soaking gauze
dressings, and the like, and mixtures thereof. Non-oral
topical compositions employ non-oral topical vehicles,
such as oils, petrolatum bases, emulsions, lotions,
creams, gels formulations, foams, ointments, sprays,
salves, and films, which are intended to be applied to
the skin or body cavity and are not intended to be taken
by mouth. Oral topical compositions employ oral
vehicles, such as mouthwashes, rinses, oral sprays,
W O 9~/10776 2 1 2 ~; 6 ~ ~3 PC~r/US92/08787
suspensions, bioadhesives, and dental gels, which are
intended to be taken by mouth but are not intended to be
ingested.
Bioadhesives are materials which adhere to live
or freshly killed mucous membranes or skin tissues.
Bioadhesives are generally water-swellable but water-
insoluble fibrous cross-linked materials. Bioadhesives
generally comprise a mucoadhesive hydrogel such as a
polyacrylic acid cross linked by a polyhydroxy compound
such as a carbohydrate (sugar, cyclitols) to form a
substantially water-insoluble hydrogel. Other
bioadhesives include carboxymethylcellulose (CMC) and
polycarbophils which are high molecular weight polymers
of acrylic acid such as CarbopolTM commercially available
from BF Goodrich Company, Cleveland, Ohio. Bioadhesi~es
are discussed in more detail in, for example, European
patent application no. 04106~6Al, to Kellaway et al., and
United States patent no. 4,615,6~7, issued to Robinson,
which disclosures are incorporated herein by ref~rence.
In one form of the invention, the therapeutic
wound healing composition is incorporated into a
pharmaceutical appliance which may be in the form of
sutures, staples, gauze, bandages, burn dressin~,
arti~icial skins, liposome or micell formulations,
microcapsules, aqueous vehicles for soaking gauze
dressings, and the like, and mixtures thereof. A variety
of traditional ingredients may optionally be included in
the pharmaceutical composition in effective amounts such
as buffers, preservatives, tonicity adjusting agents,
antioxidants, polymers for adjusting viscosity or for use
as extenders, bioadhesives, and excipients, and the like.
Specific illustrative examples of such traditional
ingredients include acetate and borate buffer~;
thimerosol, sorbic acid, methyl and propyl paraben and
chlorobutanol preservatives; sodium chloride and sugars
to adjust the tonicity; bioadhesives such as
carboxymethylcellulose (CMc), Carbopol , and
~1233~78
polycarbophil; and excipients such as mannitol, lactose
and sucrose.
In accordance with this invention,
therapeutically effective amounts of the wound healing
compositions of the present invention may be employed in
the pharmaceutical appliance. These amounts are readily
determined by those skilled in the art without the need
for undue experimentation. The exact amount of the
lo therapeutic composition employed is subject to such
factors as the type and concentration of the therapeutic
composition and the type of pharmaceutical appliance
employed. Thus, the amount of therapeutic composition
may be varied in order to obtain the result desired in
the final product and such variations are within the
capabilities of those skilled in the art without the need
for undue experimentation. In a preferred embodiment,
the pharmaceutical composition will comprise the
therapeutic composition in an amount from about 0.1% to
about 10%, by weight of the pharmaceutical composition.
In a more preferred embodiment, the pharmaceutical
composition will comprise the therapeutic composition in
an amount from about 0.1% to about 8%, by weight of the
pharmaceutical composition. In a most preferred
embodiment, the pharmaceutical composition will comp~se
the therapeutic composition in an amount from about 0.1
to about 5%, by weight of the pharmaceutical composition.
The present invention extends to methods for
making the pharmaceutical compositions. In general, a
pharmaceutical composition is made by contacting a
therapeutically effective amount of a wound healing
composition with a pharmaceutical appliance and the other
in~redients of the final desired pharmaceutical
compo~ition. The therapeutic composition may be in a
501~ent and may be absorbed onto a pharmaceutical
appliance.
W093/10776 2 1 ~ ~ ~ 7 ~ PCT/US92/08787
Other ingredients will usually be incorporated
into the wound healing composition as dictated by the
nature of the desired composition as well known by those
having ordinary skill in the art. The ultimate
pharmaceutical compositions are readily prepared using
methods generally known in the pharmaceutical arts.
In another form of the invention, the
therapeutic wound healing composition is incorporated
into a non-oral topical vehicle which may be in the form
of oils, petrolatum bases, emulsions, lotions, creams,
gels formulations, foams, ointments, sprays, salves, and
films, and the like. Typical non-toxic non-oral topical
vehicles known in the pharmaceutical arts may be used.
I The preferred non oral topical vehicles are
occlusive vehicles that minimize transepidermal water
loss from the wound site. Mi~imizing transepidermal
water loss promotes healing and reduces scarring.
Preferred non-oral topical vehicles are
petrolatum/mineral oil based products. Shark liver oil
and cod liver oil may also be included to act as
protectants to sooth and soften the tissues and minimize
transepidermal water loss. Vitamin E may also be
included to reduce scarring. Other nan-oral topi~l
vehicles such as emulsions and hydrogels may also be
employed providing that the vehicles minimize
transepidermal water loss. Other non-oral topical
vehicles include water and pharmaceutically acceptable
water-miscible organic solvents such as ethyl alcohol,
isopropyl alcohol, propylene glycol, glycerin, and the
` iike, and mixtures of these solvents.
The non-oral topical therapeutic compositions
may also contain conventional additives employed in those
products. Conventional additives include humectants,
emollients, lubricants, stabilizers, dyes, and perfumes,
providing the additives do not interfere with the
therapeutic properties of the therapeutic composition.
W093/10776 PCT/US92/08787
2123fi7(5,
Suitable humectants useful in the non-oral
topical therapeutic compositions include glycerin,
propylene glycol, polyethylene glycol, sorbitan,
fructose, and the like, and mixtures thereof~
Humectants, when employed, may be present in amounts from
about 10% to about 20%, by weight of the topical
therapeutic composition.
The coloring agents (colors, colorants) useful
in ~he non-oral topical therapeutic composition are used
in amounts effective to produce the desired color. These
coloring agents include pigments which may be
incorporated in amounts up to about 6~ by weight of the
non-oral topical therapeutic composition. A preferred
pigment, titanium dioxide, may be incorporated in amounts
up to about 2~, and preferably less than about 1%, by
weight of the non-oral topical therapeutic composition.
The coloring agents may also include natural food colors
and dyes suitable for food, drug and cosmetic
applications~ These coloring agents are known as F.D.& C.
dyes and lakes. The materials acceptable for the
foregoing uses are preferably water-soluble.
Illustrative nonlimiting examples include the indigoid
dye known as F.D.& C. Blue No.2, which is the disod~Um
8alt of 5,5-indigotindisulfonic acid. Similarly, the dye
known as F. D. & C. Green No.1 comprises a triphenylmethane
dye and is the monosodium salt of 4-~4-(N-ethyl-~-
sulfoniumbenzylamino) diphenylmethylene~-tl-(N-ethyl-N-E-
sulfoniumbenzyl)-delta-2,5-cyclohexadieneimine3. A full
recitation of a~1 F.D.& C. coloring agents and their
corresponding chemical structures may be found in the
Kirk-Othmer Encyclopedia of Chemical Technology, 3rd
Edition, in volume 5 at pages 857-884, which text is
incorporated herain by reference.
In accordance with this invention,
therapeutically effective amounts of the wound healing
compositions of the present invention may be admixed with
WO93/10776 212 3 6 ~ 8 PCT/US92/08787
a non-oral topical vehicle to form a topical therapeutic
composition. These amounts are readily determined by
those skilled in the art without the need for undue
experimentation. In a preferred embodiment, the non-oral
topical therapeutic compositions will comprise the
therapeutic composition in an amount from about 0.1% to
about 10~ and a non-oral topical vehicle in a quantity
sufficient to bring the total amount of composition to
loo~, by weight of the non-oral topical therapeutic
lo composition. In a more preferred embodiment, the non-
oral topical therapeutic compositions will comprise the
therapeutic composition in an amount from about 0.1~ to
about 10%, and in a most preferred embodiment, the non-
oral topical therapeutic compositions will comprise the
therapeutic composition in an amount from about 0.1% to
about 8%, and a non-oral topical vehicle in a quantity
sufficient to bring the total amount of composition to
100%, by weight of the non-oral topical therapeutic
composition.
The present invention extends to methods for
preparing the non-oral topical therapeutic compositions.
In such a method, the non-oral topical therapeutic
composition is prepared by admixing a therapeutically
effective amount of the wound healing composition of.~he
present invention and a non-oral topical vehicle. The
final compositions are readily prepared using standard
methods and apparatus generally known by those skilled in
the p~armaceutical arts. The apparatus useful in
accordance with the present invention comprises mixing
apparatus well known in the pharmaceutical arts, and
therefore~the selection of the specific apparatus wilI be
apparent to the artisan.
In another form of the invention, the wound
healing composition is incorporated into an oral topical
vehicle which may be in the form of a mouthwash, rinse,
oral spray, suspension, dental gel, bioadhesive, and the
like. Typical non-toxic oral vehicles known in the
WO93/10776 PCr/USs2/08787
2123 S 7 8 pharmaceutical arts may be used in the present invention.
The preferred oral vehicles are water, ethanol, and
wat2r-ethanol mixtures. The water-ethanol mixtures are
generally employed in a weight ratio ~rom about 1:1 to
about 20:1, preferably from about 3:1 to about 20:1, and
most preferably from about 3:1 to about 10:1,
respectively. The pH value of the oral vehicle is
generally from about 4 to about 7, and preferably from
about 5 to about 6.5. An oral topical vehicle having a
pH value below about 4 is generally irritating to the
oral cavity and an oral vehicle having a pH v lue greater
than about 7 generally results in an unpleasant mouth
feel.
The oral topical therapeutic compositions may
also contain conventional additives normally employed in
those products. Conventional additives include a
fluorine providing compound, a sweetening agent, a
flavoring agent, a coloring agent, a humectant, a buffer,
and an emulsifier, providing the additive~ do not
interfere with the therapeutic properties of the
therapeutic composition.
The coloring agents and humectants, and the
amounts of these additives to be employed, set out aho~e
as useful in the non-oral topical therapeutic composition
may be used in the oral topical therapeutic composition.
Fluorine providing compounds may be fully or
slightly water soluble and are characterized by their
ability to release fluoride ions or fluoride containing
ions in water and by their lack of reaction with other
components in the composition. Typical fluorine
providing compounds are inorganic fluoride salts such as
water-soluble alkali metal, alkaline earth metal, and
heavy metal salts~ for example, sodium fluoride,
potassium fluoride, ammonium fluoride, cuprous fluoride,
zinc fluoride, stannic fluoride, stannous fluoride,
barium fluoride, sodium fluorosilicate, ammonium
WO93/10776 9. ~ ? ~ - e ~. PCT/US92/0~787
~ 9
fluorosilicate, sodium fluorozirconate, sodium
monofl~orsphosphate, aluminum mono- and di-
fluorophosphates and fluorinated sodium calcium
pyrophosphate. Alkali metal fluorides, tin f luoride and
monofluorophosphates, such as sodium and stannous
fluoride, sodium mono~luorophosphate and mixtures
thereof, are preferred.
The amount of fluorine providing compound
present in the present oral topical therapeutic
composition is dependent upon the type of fluorine
providing compound employed, the solubility of the
fluorine compound, and the nature of the final oral
therapeutic composition. The amount of fluorine
lS providing compound used must be a nontoxic amount. In
general, the fluorine providing compound when used will
be present in an amount up to about 1%, preferably from
about 0.001% to about 0.1%, and most preferably from
about 0.001% to about 0.05%, by weight of the oral
topical therapeutic composition.
When sweetening agents (sweeteners) are used,
those sweeteners well known in the art, including both
natural and artificial sweeteners, may be employed. The
sweetening agent used may be selected from a wide r~ge
of materials including water-soluble sweetening agents,
water-soluble artificial sweetening agents, water-soluble
sweetening agents derived from naturally occurring water-
solu~le sweetening agents, dipeptide based sweetening
agents, and protein based sweetening agents, including
mixtures thereof~ Without being limited to particular
. sweetening agents, representative categories and examples
include:
(a) water-soluble sweetening agents such as
monosaccharides, disaccharide~ and polysaccharides such
as xylose, ribose, glucose (dextrose), mannose,
galactose, fructose (levulose), sucrose (sugar), maltose,
invert sugar (a mixture of fructose and glucose derived
from sucrose), partially hydrolyzed starch, corn syrup
WO 93tlO776 PCT/US92/08787
21~3~7~ ~
solids, dihydrochalcones, monellin, steviosides, and
glycyrrhizin, and mixtures thereof;
(b) water-soluble artificial sweeteners such
as soluble saccharin salts, i.e., sodium or calcium
saccharin salts, cyclamate salts, the sodium, ammonium or
calcium salt of 3,4-dihydro-6-methyl-1,2,3-oxathiazine-4-
one-2,2-dioxide, the potassium salt of 3,4-dihydro-6
methyl-1,2,3-oxathiazine-4-one-2,2-dioxide (Acesulfame-
K), the free acid form of saccharin, and the like;
(c) dipeptide based sweeteners, such as
L-aspartic acid derived sweeteners, such as L-aspartyl-L-
phenylalanine methyl ester (Aspartame) and materials
described in United States patent no. 3,492,131, L-alpha-
aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alanin-
amide hydrate (Alitame), methyl esters of L-aspartyl-L-
phenylglycerine and L-aspartyl-L-2,S-dihydrophenyl-
glycine, L-aspartyl-2,5-dihydro-L-phenylalanine; L-
aspartyl-L~ cyclohexen)-alanine, and the like;
(d) water-soluble sweeteners derived from
naturally occurring water-soluble sweeteners, such as
chlorinated derivatives of ordinary sugar (sucrose),
e.g., chlorodeoxysugar derivatives such as derivatives of
chlorodeoxysucrose or chlorodeoxygalactosucrose, known,
for example, under the product designation of Sucralose;
and ~-~
(e) protein based sweeteners such as
thaumaoccous danielli (Thaumatin I and II).
In general, an effective amount of sweetening
agent is utilized to provide the level of sweetness
desired in the particular oral topical therapeutic
composition, and this amount will vary with the sweetenèr
selected and the final oral therapeutic product desired.
The amount of sweetener normally present is in the range
3S from about 0.0025% to about 90%, by weight of the oral
topical therapeutic composition, depending upon the
sweetener used. The exact range of amounts for each type
of sweetener is well known in the art and is not the
subject of the present invention.
W093~10776 ~ 2 3 ;~ 7 3 PCT/US92/08787
The flavoring agents (flavors, flavorants)
which may be used include those flavors known to the
skilled artisan, such as natural and artificial flavors.
Suitable flavoring agents include mints, such as
peppermint, citrus flavors such as orange and lemon,
artificial vanilla, cinnamon, various fruit flavors, ~oth
individual and mixed, and the like.
The amount of flavoring agent employed in the
oral topical therapeutic composition is normally a matter
of preference subject to such factors as the type of
final oral therapeutic composition, the individual flavor
employed, and thé strength of flavor desired. Thus, the
lS amount of flavoring may be varied in order to obtain the
result desired in the final product and such variations
are within the capabilities of those skilled in the art
without the need ~or undue experimentation. The
flavoring agents, when used, are generally utilized in
amounts that may, for example, range in amounts from
about 0.05% to about 6%, by weight of the oral topical
therapeutic composition.
Suitable buffer solutions useful in the oral
topical therapeutic compositions include citric aç~-
sodium citrate solution, phosphoric acid-sodium phosphate
solution, and acetic acid-sodium acetate solution in
amounts up to about 1%, and preferably from about 0~05%
to about 0.5% by weight of the oral topical therapeutic
composition.
I In accordance with this invention,
therapeutically effective amounts of the wound healing
compositions of the present invention may be admixed with
an oral topical vehicle to form a topical therapeutic
composition. These amounts are readily determined by
those skilled in the art without the need for undue
experimentation. In a preferred embodiment, the oral
topical therapeutic compositions will comprise the
WO93/lQ776 PCT/US92/~8787
21236~8
therapeutic composition in an amount from about 0.1% to
about 10~ and a oral topical vehicle in a quantity
sufficient to bring the total amount of composition to
100~, by weight of the oral topical therapeutic
composition. In a more preferred embodiment, the oral
tcpical therapeutic compositions will comprise the
therapeutic composition in an amount from about 0.1% to
about 10%, and in a most preferred embodiment, the oral
topical therapeutic compositions will comprise the
therapeutic composition in an amount from about 0.1% to
about 8%, and a oral topical vehicle in a quantity
sufficient to bring the total amount of composition to
100%, by weight of the oral topical therapeutic
composition.
The present invention extends to methods for
preparing the oral topical therapeutic compositions. In
such a method, the oral topical therapeutic composition
is prepared by admixing a therapeutically effective
amount of the therapeutic composition of the present
invention and an oral topical vehicle. The final
compositions are readily prepared using standard methods
and apparatus generally known by those skilled in the
pharmaceutical arts. The apparatus useful in accordance
with the present invention comprises mixing appar.~t~s
well known in the pharmaceutical arts, and therefore the
selection of the specific apparatus will be apparent to
the artisan.
In a preferred embodiment, an oral topical
therapeutic composition is made by first dissolving
coloring agents, sweetening agents, and similar additives
in water. The therapeutic composition is then admixed
with the aqueous solution. Then sufficient water or
ethanol, or mixtures of water and ethanol, are added to
the solution with mixing until the final solution volume
is reached. In a more preferred embodiment, the
therapeutic composition is added to the solution as the
final ingredient. The final oral topical therapeutic
W093/10776 21 2 ~ v 7 8 PCT/US92/08787
. 4~
compositions are readily prepared using methods generally
known in the pharmaceutical arts.
The oral therapeutic composition may also be in
the form of ~ental gel. ~s used herein, the term "gel"
means a solid or semisolid colloid which contains
considerable quantities of water. The colloid particles
in a gel are linked together in a coherent meshwork which
immobilizes the water contained inside the meshwork.
The dental gel compositions of the present
invention may contain the conventional additives set out
above for oral topical therapeutic compositions such as
mouthwashes, rinsés, oral sprays, and suspensions and, in
addition, may contain additional additives such as a
polishing agent, a desensitizing agent, and the like,
providing the additional additives do not interfere with
the therapeutic properties of the therapeutic
composition.
In a dental gel composition, the oral vehicle
generally comprises water, typically in an amount from
about 10% to about 90%, by weight of the dental gel
composition. Polyethylene glycol, propylene glycol,
glycerin, and mixtures thereof may also be present in.the
v~hicle as humectants or binders in amounts from about
18% to about 30%, by weight of the dental gel
composition. Particularly preferred oral vehicles
comprise mixtures of water with polyethylene glycol or
water with glycerin and polypropylene glycol.
I The dental gels of the present invention
include a gelling agent (thickening agent) such as a
natural or synthetic gum or gelatin. Gelling agents such
as hydroxyethyl cellulose, methyl cellulose, glycerin,
carboxypolymethylene, and gelatin and the like, and
mixtures thereof may be used. Carboxymethylcellulose
(CMC)I CarbopolTM, and polycarbophil may also be used.
The preferred gelling agent is hydroxyethyl cellulose.
W O 93/10776 PC~r/US92/08787
212~678
Gelling agents may be used in amounts from about 0.5% to
about 5%, and preferably from about O.S~ to about 2%, by
weight of the dental gel composition.
The dental gel compositions of the present
invention may also include a polishing agent. In clear
gels, a polishing agent of colloidal silica and/or alkali
metal aluminosilicate complexes is preferred since these
materials have refractive indices close to the refractive
indices of the gelling systems commonly used in dental
gels. In non-clear gels, a polishing agent of calcium
carbonate or calcium dihydrate may be used. These
poli~hin~ agents may be used in amounts up to about 75%,
and preferably in amounts up to about 50%, by weight of
the dental gel composition.
The dental gel may also contain a desensitizing
agent such as a combination of citric acid and sodium
citrate. Citric acid may be used in an amount from about
0.1% to about 3%, and preferably from about 0.2~ to about
1%, by weight, and sodium citrate may be used in an
amount from about 0.3% to about ~, and preferably from
abo~t 0.6% to about 3%, by weight of the dental gel
composition.
~
In accordance with this invention,
therapeutically effective amounts of the wound healing
compositions of the present invention may be admixed into
the dental gel compositions. These amounts are readily
determined by those skilled in the art without the need
for undue experimentation. In a preferred embodiment,
the dental gel compositions will comprise the therapeutic
composition in an amount from about 0~1% to about 10% and
an oral topical vehicle in a quantity sufficient to bring
the total amount of composition to 100%, by weight of the
dental gel composition. In a more preferred embodiment,
the dental gel compositions will comprise the therapeutic
composition in an amount from about 0.1% to about 5%, and
in a most preferred embodiment, the dental gel
7 ~
W O 93/10776 PC~r/US92/08787
compositions will comprise the therapeutic composition in
an amount from about 0.1% to about 2%, and an oral
topical vehicle in a quantity sufficient to bring the
total amount of composition to 100%, by weight of the
dental gel composition.
The present invention extends to methods for
preparing the therapeutic dental gel compositions. In
such a method, the dental gel composition is prepared by
admixing a therapeutically effective amount of the
therapeutic compo~ition of the present invention and an
oral topical vehicle. The final compositions are readily
prepared using methods generally known by those skilled
in the dental and pharmaceutical arts. The apparatus
lS useful in accordance with the present invention comprises
mixing apparatus well known in the pharmaceutical arts,
and therefore the selection of the specific apparatus
will be apparent to the artisan.
In a preferred embodiment, a therapeutic dental
gel composition is made by first dispersing a gelling
agent in a h~mectant or water, or a mixture of both, then
admixing to the dispersion an aqueous solution of the
water-soluble additives such as the fluorine providing
compound, ~weeteners and the like, then adding ~e
polishing agent, and lastly admixing the flavoring agent
and the therapeutic composition. The final gel mixture
is then tubed or otherwise packaged. The liquids and
solids in a gel product are propor~ioned to form a creamy
or gelled mass which is extrudable from a pressuri~ed
co~tainer or from a collapsible tube. The final
therapeutic compositions are readily prepared using
methods generally known in the pharmaceutical arts.
In a specific embodiment, the invention is
directed at a wound healing pharmaceutical composition
which comprises a pharmaceutically acceptable carrier and
a therapèutically effective amount of a wound healing
composition which comprises:
WO93/10775 PCT/US92/08787
212'3fi 18 ~
(a) pyruvate selected from the group consisting
of pyruvic acid, pharmaceutically acceptable salts of
pyruvic acid, and mixtures thereof;
(b) an antioxidant; and
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
required for the repair of cellular membranes and
resuscitation of mammalian cells.
The pharmaceutically acceptable carrier may be
selected from the group consisting of pharmaceutical
appliances and topical vehicles. Preferably, the topical
vehicle is an occlusive vehicle.
In another specific embodiment, the invention
is directed at a method for preparing a pharmaceutical
composition for increasing t~e proliferation and
resuscitation ra~e of mammalian cells, which comprises
the steps of:
(A) providing a therapeutically effective amount of
a wound healing composition which comprises:
(a) pyruvate selected from the group consisting
of pyruvic acid, pharmaceutically acceptable salts of
pyruvic acid, and mixtures thereof;
(b) an antioxidant; and ~
(c) a mixture of saturated and unsaturated
fatty acids wherein the fatty acids are those fatty acids
re~uired for the repair of cellular membranes and
resuscitation of mammalian cells;
(B) providing a pharmaceutically acceptable carrier;
and
(C) admixing the wound healing composition from
step (A) and the pharmaceutically acceptable carrier from
step (B~ to form a pharmaceutical composition.
Throughout this application, various
publications have been referenced. The disclosures in
these publications are incorporated herein by reference
in order to more fully describe the state of the art.
W093/10776 2 ~ ~ ~ fi,'3 PCT/US92tO8787
~7
The present invention is further illustrated by
the following examples which are not intended to limit
the effective scope of the claims. All parts and
percentages in the examples and throughout the
specification and claims are by weight of the final
composition unless o~herwise specified.
E~AMPLE~ 1-5
These examples demonstrate a comparison of the
wound healing abilities of the therapeutic wound healing
compositions of the present invention versus conventional
wound healing Compositions.
15,".,
The wound healing compositions of Examples A-D
were prepared having the compositions set out in Table l.
Table l
Examples
Ingredient A B C D
Prep. H
sodium pyruvate -- 2%
vitamin E -- l% -- --
_ _
chicken fat -- 2% -- --
LYCD 2000 U 2400 U 2400 U --
_ _
shark liver oil 3~ 3% 3% --
!
petrolatum in 64% 66.5% 68%
mineral oil amounts 22.53% 25.03% 2~.8
paraffin totaling 5% 5% 5%
emulsifier 100%* 0.2% 0.2% 0.2%
. ~
These components are present in Preparation HTM
W093/10776 PCT/US~2/08787
21235~ Wound healing composition A was commercially
available Preparation HTM. Wound healing composition B
was a petrolatum base formulation containing live yeast
cell derivative, shark oil, and a mixture of sodium
pyruvate, vitamin E, and chicken fat~ Wound healing
composition C was a petrolatum base formulation
containing live yeast cell derivative and shark oil.
Wound healing composition D was a petrolatum base
formulation only.
Wound healing studies were carried out using
hairless mice (SKR-l, Charles River) 6-8 weeks in age.
One group of mice were untreated as a control group and
were referred to as Example E. In each group there were
15~ 6 mice for evaluation at either day 3 or day 7 for a
total number of 60 animals in the study. The mice were
anesthetized with ether and a midline 3 cm full thickness
longitudinal incision was made with a number lO scalpel
blade. Incisions were closed using steel clips at l cm
intervals. Formulations A-D set out above were applied
in a randomized blinded study to the wounds on day 0 at 2
hours following wounding and reapplied at 24 hour
intervals during the 7 days of the ~tudy. The wounds
were examined daily and scored on ~ basis of 0-5 for
closure on each day of the study, with a score of 5
representing the wound best healed.
The animals were sacrificed on day 3 and day 7
using cervical dislocation. The dorsal skin including
the inci~ion was dissected without the subcutaneous
tissue. The skin was placed in neutral buffered formalin
and subsequently sectioned and stained with hematoxylin
and eosin. The wounds were examined microscopicaIly and
representati~e tissue sections were photoyraphed.
On each day of the experiment, the score and
rank order of the formulations for closure of wounds and
speed of healing were as follows:
WO 93/10776 2 1 2 3 ~7 8 PCrtUS92/û8787
~9
B (5) >> D t4) ~> C (2) >/= E, Control (2) > A (l)
Photographs of the wounded mice on day 4 are set out in
FIGURES 1 and 2.
FIGURES 1 and 2 show that Formulation ~, which
was a petrolatum base formulation containing live yeast
cell derivative, shark oil, and a mixture of sodium
pyruvate, vitamin E, and chicken fat, was a significantly
better wound healing agent than the other formulations.
These results are supported by the subjective grading of
the wound closures and the speed of healing on each day
(1-7) of the experiment as well as on the objective
histological examination of tissue sections to measure
15~ the extent of inflammatory cell infiltrate within the
wound and the extent of epithelialization at the wound
edges. The final result was that less scar tissue was
present at day 7 on the mice treated with Formulation B.
Formulation D, which was a white petrolatum
formulation only, was judged to be significantly more
effective to promote healing than either Formulation C,
which was a petrolatum base formulation containing shark
liver oil and live yeast cell derivative, or
Formulation A, which was Preparation HTM. The superior
ability of Formulation D over Formulation C to improve
healing may result from a delay in the healing process
caused when the live yeast cell derivative is depleted
and the cells shift to an alternative nutrient source.
The presence of the mixture of sodium pyruvate,
vitamin E, and chicken fat in Formulation B apparently
j ~ offsets the depletion of the live yeast cell deri~ative.
Formulation C, which was a petrolatum base
formulation containing live yeast cell derivative and
shark oil, was judged comparable to the control
(untreated wound) in speed of wound closure and extent of
healing. Formulation A, which was Preparation HTM,
appeared to be the least effective healing formulation by
WO93/10776 PCT/US~2/08787
~`~ ~ both subjective grading of wound healing and by objective
examination of tissue sections. The superior ability of
Formulation D and Formulation c over Formulation A to
improve healing may be due to their ability to act as an
occlusive wound dressing tha~ prevents transepidermal
water loss and thus promotes healing and wound closure.
The poor ability of Formulation A to improve healing may
be due to the potential cytotoxicity of phenylmercuric
nitrate present in Preparation HTM as a preservative~
These results show that the wound healing
compositions of the present invention which comprise a
mixture of sodium pyruvate, vitamin E, and chicken fat
inrrease the proliferation and resuscitation rate of
mammalian cells. The wound healing compositions mediate
low levels of oxygen in the initial stages of healing to
suppress oxidative damage and hig~er levels of oxygen in
the later stages of healing to promote .collagen
formation.
The invention being thus described, it will be
obvious that the same may be varied in many ways. Such
variations are not to be regarded as a departure from the
spirit and scope of the invention and all such
modifications are intended to be included within the
scope of the following claims.
