CA2123678A1

CA2123678A1 - Wound healing compositions containing a pyruvate, an antioxidant and a mixture of fatty acids

Info

Publication number: CA2123678A1
Application number: CA002123678A
Authority: CA
Inventors: Alain Martin
Original assignee: Individual
Current assignee: Warner Lambert Co LLC
Priority date: 1991-11-26
Filing date: 1992-10-15
Publication date: 1993-06-10
Also published as: WO1993010776A1; NZ245275A; AU674131B2; JP3506701B2; EP0614359A1; JPH07501339A; AU2808992A; MX9206792A; ZA929155B

Abstract

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 fatty acids wherein the fatty acids are those fatty acids required for repair of cellular membranes and resuscitation of mammalian cells. The therapeutic compositions may be utilized in a wide variety of topical and ingestible pharmaceutical products. This invention also relates to methods for preparing and using the therapeutic compositions and the topical pharmaceutical products in which the therapeutic compositions may be used.

Description

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 ~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-), . 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 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.

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.

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 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 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 6 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.

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 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 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 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.

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:

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% -- --_ _ 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.

Claims

I claim:

1. 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.

2. The composition according to claim 1, wherein the mammalian cells comprise epidermal keratinocytes.

3. The composition according to claim 1, wherein 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.

4. The composition according to claim 3, wherein the pyruvate is sodium pyruvate.

5. The composition according to claim 1, wherein the antioxidant is selected from the group consisting of retinol, 3, 4-didehydroretinol, alpha-carotene, beta-carotene, gamma-carotene, delta-carotene, ascorbic acid, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, and mixtures thereof.

6. The composition according to claim 5, wherein the antioxidant is alpha-tocopherol.

7. The composition according to claim 1, wherein the mixture of saturated and unsaturated fatty acids comprises animal and vegetable fats and waxes.

8. The composition according to claim 7, wherein the mixture of saturated and unsaturated fatty acids comprises human fat, chicken fat, cow fat, sheep fat, horse fat, pig fat, and whale fat.

9. The composition according to claim 8, wherein the mixture of saturated and unsaturated fatty acids comprises 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.

10. The composition according to claim 1, wherein pyruvate is present in the therapeutic composition in an amount from about 10% to about 50%, by weight of the therapeutic composition.

11. The composition according to claim 1, wherein the antioxidant is present in the therapeutic composition in an amount from about 10% to about 50%, by weight of the therapeutic composition.

12. The composition according to claim 1, wherein the mixture of saturated and unsaturated fatty acids is present in the therapeutic composition in an amount from about 10% to about 50%, by weight of the therapeutic composition.

13. A method for preparing a therapeutic wound healing composition which comprises the steps of admixing the following ingredients:
(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.

14. 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; and (B) contacting the wound healing composition with the wound.

15. 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 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; and (B) a medicament useful for treating wounds.

16. The augmented wound healing composition according to claim 15, wherein the medicament useful for treating wounds is selected from the group consisting of anti-inflammatory agents, respiratory bursting inhibitors, inhibitors of prostaglandin synthesis, antibacterial agents, antimicrobial agents, antiseptic agents, anesthetic agents, cell nutrient media, burn relief medications, sun burn medications, acne preparations, insect bite and sting medications, wound cleansers, wound dressings, scar reducing agents, immunostimulators, and mixtures thereof.

17. A method for preparing an augmented wound healing composition 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) admixing the wound healing composition from step (A) with the medicament useful for treating wounds from step (B) to prepare the augmented wound healing composition.

18. 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 step (A) and the medicament useful for treating wounds from step (B) concurrently with the wound.

19. A wound healing pharmaceutical composition which comprises a pharmaceutically acceptable carrier and 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 required for the repair of cellular membranes and resuscitation of mammalian cells.

20. The pharmaceutical composition according to claim 19, wherein the pharmaceutically acceptable carrier is a pharmaceutical appliance.

21. The pharmaceutical composition according to claim 19, wherein the pharmaceutically acceptable carrier is a bioadhesive.

22. The pharmaceutical composition according to claim 19, wherein the pharmaceutically acceptable carrier is a topical vehicle.

23. The pharmaceutical composition according to claim 22, wherein the topical vehicle is an occlusive vehicle.

24. The pharmaceutical composition according to claim 19, wherein the wound healing composition is present in the pharmaceutical composition in an amount from about 0.1% to about 10%, by weight.

25. A method for preparing a wound healing pharmaceutical composition 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 required 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.